RU2800026C2 - MODIFIED CLOSED-ENDED DNA (scDNA) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions is described, including a non-viral capsidless DNA vector with covalently closed ends for nucleic acid delivery, an scDNA expression construct for producing a scDNA vector, a host cell producing an scDNA expression construct, a method of obtaining a scDNA vector, the use of a composition containing scDNA a vector for producing a medicinal product for delivering a therapeutic protein to a subject to correct an abnormal level and/or function of a gene product, a set for delivering a nucleic acid, and a set for producing a scDNA vector.

EFFECT: invention expands the arsenal of agents for delivering nucleic acids.

62 cl, 62 dwg, 17 tbl, 5 ex

Description

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority under 35 USC §119(e) based on Provisional Applications for US Patent No. 62/556319; No. 62/556324; No. 62/556329; No. 62/556331; 62/556281 and 62/556335, each filed September 8, 2017, the contents of each of which are hereby incorporated by reference in their entirety.

SEQUENCE LIST

[0002] This application includes a sequence listing filed electronically in ASCII format and incorporated herein in its entirety by reference. The specified ASCII copy, created on September 7, 2018, is named 080170-090580WOPT SL.txt and is 205991 bytes in size.

FIELD OF TECHNOLOGY

[0003] The present invention relates to the field of gene therapy, including the delivery of exogenous DNA sequences to a target cell, tissue, organ, or organism.

BACKGROUND OF THE INVENTION

[0004] The goal of gene therapy is to improve clinical outcomes for patients suffering from genetic mutations or acquired diseases caused by aberrations in the gene expression profile. Gene therapy includes the treatment or prevention of medical conditions due to defective genes or abnormal regulation or expression, such as underexpression or overexpression, which can lead to a disorder, disease, cancer, and the like. For example, treatment, prevention, or alleviation of a disease or disorder caused by a defective gene may be accomplished by delivering corrective genetic material to a patient resulting in therapeutic expression of said genetic material in said patient. Gene therapy is based on providing a transcriptional cassette with an active gene product (sometimes referred to as a transgene), for example, which can lead to a positive gain-of-function effect, a negative loss-of-function effect, or another outcome such as, for example, an oncolytic effect. Gene therapy may also be used to treat disease or cancer caused by other factors. Treatment of human monogenic disorders can be carried out by delivery and expression of a normal gene into target cells. Delivery and expression of the corrective gene in target cells (target cells) of the patient can be carried out using numerous methods, including the use of engineered viruses and viral vectors for gene delivery. Among the many viral-derived vectors available (eg, from recombinant retrovirus, recombinant lentivirus, recombinant adenovirus, etc.), recombinant adeno-associated virus (rAAV) is gaining popularity as a multipurpose gene therapy vector.

[0005] Adeno-associated viruses (AAV) belong to the Parvoviridae family; more specifically, they constitute the genus Dependoparvovirus. The AAV genome consists of a linear single-stranded DNA molecule that contains approximately 4.7 kb. (kb) and consists of two main open reading frames (ORFs) encoding the non-structural protein Rep (replication) and the structural protein Cap (capsid). A second ORF has been identified in the cap gene that encodes an assembly activating protein (AΑΡ). The DNA flanking the AAV coding regions are two cis-acting inverted terminal repeat (ITR) sequences of approximately 145 nucleotides in length, with discontinuous palindromic sequences that can fold into energetically stable hairpin structures that function as primers for DNA replication. Along with a role in DNA replication, these ITR sequences have been shown to be involved in viral DNA integration into the cellular genome, rescue from the host genome or plasmid, and capsid capsidization of the viral nucleic acid in mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129).

[0006] AAV-derived vectors (i.e., recombinant AAV-(rAVV) or AAV-vectors) are an attractive method for delivering genetic material because (i) they are able to infect (transduce) a wide range of non-dividing and dividing cell types, including myocytes and neurons; (ii) they lack viral structural genes, resulting in reduced host cell responses to viral infection, such as interferon-mediated responses; (iii) wild-type viruses are considered non-pathogenic to humans; (iv) unlike wild-type AAV, which is capable of integrating into the host cell genome, replication-defective AAV vectors lack the rep gene and typically persist as episomes, limiting the risk of insertional mutagenesis or genotoxicity; and (v) compared to other vector systems, AAV vectors are generally considered to be relatively weak immunogens and, accordingly, do not trigger a significant immune response (see ii), thus increasing vector DNA persistence and potentially long-term expression of therapeutic transgenes. AAV vectors can also be produced and formulated at high titers and delivered via intra-arterial, intravenous, or intraperitoneal injections, allowing vector distribution and gene transfer to significant muscle regions by a single injection in rodents (Goyenvalle et al., 2004; Fougerousse et al., 2007; Koppanati et al., 20 10; Wang et al., 2009) and dogs. In a clinical trial for the treatment of spinal muscular dystrophy type 1, AAV vectors were delivered systemically to target the brain, resulting in visible clinical improvements.

[0007] However, the use of AAV particles as a gene delivery vector has a number of serious disadvantages. One major disadvantage associated with rAAV is the limited viral packaging capacity of approximately 4.5 kb of heterologous DNA (Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010). As a result, the use of AAV vectors has been limited to encoding less than 15,000 Da of protein. The second disadvantage is that, due to the prevalence of wild-type AAV infection in the population, candidates for rAAV gene therapy must be screened for the presence of neutralizing antibodies that eliminate the vector from the patient's body. A third disadvantage is related to the immunogenicity of the capsid, which prevents re-administration in patients who are not excluded from the primary treatment. The patient's immune system can respond to a vector that effectively acts as a "booster" to stimulate the immune system to generate high titers of anti-AAV antibodies, precluding future treatment. Some recent reports have described problems with immunogenicity in high dose situations. Another notable drawback is the relatively slow onset of AAV-mediated gene expression, given that single-stranded AAV DNA must be converted to double-stranded DNA prior to heterologous gene expression. Although attempts have been made to circumvent this problem by constructing double-stranded DNA vectors, this strategy further limits the size of the transgene expression cassette that can be integrated into an AAV vector (McCarty, 2008; Varenika et al., 2009; Foust et al., 2009).

[0008] In addition, standard AAV virions with capsids are obtained by introducing a plasmid or plasmids containing the AAV genome, rep genes and cap genes (Grimm et al., 1998). With the introduction of these helper (auxiliary) plasmids in the trans position, the AAV genome is “rescued” (i.e., released with subsequent amplification) from the host genome, and further enclosed in a capsid (viral capsids) to obtain biologically active AAV vectors. However, such capsid-encapsulated viral AAV vectors have been found to inefficiently transduce certain types of cells and tissues. These capsids also induce an immune response.

[0009] Accordingly, the use of adeno-associated virus (AAV) vectors for gene therapy is limited to single administration to patients (due to immune response in patients), the limited range of transgenic genetic material suitable for delivery in AAV vectors due to the minimal viral packaging capacity (approximately 4.5 kb) of the associated AAV capsid, as well as slow AAV-mediated gene expression. The use of clinical gene therapy using rAAV is further hampered by interpatient variability, which cannot be predicted based on dose dependence in syngeneic mouse models or other model species.

[0010] Recombinant capsidless AAV vectors can be generated as an isolated linear nucleic acid molecule containing an expressed transgene and promoter regions flanked by two wild-type AAV inverted terminal repeat (ITR) sequences, including a Rep binding site and a terminal clearance site. Said recombinant AAV vectors lack the coding sequences for the AAV capsid protein, and may be single stranded, double stranded or duplex, with one or both ends covalently linked via two wild-type ITR palindromic sequences (e.g., WO 2012/123430, US Pat. No. 9,598,703). They avoid many of the problems of AAV-mediated gene therapy, in terms of significantly greater transgene capacity, rapid onset of transgene expression, and actual recognition by the patient's immune system of DNA molecules as a virus to be eliminated. However, persistent transgene expression may not be desirable in all cases, and canonical wild-type AAV ITRs may not be optimized for ccDNA functions. Therefore, there remains an important unmet need for controlled recombinant DNA vectors with improved production and/or expression characteristics.

BRIEF DESCRIPTION OF THE INVENTION

[0011] The invention described herein relates to a non-viral, non-capsid DNA vector with covalently closed ends (referred to herein as a "closed-ended DNA vector" or "scDNA vector"). The scDNA vectors described herein are capsidless linear duplex DNA molecules formed by a continuous chain of complementary DNA with covalently closed ends (linear, continuous and capsidless structure) that contain an inverted 5'-terminal repeat (ITR) sequence and a 3'-ITR sequence that are different from each other, or asymmetric relative to each other.

[0012] The technology described herein relates to a ccDNA vector containing at least one modified AAV inverted terminal repeat (ITR) sequence and an expressible transgene. The ccDNA vectors as described herein can be produced in eukaryotic cells and are therefore free from prokaryotic DNA modifications and bacterial endotoxin contamination in insect cells.

[0013] In one aspect, non-viral capsidless DNA vectors with covalently closed ends are preferably linear duplex molecules, and can be derived from a vector polynucleotide that encodes a heterologous nucleic acid functionally (i.e. capable of exercising its function) located between two different sequences of inverted terminal repeats (ITR) (for example, ITR AAV), characterized in that at least one of the specified I The TR contains an end resolution site and a replication protein (RPS) binding site (sometimes referred to as a replicative protein binding site), eg, a Rep binding site; and one of the ITRs contains a deletion, insertion, or substitution with respect to the other ITR, i.e., one of the ITRs is asymmetric with respect to the other ITR. In one embodiment, at least one of the ITRs is an AAV ITR, such as a wild-type AAV ITR or a modified AAV ITR. In one embodiment, at least one of the ITRs is an ITR modified from the other ITR—that is, the scDNA contains ITRs that are asymmetric with respect to each other. In one embodiment, at least one of the ITRs is a non-functional ITR.

[0014] In some embodiments, said scDNA vector contains: (1) an expression cassette containing a cis regulatory element, a promoter, and at least one transgene; or (2) a promoter operably linked to at least one transgene, and (3) two self-complementary sequences, eg, ITRs, flanking said expression cassette, said scDNA vector not associated with a capsid protein. In some embodiments, said scDNA vector contains two self-complementary sequences found in the AAV genome, of which at least one contains a functional Rep binding element (RBE) (also sometimes referred to herein as "RBS") and an AAV terminal clearance site (trs), or a functional RBE variant, and one or more cis-regulatory elements operably linked to the transgene. In some embodiments, said scDNA vector contains additional components for regulating transgene expression, for example, regulatory switches, which are described herein in the "Regulatory Switches" section, for controlling and regulating the expression of said transgene, and may include a regulatory switch, for example, a "safety switch", providing controlled cell death of the cell containing the scDNA vector.

[0015] In some embodiments, the two self-complementary sequences may be ITR sequences from any known parvovirus, eg, dependovirus, such as AAV (eg, AAV1-AAV12). Any AAV serotype can be used, including, but not limited to, a modified AAV2 ITR sequence that retains a Rep binding site (RBS) such as 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) and a terminal clearance site (trs), along with a variable palindromic sequence that provides a hairpin secondary structure. In some embodiments, said ITR is a synthetic ITR sequence that retains a functional Rep binding site (RBS) such as 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) and a terminal resolution site (TRS), along with a variable palindromic sequence that provides a hairpin secondary structure. In some examples, the modified ITR sequence retains the RBS sequence, trs, and the structure and position of the Rep binding element forming the terminal loop portion of the hairpin secondary structure of one of the ITRs from the corresponding wild-type AAV2 ITR sequence.

[0016] Exemplary ITR sequences for use in ccDNA vectors are disclosed in any one or more of Tables 2-10A and 10B, or in SEQ ID NOs: 2, 52, 101-499, and 545-547, or in the partial ITR sequences shown in FIG. 26A-26B. In some embodiments, said scDNA vectors do not contain an ITR that contains any sequence selected from SEQ ID NOs: 500-529.

[0017] In some embodiments, the scDNA vector may comprise an ITR with a modification in the ITR corresponding to any of the modifications in the ITR sequences or partial ITR sequences shown in any one or more of Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A, and 10B herein.

[0018] In an illustrative example, the present invention provides a closed-ended DNA vector containing a promoter operably linked to a transgene, wherein the scDNA contains no capsid proteins and: (a) is derived from an scDNA plasmid (e.g., see Examples 1-2 and/or FIGS. 1A-B) that encodes a mutated AAV2 right-ITR with the same number of intramolecular duplex base pairs as and SEQ ID NO: 2, or a mutated AAV2 left ITR with the same number of intramolecular duplex base pairs as SEQ ID NO: 51, in a hairpin secondary configuration (preferably excluding the deletion of any terminal loop of AAA or TTT in said configuration relative to said reference sequences); and (b) identified as ccDNA using native and denaturing agarose gel electrophoresis assay to identify ccDNA according to Example 1. Examples of such modified ITR sequences are shown in Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A and 10B herein.

[0019] The technology described herein also relates to a scDNA vector that can deliver to a target cell and encode in the target cell one or more transgenes, e.g., a scDNA vector that contains a multicistronic sequence, or a scDNA vector into which the transgene is inserted along with its natural genomic context (e.g., transgene, introns, and endogenous untranslated regions). Transgenes can be protein-coding transcripts, non-coding transcripts, or both. Said ccDNA vector may contain multiple coding sequences and a non-canonical translation initiation site, or more than one promoter for the expression of protein-coding transcripts, non-coding transcripts, or both. Said transgene may contain a sequence encoding more than one protein, or may be a sequence of a non-coding transcript. The expression cassette may contain, for example, more than 4,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any number in the range from about 4,000 to 10,000 nucleotides, or from 10,000 to 50,000 nucleotides, or more than 50,000 nucleotides. These scDNA vectors are not size limited as are the capsid-encapsulated AAV vectors and thus allow delivery of a large size expression cassette to ensure efficient expression of the transgenes. In some embodiments, said scDNA vector lacks specific prokaryotic methylation.

[0020] Said expression cassette may also contain an internal ribosome entry site (IRES) and/or a 2A element. Cis-regulatory elements include, but are not limited to, a promoter, riboswitch, insulator, miRNA-regulated element, post-transcriptional regulatory element, tissue-specific and cell-specific promoter, and enhancer. In some embodiments, said ITR may act as a promoter for the transgene. In some embodiments, said scDNA vector contains additional components to regulate expression of the transgene. For example, said additional regulatory component may be a regulatory switch as described herein, including, but not limited to, a "kill switch" that can kill (kill) an infected ccDNA cell when needed, and other inducible and/or repressible elements.

[0021] The technology described herein also relates to novel methods for the delivery and efficient and selective expression of one or more transgenes using scDNA vectors. The zcDNA vector can be taken up by host cells and also transported into the nucleus in the absence of an AAV capsid. In addition, the scDNA vectors described herein lack a capsid and thus avoid the immune response that can be elicited in response to capsid-containing vectors.

[0022] Aspects of the present invention relate to methods for producing ccDNA vectors described herein. Other implementation options relate to the scDNA vector obtained using the method proposed according to the present invention. In one embodiment, said capsid-free, non-viral DNA vector (scDNA vector) is derived from a plasmid (referred to herein as a "scDNA plasmid") containing an expression construct polynucleotide template containing, in this order: a first inverted 5'-terminal repeat (eg, AAV ITR); expression cassette; and 3'-ITRs (e.g., AAV ITRs), wherein at least one of the 5'- and 3'-ITRs is a modified ITR, or if both 5' and 3' ITRs are modified, they contain different modifications and their sequences are not the same.

[0023] An scDNA vector as described herein can be obtained using a number of methods known to one of skill in the art after reading this description. For example, the expression construct polynucleotide template used to generate the scDNA vectors of the present invention may be a scDNA plasmid (eg, see Table 12 or FIG. 10B), an scDNA bacmid, and/or an scDNA baculovirus. In one embodiment, said zcDNA plasmid contains a cloning restriction site (e.g., SEQ ID NO: 7) operably located between the ITR, where an expression cassette can be inserted containing, e.g., a promoter operably linked to the transgene, e.g., a reporter gene and/or a therapeutic gene). In some embodiments, scDNA vectors are derived from a polynucleotide template (e.g., scDNA plasmids, scDNA bacmids, scDNA baculovirus) containing an ITR modified from the corresponding wild-type AAV3 or AAV2 ITR flanking ITR sequence, wherein the modification is any one or more of a deletion, insertion, and/or substitution.

[0024] In a permissive host cell, in the presence of, for example, Rep, a polynucleotide template containing at least one modified ITR is replicated to produce scDNA vectors. Obtaining the scDNA vector proceeds in two stages: firstly, excision (“rescue”) of the template from the backbone of the template (for example, scDNA plasmids, scDNA bacmids, scDNA baculovirus genome, etc.) by Rep proteins, and secondly, Rep-mediated replication of the excised scDNA vector. The Rep proteins and Rep binding sites of the various AAV serotypes are well known to those skilled in the art. The skilled artisan knows how to select a Rep protein from a serotype that binds and replicates a nucleic acid sequence based on at least one functional ITR. For example, if a replication-competent ITR is from AAV serotype 2, the corresponding Rep must be from an AAV serotype that works with said serotype, e.g. AAV2 ITR with Rep AAV2 or AAV4, but not with Rep AAV5, which will not work. After replication, the covalently closed scDNA vector continues to accumulate in permissive cells, and the scDNA vector is preferably sufficiently stable over time in the presence of the Rep protein under standard conditions for replication, e.g., to accumulate at least 1 pg/cell, preferably at least 2 pg/cell, preferably at least 3 pg/cell, more preferably at least 4 pg/cell, even more preferably at least 5 pg/cell.

[0025] Accordingly, according to one aspect, the present invention relates to a process comprising the following steps: a) incubation of a population of host cells (e.g., insect cells) bearing a polynucleotide template expression construct (e.g., scDNA plasmid, scDNA bacmid, and/or scDNA baculovirus) that does not contain viral capsid coding sequences, in the presence of a Rep protein under conditions effective for, and over time, sufficient to induce the production of said zcDNA vector in host cells, wherein said host cells do not contain viral capsid coding sequences; and b) collecting and isolating the scDNA vector from host cells. The presence of the Rep protein induces replication of the ITR-modified vector polynucleotide to produce a scDNA vector in the host cell. However, viral particles (eg AAV virions) are not expressed. Accordingly, there are no size restrictions due to virions.

[0026] The presence of a host-derived scDNA vector can be confirmed by digesting the host cell-derived DNA with a restriction enzyme having a single recognition site on said scDNA vector and analyzing the digested DNA material on denaturing and non-denaturing gels to confirm the presence of characteristic linear and continuous DNA bands that are distinct from linear and discontinuous DNA bands.

[0027] In some embodiments, the present invention may be described by any of the following:

1. A non-viral capsid-free DNA vector with covalently closed ends (scDNA vector), characterized in that said scDNA vector contains at least one heterologous nucleotide sequence functionally located between the sequences of asymmetric inverted terminal repeats (asymmetric ITRs), and at least one of the asymmetric ITRs contains a functional terminal resolution site and a Rep binding site.

2. The zcDNA vector of claim 1, wherein said zcDNA vector, when digested with a restriction enzyme having a single recognition site on said zcDNA vector and analyzed using both native and denaturing gel electrophoresis, exhibits characteristic linear and continuous DNA bands that differ from linear and discontinuous control DNA bands.

3. The zcDNA vector of items 1 or 2, wherein one or more of the asymmetric ITR sequences are from a virus selected from parvovirus, dependdovirus, and adeno-associated virus (AAV).

4. ZcDNA vector according to item 3, where said asymmetric ITRs are taken from different viral serotypes.

5. The zcDNA vector of claim 4, wherein said one or more asymmetric ITRs are from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

6. The zcDNA vector according to any one of 1-3, wherein one or more of the asymmetric ITR sequences are synthetic.

7. The zcDNA vector of any one of 1-3 and 6, wherein one or more of said ITRs is not a wild-type ITR.

8. The zcDNA vector according to any one of paragraphs 1-7, wherein one or both asymmetric ITRs are modified by deletion, insertion and/or replacement of at least one of the ITR regions selected from A, A', B, B', C, C', D and D'. In some embodiments, one or both of the asymmetric ITRs may include a deletion, insertion, and/or substitution in any combination of the B, B', C, or C' regions as described in Table 1. In some embodiments, one or both of the asymmetric ITRs may include a deletion, insertion, and/or substitution in the B region and/or the B' region and/or the C region and/or the C' region. In some embodiments, one or both asymmetric ITRs may contain one or more deletions, insertions, and/or substitutions in region A and/or region A' and/or region B and/or region B' and/or region C and/or region C' and/or region D and/or region D'. For example, in some embodiments, the modified ITR may include removing or deleting all or part of a particular arm, e.g., all or part of A-A', or all or part of B-B', or all or part of C-C', or alternatively, deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs forming the stem of the loop, as long as the last loop at the top of the stem is still present ( eg one shoulder) (eg see ITR-6). In some embodiments, the modified ITR may include deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from the B-B' arm. In some embodiments, the modified ITR may include deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from the C-C' arm. In some embodiments, the modified ITR may include deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from the C-C' arm and deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from the B-B' arm. Any combination of deletion of base pairs is contemplated. In some embodiments, the modified ITR lacks the B-B' arm. In some embodiments, the modified ITR lacks the C-C' arm.

9. The zcDNA vector of claim 8, wherein said deletion, insertion, and/or substitution results in the deletion of all or part of the stem loop structure normally formed by regions A, A', B, B' C or C'. For example, in some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more complementary base pairs are removed from each of the C and C' portions of the C-C' arm such that the C-C' arm is truncated, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9 or more complementary base pairs are removed from each of the B and B' arm portions. and B-B', so that the arm C-C' and/or the arm B'-B is truncated. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs are removed from the C portion of the C-C' arm such that only the C' portion of the arm remains. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs are removed from the C' portion of the C-C' arm such that only the C portion of the arm remains. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs are removed from the B portion of the B-B' arm such that only the B' portion of the arm remains. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs are removed from the B' portion of the B-B' arm such that only the B portion of the arm remains. In some embodiments, any deletion in region C, region C', region B, or region B' still retains a stem-loop terminal loop. In some embodiments, the terminal loop contains at least three amino acids. In some embodiments, the end loop of the C-C' arm and/or the B-B' arm contains at least three consecutive TTTs or three consecutive AAAs.

10. A zcDNA vector according to item 8 or item 9, wherein one or both of the asymmetric ITRs are modified by a deletion, insertion and/or substitution that results in the deletion of all or part of the stem-loop structure normally formed by the B and B' regions.

11. The zcDNA vector of any one of 8-10, wherein one or both of the asymmetric ITRs are modified by a deletion, insertion, and/or substitution that results in the deletion of all or part of the stem-loop structure normally formed by the C and C' regions.

12. ZcDNA vector according to item 10 or item 11, where one or both asymmetric ITRs are modified by a deletion, insertion and/or substitution, which leads to the deletion of part of the stem loop structure normally formed by regions B and B', and/or part of the stem loop structure normally formed by regions C and C'.

13. The zcDNA vector according to any one of items 1-12, wherein one or both of the asymmetric ITRs contain one stalk loop in a region that normally contains a first stalk loop formed by regions B and B' and a second stalk loop formed by regions C and C'.

14. The zcDNA vector of item 13, wherein one or both asymmetric ITRs contain one stem and two loops in a region that normally contains a first stem loop structure formed by regions B and B' and a second stem loop structure formed by regions C and C'.

15. The zcDNA vector according to item 13 or item 14, wherein one or both asymmetric ITRs contain one stem and one loop in a region that normally contains a first stem loop structure formed by regions B and B' and a second stem loop structure formed by regions C and C'.

16. The zcDNA vector according to any one of items 1-15, wherein at least one asymmetric ITR is a modified AAV2 ITR containing a nucleotide sequence selected from: the ITR in FIG. 26A or 26B, SEQ ID NOs: 101-499 or 545-547, an ITR having a sequence at least 95% identical to that of the ITR in FIG.

17. The zcDNA vector according to any one of items 1-16, wherein at least one asymmetric ITR is a modified AAV2 ITR comprising the nucleotide sequence of SEQ ID NO. 2, 52, 63 or 64, or a nucleotide sequence at least 95% identical to SEQ ID NO:. 2, 52, 63 or 64.

18. The zcDNA vector according to any one of items 1-16, wherein the 5'-ITR is a wild-type AAV ITR and the 3'-ITR contains a sequence selected from SEQ ID NOs: 2, 64, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 12 4, 126, 128,130, 132, 134, 469-483, and 546 and the ITR sequences shown in FIG. 26A and sequences at least 95% identical to any of the above sequences.

19. The zcDNA vector according to any one of items 1-16, wherein the 3'-ITR is a wild-type AAV ITR and the 5'-ITR contains a sequence selected from SEQ ID NOs: 52, 63, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 1 23, 125, 127, 129, 131, 133, 484^199, 545 and 547 and the ITR sequences shown in FIG. 26B and sequences at least 95% identical to any of the above sequences.

20. ZcDNA vector according to any one of items 1 to 16, where 5'-ITR contains a sequence selected from SEQ ID NO: 52, 63, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 , 131, 133, 484-499, 545 and 547 and the ITR sequences shown in FIG. 26B and sequences at least 95% identical to any of the above sequences; and the 3'-ITR contains a sequence selected from SEQ ID NOs: 2, 64, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 469-483 and 5 46 and the ITR sequences shown in FIG. 26A and sequences at least 95% identical to any of the above sequences.

21. ZkDNA vector according to paragraph 1, containing at least two asymmetric ITR selected from: (a) SEQ ID NO: 1 and SEQ ID NO: 52; and (b) SEQ ID NO: 2 and SEQ ID NO: 51.

22. ZkDNA vector according to paragraph 1, containing a pair of asymmetric ITR selected from: (a) SEQ ID NO: 1 and SEQ ID NO: 52; and (b) SEQ ID NO: 2 and SEQ ID NO: 51.

23. The zcDNA vector according to any one of items 1-20, wherein one or both of the asymmetric ITRs contain a sequence other than SEQ ID NOS: 2, 52, 63 64, 113, 114 and 557.

24. A zcDNA vector according to any one of items 1-24, wherein all or part of the heterologous nucleotide sequence is under the control of at least one regulatory switch.

25. The zcDNA vector of claim 24, wherein at least one regulatory switch is selected from any of the regulatory switches or combination of regulatory switches listed in Table 11 or in the Regulatory Switches section herein. For example, a regulatory switch serves to fine-tune the expression of a heterologous nucleotide sequence, such as a transgene, and may, in some embodiments, function as a biological containment of the scDNA vector. In some embodiments, the control switch is an on/off (on/off) switch. Without wishing to be bound by a particular theory, the on/off switch is designed to start or stop (ie stop) the expression of the heterologous nucleotide sequence or transgene expressed from the scDNA vector in a controlled and regulated manner. In some embodiments, said regulatory switch is a "kill switch" that can cause a cell containing said scDNA vector to enter programmed cell death upon activation of the switch. In some embodiments, said regulatory switch is selected from any of the following: a binary switch (e.g., inducible promoters, cofactors, or exogenous agents derepress transcription), a small molecular weight regulatory switch (e.g., drug inducible or prodrugs activate or stop transcription), a "password" regulatory switch (e.g., a combination of conditions necessary for transgene expression and/or repression to occur), a regulator a nucleic acid-based switch (e.g., a nucleic acid-based mechanism to control expression and/or repression), a post-translational and/or post-transcriptional regulatory switch (e.g., transgenes expressed with signal response elements (SREs) or destabilizing domains (DDs) preventing functionality of the transgenes until post-translational modification) or a "kill switch" (e.g., a switch to induce programmed cell death as a way to permanently remove the introduced ccDNA -vector from the subject's system).

26. The zcDNA vector according to any one of items 1-25, wherein said vector is in a nanocarrier.

27. ZcDNA vector according to item 26, wherein said nanocarrier contains a lipid nanoparticle (LNP).

28. A non-viral capsid-free DNA vector with covalently closed ends (scDNA vector) according to any one of items 1-25, which is obtained by a method comprising the following steps: (a) incubating a population of insect cells bearing an scDNA expression construct in the presence of at least one Rep protein, and said scDNA expression construct encodes the scDNA vector, under conditions effective for, and over time, sufficient to induce the production of said scDNA vector in insect cells; and (b) isolating said ccDNA vector from insect cells.

29. The zcDNA vector according to item 28, wherein said zcDNA expression construct is selected from plasmid zcDNA, bacmida zcDNA, and baculovirus zcDNA.

30. ZcDNA vector according to item 28 or item 29, wherein said insect cell expresses at least one Rep protein.

31. The zcDNA vector of item 30 wherein at least one Rep protein is from a virus selected from parvovirus, dependdovirus, and adeno-associated virus (AAV).

32. The zcDNA vector of item 31 wherein at least one Rep protein is from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

33. An scDNA expression construct that encodes an scDNA vector according to any one of items 1-25.

34. The scDNA expression construct of item 33, which is a scDNA plasmid, scDNA bacmid, or scDNA baculovirus.

35. Host cell containing the ccDNA expression construct of item 33 or item 34.

36. The host cell of claim 35 that expresses at least one Rep protein.

37. The host cell of claim 36, wherein at least one Rep protein is from a virus selected from parvovirus, dependovirus, and adeno-associated virus (AAV).

38. The host cell according to claim 37, wherein at least one Rep protein is taken from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

39. The host cell according to any one of paragraphs 35-38, which is an insect cell.

40. The host cell of item 39, which is an Sf9 cell.

41. A method of obtaining scDNA vector, including: (a) incubation of the host cell according to any one of paragraphs 35-40 under conditions effective for, and for a time sufficient to induce the production of scDNA vector; and (b) isolating said ccDNA from host cells.

42. A method for treating, preventing, alleviating, monitoring or diagnosing a disease or disorder in a subject, comprising: administering to a subject in need of a composition containing the scDNA vector according to any one of paragraphs 1-25, where at least one heterologous nucleotide sequence is selected for treating, preventing, alleviating, diagnosing or monitoring said disease or disorder.

43. The method of claim 42, wherein said at least one heterologous nucleotide sequence, when transcribed or translated, corrects for an abnormal amount of endogenous protein in said subject.

44. The method of claim 42, wherein said at least one heterologous nucleotide sequence, when transcribed or translated, corrects the abnormal function or activity of an endogenous protein or pathway in said subject.

45. The method of any one of items 42-44, wherein said at least one heterologous nucleotide sequence encodes or contains a nucleotide molecule selected from RNA interference (RNAi), miRNA, microRNA, dncRNA, and an antisense oligo or polynucleotide.

46. The method of any one of items 42-44, wherein said at least one heterologous nucleotide sequence encodes a protein.

47. The method of claim 42, wherein said at least one heterologous nucleotide sequence encodes a marker protein (eg, a reporter protein).

48. The method of any one of items 42-46, wherein said at least one heterologous nucleotide sequence encodes an agonist or antagonist of an endogenous protein or pathway associated with said disease or disorder.

49. The method of any one of items 42-46, wherein said at least one heterologous nucleotide sequence encodes an antibody.

50. The method according to any one of paragraphs 42-49, wherein said disease or disorder is selected from: a metabolic disease or disorder, a CNS disease or disorder, an ocular disease or disorder, a blood system disease or disorder, a liver disease or disorder, an immune disease or disorder, an infectious disease, a muscle disease or disorder, cancer, and a disease or disorder based on an abnormal level and/or function of a gene product.

51. The method of claim 50, wherein said metabolic disease or disorder is selected from diabetes, a lysosomal storage disorder, a mucopolysaccharidosis disorder, a urea cycle disease or disorder, and a glycogen storage disease or disorder.

52. The method of claim 51 wherein said lysosomal storage disorder is selected from Gaucher disease, Pompe disease, metachromatic leukodystrophy (MLD), phenylketonuria (PKU), and Fabry disease.

53. The method of claim 51 wherein said disease or urea cycle disorder is ornithine transcarbamylase (OTC) deficiency.

54. The method of claim 51, wherein said mucopolysaccharidosis disorder is selected from Sly's syndrome, Hurler's syndrome, Schie's syndrome, Hurler-Scheie's syndrome, Gunter's syndrome, Sanfilippo's syndrome, Morquio's syndrome, and Maroteau-Lami's syndrome.

55. The method of claim 50, wherein said CNS disease or disorder is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma resulting from spinal cord or head injuries, Tay-Sachs disease, Lesch-Nychen disease, epilepsy, cerebral infarction, psychiatric disorders, schizophrenia, drug or drug addiction, neurosis, psychosis, dementia, paranoia, attention deficit disorder, sleep disorders, pain-related disorders, nutritional or body weight disorders, as well as cancers and tumors of the central nervous system.

56. The method of claim 50, wherein said ocular disease or disorder is selected from ophthalmic disorders affecting the retina, posterior tract, and/or optic nerve.

57. Способ по пункту 55, где указанное офтальмологическое нарушение, затрагивающее сетчатку, задний путь и/или зрительный нерв, выбрано из диабетической ретинопатии, макулярной дегенерации, в том числе возрастной макулярной дегенерации, географической атрофии, сосудистой или «влажной» макулодистрофии, глаукомы, увеита, пигментного ретинита, болезни Штаргардта, врожденного амавроза Лебера (LCA), синдрома Ушера, эластической псевдоксантомы (РХЕ), Х-сцепленного пигментного ретинита (XLRP), Х-сцепленного расслоения сетчатки (XLRS), хороидеремии, врожденной нейропатии зрительного нерва Лебера (LHON), ахроматопсии, палочко-колбочковой дистрофии, эндотелиальной дистрофии роговицы Фукса, диабетического макулярного отека; and cancers and eye tumors.

58. The method of claim 50, wherein said disease or disorder of the blood system is selected from hemophilia A, hemophilia B, thalassemia, anemia, and blood cancer.

59. The method of claim 50, wherein said liver disease or disorder is selected from progressive familial intrahepatic cholestasis (FIC), and liver cancers and tumors.

60. The method of claim 42 wherein said disease or disorder is cystic fibrosis.

61. The method according to paragraphs 42-60, wherein said scDNA vector is administered in combination with a pharmaceutically acceptable carrier.

62. A method for delivering a therapeutic protein to a subject, comprising administering to the subject a composition comprising an scDNA vector according to any one of items 1-25, wherein at least one heterologous nucleotide sequence encodes a therapeutic protein.

63. The method of claim 62, wherein said therapeutic protein is a therapeutic antibody.

64. The method of claim 62, wherein said therapeutic protein is selected from an enzyme, erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, cytokine, cystic fibrosis transmembrane conductance regulator (CFTR), peptide growth factor, and hormone.

65. A kit containing an scDNA vector according to any one of items 1-25 and a nanocarrier packaged in a container with a package insert.

66. A kit for producing a scDNA vector, comprising an expression construct containing at least one restriction site for insertion of at least one heterologous nucleotide sequence, or a regulatory switch, or both the first and the second, wherein said at least one restriction site is functionally located between sequences of asymmetric inverted terminal repeats (asymmetric ITRs), and at least one of the asymmetric ITRs contains a functional end resolution site and a site binding Rep.

67. The kit according to item 66, which is suitable for obtaining an scDNA vector according to any one of items 1-25.

68. The kit of item 66 or item 67, further comprising a population of insect cells that does not contain viral capsid coding sequences, where production of the scDNA vector can be induced in the presence of the Rep protein.

69. The kit of any one of items 66-68, further comprising a vector containing a polynucleotide sequence that encodes at least one Rep protein, wherein said vector is suitable for expressing said at least one Rep protein in an insect cell.

[0028] In some embodiments, one aspect of the technology described herein relates to a non-viral capsidless DNA vector with covalently closed ends (ccDNA vector), characterized in that it contains at least one heterologous nucleotide sequence operably located between sequences of asymmetric inverted terminal repeats (asymmetric ITRs), wherein at least one of the asymmetric ITRs contains a functional end resolution site and a Rep binding, and optionally said heterologous nucleic acid sequence encodes a transgene and said vector is not enclosed in a viral capsid.

[0029] These and other aspects of the present invention are described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1A shows an example of the structure of an scDNA vector. In this embodiment, an exemplary scDNA vector includes an expression cassette containing the CAG promoter, WPRE and BGHpA. An open reading frame (ORF) encoding a luciferase transgene is inserted into a cloning site (R3/R4) between the CAG promoter and WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs), the wild-type AAV2 ITR at the start (at the 5' end) and the modified ITR at the end (at the 3' end) of the expression cassette, respectively, these two ITRs flanking the expression cassette are asymmetric with respect to each other.

[0031] In FIG. 1B shows an exemplary structure of a scDNA vector with an expression cassette containing the C promoter of AG, WPRE and BGHpA. An open reading frame (ORF) encoding a luciferase transgene is inserted into a cloning site between the CAG promoter and WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs), a modified ITR at the start (at the 5' end) and a wild-type ITR at the end (at the 3' end) of the expression cassette.

[0032] FIG. 1C depicts an exemplary structure of a scDNA vector with an expression cassette containing an enhancer/promoter, an open reading frame (ORF) for transgene insertion, a post-transcriptional element (WPRE), and a poly(A) signal. An open reading frame (ORF) allows insertion of a transgene into a cloning site between the CAG promoter and WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs) that are asymmetrical with respect to each other; a modified ITR at the start (at the 5' end) and a modified ITR at the end (at the 3' end) of the expression cassette, wherein both the 5'-ITR and the 3TTR are modified but contain different modifications (i.e., do not contain the same modifications).

[0033] FIG. 2A shows the T-loop-on-stem structure of the left AAV2 wild-type ITR (SEQ ID NO: 538) with the A-A' arm, B-B' arm, C-C' arm, two Rep binding sites (RBE and RBE') identified, and the terminal resolution site (trs). RBE contains a series of 4 duplex tetramers that are believed to interact with either Rep 78 or Rep 68. In addition, RBE' is also expected to interact with the Rep complex assembled on wild-type ITR or mutated ITR in this construct. The D and D' regions contain transcription factor binding sites and other conserved structures. In FIG. 2B shows putative Rep-catalyzed nicking and ligating activity in the left wild-type ITR (SEQ ID NO: 539), including the loop-on-stem T-shaped structure of the AAV2 left wild-type ITR with the identified A-A' arm, B-B' arm, C-C' arm, two Rep binding sites (RBE and RBE'), and shows the terminal resolution site (trs) and region D and D' containing several binding sites for transcription factors and other conserved structures.

[0034] FIG. 3A shows the primary structure (polynucleotide sequence) (left) and secondary structure (right) of the RBE-containing portions of the A-A' arm, and the C-C' arm and the B-B' arm of the left AAV2 wild-type ITR (SEQ ID NO: 540). In FIG. 3B shows an example sequence of a mutated ITR (also referred to as modified ITR) for a left ITR. Shown are the primary structure (left) and the predicted secondary structure (right) of the RBE portion of arm A-A', arm C, and arm B-B' from the mutated left ITR example (ITR-1, left) (SEQ ID NO: 113). In FIG. 3C shows the primary structure (left) and secondary structure (right) of the RBE-containing portion of the A-A' loop, and the B-B' and C-C' arms of the wild-type right AAV2 ITR (SEQ ID NO: 541). In FIG. 3D shows an example of a right modified ITR. Shown are the primary structure (left) and the predicted secondary structure (right) of the RBE-containing portion of arm A-A', B-B', and arm C from an example of mutant right ITR (ITR-1, right) (SEQ ID NO: 114). Any combination of left and right ITRs (eg, AAV2 or other viral serotype ITRs, or synthetic ITRs) may be used, provided that the left ITR is asymmetric to or different from the right ITR. On each of the FIGS. 3A-3D, the polynucleotide sequences refer to the sequence used in the plasmid or baculovirus baculovirus genome used to generate scDNA as described herein. Also in each of Figs. 3A-3D show the respective secondary structures of scDNA derived from the configurations of the scDNA vector in the plasmid or baculovirus baculovirus genome and predicted Gibbs free energies.

[0035] FIG. 4A is a schematic of an upstream process for producing baculovirus-infected insect cells (BIS) that are suitable for producing scDNA by the method schematically shown in FIG. 4B. In FIG. 4B is a schematic representation of an exemplary method for obtaining scDNA, and FIG. 4C shows the biochemical method and process for confirming the production of the scDNA vector. In FIG. 4D and FIG. 4E are schematic diagrams describing the process for identifying the presence of ccDNA in DNA collected from cell pellets obtained during the ccDNA preparation processes of FIG. 4B. In FIG. 4E shows DNA with a discontinuous pattern. The zcDNA can be cut with a restriction endonuclease having a single recognition site on the scDNA vector to produce two DNA fragments of different sizes (1 kb and 2 kb) under both neutral and denaturing conditions. In FIG. 4E also shows scDNA having a linear and continuous structure. Said scDNA vector can be cut with a restriction endonuclease to produce two DNA fragments migrating as 1 kb stretches. and 2 kb. under neutral conditions, however, under denaturing conditions, the chains remain connected and produce single chains migrating as 2 kb stretches. and 4 kb. In FIG. 4D is a schematic representation of the expected bands for an example of scDNA uncleaved or restriction endonuclease cleaved and then subjected to electrophoresis on either native or denaturing gel. The leftmost schematic image shows a native gel with several bands suggesting that in duplex and uncut form, ccDNA exists in at least monomeric and dimeric states, which are seen as a smaller faster migrating monomer and a slower migrating dimer that is 2 times the size of the monomer. The second schematic from the left shows that when scDNA is cut with a restriction endonuclease, the original bands disappear and bands of more rapidly migrating fragments (eg, smaller) appear, corresponding to the expected sizes of the fragments remaining after cleavage. Under denaturing conditions, the original duplex DNA is single-stranded and migrates as a compound twice as large as that observed on native gel, since the complementary strands are covalently linked. Accordingly, in the second schematic image on the right, cleaved scDNA shows a similar distribution of bands compared to that observed on native gel, however, the bands correspond to the migration of fragments that are twice the size of their native gel equivalents. The rightmost schematic image shows that uncut scDNA under denaturing conditions migrates as a single-stranded open ring, and, accordingly, the size of the observed bands is twice the size of those observed under native conditions with an unopened ring. In the specified drawing "t.p.o." refers to the relative size of nucleotide molecules based, depending on the context, either on the length of the nucleotide chain (for example, for single-stranded molecules observed under denaturing conditions) or on the number of base pairs (for example, for double-stranded molecules observed under native conditions).

[0036] FIG. 5 is an exemplary denaturing gel run of examples of ccDNA vectors with (+) or without (-) endonuclease cleavage (EcoRI for ccDNA constructs 1 and 2; BamH1 for ccDNA constructs 3 and 4; SpeI for ccDNA constructs 5 and 6; and XhoI for ccDNA constructs 7 and 8). The dimensions for the bands highlighted with an asterisk are defined and shown at the bottom of the drawing.

[0037] FIG. Figure 6A shows the results of an in vitro protein expression assay measuring luciferase activity (Y-axis, RQ(Luc)) in HEK293 cells 48 hours post-transfection for 400 ng (black), 200 ng (grey) or 100 ng (white) of the constructs identified on the x-axis (construct-1, construct-3, construct-5, construct-7 (Table 12). 6B shows luciferase activity (Y-axis, RQ (Luc)) measured in HEK293 cells 48 hours after transfection with 400 ng (black), 200 ng (grey), or 100 ng (white) constructs identified on the x-axis (construct-2, construct-4, construct-6, construct-8) (Table 12). in HEK293 cells treated with Fugene without any plasmids ("Fugene"), or in untreated HEK293 cells ("untreated").

[0038] FIG. 7A shows the viability of HEK293 cells (y-axis) 48 hours after transfection with 400 ng (black), 200 ng (grey) or 100 ng (white) of the constructs identified on the x-axis (construct-1, construct-3, construct-5, construct-7). In FIG. 7B shows the viability of HEK293 cells (y-axis) 48 hours after transfection with 400 ng (black), 200 ng (grey) or 100 ng (white) of the constructs identified on the x-axis (construct-2, construct-4, construct-6, construct-8).

[0039] FIG. 8A shows an example of a Rep bacmid in plasmid pFBDLSR containing the nucleic acid sequences for the Rep proteins Rep52 and Rep78. This example Rep-bakmida contains: fragment of the IE1 promoter (SEQ ID NO: 66); a Rep78 nucleotide sequence, including a Kozak sequence (SEQ ID NO: 67), a polyhedrin promoter sequence for Rep52 (SEQ ID NO: 68), and a Rep58 nucleotide sequence starting from the Kozak sequence gccgccacc) (SEQ ID NO: 69). In FIG. 8B is a schematic representation of an example zcDNA plasmid-1, with wt-L ITR, CAG promoter, luciferase transgene, WPRE, and polyadenylation sequence; and mod-R (modified right) ITR.

[0040] FIG. 9A shows the predicted structure with the lowest energy of the RBE-containing portion of the A-A' arm, and the C-C' arm of an example of a modified left ITR ("ITR-2 (left)" SEQ OD NO: 101), and FIG. 9B shows the predicted structure with the lowest energy of the RBE-containing portion of the A-A' arm, and the C-C' arm of an example of a modified right ITR ("ITR-2 (right)" SEQ ID NO: 102). They predicted the formation of a structure with a single arm (С-С') and a single unpaired loop. Gibbs free energy predicted for them during deployment: -72.6 kcal/mol.

[0041] In FIG. 10A shows the predicted structure with the lowest energy of the RBE-containing part of the arm A-A', and arm B-B' of an example of a modified left ITR ("ITR-3 (left)" SEQ ID NO: 103), and FIG. 10B shows the predicted structure with the lowest energy RBE-co of the holding portion of the arm A-A', and the arm B-B' of an example of a modified right ITR ("ITR-3 (right)" SEQ ID NO: 104). They predicted the formation of a structure with a single shoulder (B-B') and a single unpaired loop. Gibbs free energy predicted for them during deployment: -74.8 kcal/mol.

[0042] FIG. 11A shows the predicted structure with the lowest energy of the RBE-containing part of the arm A-A', and arm C-C' of an example of a modified left ITR ("ITR-4 (left)" SEQ ID NO: 105), and FIG. 11B shows the predicted structure with the lowest energy of the RBE-containing part of the A-A' arm, and the C-C' arm of an example of a modified right ITR ("ITR-4 (right)" SEQ ID NO: 106). They predicted the formation of a structure with a single arm (С-С') and a single unpaired loop. Gibbs free energy predicted for them during deployment: -76.9 kcal/mol.

[0043] FIG. 12A shows the predicted lowest energy structure of the RBE-containing portion of the A-A' arm, and the C-C' and B-B' portions of an example of a modified left ITR, showing the complementary base pairing of the C-B' and C'-B portions ("ITR-10 (left)" SEQ ID NO: 107), and FIG. 12B shows the predicted lowest energy structure of the RBE-containing portion of the A-A' arm, and the B-B' and C-C' portions of an example of a modified right ITR, showing the complementary base pairing of the B-C and B'-C portions ("ITR-10 (right)" SEQ ID NO: 108). They predicted the formation of a structure with a single arm (part C'-B and C-B', or part B'-C and B-C') and a single unpaired loop. Gibbs free energy predicted for them during deployment: -83.7 kcal/mol.

[0044] FIG. 13A shows the predicted structure with the lowest energy RBE-containing arm portion A-A' and portions C-C' and B-B' of an example of a modified left ITR ("ITR-17 (left)" SEQ ID NO: 109), and FIG. 13B shows the predicted structure with the lowest energy RBE-containing arm portion A-A' and the C-C' and B-B' portions of an example of a modified right ITR ("ITR-17 (right)" SEQ ID NO: 110). For both ITR-17 (left) and ITR-17 (right), the formation of a structure with a single arm (B-B') and a single unpaired loop is predicted. Gibbs free energy predicted for them during deployment: -73.3 kcal/mol.

[0045] FIG. 14A shows the predicted structure with the lowest energy of the RBE-containing arm portion A-A' of an example of a modified ITR ("ITR-6 (left)" SEQ ID NO: 111), and FIG. 14B shows the predicted structure with the lowest energy of the RBE-containing arm portion A-A' of an example of a modified ITR ("ITR-6 (right)" SEQ ID NO: 112). For both ITR-6 (left) and ITR-6 (right), the formation of a structure with a single shoulder is predicted. Gibbs free energy predicted for them during deployment: -54.4 kcal/mol.

[0046] FIG. 15A shows the predicted structure with the lowest energy RBE containing portion of the A-A' arm, C arm, and B-B' arm of an example of a modified left ITR ("ITR-1 (left)" SEQ ID NO: 113), and FIG. 15B shows the predicted lowest energy structure of the RBE containing portion of the A-A' arm, C arm, and B-B' arm of an example of a modified right ITR ("ITR-1 (right)" SEQ ID NO: 114). For both ITR-1 (left) and ITR-1 (right), the formation of a structure with two arms, one of which is truncated, is predicted. Gibbs free energy predicted for them during deployment: -74.7 kcal/mol.

[0047] FIG. 16A shows the predicted structure with the lowest energy of the RBE-containing portion of the A-A' arm; arm C' and arm B-B' of an example of a modified left ITR ("ITR-5 (left)" SEQ ID NO: 545), and in FIG. 16B shows the predicted structure with the lowest energy RBE-containing portion of the A-A' arm, B-B' arm, and C' arm of an example of a modified right ITR ("ITR-5 (right)" SEQ ID NO: 116). Both ITR-5 (left) and ITR-5 (right) are predicted to form a structure with two arms, one of which (eg, arm C') is truncated. Gibbs free energy predicted for them during deployment: -73.4 kcal/mol.

[0048] FIG. 17A shows the predicted structure with the lowest energy RBE-containing portion of the A-A' arm, C-C' arm, and B-B' arm of an example of a modified left ITR ("ITR-7 (Left)" SEQ ID NO: 117), and FIG. 17B shows the predicted structure with the lowest energy RBE containing portion of the A-A' arm, B-B' arm, and C-C' arm of an example of a modified right ITR ("ITR-7 (right)" SEQ ID NO: 118). Both ITR-17 (left) and ITR-17 (right) are predicted to form a structure with two arms, one of which (eg arm B-B') is truncated. Gibbs free energy predicted for them during deployment: -89.6 kcal/mol.

[0049] FIG. 18A shows the predicted structure with the lowest energy RBE containing portion of the A-A' arm, C-C' arm, and B-B' arm of an example of a modified left ITR ("ITR-8 (left)" SEQ ID NO: 119), and FIG. 18B shows the predicted lowest energy structure of the RBE containing portion of the A-A' arm, B-B' arm, and C-C' arm of an example of a modified right ITR ("ITR-8 (right)" SEQ ID NO: 120). For both ITR-8 (left) and ITR-8 (right), the formation of a structure with two arms, one of which is truncated, is predicted. Gibbs free energy predicted for them during deployment: -86.9 kcal/mol.

[0050] FIG. 19A shows the predicted structure with the lowest energy RBE-containing portion of the A-A' arm, C-C' arm, and B-B' arm of an example of a modified left ITR ("ITR-9 (left)" SEQ ID NO: 121), and FIG. 19B shows the predicted lowest energy structure of the RBE containing portion of the A-A' arm, B-B' arm, and C-C' arm of an example of a modified right ITR ("ITR-9 (right)" SEQ ID NO: 122). For both ITR-9 (left) and ITR-9 (right), the formation of a structure with two arms, one of which is truncated, is predicted. Gibbs free energy predicted for them during deployment: -85.0 kcal/mol.

[0051] FIG. 20A shows the predicted structure with the lowest energy RBE containing portion of the A-A' arm, C-C' arm, and B-B' arm of an example of a modified left ITR ("ITR-11 (left)" SEQ ID NO: 123), and FIG. 20B shows the predicted structure with the lowest energy of the RBE-containing portion of the A-A' arm, B-B' arm, and C-C' arm of an example of a modified right ITR ("ITR-11 (right)" SEQ ID NO: 124). For both ITR-11 (left) and ITR-11 (right), the formation of a structure with two arms, one of which is truncated, is predicted. Gibbs free energy predicted for them during deployment: -89.5 kcal/mol.

[0052] FIG. 21A shows the predicted structure with the lowest energy RBE-containing portion of the A-A' arm, C-C' arm, and B-B' arm of an example of a modified left ITR ("ITR-12 (left)" SEQ ID NO: 125), and FIG. 21B shows the predicted structure with the lowest energy RBE-containing portion of the A-A' arm, B-B' arm, and C-C' arm of an example of a modified right ITR ("ITR-12 (right)" SEQ ID NO: 126). For both ITR-12 (left) and ITR-12 (right), the formation of a structure with two arms, one of which is truncated, is predicted. Gibbs free energy predicted for them during deployment: -86.2 kcal/mol.

[0053] FIG. 22A shows the predicted structure with the lowest energy of the RBE-containing portion of the A-A' arm, C-C' arm, and B-B' arm of an example of a modified left ITR ("ITR-13 (left)" SEQ ID NO: 127), and FIG. 22B shows the predicted lowest energy structure of the RBE containing portion of the A-A' arm, B-B' arm, and C-C' arm of an example of a modified right ITR ("ITR-13 (right)" SEQ ID NO: 128). Both ITR-13 (left) and ITR-13 (right) are predicted to form a structure with two arms, one of which (eg, the C-C' arm) is truncated. Gibbs free energy predicted for them during deployment: -82.9 kcal/mol.

[0054] FIG. 23A shows the predicted structure with the lowest energy of the RBE-containing portion of the A-A' arm, C-C' arm, and B-B' arm of an example of a modified left ITR ("ITR-14 (left)" SEQ ID NO: 129), and FIG. 23B shows the predicted structure with the lowest energy RBE containing portion of the A-A' arm, B-B' arm, and C-C' arm of an example of a modified right ITR ("ITR-14 (right)" SEQ ID NO: 130). Both ITR-14 (left) and ITR-14 (right) are predicted to form a structure with two arms, one of which (eg, the C-C' arm) is truncated. Gibbs free energy predicted for them during deployment: -80.5 kcal/mol.

[0055] FIG. 24A shows the predicted structure with the lowest energy RBE-containing portion of the A-A' arm, C-C' arm, and B-C' arm of an example of a modified left ITR ("ITR-15 (left)" SEQ ID NO: 131), and FIG. 24B shows the predicted structure with the lowest energy RBE-containing portion of the A-A' arm, B-B' arm, and C-C' arm of an example of a modified right ITR ("ITR-15 (right)" SEQ ID NO: 132). Both ITR-15 (left) and ITR-15 (right) are predicted to form a structure with two arms, one of which (eg, the C-C' arm) is truncated. Gibbs free energy predicted for them during deployment: -77.2 kcal/mol.

[0056] FIG. 25A shows the predicted structure with the lowest energy RBE-containing portion of the A-A' arm, C-C' arm, and B-C' arm of an example of a modified left ITR ("ITR-16 (left) SEQ ID NO: 133), and FIG. 25B shows the predicted structure with the lowest energy RBE-containing portion of the A-A' arm, B-B' arm, and C-C' arm of an example of a modified right ITR ("ITR-16 (right)" SEQ ID NO: 134). Both ITR-16 (left) and ITR-16 (right) are predicted to form a structure with two arms, one of which (eg, the C-C' arm) is truncated. Gibbs free energy predicted for them during deployment: -73.9 kcal/mol.

[0057] FIG. 26A shows the predicted structures of the RBE-containing arm portion A-A', and the modified arm B-B' and/or the modified arm C-C' of the modified right ITR examples shown in Table 10A. In FIG. 26B shows the predicted structures of the RBE-containing portion of arm A-A', and modified arm C-C' and/or modified arm B-B' of the modified left ITR examples shown in Table 10B. The structures shown are the predicted structures with the lowest free energy. Conditional colors: red = >99% probability; orange = 99% - 95% chance; beige = 95-90% chance; dark green 90% - 80%; bright green = 80% - 70%; blue = 70% - 60%; dark blue 60% - 50%; and pink = <50%.

[0058] FIG. 27 shows luciferase activity in Sf9 GlycoBac insect cells transfected with selected asymmetric ITR mutant variants from Tables 10A and 10B. The zcDNA vector contained a luciferase gene flanked by a wild type (wt) ITR and a modified asymmetric ITR selected from Tables 10A or 10B. "ITR-50 R without rep" is a known salvageable mutant without co-infection with Rep-containing baculovirus. "Sham transfection" only transfection reagents without donor DNA.

[0059] FIG. 28 shows a native agarose gel (1% agarose, 1x TAE buffer) of representative crude scDNA extracts from Sf9 insect cell cultures transfected with scDNA plasmids containing a left wt-ITR and another ITR selected from the various mutant right ITRs shown in Table 10A. each lane was loaded with 2 µg of the total extract. From left to right: track 1) 1 Kb Plus Ladder, track 2) ITR-18, right, track 3) ITR-49, right, track 4) ITR-19, right, track 5) ITR-20, right, track 6) ITR-21, right, track 7) ITR-22, right, track 8) ITR -23, right, track 9) ITR-24, right, track 10) ITR-25, right, track 11) ITR-26, right, track 12) ITR-27, right, track 13) ITR-28, right, track 14) ITR-50, right, track 15) 1 Kb Plus Ladder.

[0060] FIG. 29 shows a denaturing gel (0.8% alkaline agarose) for representative constructs from the ITR mutant library. The zcDNA vector is generated from plasmid constructs containing a left wt-ITR and another ITR selected from the various mutant right ITRs shown in Table 10A. From left to right: lane 1) DNA ladder 1 Kb Plus Ladder, lane 2) ITR-18, right, uncut, lane 3) ITR-18, right, restriction digest, lane 4) ITR-19, right, uncut, lane 5) ITR-19, right, restriction digest, lane 6) ITR-21 , right, uncut, lane 7) ITR-21, right, restriction digest, lane 8) ITR-25, right, uncut, lane 9) ITR-25, right, restriction digest. Extracts were treated with EcoRI restriction endonuclease. It is assumed that each scDNA mutant contains a single EcoRI recognition site to obtain two characteristic fragments, ~2000 bp. and ~3000 bp, which will move as ~4000 and ~6000 bp fragments, respectively, under denaturing conditions. Raw scDNA extracts contain ~5000 bp fragments. and, presumably, will migrate as ~11000 bp fragments. under denaturing conditions.

[0061] FIG. 30 shows in vitro luciferase activity in HEK293 cells with mutant ITRs, right ITR-18, right ITR-19, right ITR-21 and right ITR-25, and ITR-49, with the left ITR in the scDNA vector represented by the WT ITR. "Sham transfection" is only transfection reagents without donor DNA, and "raw" is the negative control.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

[0062] Unless otherwise indicated herein, scientific and technical terms used in connection with the present invention have the meanings commonly implied by those skilled in the art to which the present invention pertains. It should be understood that the present invention is not limited to the particular methodology, protocols, and reagents, and the like described herein, and such methods, protocols, and reagents may therefore vary. The terminology used herein serves only to describe specific embodiments, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common immunology and molecular biology terms can be found in the following sources: The Merck Manual of Diagnosis and Therapy, 19th ed., published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), Fields Virology, ^6th ed., Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013), Knipe, DM and Howley, PM (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, Elsevier edition, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414); Davis et al, Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are hereby incorporated by reference in their entirety.

[0063] As used herein, the terms "heterologous nucleotide sequence" and "transgene" are used interchangeably and refer to a nucleic acid of interest (which is not a nucleic acid encoding a capsid polypeptide) that is included in an scDNA vector and can be delivered and expressed by the scDNA vector as described herein. Transgenes of interest include, but are not limited to, nucleic acids encoding polypeptides, preferably therapeutic (eg for medical, diagnostic or veterinary use) or immunogenic polypeptides (eg for vaccines). In some embodiments, nucleic acids of interest include nucleic acids transcribed into therapeutic RNA. Provided transgenes for use in ccDNA vectors of the present invention include, but are not limited to, transgenes that express or encode one or more polypeptides, peptides, ribozymes, aptamers, peptide nucleic acids, siRNA, RNAi (RNA interference), siRNA, dncRNA, antisense oligo- or polynucleotides, antibodies, antigen-binding fragments, or any combination of these leg.

[0064] As used herein, the terms "expression cassette" and "transcriptional cassette" are used interchangeably and refer to a linear stretch of nucleic acids that includes a transgene that is operably linked to one or more promoters or other regulatory sequences sufficient to direct transcription of said transgene, but does not include capsid coding sequences, other vector sequences, or inverted terminal repeat regions. The expression cassette may further comprise one or more cis-acting sequences (eg, promoters, enhancers, or repressors), one or more introns, and one or more post-transcriptional regulatory elements.

[0065] As used herein, the term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that contains at least one minimum required origin of replication and a region containing a palindromic hairpin structure. The Rep binding sequence ("RBS") and the terminal resolution site ("TRS") together constitute the "minimum required origin of replication" and, accordingly, the terminal repeat (TR) contains at least one RBS and at least one TRS. All TRs that are reversely complementary to each other within a given stretch of polynucleotide sequence are generally referred to as "inverted terminal repeats" or "ITRs". In the context of a virus, ITRs mediate viral replication, packaging, integration, and provirus rescue. As was surprisingly found by the inventors of the present invention, TRs that are not reverse complementary throughout can still perform traditional ITR functions, and accordingly, the term ITR herein refers to a TR in a scDNA genome or scDNA vector that is capable of mediating replication of the scDNA vector. One skilled in the art will recognize that more than two ITRs or pairs of asymmetric ITRs may be present in complex ccDNA configurations. The ITR may be an AAV ITR or a non-AAV ITR, or may be derived from an AAV ITR or a non-AAV ITR. For example, the ITR can be derived from a virus of the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, murine parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin, which serves as the origin of SV40 replication, can be used as an ITR, which can be further modified by truncation, substitution, deletion, insertion, and/or addition. Viruses of the family Parvoviridae consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses capable of replication in vertebrate hosts, including, but not limited to, human, primate, bovine, canine, equine, and ovine species.

[0066] As used herein, the term "asymmetric ITRs" refers to a pair of ITRs within the same scDNA genome or scDNA vector that are not reversely complementary throughout. The difference in the sequences of the two ITRs can be due to the addition of nucleotides, deletion, truncation or point mutation. In one embodiment, one ITR of a pair may be a wild-type AAV sequence and the other a non-wild-type or synthetic sequence. In another embodiment, neither ITR of the pair is a wild-type AAV sequence, and the sequences of the two ITRs are different. For convenience, herein the ITR located 5' to the expression cassette (upstream) in the scDNA vector is referred to as the "5'-ITR" or "left ITR", and the ITR located 3' to the expression cassette (down) in the scDNA vector is referred to as the "3'-ITR" or "right ITR".

[0067] As used herein, the term "ccDNA genome" refers to an expression cassette that further includes at least one inverted terminal repeat region. The zcDNA genome may further comprise one or more spacer regions. In some embodiments, said scDNA genome is incorporated as a DNA polynucleotide with intermolecular duplex structures into a plasmid or viral genome.

[0068] As used herein, the term "scDNA spacer region" refers to an intermediate sequence that separates functional elements in said scDNA vector or scDNA genome. In some embodiments, the scDNA spacer regions hold the two functional elements at the required distance for optimal functionality. In some embodiments, spacer regions of the ccDNA provide or increase the genetic stability of the ccDNA genome in, for example, a plasmid or baculovirus. In some embodiments, scDNA spacer regions facilitate prepared genetic manipulation of the scDNA genome by providing a convenient location for cloning sites and the like. For example, in certain aspects, an oligonucleotide "polylinker" containing multiple endonuclease restriction sites or a sequence not in the open reading frame, designed to contain no known protein binding sites (e.g., a transcription factor), can be placed in the ccDNA genome to separate cis-acting factors, e.g., 6-mer, 12-mer, 18-mer, 24-mer, 48-mer, 8 6-mer, 176-mer, etc., between the terminal clearance site and the 5' transcriptional regulatory element. Similarly, a spacer can be included between the polyadenylation signal sequence and the 3' clearance site.

[0069] As used herein, the terms "Rep binding site", "Rep binding element", "RBE", and "RBS" are used interchangeably and refer to a binding site for a Rep protein (e.g., Rep 78 AAV or Rep 68 AAV) that, when bound by the Rep protein, allows said Rep protein to exhibit the appropriate site-specific endonuclease activity on the sequence comprising RBS. The RBS sequence and its reverse complementary sequence together form one RBS. RBS sequences are known in the art and include, for example, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531), an RBS sequence identified in AAV2. Any known RBS sequence may be used in accordance with embodiments of the present invention, including other known AAV RBS sequences and other known natural or synthetic RBS sequences. Without wishing to be bound by any theory, it is believed that the nuclease domain of the Rep protein binds to the duplex nucleotide sequence of GCTC, and, accordingly, the duplex oligonucleotide 5'-(GCGC)(GCTC)(GCTC)(GCTC)-3' (SEQ ID NO: 531) directly binds and stably assembles two known AAV Rep proteins. In addition, soluble aggregated conformers (ie, an indefinite number of interconnected Rep proteins) dissociate and bind to oligonucleotides that contain Rep binding sites. Each Rep protein interacts with both nitrogenous bases and a phosphodiester backbone on each strand. Interactions with nitrogenous bases provide sequence specificity, while interactions with the phosphodiester backbone are nonspecific or less sequence specific and stabilize the protein-DNA complex.

[0070] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably and refer to the region in which Rep forms via a tyrosine-phosphodiester bond with 5'-thymidine to form a 3'-OH, which serves as a substrate for DNA completion by a cellular DNA polymerase, e.g., DNA polymerase delta or DNA polymerase epsilon. Alternatively, the Rep-thymidine complex may be involved in a coordinated ligation reaction. In some embodiments, the TRS encompasses at least unpaired thymidine. In some embodiments, the nicking efficiency of the TRS can be controlled, at least in part, by the distance between it and the RBS within a single molecule. If the acceptor substrate is a complementary ITR, the final product is an intramolecular duplex. TRS sequences are known in the art and include, for example, 5'-GGTTGA-3' (SEQ ID NO: 45), a hexanucleotide sequence identified in AAV2. Any known TRS sequence may be used in accordance with embodiments of the present invention, including other known AAV TRS sequences and other known natural or synthetic TRS sequences such as AGTT (SEQ ID NO: 46), GGTTGG (SEQ ID NO: 47), AGTTGG (SEQ ID NO: 48), AGTTGA (SEQ ID NO: 49); and other motifs such as RRTTRR (SEQ ID NO: 50).

[0071] As used herein, the term "ccDNA plasmid" refers to a plasmid that contains the scDNA genome as an intermolecular duplex.

[0072] As used herein, the term "scDNA bacmida" refers to an infectious baculovirus genome comprising the scDNA genome as an intermolecular duplex capable of replicating in E. coli as a plasmid and thus functioning as a shuttle vector for baculovirus.

[0073] As used herein, the term "ccDNA baculovirus" refers to a baculovirus that contains the scDNA genome as an intermolecular duplex within the baculovirus genome.

[0074] As used herein, the terms "baculovirus zcDNA-infected insect cell" and "baculovirus zcDNA-infected cell" are used interchangeably and refer to an invertebrate host cell (including, but not limited to, an insect cell (e.g., Sf9 cell)) infected with baculovirus zcDNA.

[0075] As used herein, the terms "closed-ended DNA vector", "scDNA vector", and "scDNA" are used interchangeably and refer to a non-viral capsidless DNA vector with at least one covalently closed end (i.e., containing an intramolecular duplex). In some embodiments, the scDNA contains two covalently closed ends.

[0076] As defined herein, "reporters" refers to proteins that can be used to provide detectable readings. Reporters typically produce a measurable signal, such as fluorescent, color, or luminescent. Reporter protein coding sequences encode proteins whose presence in a cell or organism is readily observable. For example, fluorescent proteins cause the cell to fluoresce when excited by light of a particular wavelength, luciferases cause the cell to catalyze a reaction that generates light, and enzymes such as β-galactosidase convert the substrate into a colored product. Examples of reporter polypeptides suitable for experimental or diagnostic purposes include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP) and other fluorescent proteins, chloramphenicol acetyl transferase (CAT), luciferase, and others well known in the art. .

[0077] As used herein, the term "effector protein" refers to a polypeptide that provides a detectable indication, either, for example, as a reporter polypeptide, or, more preferably, as a cell-killing polypeptide, for example, a toxin, or an agent that renders a cell susceptible to being killed by a specified agent or in the absence of a specified agent. Effector proteins include any protein or peptide that directly targets or damages the host cell's DNA and/or RNA. For example, effector proteins may include, but are not limited to, a restriction endonuclease that targets a host cell's DNA sequence (genomic or extrachromosomal element), a protease that degrades a polypeptide target essential for cell survival, a DNA gyrase inhibitor, and a ribonuclease-type toxin. In some embodiments, expression of an effector protein controlled by a synthetic biological circuit as described herein can be factored into another synthetic biological circuit, thereby expanding the range and complexity of the response of the biological circuit system.

[0078] The term "transcriptional regulators" refers to transcriptional activators and repressors that either activate or repress the transcription of a gene of interest. Promoters are regions of a nucleic acid that initiate the transcription of a particular gene. Transcriptional activators typically bind in the vicinity of transcriptional promoters and recruit RNA polymerase to initiate transcription directly. Repressors bind to transcription promoters and sterically hinder transcription initiation by RNA polymerase. Other transcriptional regulators can serve as either an activator or a repressor, depending on their binding site and cellular and environmental conditions. Non-limiting examples of classes of transcriptional regulators include, but are not limited to, homeodomain proteins, zinc finger proteins, winged helix proteins (Forkhead proteins), and leucine zipper proteins.

[0079] As used herein, a "repressor protein" or "inducer protein" is a protein that binds to a regulatory sequence element and represses or activates, respectively, the transcription of sequences operably linked to said regulatory sequence element. Preferred repressor proteins and inducer proteins as described herein are sensitive to the presence or absence of at least one input agent or environmental input. Preferred proteins as described herein are modular in form, containing, for example, separable DNA-binding and input-binding or responsive elements or domains.

[0080] As used herein, "carrier" includes any solvents, dispersion media, bases, coatings, diluents, antibacterial and antimycotic agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Additional active ingredients may also be included in these compositions. The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not result in a toxic, allergic or similar adverse reaction when administered to a host.

[0081] As used herein, an "input agent-responsive domain" is a transcription factor domain that binds or otherwise responds to a condition or input agent in such a way as to cause its associated DNA-binding fusion domain to respond to the presence of said condition or input action. In one embodiment, the presence of said condition or input action results in a conformational change in the input agent-responsive domain or protein to which it is fused, which modifies the transcription-modulating activity of the transcription factor.

[0082] The term "in vivo" refers to assays or processes that occur in or within an organism, such as a multicellular animal. According to some aspects of the present invention, the method or application may be said to be carried out "in vivo" if a single-celled organism, such as a bacterium, is used. The term "ex vivo" refers to methods and uses that are carried out using a living cell with an intact membrane that is outside the body of a multicellular animal or plant, such as explants, cultured cells, including primary cells and cell lines, transformed cell lines, and extracted tissues or cells, including blood cells, and the like. The term "in vitro" refers to assays and methods that do not require a cell with an intact membrane, such as on cell extracts, and may refer to the introduction of a programmable synthetic biological circuit into a non-cellular system, such as an environment free of cells or cell systems, such as cell extracts.

[0083] The term "promoter" as used herein refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by directing the transcription of said nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. Promoters can be constitutive, inducible, repressible, tissue-specific, or any combination of the above. A promoter is a control region of a nucleic acid sequence in which initiation takes place and the level of transcription of the rest of the nucleic acid sequence is determined. The promoter may also contain genetic elements in which binding of regulatory proteins and molecules such as RNA polymerase and other transcription factors can occur. In some embodiments of aspects of this document, a promoter may direct the expression of a transcription factor that regulates the expression of the promoter itself, or a transcription factor of another promoter used in another modular component of the synthetic biological circuits described herein. The promoter sequence contains a transcription initiation site, as well as protein-binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes. Various promoters, including inducible promoters, can be used to drive the expression of transgenes in the ccDNA vectors described herein.

[0084] The term "enhancer" as used herein refers to a cis-acting regulatory sequence (e.g., 50-1500 bp) that binds one or more proteins (e.g., activator proteins or a transcription factor) to increase transcriptional activation of the nucleic acid sequence. Enhancers can be located up to 1,000,000 base pairs upstream of the gene start site or downstream of the gene start site they regulate. An enhancer can be located in the intron region or in the exon region of an unrelated gene.

[0085] A promoter can be said to direct expression or direct transcription of the nucleic acid sequence it regulates. The terms "operably linked", "operably located", "under control" and "under transcriptional control" indicate that the promoter is in the correct functional position and/or orientation relative to the nucleic acid sequence it regulates to control the initiation of transcription and/or expression of said sequence. "Inverted promoter" as used herein refers to a promoter whose nucleic acid sequence is reversed such that the coding strand becomes non-coding, and vice versa. Inverted promoter sequences can be used in various embodiments to regulate the state of a switch. In addition, in various embodiments, a promoter may be used in combination with an enhancer.

[0086] A promoter can be a naturally associated gene or sequence promoter that can be obtained by isolating non-coding 5' sequences located upstream of the coding segment and/or exon of a particular gene or sequence. Such a promoter may be referred to as "endogenous". Similarly, in some embodiments, an enhancer may be a naturally associated enhancer with a nucleic acid sequence located either 3' or 5' from said sequence.

[0087] In some embodiments, a nucleic acid coding segment is placed under the control of a "recombinant promoter" or "heterologous promoter", where both terms refer to a promoter that is not normally associated with the nucleic acid coding sequence to which it is operably linked under natural environmental conditions. Recombinant or heterologous enhancer refers to an enhancer not normally associated with a specific nucleic acid sequence under natural environmental conditions. Such promoters or enhancers may include promoters or enhancers of other genes; promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell; and synthetic promoters or enhancers that are not "naturally occurring" i. containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression introduced using genetic engineering methods known in the art. In addition to producing promoter and enhancer nucleic acid sequences synthetically, promoter sequences can be obtained using recombinant cloning and/or nucleic acid amplification techniques, including PCR, in relation to synthetic biological circuits and modules as described herein (see, for example, US Patent No. 4,683,202, US Patent No. 5,928,906, each of which is incorporated herein by reference). In addition, it is also possible to use control sequences that direct the transcription and/or expression of sequences in non-nuclear organelles such as mitochondria, chloroplasts, and the like.

[0088] According to the present disclosure, an "inducible promoter" is a promoter that is characterized by the initiation or enhancement of transcriptional activity in the presence of, upon exposure to, or upon contact with an inducer or inducing agent. An "inducer" or "inducing agent" as defined herein can be a normally endogenous or exogenous compound or protein that is or is administered in such a manner as to be active in terms of inducing the transcriptional activity of an inducible promoter. In some embodiments, said inductor or inducing agent, i. the chemical, compound, or protein may itself be derived from the transcription or expression of a nucleic acid sequence (i.e., the inducer may be an inducer protein expressed by another component or module), which may itself be under the control of an inducible promoter. In some embodiments, induction of an inducible promoter occurs in the absence of certain agents, such as a repressor. Examples of inducible promoters include, but are not limited to, tetracycline, metallothione, ecdysone, mammalian viruses (e.g., adenovirus late promoter; and mouse mammary tumor virus long terminal repeat (MMTV-LTR)) and other steroid-responsive promoters, rapamycin-responsive promoters, and the like.

[0089] The term "subject" as used herein refers to a human or animal being treated, including prophylactic treatment, with an scDNA vector according to the present invention. Typically, said animal is a vertebrate, such as, but not limited to, a primate, rodent, domestic animal, or game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, coats, and macaques such as the rhesus monkey. Rodents include mice, rats, marmots, ferrets, rabbits and hamsters. Domestic and game animals include, but are not limited to, cows, horses, pigs, deer, bison, buffalo, felines such as the domestic cat, canine species such as the dog, fox, wolf, bird species such as chicken, emu, ostrich, and fish such as trout, catfish and salmon. In certain embodiments of aspects of the present invention, said subject is a mammal, such as a primate or a human. The subject may be male or female. In addition, the subject may be an infant or child. In some embodiments, said subject may be a neonatal or unborn subject, such as an in utero subject. Preferably, said subject is a mammal. Said mammal may be, but is not limited to, a human, non-human primate, mouse, rat, dog, cat, horse, or cow. Non-human mammals can be successfully used as subjects for animal models of diseases and disorders. In addition, the methods and compositions described herein can be applied to domesticated and/or companion animals. The human subject can be of any age, gender, race or ethnicity, such as Caucasian (White), Asian, African, Black, African American, African European, Hispanic, Middle Eastern, etc. In some embodiments, said subject may be a patient or other subject in a clinical setting. In some embodiments, said subject is already undergoing treatment.

[0090] As used herein, the term "antibody" is used in its broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. "Antibody fragment" refers to a non-intact antibody molecule that contains a portion of an intact antibody that binds the same antigen to which said intact antibody binds. In one embodiment, the antibody or antibody fragment comprises an immunoglobulin chain or antibody fragment and at least one immunoglobulin variable domain sequence. Examples of antibodies or fragments thereof include, but are not limited to, Fv-, scFv-, Fab-fragment, Fab'-, F(ab') ₂ , Fab'-SH, single domain antibody (dAb), heavy chain, light chain, heavy and light chain, complete antibody (for example, including all of the following: Fc, Fab, heavy chains, light chains, variable regions, etc.), bispecific antibodies o, diabody, linear antibody, single chain antibody, intrabody, monoclonal antibody, chimeric antibody, multispecific antibody, or multimeric antibody. An antibody or fragment thereof may be of any class, including, but not limited to, IgA, IgD, IgE, IgG, and IgM, and any subclass thereof, including, but not limited to, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In addition, the antibody may be derived from any mammalian body, such as primates, humans, rats, mice, horses, goats, and the like. In one embodiment, said antibody is a human antibody or a humanized antibody. In some embodiments, said antibody is a modified antibody. In some embodiments, the components of an antibody may be expressed separately, such that said antibody is self-assembled upon expression of the protein components. In some embodiments, said antibody is "humanized" to reduce immunogenic responses in humans. In some embodiments, said antibody has a desired function, such as interacting with and inhibiting a desired protein to treat a disease or symptom of a disease. In one embodiment, said antibody or antibody fragment contains a framework region or Fc region.

[0091] As used herein, the term "antigen-binding domain" of an antibody molecule refers to the portion of an antibody molecule, such as an immunoglobulin (Ig) molecule, that is involved in antigen binding. In embodiments, the antigen binding site is formed by the amino acid residues of the heavy (H) and light (L) chain variable (V) regions. Three very different segments in the composition of the variable regions of heavy and light chains, called hypervariable regions, are located between more conservative flanking segments, called "framework regions" (FR). FRs are amino acid sequences that are naturally found in immunoglobulins between and adjacent to hypervariable regions. In embodiments, in an antibody molecule, three light chain hypervariable regions and three heavy chain hypervariable regions are arranged relative to each other in three-dimensional space to form an antigen-binding surface that is complementary to the three-dimensional surface of the bound antigen. The three hypervariable regions from each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs". The framework region and CDR have been defined and described, for example, in Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fife ed., U.S. Department of Health and Human Services, NTH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (e.g., variable heavy chain and variable light chain) is typically composed of three CDRs and four FRs, whose amino-terminal to carboxyl-terminal amino acid sequences are in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0092] As used herein, the term "full-length antibody" refers to an immunoglobulin (Ig) molecule (eg, IgG antibodies), for example, naturally occurring, and formed by fragments of immunoglobulin genes during normal recombinatory processes.

[0093] As used herein, the term "functional antibody fragment" refers to a fragment that binds to the same antigen that an intact (eg, full length) antibody recognizes. The terms "antibody fragment" or "functional fragment" also include isolated fragments consisting of variable regions, such as "Fv" fragments consisting of heavy and light chain variable regions, or recombinant single chain polypeptide molecules in which the heavy and light chain variable regions are connected by a peptide linker ("scFv proteins"). In some embodiments, the antibody fragment does not contain antibody portions without antigen-binding activity, such as Fc fragments or single amino acid residues.

[0094] As used herein, "an immunoglobulin variable domain sequence" refers to an amino acid sequence that can form an immunoglobulin variable domain structure. For example, said sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the specified sequence may or may not include one, two or more N- or C-terminal amino acids, or may include other changes that are compatible with the formation of the protein structure.

[0095] As used herein, the term “comprising” or “comprises” is used to refer to compositions, methods, and their respective component (or components) that are or are essential to said method or composition, but may or may not include elements that are not indicated.

[0096] As used herein, the term "consisting essentially of" refers to the elements required for a particular implementation. The specified term allows the presence of elements that do not significantly affect the main and new or functional characteristic or characteristics of the specified implementation option.

[0097] The term "consisting of" refers to compositions, methods, and their respective components as described herein, excluding any element not mentioned in the description of the specified implementation option.

[0098] As used herein and in the appended claims, the singular forms, including those accompanied by the definition "specified", include the corresponding plural forms, unless otherwise clearly follows from the context. Accordingly, for example, reference to "said method" includes one or more methods and/or steps of the type described herein, and/or methods and/or steps that will be apparent to those skilled in the art upon reading the present description, etc. Likewise, the term "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. The expression "for example" is used herein to describe a non-limiting example.

[0099] With the exception of working examples or otherwise specified, all numbers reflecting the amounts of ingredients or reaction conditions according to the present invention, in all cases should be construed as modified by the term "approximately". The term "approximately" in relation to percentages can mean ±1%. The present invention is further explained in more detail in the following examples, however, the scope of the present invention is not limited thereto.

[00100] It should be understood that the present invention is not limited to the specific methodology, protocols and reagents, and the like described herein, and thus may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

II. zcDNA-vector

[00101] The present invention provides novel non-viral capsidless scDNA molecules with covalently closed ends (scDNA). Said non-viral capsidless scDNA molecules can be produced in permissive host cells with an expression construct (e.g., scDNA plasmids, scDNA bacmids, scDNA baculovirus, or cell line in an integrated state) containing a heterologous gene (transgene) located between two different inverted terminal repeat (ITR) sequences, wherein the ITRs are different from each other. In some embodiments, one of the ITRs is modified by a deletion, insertion, and/or substitution from a wild-type ITR sequence (eg, an AAV ITR); and at least one of the ITRs contains a functional end resolution site (trs) and a Rep binding site. Said scDNA vector is preferably duplex, eg self-complementary, over at least a portion of a molecule, such as an expression cassette (eg, scDNA is not a double-stranded circular molecule). The 3cDNA vector has covalently closed ends and is therefore resistant to cleavage by an exonuclease (eg, exonuclease I or exonuclease III), for example, for one hour at 37°C.

[00102] ZcDNA vectors as described herein do not have packaging restrictions imposed by limited space within the viral capsid. ZcDNA vectors represent a viable alternative for production in eukaryotes instead of production in prokaryotes from plasmid DNA vectors, as opposed to encapsulated AAV genomes. This allows the insertion of control elements, eg, regulatory switches as described herein, large transgenes, multiple transgenes, and the like.

[00103] According to one aspect, the scDNA vector contains, in the 5' to 3' direction: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein) and a second AAV ITR, wherein the first ITR and the second ITR are asymmetric with respect to each other, that is, they are different from each other. In an exemplary embodiment, the first ITR may be a wild-type ITR and the second ITR may be a mutated or modified ITR. In some embodiments, the first ITR may be a mutated or modified ITR and the second ITR may be a wild-type ITR. In another embodiment, both the first ITR and the second ITR are modified but have different sequences, or contain different modifications, or are non-identically modified ITRs. In other words, these ITRs are asymmetric, in the sense that any changes in one ITR are not reflected in the other ITR; or, alternatively, where the ITRs differ from each other. Examples of ITRs in said scDNA vector and for use in the production of scDNA plasmids are discussed below in the section entitled "ITR".

[00104] Wild-type ITR sequences, or mutated or otherwise modified ITR sequences of the present invention are DNA sequences included in an expression construct (e.g., scDNA plasmid, scDNA bacmid, scDNA baculovirus) to produce an scDNA vector. Accordingly, the ITR sequences actually contained in said ccDNA vector produced from an ccDNA plasmid or other expression construct may or may not be identical to the proposed ITR sequences of the present invention due to natural variations occurring during the production process (e.g. replication error).

[00105] In some embodiments, the scDNA vector described herein contains an expression cassette with a transgene, which can be, for example, a regulatory sequence, a nucleic acid coding sequence (such as miR or an antisense sequence), or a polypeptide coding sequence (such as a transgene). In one embodiment, said transgene may be operably linked to one or more regulatory sequences that allow or control the expression of the transgene. In one embodiment, said polynucleotide comprises a first ITR sequence and a second ITR sequence, wherein the nucleotide sequence of interest is flanked by said first and second ITR sequences, and said first and second ITR sequences are asymmetric with respect to each other.

[00106] In one embodiment, in each of these aspects, the expression cassette is located between two ITRs contained together with one or more of the following, in the following order: promoter operably linked to the transgene, post-transcriptional regulatory element; and a polyadenylation and termination signal. In one embodiment, said promoter is regulable: inducible or repressible. Said promoter may be any sequence that facilitates transcription of the transgene. In one embodiment, said promoter is a CAG promoter (eg, SEQ ID NO: 03) or a variant thereof. The specified post-transcriptional regulatory element is a sequence that modulates the expression of a transgene, in a non-limiting example, any sequence that forms a tertiary structure that enhances the expression of the transgene.

[00107] In one embodiment, said post-transcriptional regulatory element comprises a WPRE (eg, SEQ ID NO: 08). In one embodiment, said polyadenylation and termination signal comprises poly(A) BGH (eg, SEQ ID NO: 09). Additionally, any cis-regulatory element known in the art, or a combination thereof, may be used, for example, the SV40 late polyadenylation signal 5' enhancer sequence (USE), or other post-transcriptional processing elements, including, but not limited to, the thymidine kinase gene of the herpes simplex virus, or hepatitis B virus (HBV). In one embodiment, the length of the expression cassette in the 5'-3' direction is greater than the known maximum length for the AAV virion capsid sequence. In one embodiment, said length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb. Examples of various expression cassettes are provided herein.

[00108] Said expression cassette may contain more than 4,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any number from about 4,000 to 10,000 nucleotides, or 10,000 -50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, said expression cassette may contain a transgene or nucleic acid in the range of 500 to 50,000 nucleotides in length. In some embodiments, said expression cassette may contain a transgene or nucleic acid in the range of 500 to 75,000 nucleotides in length. In some embodiments, said expression cassette may contain a transgene or nucleic acid in the range of 500 to 10,000 nucleotides in length. In some embodiments, said expression cassette may contain a transgene or nucleic acid in the range of 1,000 to 10,000 nucleotides in length. In some embodiments, said expression cassette may contain a transgene or nucleic acid in the range of 500 to 5,000 nucleotides in length. The zcDNA vectors are not size limited as are the capsid-encapsulated AAV vectors and thus allow the delivery of a large size expression cassette to enable efficient expression of the transgenes. In some embodiments, said scDNA vector is devoid of specific prokaryotic methylation.

[00109] Said expression cassette may also contain an internal ribosome entry site (DIES) and/or a 2A element. Cys regulatory elements include, but are not limited to, a promoter, a riboswitch, an insulator, an miRNA-regulated element, a post-transcriptional regulatory element, a tissue-specific and cell-specific promoter, and an enhancer. In some embodiments, said ITR may act as a promoter for the transgene. In some embodiments, said scDNA vector contains additional components for regulating the expression of said transgene, for example, a regulatory switch, which is described herein in the "Regulatory Switches" section, to control and regulate the expression of said transgene, and may contain, if required, a regulatory switch, which is a "safety switch" that allows for controlled cell death of a cell containing the scDNA vector.

[00110] FIGS. 1A-1C are schematic representations of non-limiting examples of scDNA vectors, or the corresponding sequence of scDNA plasmids. ZcDNA vectors are capsidless and can be generated from a plasmid encoding, in this order: the first ITR, the expressed transgene cassette, and the second ITR, wherein at least one of the first and/or second ITR sequences is mutated relative to the corresponding wild-type AAV2 ITR sequence. Said transgenic cassette to be expressed preferably includes, in that order, one or more of the following: enhancer/promoter, ORF reporter (transgene), post-transcriptional regulatory element (eg, WPRE); and a polyadenylation and termination signal (eg, poly(A) BGH).

[00111] Said expression cassette may contain any transgene of interest. Transgenes of interest include, but are not limited to, nucleic acids encoding polypeptides or non-coding nucleic acids (e.g., RNAi, miRNA, etc.), preferably therapeutic (e.g., for medical, diagnostic, or veterinary use) or immunogenic (e.g., for vaccines) polypeptides. In some embodiments, the transgenes in the expression cassette encode one or more of polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAi, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments; or any combination of the above. In some embodiments, said transgene is a therapeutic gene or marker protein. In some embodiments, said transgene is an agonist or antagonist. In some embodiments, said antagonist is a mimetic or an antibody, or an antibody fragment, or an antigen-binding fragment thereof, such as a neutralizing antibody or antibody fragment, and the like. In some embodiments, said transgene encodes an antibody, including a full length antibody or antibody fragment as defined herein. In some embodiments, said antibody is an immunoglobulin antigen-binding domain or variable domain sequence as defined herein.

[00112] In particular, said transgene may encode one or more therapeutic agents, including but not limited to, for example, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen-binding fragments, and variants and/or active fragments thereof, for use in the treatment, prevention, and/or alleviation of one or more symptoms of a disease, dysfunction, injury, and/or disorder. Examples of transgenes are described herein in the "Method of Treatment" section.

[00113] Many structural features distinguish ccDNA vectors from plasmid-based expression vectors. ZcDNA vectors may be characterized by one or more of the following: absence of original (i.e., not inserted) bacterial DNA, absence of a prokaryotic origin of replication, autonomy, i.e. they do not require the presence of any sequences other than two ITRs, including a Rep-binding site and a terminal clearance site (RBS and TRS), and an exogenous sequence between these ITRs, the presence of ITR sequences that form hairpins of eukaryotic origin (i.e., they are produced in eukaryotic cells); and the absence of bacterial-type DNA methylation, or any other methylation perceived as abnormal by the mammalian host. In general, it is preferred that the proposed vectors do not contain any prokaryotic DNA, however, it is possible to insert any prokaryotic DNA as an exogenous sequence, according to a non-limiting example, in the promoter or enhancer region. Another important feature that distinguishes scDNA vectors from plasmid expression vectors is that scDNA vectors are single-stranded linear DNA with closed ends, while plasmids are always double-stranded DNA.

[00114] scDNA vectors produced by the methods of the present invention preferably have a linear and continuous structure, rather than a discontinuous structure, as assessed based on the results of a restriction enzyme digestion assay (FIG. 4D). A linear and continuous structure is considered more stable when attacked by cellular endonucleases, and is also less likely to undergo recombination and cause mutagenesis. Accordingly, an scDNA vector having a linear and continuous structure is a preferred embodiment. A continuous, linear, single-stranded ccDNA vector with intramolecular duplexes may contain covalently linked ends, without sequences encoding AAV capsid proteins. These scDNA vectors are structurally different from plasmids (including the scDNA plasmids described herein), which are circular duplex nucleic acid molecules of bacterial origin. Complementary strands of plasmids can be separated after denaturation to produce two nucleic acid molecules, whereas scDNA vectors, on the contrary, although they contain complementary strands, are one DNA molecule and, accordingly, even after denaturation remain in the form of one molecule. In some embodiments, scDNA vectors as described herein can be generated without methylation of prokaryotic-type DNA bases, unlike plasmids. Accordingly, scDNA vectors and scDNA plasmids differ both in structure (in particular, linear or circular), and in the methods used to obtain and purify these different entities (see below), as well as in DNA methylation, which occurs in the prokaryotic type in scDNA plasmids and in the eukaryotic type in the scDNA vector e.

[00115] Some of the advantages of the zcDNA vector described herein over plasmid-based expression vectors include, but are not limited to: 1) plasmids contain bacterial DNA sequences and undergo specific prokaryotic methylation, e.g. and do not undergo specific prokaryotic methylation; as a result, capsidless AAV vectors are less likely to induce inflammatory and immune responses than plasmids; 2) plasmids require the presence of a resistance gene during production, but scDNA vectors do not; 3) the circular plasmid is not delivered to the nucleus when introduced into the cell and requires an excess load to bypass degradation by cellular nucleases, and scDNA vectors contain viral cis elements, i.e. ITRs conferring nuclease resistance and can be engineered to target and deliver to the nucleus. It has been hypothesized that the minimum defining elements required for ITR function are the Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) for AAV2) and the terminal clearance site (TRS; 5'-AGTTGG-3' (SEQ ID NO: 48) for AAV2), plus a variable palindromic sequence providing hairpin formation; and 4) ccDNA vectors do not feature the over-abundance of CpG dinucleotides often found in prokaryotic-derived plasmids, which are reported to bind to a member of the Toll-like receptor family to elicit a T-cell mediated immune response. Conversely, transduction with capsidless AAV vectors as described herein can effectively target cell and tissue types that are difficult to transduce with standard AAV virions using a variety of delivery reagents.

III. ITR

[00116] According to the present disclosure, scDNA vectors contain a heterologous gene located between two inverted terminal repeat (ITR) sequences that differ from each other (i.e., are asymmetric ITRs). In some embodiments, at least one of the ITRs is modified by a deletion, insertion, and/or substitution from a wild-type ITR sequence (eg, an AAV ITR); and at least one of the ITRs contains a functional Rep binding site (RBS; e.g., 5'-GCGCGCTCGCTCGCTC-3' for AAV2, SEQ ID NO: 531) and a functional terminal resolution site (TRS; e.g., 5'-AGTT-3', SEQ ID NO: 46.) In one embodiment, at least one of the ITRs is a non-functional ITR. In one embodiment, said different ITRs are not wild-type ITRs from different serotypes.

[00117] Although the exemplary ITRs in the description and examples herein are AAV2 ITRs, one skilled in the art will recognize that, as noted above, the ITRs of any known parvovirus, e.g. AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ and AAV-DJ8 For example, from NCBI: NC 002077; NC 001401; NC001729; NC001829; NC006152; NC 006260; NC 006261), chimeric ITR or ITR from any synthetic A A.V. In some embodiments, said AAV can infect warm-blooded animals such as avian (AAAV), bovine (BAAV), canine, equine, and ovine adeno-associated viruses. In some embodiments, said ITR is from Parvovirus B19 (GenBank Accession No. NC 000883), Small Mouse Virus (MVM) (GenBank Accession No. NC 001510); geese parvovirus (GenBank Accession No. NC 001701); serpentine parvovirus 1 (GenBank Accession No. NC 006148).

[00118] In some embodiments, the ITR sequence can be taken from viruses in the Parvoviridae family, which includes two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect insects, the Parvovirinae subfamily (referred to as "parvoviruses") includes the genus Dependovirus, members of which, under most conditions, require coinfection with a helper virus, such as adenovirus or herpesvirus, for a productive infection. The genus Dependovirus includes adeno-associated virus (AAV), which commonly infects humans (eg, serotypes 2, 3A, 3B, 5, and 6) or primates (eg, serotypes 1 and 4), and related viruses that infect other warm-blooded animals (eg, bovine, canine, equine, and ovine adeno-associated viruses). Parvoviruses and other members of the Parvoviridae family are generally described in: Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996).

[00119] One of ordinary skill in the art will recognize that ITR sequences are similar in structure and contain a double-stranded Holliday structure, which is typically a T-shaped or Y-shaped hairpin structure (see, for example, FIGS. 2A and FIG. 3A), with each ITR formed by two palindromic arms or loops (B-B' and C-C') immersed in a larger palindromic arm (A-A'), and single strands sequence D (where the order of the indicated palindromic sequences determines the orientation rotation of the ITR); appropriate modified ITR sequences from any AAV serotype can be easily determined for use in a ccDNA vector or ccDNA plasmid based on the exemplary AAV2 ITR sequences provided by the present invention. See, for example, a description of the structural analysis and comparison of ITR sequences from different AAV serotypes (AAV1-AAV6) in Grimm et al., J. Virology, 2006; 80(1); 426-439; Yan et al, J. Virology, 2005; 364-379; Duan et al., Virology 1999; 261; 8-14.

[00120] Specific ITR changes and mutations are detailed herein, but in the context of an ITR, "altered" or "mutated" indicates that nucleotides have been inserted, deleted, and/or substituted relative to a wild-type ITR sequence, a reference or parent ITR sequence, and may be altered relative to another flanking ITR in a scDNA vector containing two flanking ITRs. The altered or mutated ITR may be an engineered ITR. As used herein, "engineered" refers to an aspect that has been manipulated by human hands. For example, a polypeptide is considered "engineered" if, in at least one aspect, said polypeptide, such as its sequence, has been manipulated by human hands in such a way that it differs from naturally occurring in said aspect.

[00121] In some embodiments, the ITR may be synthetic. In one embodiment, the synthetic ITR is based on ITR sequences from more than one AAV serotype. In another embodiment, the synthetic ITR does not include an AAV-based sequence. In yet another embodiment, the synthetic ITR retains the ITR structure described above, although it contains only some or no sequences taken from AAV. In some aspects, the synthetic ITR may interact preferentially with a wild-type or serotype-specific Rep, or, in some cases, it will not recognize a wild-type Rep but only a mutated Rep.

[00122] ITR sequences are similar in structure and contain a double-stranded Holliday structure, which is typically a T-shaped or Y-shaped hairpin structure (see, for example, figa and figa), with each ITR formed by two palindromic arms or loops (B-B' and C-C'), immersed in a larger palindromic arm (A-A'), and a single-stranded sequence D (where the order of the specified palindrome sequences determines the rotation of the ITR orientation). One of ordinary skill in the art can easily determine ITR sequences or modified ITR sequences from any AAV serotype for use in a ccDNA vector or ccDNA plasmid based on the exemplary AAV2 ITR sequences provided by the present invention. See, for example, the comparison of ITR sequences from different AAV serotypes (AAV1 - AAV6, avian AAV (AAAV) and bovine AAV (BAAV)) described in: Grimm et al., J. Virology, 2006; 80(1); 426-439; where % identity of AAV2 left ITR and left ITR from other serotypes is given: AAV-1 (84%), AAV-3 (86%), AAV-4 (79%), AAV-5 (58%), AAV-6 (left ITR) (100%) and AAV-6 (right ITR) (82%).

[00123] Accordingly, while AAV2 ITRs are used as examples of ITRs in ccDNA vectors of the present invention, a ccDNA vector as described herein can be derived from or based on the ITR of any known AAV serotype, including, for example, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), serotype AAV 6 (AAV6), serotype AAV 7 (AAV7), serotype AAV 8 (AAV8), serotype AAV 9 (AAV9), serotype AAV 10 (AAV10), serotype AAV 11 (AAV11), or serotype AAV 12 (AAV12). The person skilled in the art can determine the corresponding sequence in other serotypes using known methods, for example, by locating the change: in region A, A', B, B', C, C, or D, and determining the corresponding region in another serotype. BLAST® (Basic Local Alignment Search Tool) or other program for aligning homologous sequences in the default state can be used to determine the appropriate sequence. The present invention also provides populations and populations of ccDNA vectors containing ITRs from a combination of different AAV serotypes, i.e. one ITR can be taken from one AAV serotype and another ITR can be taken from another serotype. Without wishing to be bound by theory, in one embodiment, one ITR may be taken from or based on the AAV2 ITR sequence, and the other ITR of said ccDNA vector may be taken from or based on any one or more ITR sequences of AAV serotype 1 (AAV1), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), serotype a AAV 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV 10 serotype (AAV10), AAV 11 serotype (AAV11), or AAV 12 serotype (AAV12).

[00124] Any ITR parvovirus can be used as an ITR or as an ITR base for modification. Preferably, said parvovirus is a dependovirus. More preferably, it is AAV. The choice of serotype may be based on the tissue tropism of said serotype. AAV2 has a tropism for a wide range of tissues, AAV1 preferentially targets neural tissue and skeletal muscle, and AAV5 preferentially targets neural tissue, retinal pigment epithelium, and photoreceptors. AAV6 predominantly targets skeletal muscle and lungs. AAV8 predominantly targets the liver, skeletal muscle, heart, and pancreatic tissue. AAV9 predominantly targets the liver, skeletal tissues, and lung tissues. In one embodiment, the modified ITR is based on the AAV2 ITR. For example, it is selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO: 52. In one embodiment of each of these aspects, the vector polynucleotide contains an ITR pair selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 52; and SEQ ID NO: 2 and SEQ ID NO: 51. In one embodiment of each of these aspects, the vector polynucleotide or non-viral capsidless DNA vectors with covalently closed ends contain a pair of different ITRs selected from the group consisting of: SEQ ID NO: 101 and SEQ ID NO: 102; SEQ ID NO: 103 and SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106; SEQ ID NO: 107 and SEQ ID NO: 108; SEQ ID NO: 109 and SEQ ID NO: 110; SEQ ID NO: 111 and SEQ ID NO: 112; SEQ ID NO: 113 and SEQ ID NO: 114; and SEQ ID NO: 115 and SEQ ID NO: 116. In some embodiments, the modified ITR is selected from any of the ITR or partial ITR sequences from SEQ ID NO: 2, 52, 63, 64, 101-499, or 545-547.

[00125] In some embodiments, the scDNA vector may comprise an ITR with an ITR modification corresponding to any of the modifications in the ITR sequences or partial ITR sequences shown in any one or more of Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A, and 10B herein, or the sequences shown in Figures 26A or 26B.

[00126] In some embodiments, scDNAs may form an intramolecular duplex secondary structure. Examples of the secondary structure of the first ITR and the asymmetric second ITR are presented in the context of wild-type ITR structures (see, eg, FIGS. 2A, 3A, 3C) and modified ITRs (see, eg, FIGS. 2B and 3B, 3D). Secondary structures are derived or predicted based on the ITR sequences of the plasmid used to generate the scDNA vector. Examples of secondary structures of modified ITRs where part of the stem-loop structure is removed are shown in FIGS. 9A-25B and FIGS. 26A-26B, and are also shown in Tables 10A and 10B. Examples of secondary structures of modified ITRs containing a single stem and two loops are shown in FIGS. 9A to 13B. An example of the secondary structure of a modified ITR with a single stem and a single loop is shown in FIG. In some embodiments, the secondary structure can be inferred as described herein using thermodynamic techniques based on nearest neighbor rules predicting structure stability by quantifying stacking free energy change. For example, a structure can be predicted by finding the structure with the lowest free energy. Some implementations may use the algorithm described in Reuter, J. S., & Mathews, D. H. (2010) RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics. 11,129 and implemented in the RNAstructure software (available on the Internet at: "rna.urmc.rochester.edu/RNAstructureWeb/index.html") to predict the ITR structure. This algorithm may also include both free energy change measures at 37° C. and enthalpy change measures from published experimental data to predict conformational stability at an arbitrary temperature. Using the RNAstructure software, some of the modified ITR structures can be predicted as modified stem-loop T-structures with the estimated Gibbs free energy (AG) change for physiological unfolding shown in FIGS. 3A-13D. Using the RNAstructure software, three types of modified ITRs have a predicted Gibbs free energy for unfolding that is greater than that of the wild-type AAV2 ITR (-92.9 kcal/mol) and are represented by the following ITRs: (a) A modified ITR having the single arm/single unpaired loop structure proposed by the present invention, with a predicted Gibbs free energy for unfolding ranging from -85 to -70 kcal/mol. (b) Modified ITR having a single hairpin structure according to the present invention with a predicted unfolding free energy ranging from -70 to -40 kcal/mol. (c) Modified ITR having the double arm structure of the present invention with a predicted Gibbs free energy for unfolding ranging from -90 to -70 kcal/mol. Without wishing to be bound by any theory, structures with higher Gibbs free energy are more readily unfolded for replication by the action of the Rep 68 or Rep 78 replication proteins. Accordingly, modified ITRs having higher Gibbs free energies for unfolding - e.g. single arm/single unpaired loop structure, single hairpin structure, truncated structure tend to replicate more efficiently than wild-type ITRs.

[00127] In one embodiment, the left ITR of said scDNA vector is modified or mutated relative to the wild-type (wt) AAV ITR structure, and the right ITR is the wild-type AAV ITR. In one embodiment, the right ITR of said scDNA vector is modified relative to the structure of the wild-type AAV ITR and the left-hand ITR is the wild-type AAV ITR. In such an embodiment, a modification of the ITR (eg, left or right ITR) can be obtained by deletion, insertion, or substitution of one or more wild-type ITR nucleotides derived from the AAV genome.

[00128] The ITRs used according to the present invention can be resolvable and non-resolvable, and AAV sequences are preferably selected for use in ccDNA vectors, with serotypes 1, 2, 3, 4, 5, 6, 7, 8, and 9 being preferred. insertions, deletions, truncations, and/or missense mutations), so long as the terminal repeat mediates the required functions, e.g., viral replication, virus packaging, virus integration and/or provirus rescue, etc. Typically, though not necessarily, the ITRs are from a single AAV serotype, e.g., both ITR sequences of said scDNA vector are from AAV2. Said ITRs may be synthetic sequences that function as inverted terminal repeats of AAV, such as "double D sequence" as described in US Pat. No. 5,478,745 to Samulski et al. Although not required, the ITRs may be from the same parvovirus, eg both ITR sequences are from AAV2.

[00129] In one embodiment, the scDNA may include an ITR structure mutated relative to one of the wild-type ITRs as described herein, however, the mutated or modified ITR retains a functional Rep binding site (RBE or RBE') and terminal clearance site (trs). In one embodiment, said ITR ccDNA mutant includes a functional site for a replication protein (RPS-1) and a replication competent protein is used in production that binds said RPS-1 site.

[00130] In one embodiment, at least one of the ITRs is a defective ITR in relation to Rep binding and/or Rep nicking. In one embodiment, said defect is at least a 30% reduction relative to wild-type ITR, in other embodiments, it is at least a 35%..., 50%..., 65%..., 75%..., 85%..., 90%..., 95% reduction …, 98%…, or no function at all, or any degree of reduction in between. Host cells do not express viral capsid proteins and the vector polynucleotide template does not contain any viral capsid coding sequences. In one embodiment, the vector polynucleotide template and host cells that do not contain the AAV capsid genes, and the resulting protein also does not encode or express the capsid genes of other viruses. In addition, according to a specific implementation variant, the nucleic acid molecule also does not contain coding sequences for the Rep AAV protein.

[00131] In some embodiments, the ITR structural element can be any structural element that is involved in the functional interaction of the ITR with a large Rep protein (eg, Rep 78 or Rep 68). In some embodiments, said structural element provides selectivity for the interaction of the ITR with the large Rep protein, i.e. determines, at least in part, which Rep protein functionally interacts with the ITR. In other embodiments, said structural element physically interacts with a large Rep protein upon binding said Rep protein to the ITR. Each structural element can be, for example, an ITR secondary structure, an ITR nucleotide sequence, a gap between two or more elements; or a combination of any of the listed elements. In one embodiment, said building blocks are selected from the group consisting of arm A and arm A', arm B and arm B', arm C and arm C', arm D, Rep binding site (RBE) and RBE' (i.e. complementary to the RBE sequence), and terminal clearance site (trs).

[00132] More specifically, the ability of a structural element to functionally interact with a particular large Rep protein can be changed by modifying said structural element. For example, the nucleotide sequence of a structural element may be modified relative to the wild-type ITR sequence. In one embodiment, said ITR building block (e.g., arm A, arm A', arm B, arm B', arm C, arm C', arm D, RBE, RBE', and trs) of the ITR can be removed and replaced with a wild-type building block from another parvovirus. For example, the replacement structure can be taken from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV 13, serpentine parvovirus (e.g., king python parvovirus), bovine parvovirus, goat parvovirus, avian parvovirus, canine parvovirus, equine parvovirus , shrimp parvovirus, porcine parvovirus, or insect AAV. For example, the ITR may be an ITR of AAV2 and the arm A or A' or RBE may be replaced by a building block from AAV5. According to another example, the ITR may be an AAV5 ITR and the C or C, RBE and trs arms may be replaced by a building block from AAV2. According to another example, the AAV ITR may be an AAV5 ITR, where the B and B' arms are replaced by the AAV2 ITR arms B and B'.

[00133] For example, Table 1 provides examples of at least one nucleotide modifications (e.g., deletions, insertions, and/or substitutions) in regions of modified ITRs, where X indicates at least one nucleic acid modification (e.g., deletion, insertion, and/or substitution) in the specified region relative to the corresponding wild-type ITR. In some embodiments, any modification of at least one nucleotide (e.g., deletions, insertions, and/or substitutions) in any of the C and/or C and/or B and/or B' regions retains three consecutive T (i.e., TTT) nucleotides in at least one terminal loop. For example, if the modification results in any of: a single-arm ITR (e.g., a single C-C' arm or a single B-B' arm), or a modified C-B' arm or a C'-B arm, or a two-arm ITR containing at least one truncated arm (e.g., a truncated C-C' arm and/or a truncated B-B' arm) in at least said single arm, or in at least one of the ITR arms with two arms (where one arm may be truncated), three consecutive T nucleotides (i.e., TTT) are retained in at least one terminal loop. In some embodiments, the truncated C-C arm and/or the truncated B-B' arm contains three consecutive T nucleotides (ie, TTT) in the terminal loop.

[00134] Table 1: Examples of combinations of at least one nucleotide modifications (e.g., deletions, insertions, and/or substitutions) in different regions or arms B-B' and C-C' of the ITR (X indicates a nucleotide modification, e.g., addition, deletion, or substitution of at least one nucleotide in a region).

[00135] In some embodiments, a modified ITR for use according to the present invention may comprise any of the combinations of modifications shown in Table 1, as well as a modification of at least one nucleotide in any one or more of the regions selected from: the region between A' and C, between C and C', between C' and B, between B and B', and between B' and A. In some embodiments, any modification of at least one nucleotide (e.g., deletions, insertions and/or substitution) in areas C, or C', or B, or B', the terminal loop "loop on the stem" is still preserved. In some embodiments, any modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) between C and C' and/or B and B' retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. In alternative embodiments, any modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) between C and C' and/or B and B' retains three consecutive A (i.e., AAA) nucleotides in at least one terminal loop. In some embodiments, a modified ITR for use according to the present invention may contain any of the combinations of modifications shown in Table 1, as well as a modification of at least one nucleotide (e.g., a deletion, insertion, and/or substitution) in any one or more of the regions selected from: A', A, and/or D. at least one nucleotide (e.g., deletion, insertion, and/or substitution) in region A. In some embodiments, a modified ITR for use according to the present invention may comprise any of the combinations of modifications shown in Table 1, as well as at least one nucleotide modification (e.g., deletion, insertion, and/or substitution) in region A'. In some embodiments, a modified ITR for use according to the present invention may comprise any of the combinations of modifications shown in Table 1, as well as at least one nucleotide modification (e.g., deletion, insertion, and/or substitution) in the A and/or A' region. In some embodiments, a modified ITR for use according to the present invention may contain any of the combinations of modifications shown in Table 1, as well as at least one nucleotide modification (e.g., deletion, insertion, and/or substitution) in region D.

[00136] In one embodiment, the nucleotide sequence of a structural element can be modified (e.g., by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides, or any number of nucleotides in the available range) to obtain modified structures element. Examples of specific ITR modifications according to one embodiment are provided herein (eg, SEQ ID NOs: 2, 52, 63, 64, 101-499, or 545,547). In some embodiments, the ITR may be modified (e.g., by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides, or any number of nucleotides in the available range). In other embodiments, the sequence of said ITR may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the sequence of one of the modified ITRs of SEQ ID NO: 469499 or 545-547, or R BE-containing section of the arm A-A' and arms C-C' and B-B' from SEQ ID NO: 101-134 or 545-547.

[00137] In some embodiments, the modified ITR may, for example, include deletion or deletion of all of a particular arm, e.g., all or part of arm A-A', or all of or part of arm B-B', or all of or part of arm C-C', or alternatively, deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs forming a loop stem, provided that the last loop on the top of the stem (eg one shoulder) is still present (eg see ITR-6). In some embodiments, the modified ITR may include deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from the B-B' arm. In some embodiments, the modified ITR may include deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from the C-C' arm. In some embodiments, the modified ITR may include deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from the C-C' arm and deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from the B-B' arm. Any combination of base pair removal is contemplated, for example, 6 base pairs in the C-C' arm and 2 base pairs in the B-B' arm can be removed. In an illustrative example, FIGS. 13A-13B show an example of a modified ITR where at least 7 base pairs are deleted from both part C and part C', a nucleotide is changed in the loop between regions C and C', and at least one base pair is deleted from both part B and part B', such that said modified ITR contains two arms, at least one of which (e.g., C-C') is truncated. Note that in this example, since the modified ITR contains a deletion of at least one base pair in each of the B and B' regions, the B-B' arm is also truncated relative to the WT ITR.

[00138] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more complementary base pairs are removed from both part C and part C' of the C-C' arm in such a way that the C-C' arm is truncated, i.e. if the base is removed in the C part of the C-C' arm, the complementary base pair in part C is also removed, thus truncating the C-C arm '. In such embodiments, 2, 4, 6, 8 or more base pairs are removed from the C-C' arm such that the C-C' arm is truncated. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs are removed from the C portion of the C-C' arm such that only the C' portion of the arm remains. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs are removed from the C' portion of the C-C' arm such that only the C portion of the arm remains.

[00139] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more complementary base pairs are removed from each of the B and B' portions of the B-B' arm such that the B-B' arm is truncated, i.e. if the base is removed at the B portion of the B-B' arm, the complementary base pair at the B' portion is also removed, thereby truncating the B-B' arm . In such embodiments, 2, 4, 6, 8 or more base pairs are removed from the B-B' arm such that the B-B' arm is truncated. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs are removed from the B portion of the B-B' arm such that only the B' portion of the arm remains. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs are removed from the B' portion of the B-B' arm such that only the B portion of the arm remains.

[00140] In some embodiments, the modified ITR may comprise 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotide deletions relative to the full length wild-type ITR sequence. In some embodiments, the modified ITR may contain 1 to 30 nucleotide deletions relative to the sequence of the full length WT ITR. In some embodiments, the modified ITR contains 2 to 20 nucleotide deletions relative to the sequence of the full length wild type ITR.

[00141] In some embodiments, the modified ITR forms two opposing longitudinally asymmetric stem loops, eg, the length of the C-C' loop is different from the length of the B-B' loop. In some embodiments, the length of the C-C' and/or B-B' stem portion of one of the opposing longitudinally asymmetric stem loops of the modified ITR is in the range of 8 to 10 base pairs, and the loop portion (e.g., between C-C' or between B-B') contains 2-5 unpaired deoxyribonucleotides. In some embodiments, one of the longitudinally asymmetric stem loops of the modified ITR contains a C-C' and/or B-B' portion of the stem less than 8, or less than 7, 6, 5, 4, 3, 2, 1 base pairs long, and the loop portion (e.g., between C-C' or between B-B') contains 0 5 nucleotides. In some embodiments, the modified ITR with a longitudinally asymmetric stem loop contains a C-C' and/or B-B' portion of the stem less than 3 bp in length.

[00142] In some embodiments, the modified ITR does not contain any nucleotide deletions in the RBE-containing portion of the A or A regions so as not to interfere with DNA replication (eg, binding to the RBE of the Rep protein, or nicking at the terminal clearance site). In some embodiments, the modified ITR provided for use in the present invention contains one or more deletions in the B, B', C, and/or C region as described herein. Some non-limiting examples of modified ITRs are shown in FIGS. 9A-26B.

[00143] In some embodiments, the modified ITR may include a deletion of the B-B' arm so that the C-C' arm remains, e.g., see examples of ITR-2 (left) and ITR-2 (right) in FIGS. 9A-9B, and ITR-4 (left), and for ITR-4 (right) (FIGS. 11A-11B). In some embodiments, the modified ITR may include a deletion of the C-C' arm so that the B-B' arm remains, for example, see examples for ITR-3 (left), and for ITR-3 (right), shown in Figures 10A-10B. In some embodiments, the modified ITR may include a deletion of the B-B' arm and the C-C' arm so that a single stem loop remains, e.g. 14A-14B, as well as ITR-21 and ITR-37. In some embodiments, the modified ITR may include a deletion of the C' region such that a truncated C-loop and B-B' arm remains, for example, see examples of ITR-1 (left) and ITR-1 (right) shown in FIG. 15A-15B. Similarly, in some embodiments, the modified ITR may include a deletion of the C region such that a truncated C'-loop and B-B' arm remains, for example, see examples of ITR-5 (left) and ITR-5 (right) shown in FIG. 16A-16B.

[00144] In some embodiments, the modified ITR may include a base pair deletion in any one or more of the C, C', B, or B' portions such that complementary base pairing occurs between the C-B' portions and the C'-B portions to form a single arm, for example, see ITR-10 (right) and ITR-10 (left) (FIGS. 12A-12B).

[00145] In some embodiments, in addition to modifying one or more nucleotides in regions C, C', B, and/or B', a modified ITR for use according to the present invention may include a modification (e.g., deletion, substitution, or addition) of at least 1, 2, 3, 4, 5, 6 nucleotides in any one or more regions selected from: the region between A' and C, between C and C', between C and B, between B and B', and between B ' and A. For example, the nucleotide between B' and C in the right-modified ITR may be changed from nA to G, C, or A, or deleted, or one or more nucleotides may be added; the nucleotide between C and B in the modified left ITR may be changed from T to G, C or A, or deleted, or one or more nucleotides may be added.

[00146] According to some embodiments of the present invention, the scDNA vector does not contain a modified ITR consisting of a nucleotide sequence selected from any of SEQ ID NOs: 550-557. According to some embodiments of the present invention, the scDNA vector does not contain a modified ITR containing a nucleotide sequence selected from any of SEQ ID NOs: 550-557.

[00147] In some embodiments, said scDNA vector comprises a regulatory switch as described herein and a selected modified ITR with a nucleotide sequence selected from any sequence from the group consisting of: SEQ ID NO: 550,557.

[00148] According to another implementation variant, the structure of the structural element can be modified. For example, the height of the stem and/or the number of nucleotides in the loop is changed in the structural element. For example, the stem height may be approximately 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides or more, or any number within the available range. In one embodiment, the stem may be from about 5 nucleotides to about 9 nucleotides in height and is operatively interacting with Rep. In another embodiment, the stem may be approximately 7 nucleotides high and operably interacts with Rep. According to another example, the loop may contain 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides or more, or any number of nucleotides in the available range.

[00149] In another embodiment, the number of GAGY binding sites or GAGY-related binding sites within an RBE or an extended RBE may be increased or decreased. According to one example, the RBE or extended RBE may contain 1, 2, 3, 4, 5, or 6 or more GAGY binding sites, or any number of binding sites in the available range. Each GAGY binding site can independently be the exact GAGY sequence or a GAGY-like sequence, provided that the sequence is sufficient to bind the Rep proteins.

[00150] In another embodiment, the spacing between two elements (eg, but not limited to RBE and hairpin) can be changed (eg, increased or decreased) to change the functional interaction with the large Rep protein. For example, said span may be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides or more, or contain any number of nucleotides in the available range.

[00151] The scDNA vector described herein may include an ITR structure modified from the wild-type AAV2 ITR structure as described herein, but still containing the RBE, trs, and RBE' functional portion. In FIG. 2A and FIG. 2B shows one possible mechanism of action for the trs site within the structure of the wild-type ITR portion of the scDNA vector. In some embodiments, said scDNA vector contains one or more functional ITR polynucleotide sequences that contain a Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' (SEQ GO NO: 531) for AAV2) and a terminal resolution site (TRS; 5'-AGTT (SEQ ID NO: 46)). In some embodiments, at least one ITR (wt or modified ITR) is functional. In alternative embodiments, when the scDNA vector contains two modified ITRs that are different or asymmetric from each other, at least one modified ITR is functional and at least one modified ITR is non-functional.

[00152] In some embodiments, the scDNA vector does not contain a modified ITR selected from any sequence consisting or consisting essentially of: SEQ ID NO: 500-529 as described herein. In some embodiments, the scDNA vector does not contain an ITR selected from any sequence selected from SEQ ID NOs: 500-529.

[00153] In some embodiments, the modified ITR (eg, left or right ITR) of the scDNA vector described herein contains modifications in the loop arm, truncated arm, or spacer. Examples of ITR sequences with modifications in the loop arm, truncated arm, or spacer are shown in Table 2.

[00154] In some embodiments, the modified ITR (eg, left or right ITR) of the scDNA vector described herein contains modifications in the loop arm and the truncated arm. Examples of ITR sequences with modifications in the loop arm and truncated arm are shown in Table 3.

[00155] In some embodiments, the modified ITR (eg, left or right ITR) of the scDNA vector described herein contains modifications in the loop arm and spacer. Examples of ITR sequences with modifications in the loop arm and spacer are shown in Table 4.

[00156] In some embodiments, the modified ITR (eg, left or right ITR) of the scDNA vector described herein contains modifications to the truncated arm and spacer. Examples of ITR sequences with modifications in the truncated arm and spacer are shown in Table 5.

[00157] In some embodiments, the modified ITR (eg, left or right ITR) of the scDNA vector described herein contains modifications in the loop arm, truncated arm, and spacer. Examples of ITR sequences with modifications in the loop arm, truncated arm, and spacer are shown in Table 6.

[00158] In some embodiments, the ITR (eg, left or right ITR) is modified to have the lowest unfolding energy ("low energy structure"). The low energy corresponds to a reduced Gibbs free energy compared to the wild-type ITR. Examples of ITR sequences modified to achieve low (ie, reduced) unfolding energy are provided herein in Tables 7-9.

[00159] In some embodiments, the modified ITR is selected from any ITR or combination of ITRs shown in Tables 2-9, 10A or 10B.

[00160] Table 2: ITR sequences with modifications in loop arm, truncated arm, or spacer. They include the RBS sequence GCGCGCTCGCTCGCTC (SEQ ID NO: 531) at the 5' end and the complementary sequence RBE' GAGCGAGCGAGCGCGC (SEQ ID NO: 536) at the 3' end.

[00161] According to the present disclosure, a modified ITR can be obtained that includes the deletion, insertion, or substitution of one or more nucleotides from a wild-type ITR derived from the AAV genome. The modified ITR can be obtained by genetic modification by propagation in a plasmid in Escherichia coli, or as a baculovirus genome in Spodoptera frugiperda cells, or by other biological methods, for example, in vitro using a polymerase chain reaction, or by chemical synthesis.

[00162] In some embodiments, the modified ITRs include deletion, insertion, or substitution of one or more nucleotides from AAV2 wild-type (left) ITR (SEQ ID NO: 51) or AAV2 wild-type (right) ITR (SEQ ID NO: 1). In particular, one or more nucleotides from the B-C' or C-C' T-shaped stem loop structure are deleted, inserted or replaced. Further, the modified ITR does not include modifications in the Rep-binding elements (RBE) and terminal clearance site (trs) of the AAV2 wild-type ITR, although RBE' (TTT) may or may not be present depending on whether the template has undergone one round of replication, thus converting the AAA triplet to the complementary RBE'-TTT triplet.

[00163] Examples of three types of modified ITRs are provided (1) modified ITR having the lowest energy structure containing a single arm and a single unpaired loop ("single arm structure/single unpaired loop"); (2) a modified ITR having the lowest energy single hairpin structure ("single hairpin structure"); and (3) a modified ITR having the lowest energy structure with two arms, one of which is truncated ("truncated structure").

[00164] Modified ITR having single arm/single unpaired loop structure

[00165] A wild-type ITR can be modified to form a secondary structure containing a single arm and a single unpaired loop (i.e., "single arm/single unpaired loop structure"). The change in Gibbs free energy (ΔG) for unfolding said structure can range from -85 kcal/mol to -70 kcal/mol. Examples of modified ITR structures are proposed.

[00166] Modified ITRs that are predicted to form a single arm/single unpaired loop structure may include deletion, insertion, or substitution of one or more nucleotides from the wild-type ITRs in the sequences forming the B and B' arm and/or the C and C' arm. The modified ITR may be obtained by genetic modification or by biological and/or chemical synthesis.

[00167] For example, the ITR-2 left and right shown in FIG. 9A-9B (SEQ ID NOS: 101 and 102) were generated to include a two nucleotide deletion from the C-C' arm and a 16 nucleotide deletion from the B-B' arm in the AAV2 wild-type ITR. The three remaining nucleotides in the B-B' arm of the modified ITR do not undergo complementary pairing. Accordingly, ITR-2, left and right, have the lowest energy structure with a single C-C' arm and a single unpaired loop. The predicted change in Gibbs free energy for the unfolding of this structure is approximately -72.6 kcal/mol.

[00168] ITR-3 left and right shown in FIG. 10A and 10B (SEQ ID NOs: 103 and 104) were generated to include 19 nucleotide deletions in the C-C arm of the AAV2 wild-type ITR. The three remaining nucleotides in the B-B' arm of the modified ITR do not undergo complementary pairing. Accordingly, ITR-3 left and right have the lowest energy structure with a single B-B' arm and a single unpaired loop. The predicted Gibbs free energy change for the unfolding of this structure is approximately -74.8 kcal/mol.

[00169] ITR-4 left and right shown in FIG. 11A and 11B (SEQ ID NOs: 105 and 106) were generated to include 19 nucleotide deletions in the B-B' arm of the AAV2 wild-type ITR. The three remaining nucleotides in the B-B' arm of the modified ITR do not undergo complementary pairing. Accordingly, ITR-4 left and right have the lowest energy structure with a single C-C' arm and a single unpaired loop. The predicted Gibbs free energy change for the unfolding of this structure is approximately -76.9 kcal/mol.

[00170] ITR-10 left and right shown in FIG. 12A and 12B (SEQ ID NOs: 107 and 108) were generated to include 8 nucleotide deletions in the B-B' arm of the AAV2 wild-type ITR. The remaining nucleotides in the B-B' and C-C' arms form new complementary bonds between the B and C' motifs (ITR-10, left) or between the C and B' motifs (ITR-10, right). Accordingly, ITR-10 left and right have the lowest energy structure with a single B-C' or C-B' arm and a single unpaired loop. The predicted Gibbs free energy change for the unfolding of this structure is approximately -83.7 kcal/mol.

[00171] ITR-17 left and right shown in FIG. 13A and 13B (SEQ ID NOs: 109 and 110) were prepared to include 14 nucleotide deletions in the C-C' arm of the AAV2 wild-type ITR. The remaining eight nucleotides in the C-C' arm do not form complementary bonds. As a result, ITR-17, left and right, have the lowest energy structure with a single B-B' arm and a single unpaired loop. The predicted change in Gibbs free energy for the unfolding of this structure is approximately -73.3 kcal/mol.

[00172] The sequences of wild-type left or right ITRs (top) and various modified left or right ITRs (bottom) predicted to form a single arm/single unpaired loop structure are aligned; the alignment is shown in Table 7 below.

[00173] Table 7: Alignment of wt-ITR and modified ITR (ITR-2, ITR-3, ITR-4, ITR-10 and ITR-17) having single arm/single unpaired loop structure.

[00174] Modified ITR having a single hairpin structure

[00175] The wild type ITR can be modified to have the lowest energy structure containing the single hairpin structure. The change in Gibbs free energy (AG) for unfolding said structure can range from -70 kcal/mol to -40 kcal/mol. Examples of modified ITR structures are provided in FIG. 14A and 14B.

[00176] Modified ITRs that are predicted to form a single hairpin structure may include deletion, insertion, or substitution of one or more nucleotides from the wild-type ITRs in the sequences forming the B and B' arm and/or the C and C' arm. The modified ITR may be obtained by genetic modification or by biological and/or chemical synthesis.

[00177] For example, the ITR-6 left and right shown in FIG. 14A and 14B (SEQ ID NOS: 111 and 112) include 40 nucleotide deletions in the B-B' and C-C' arms of the AAV2 wild-type ITR. The remaining nucleotides in the modified ITR are predicted to form a single hairpin structure. The change in Gibbs free energy for unfolding said structure is approximately -54.4 kcal/mol.

[00178] The wild-type and ITR-6 (both left and right) sequences are aligned; the alignment is shown in Table 8 below.

[00179] Table 8: Alignment of wt-ITR and modified ITR-6 having a single hairpin structure.

[00180] Modified ITR with truncated structure

[00181] The wild-type ITR can be modified to have the lowest energy structure containing two arms, one of which is truncated. Their Gibbs free energy (AG) change for unfolding ranges from -90 to -70 kcal/mol. Accordingly, their Gibbs free energy values for unfolding are lower than for wild-type AAV2 ITRs.

[00182] The modified ITR may include deletion, insertion, or substitution of one or more nucleotides from the wild-type ITR in sequences forming the B and B' arm and/or the C and C' arm. In some embodiments, the modified ITR may, for example, include the removal of a particular loop entirely, such as the A-A' loop, the B-B' loop, or the C-C' loop, or alternatively, the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs forming the stem of the loop, provided that the last loop at the end of the stem still remains. The modified ITR may be obtained by genetic modification or by biological and/or chemical synthesis.

[00183] Examples of modified ITR structures with a truncated structure are shown in FIG. 15A-15B.

[00184] The sequences of various modified ITRs that are predicted to form a truncated structure are aligned with the wild-type ITR sequence; the alignment is shown in Table 9 below.

[00185] Table 9: Alignment of wt-ITR and modified ITR (ITR-5, ITR-7, ITR-8, ITR-9, ITR-11, ITR-12, ITR-13, ITR-14, ITR-1 and ITR-16) with a truncated structure.

[00186] Additional examples of modified ITRs in each of the above classes for use in accordance with the present invention are shown in Tables 10A and 10B. The predicted secondary structure of the right modified ITR from Table 10A is shown in FIG. 26A, and the predicted secondary structure of the left modified ITRs from Table 10B is shown in FIG. 26V.

[00187] Table 10A and Table 10B provide examples of right and left modified ITRs.

[00188] Table 10A: Modified Right ITR Examples. These examples of modified right ITRs may comprise RBE GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531), spacer ACTGAGGC (SEQ ID NO: 532), spacer complement GCCTCAGT (SEQ ID NO: 535), and RBE' (i.e. RBE complement) GAGCGAGCGAGCGCGC (SEQ ID NO: 536).

[00189] TABLE 10B: Examples of modified left ITRs. These examples of modified left ITRs may contain RBE GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531), spacer ACTGAGGC (SEQ ID NO: 532), spacer complement GCCTCAGT (SEQ ID NO: 535), and complement RBE (RBE') GAGCGAGCGAGCGCGC (SEQ ID NO: 536).

[00190] According to embodiments of the present invention, the scDNA vector as described herein does not contain a modified ITR with a nucleotide sequence selected from any sequence from the group of SEQ ID Nos: 550, 551, 552, 553, 553, 554, 555, 556, 557.

[00191] Provided that the scDNA vector contains a modified ITR with one of the modifications in region B, B', C, or C' as described in SEQ ID NO: 550-557, as defined in any one or more claims of the present invention, or any other invention as defined in the amended claims that may hereafter be filed with this application, or in any derivative patent, and under the laws of any participating state or states where any claim is claimed, the present inventors reserve the right to waive said disclosure in the claims appended to this application or any derivative patent to the extent necessary to prevent the invalidity of this application or any derivative patent.

[00192] For example, and without limitation, the authors of the present invention reserve the right to disclaim any of the following objects of the invention from any claim of the present invention, in the current version or as amended in the future, or from any derivative patent:

[00193] A. A modified ITR selected from any sequence from the group consisting of: SEQ ID NOs: 2, 52, 6364, 113, 114, 550, 551; 552, 553, 553, 554, 555, 556, 557 used in scDNA vector without regulatory switch

[00194] B. The modified A. ITR described above, in a ccDNA vector without a regulatory sequence, wherein the heterologous nucleic acid encodes ABCA4, USA2A var1, VEGFR, CEP290, BDD factor VIII (FVIII), factor VIII, vWF_His, vWF, lecithincholesterol acyltransferase, PAH, G6PC, or CFTR

[00195] Without limitation, the authors of the present invention argue that the above right of withdrawal applies to at least claims 1-57 in this application and to all claims in the description, including, but not limited to, the claims in [0027] and [00397].

IV. regulatory elements.

[00196] These scDNA vectors can be generated from expression constructs that additionally contain a specific combination of cis regulatory elements. These cis-regulatory elements include, but are not limited to, a promoter, a riboswitch, an insulator, an miRNA-regulated element, a post-transcriptional regulatory element, a tissue-specific and cell-specific promoter, and an enhancer. In some embodiments, said ITR may act as a promoter for the transgene. In some embodiments, said scDNA vector contains additional components to regulate the expression of said transgene, such as regulatory switches as described herein to regulate the expression of said transgene, or a "kill switch" that can cause the cell containing the scDNA vector to die.

[00197] ZcDNA vectors can be derived from expression constructs that further contain a specific combination of cis regulatory elements such as the WHP post-transcriptional regulatory element (WPRE) (e.g., SEQ ID NO: 8) and poly(A) BGH (SEQ ID NO: 9). Suitable expression cassettes for use in expression constructs are not limited by packaging restrictions imposed by the viral capsid. The expression cassettes of the present invention include a promoter that can influence overall expression levels as well as cell specificity. In the case of transgene expression, they may include a highly active immediate early promoter of viral origin. Expression cassettes may contain tissue-specific eukaryotic promoters to limit transgene expression to specific cell types and reduce toxic effects and immune responses caused by unregulated ectopic expression. In preferred embodiments, the expression cassette may contain a synthetic regulatory element such as the CAG promoter (SEQ ID NO 3). The CAG promoter contains (i) a cytomegalovirus (CMV) early enhancer element, (ii) a promoter, the first exon and the first intron of the chicken beta-actin gene, and (iii) a splicing acceptor of the rabbit beta-globin gene. Alternatively, the expression cassette may contain an alpha-1-antitrypsin (AAT) promoter (SEQ ID NO: 4 or SEQ ID NO: 74), a liver-specific (LP1) promoter (SEQ ID NO: 5 or SEQ ID NO: 16), or a human extension factor-l-alpha (EF1a) promoter (e.g., SEQ ID NO: 6 or SEQ ID NO: 15). In some embodiments, said expression cassette comprises one or more constitutive promoters, e.g., Rous sarcoma virus (RSV) retroviral LTR promoter (optionally with an RSV enhancer), or cytomegalovirus (CMV) immediate early promoter (optionally with a CMV enhancer, e.g., SEQ ID NO: 22). Alternatively, an inducible promoter, a natural transgene promoter, a tissue-specific promoter, or various promoters known in the art can be used.

[00198] Suitable promoters, including those described above, may be derived from viruses and may accordingly be referred to as viral promoters, or they may be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression using any RNA polymerase (eg pol I, pol II, pol III). Examples of promoters include, but are not limited to, the SV40 early promoter, the promoter from the mouse mammary tumor virus (LTR) long terminal repeat; adenovirus large late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter (CMVIE) region, Rous sarcoma virus (RSV) promoter, human small nuclear RNA U6 promoter (U6, e.g. Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), human H1 promoter (H1) (e.g., SEQ ID NO: 19), CAG promoter, human alpha-1 antitrypsin (HAAT) promoter (e.g., SEQ ID NO: 21), and the like. In embodiments, said promoters are modified at the 3'-intron-containing end to include one or more nuclease cleavage sites. In embodiments, the DNA containing the nuclease cleavage site(s) is foreign to the DNA promoter.

[00199] The promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and/or to change the spatial and/or temporal characteristics of their expression. The promoter may also contain distal enhancer or repressor elements, which may be located up to several thousand base pairs from the transcriptional start site. The promoter may be derived from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter may regulate the expression of a gene component constitutively or selectively in relation to the cell, tissue, or organ where expression occurs, or in consideration of the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lactose operator promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the SV40 early or SV40 late promoter, and the CMV IE promoter, as well as the promoters listed below. . Such promoters and/or enhancers may be used to express any gene of interest, eg, genome editing molecules, donor sequence, therapeutic proteins, and the like). For example, the vector may contain a promoter that is operably linked to a nucleic acid sequence encoding a therapeutic protein. Said promoter operably linked to the therapeutic protein coding sequence may be the simian virus 40 (SV40) promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HTV) promoter, e.g. ), such as the CMV immediate early (IE) promoter, the Epstein-Barr virus (EBV) promoter, or the Rous sarcoma virus (RSV) promoter. Said promoter may also be a human gene promoter, for example, human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. Said promoter may also be a tissue-specific promoter such as a liver-specific promoter, eg human alpha-1 antitrypsin (HAAT), natural or synthetic. In one embodiment, delivery to the liver can be achieved using endogenous ApoE-specific targeting of the composition containing the ccDNA vector to hepatocytes via a low-density lipoprotein (LDL) receptor present on the surface of the hepatocyte.

[00200] In one embodiment, the promoter used is the natural promoter of the gene encoding the therapeutic protein. Promoters and other regulatory sequences of the relevant genes encoding therapeutic proteins are known and have been characterized. The promoter region used may further include one or more additional (eg, natural) regulatory sequences such as enhancers (eg, SEQ ID NO: 22 and SEQ ID NO: 23).

[00201] Non-limiting examples of suitable promoters for use in accordance with the present invention include the CAG promoter, e.g. (SEQ ID NO: 3), the HAAT promoter (SEQ ID NO: 21), the human EF1-α promoter (SEQ ID NO: 6) or a fragment of the EF1a promoter (SEQ ID NO: 15), the IE2 promoter (e.g., SEQ ID NO: 20), and the rat EF1-α promoter (SEQ ID NO: 24).

[00202] Polyadenylation sequences: A sequence encoding a polyadenylation sequence can be included in the scDNA vector to stabilize the mRNA expressed from said scDNA vector and to promote nuclear export and translation. In one embodiment, said scDNA vector does not contain a polyadenylation sequence. In other embodiments, said vector comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50 or more adenine dinucleotides. In some embodiments, said polyadenylation sequence is about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any number of nucleotides in the ranges indicated.

[00203] Expression cassettes may include a polyadenylation sequence known in the art, or a variant thereof, such as a naturally occurring sequence isolated from bovine growth hormone BGHpA (e.g., SEQ ID NO: 74) or SV40pA virus (e.g., SEQ ID NO: 10), or a synthetic sequence (e.g., SEQ ID NO: 27). Some expression cassettes may also include the SV40 late poly(A) signal (USE) 5' enhancer sequence. In some embodiments, USE may be used in combination with SV40pA or a heterologous poly(A) signal.

[00204] Expression cassettes may also include a post-transcriptional element to increase expression of the transgene. In some embodiments, a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) is used to increase transgene expression (eg, SEQ ID NO: 8). Other post-transcriptional processing elements may be used, such as a post-transcriptional element from the herpes simplex virus or hepatitis B virus (HBV) thymidine kinase gene. Secretory sequences may be fused to the transgenes, for example the VH-02 and VK-A26 sequences, for example SEQ ID NO: 25 and SEQ ID NO: 26.

V. Regulator switches

[00205] A molecular regulatory switch generates a measurable state change in response to a signal. Such regulatory switches can be successfully combined with the scDNA vectors described herein to control the results of using the scDNA vector. In some embodiments, said scDNA vector contains a regulatory switch that serves to fine-tune the expression of the transgene. For example, it may serve to provide the biological containment function of the scDNA vector. In some embodiments, said switch is an on/off switch that is designed to start or stop (ie stop) expression of a gene of interest in the ccDNA in a controlled and regulated manner. In some embodiments, said switch may include a "kill switch" that may cause a cell containing said scDNA vector to enter programmed cell death upon activation of the switch.

[00206] A. Binary Regulator Switches

[00207] In some embodiments, the scDNA vector contains a regulatory switch that can be used to control the expression of the transgene. In such an embodiment, an expression cassette located between the ITRs of said ccDNA vector may further comprise a regulatory region, e.g., a promoter, cis element, repressor, enhancer, etc., operably linked to the gene of interest, said regulatory region being regulated by one or more cofactors or exogenous agents. Accordingly, in one embodiment, transcription and expression of the gene of interest from the scDNA vector will only occur if said one or more cofactors or exogenous agents are present in the cell. In another embodiment, one or more cofactors or exogenous agents may be used to derepress transcription and expression of a gene of interest.

[00208] Any nucleic acid regulatory regions known to those skilled in the art can be used in a scDNA vector designed to include a regulatory switch. For example, regulatory regions can modulate small molecular switches, or inducible or repressible promoters. Non-limiting examples of inducible promoters are hormone inducible or metal inducible promoters. Other examples of inducible promoter/enhancer elements include, but are not limited to, RU486 inducible promoter, ecdysone inducible promoter, rapamycin inducible promoter, and metallothionein promoter. Classic switches based on tetracycline or other antibiotics are contemplated, including those described in Fussenegger et al., Nature Biotechnol. 18:1203-1208 (2000).

[00209] B. Low molecular weight regulatory switches

[00210] Various small molecule regulatory switches are known in the art and can be combined with scDNA vectors as described herein to produce a switch-controlled scDNA vector. In some embodiments, said regulatory switch may be selected from any one or combination of the following: an orthogonal ligand/nuclear receptor pair, such as a retinoid receptor variant/LG335 and GRQCIMFI, along with an artificial promoter that controls the expression of an operably linked transgene, such as as described in Taylor, et al. Navy Biotechnology 10 (2010): 15; engineered steroid receptors, for example, a modified progesterone receptor truncated at the C-terminus, which is not able to bind progesterone, but binds RU486 (mifepristone) (US patent No. 5364791); an ecdysone receptor from Drosophila and its ecdysteroid ligands (Saez, et al., PNAS, 97(26)(2000), 14512-14517; or a switch controlled by the antibiotic trimethoprim (TMP) as described in Sando R 3 ^rd ; Nat Methods. 2013, 10(11): 1085-8.

[00211] Other small molecule regulatory switches known to those skilled in the art are also contemplated for use in controlling transgenic expression of ccDNA, including but not limited to those described in: Buskirk et al., Cell; Chem and Biol., 2005; 12(2); 151-161; abscisic acid sensitive positive switch; such as those described in the source: Liang, F.-S., et al., (2011) Science Signaling, 4(164); exogenous L-arginine sensitive positive switches, such as those described in Hartenbach, et al. Nucleic Acids Research, 35(20), 2007, synthetic bile acid sensitive positive switches such as those described in: Rössger et al., Metab Eng. 2014, 21: 81-90; biotin-sensitive positive switches, such as those described in Weber et al., Metab. Eng. 2009 Mar; 11(2): 117-124; benzoate/vanillin dual stimulation sensitive regulatory switches such as those described in Xie et al., Nucleic Acids Research, 2014; 42(14); e116; 4-hydroxy tamoxifen sensitive switches such as those described in: Giuseppe et al., Molecular Therapy, 6(5), 653-663; and flavinoid (phloretin) sensitive regulatory switches such as those described in Gitzinger et al., Proc. Natl. Acad. sci. USA. 2009 Jun 30; 106(26): 10638-10643.

[00212] In some embodiments, said regulatory switch, for controlling the transgene or expressed by the ccDNA vector, is a prodrug activation switch, such as that described in US Pat. Nos. 8,771,679 and 6,339,070.

[00213] Examples of regulatory switches for use in scDNA vectors include, but are not limited to, those listed in Table 11.

[00214] C. Regulatory switches with "password"

[00215] In some embodiments, said regulatory switch may be a "password switch" or "password loop". Password switches allow fine-tuning of the control of the expression of the specified transgene from the scDNA vector when specific conditions occur - that is, a combination of conditions that are required for transgene expression and/or repression to occur. For example, expression of a transgene requires at least conditions A and B. A regulatory password switch can be any number of conditions, such as at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7 or more conditions, necessary for transgene expression to occur. Some implementations require at least 2 conditions (eg, conditions A, B), some implementations require at least 3 conditions (eg, A, B, and C, or A, B, and D). By way of example only, conditions A, B, and C must be present for gene expression to occur with ccDNA containing the regulatory switch password "ABS". Conditions A, B, and C may be as follows; condition A is represented by the presence of a condition or disease, condition B is a hormonal response, and condition C is a response to transgene expression. In a purely illustrative example, when the transgene is insulin, condition A is met if the subject has diabetes, condition B is met when blood sugar is high, and condition C is represented by a level of endogenous insulin not expressed in the required amounts. After the sugar level drops or the desired insulin level is reached, the transgene (eg insulin) is turned off again until 3 conditions are met to turn it on again. In another illustrative example, when the transgene is an EPO, condition A is represented by the presence of chronic kidney disease (CKD), condition B is met by the presence of a hypoxic condition in the subject's kidneys, condition C is represented by impaired recruitment of erythropoietin-producing cells (EPC) in the kidney; or, alternatively, impaired HIF-2 activation. After oxygen levels increase or the desired EPO level is reached, the transgene (eg, EPO) is turned off again until 3 conditions are met to turn it on again.

[00216] Regulatory password switches are suitable for fine tuning the expression of a transgene from an scDNA vector. For example, a regulatory password switch can be modular, that is, contain several switches, such as a tissue-specific inducible promoter that is turned on only in the presence of a certain level of metabolite. According to this embodiment, for transgene expression to occur from the scDNA vector, an inducible agent must be present (condition A) in the desired cell type (condition B), and the metabolite level must be equal to a certain threshold value, or be above or below a certain threshold value (condition C). In alternative embodiments, said regulatory password switch can be designed such that transgene expression is turned on when conditions A and B are met, but turned off when condition C is met. Such an implementation is useful if condition C is a direct result of the expressed transgene - that is, condition C serves as a positive feedback in a loop to turn off transgene expression from the scDNA vector after the transgene has provided the desired therapeutic effect of sufficient magnitude.

[00217] In some embodiments, a regulatory password switch for use in said scDNA vector is disclosed in WO 2017/059245, which describes a switch referred to as a "password switch" or "password network" or "password kill switch", which is a synthetic biological circuit employing hybrid transcription factors (TFs) to construct complex environmental requirements for cell survival. . The regulatory password switches described in WO 2017/059245 are particularly suitable for use in ccDNA vectors because they are modular and configurable with respect to both environmental conditions that control loop activation and output modules that control cell fate. In addition, the password loop is particularly useful for use in ccDNA vectors because, without suitable "password" molecules, it only allows transgenic expression in the presence of the required predetermined conditions. If something unforeseen happens to the cell, or if for some reason additional transgene expression is not required, the associated "kill switch" (i.e. blocker) can be triggered.

[00218] In some embodiments, the regulatory password switch or "password loop" provided for use in said scDNA vector contains hybrid transcription factors (TFs) to increase the range and complexity of environmental signals used to set biocontainment conditions. Unlike a blocker that triggers cell death upon the presence of a predetermined condition, a "password circuit" allows for cell survival or transgene expression in the presence of a specific "password", and can be easily reprogrammed to allow transgene expression and/or cell survival only in the presence of a predetermined environmental condition or password.

[00219] According to one aspect, a system with a “password”, which limits the growth of cells, the condition for the presence of a pre-set set of at least two selected agents, includes one or more structures of nucleic acids, encoding expression modules containing: I) toxin expression, which encodes toxin, toxic to the observation cell, where the sequence indicated by the indicated current of the current , functionally associated with the promotor P1, which is repressed by the binding of the first hybrid protein-consulter HRP1; ii) an expression module of a first fusion repressor protein that encodes a first hRP1 fusion repressor protein, wherein hRP1 expression is controlled by a logical addition scheme formed by two fusion transcription factors hTF1 and hTF2 whose binding or activity is responsive to agents A1 and A2, respectively, such that both agents A1 and A2 are required for hRP1 expression, and in the absence of either of A1 and A2, hRP1 expression is insufficient for repression the P1 toxin promoter module and toxin production, thus the host cell dies. In this system, each of the hybrid factors hTF1, hTF2 and hRP1 contains an environmentally sensitive module from one transcription factor and a DNA recognition module from another transcription factor, which makes the binding of the corresponding regulatory password switch sensitive to the presence of an environmental agent, A1 or A2, different from that with which the respective subunits normally bind in nature.

[00220] Accordingly, the scDNA vector may contain a "password regulatory circuit" that requires the presence and/or absence of specific molecules to activate the output module. In some embodiments, if genes that encode cellular toxins are placed in the output module, the password-protected regulatory circuit can be used not only to regulate transgene expression, but also to create a "safety switch" mechanism, where the specified circuit kills the cell if the cell behaves in an undesirable way (for example, leaves a specific environment set by sensory domains, or differentiates into another type of cell). According to one non-limiting example, the hybrid transcription factor modular structure, circuit architecture, and output module allow the circuit to be reconfigured to sense other environmental cues, respond to said environmental cues in a different manner, and control other functions in the cell along with induced cell death, as is known in the art.

[00221] All combinations of regulatory switches as described herein, e.g., small molecule switches, nucleic acid switches, small molecule/nucleic acid hybrid switches, transgene post-transcriptional regulatory switches, post-translational regulation, radiation-controlled switches, hypoxia-mediated switches, and other regulatory switches known to those skilled in the art as described herein, can be used in a regulatory switch with a password according to description in this document. Regulatory switches intended for use are also discussed in the review article by Kis et al. (Kis et al., J R Soc Interface. 12: 20141000 (2015)), and are summarized in Table 1 of the Kis article. In some embodiments, the regulatory switch for use in a password system may be selected from any of the switches or combinations thereof from Table 11.

[00222] D. Nucleic acid-based regulatory switches for controlling transgene expression

[00223] In some embodiments, a regulatory switch for controlling a transgene expressed by ccDNA is based on a nucleic acid-based control mechanism. Examples of nucleic acid-based control mechanisms are known in the art and are contemplated for use. For example, such mechanisms include riboswitches such as those described, for example, in US 2009/0305253, US 2008/0269258, US 2017/0204477, WO 2018026762 A1, in US patent 9222093 and European patent application EP 288071, as well as in the review by Villa et al. ami (Villa JK et al., Microbiol Spectr. 2018 May; 6(3)). Also included are metabolite-responsive transcriptional biosensors such as those described in WO 2018/075486 and WO 2017/147585. Other mechanisms known in the art for use include silencing said transgene by an siRNA or RNAi molecule (eg, miR, shRNA). For example, said scDNA vector may contain a regulatory switch that encodes an RNA interference (RNAi) molecule complementary to the transgene expressed by said scDNA vector. When such RNAi is expressed, even when the transgene is expressed by the scDNA vector, it is silencing due to the action of the complementary RNA interference molecule, and in the absence of RNAi expression, if the indicated transgene is expressed by the scDNA vector, the indicated transgene is not silencing due to RNA interference. Such an example of an RNAi molecule controlling gene expression, or a regulatory switch, is disclosed in US 2017/0183664. In some embodiments, said regulatory switch contains a repressor that blocks the expression of said transgene from the scDNA vector. In some embodiments, said on-off switch is a small transcription-activating RNA (STAR) based switch, such as those described in Chappell J. et al., Nat Chem Biol. March 2015; 11(3): 214-20; and Chappell et al., Microbiol Spectr. May 2018; 6(3. In some embodiments, said regulatory switch is a holding switch, such as those described in: US 2009/0191546, US 2016/0076083, WO 2017/087530, US 2017/0204477, WO 2017/075486; and Green et al, Cell, 2014; 15 9(4);925-939.

[00224] In some embodiments, said regulatory switch is a tissue-specific self-inactivating regulatory switch, e.g., as described in US 2002/0022018, that specifically turns off transgene expression at a site where transgene expression may, on the contrary, be unfavorable. In some embodiments, said regulatory switch is a recombinase reversible gene expression system, such as as described in US 2014/0127162 and US Pat. No. 8,324,436.

[00225] In some embodiments, said regulatory switch for controlling the transgene or gene of interest expressed by the scDNA vector is a hybrid of a nucleic acid-based control mechanism and a low molecular weight regulatory system. Such systems are well known to those skilled in the art and are envisaged for use in accordance with the present invention. Examples of such regulatory switches include, but are not limited to, the LTRi system or the "Lac-Tet-RNAi" system, for example, as described in the sources: US 2010/0175141, Deans T. et al., Cell, 2007, 130(2); 363-372, WO 2008/051854 and US Pat. No. 9,388,425.

[00226] In some embodiments, said regulatory switch for controlling the transgene or gene of interest expressed by the scDNA vector comprises a circular permutation as described in US Pat. No. 8,338,138. In such an embodiment, said molecular switch is multistable, i. able to switch between at least two states, or alternatively, bistable, i.e. is in either an "on" or "off" state, for example, with respect to the ability or inability to emit light, the ability or inability to bind, the ability or inability to catalyze, the ability or inability to transfer electrons, etc. According to another aspect, said molecular switch activates a fusion molecule, respectively, said switch is capable of switching between more than two states. For example, in response to a particular threshold state exhibited by an insertion sequence or an acceptor sequence, a corresponding other fusion sequence may exhibit a range of states (eg, range of binding activity, range of enzymatic catalysis, etc.). Accordingly, instead of switching between "on" or "off" states, a fusion molecule may exhibit a stepwise response to a stimulus.

[00227] In some embodiments, the nucleic acid regulatory switch may be selected from any switch or combination thereof from Table 11.

[00228] E. Post-transcriptional and post-translational regulatory switches.

[00229] In some embodiments, said regulatory switch for controlling the transgene or gene of interest expressed by the scDNA vector is a post-transcriptional modification system. For example, such a regulatory switch may be a tetracycline or theophylline sensitive riboswitch-aptazim as described in US 2018/0119156, GB 201107768, WO 2001/064956 A3, EP 2707487 and Beilstein et al., ACS Synth. Biol, 2015, 4(5), pp 526-534; Zhong et al, Elife. 2016 Nov 2;5. pii: e18858. In some embodiments, it is contemplated that one skilled in the art can encode both a transgene and an inhibitory siRNA that contains a ligand-responsive (negative switch) aptamer, resulting in a ligand-responsive positive switch.

[00230] In some embodiments, said regulatory switch for controlling the transgene or gene of interest expressed by the scDNA vector is a post-translational modification system. In alternative embodiments, said gene or protein of interest is expressed as a proprotein or pre-proprotein, or contains a signal response element (SRE) or destabilizing domain (DD) attached to the expressed protein that prevents the protein from folding and/or activity prior to post-translational modification. In the case of a destabilizing domain (DD) or SRE, said destabilizing domain is post-translationally cleaved in the presence of an exogenous agent or small molecule. One skilled in the art can use such controls as described in US Pat. No. 8,173,792 and PCT Application WO 2017180587. Other post-transcriptional control switches contemplated for use in said scDNA vector to control the activity of a functional transgene are described in Rakhit et al, Chem Biol. 2014;21(9): 1238-52 and Navarro et al, ACS Chem Biol. 2016; 19; 11(8): 2101-2104A.

[00231] In some embodiments, the regulatory switch for controlling the transgene or gene of interest expressed by the ccDNA vector is a post-translational modification system that includes ligand-responsive inteins in the coding sequence of the transgene such that said transgene or expressed protein is inhibited prior to splicing. For example, this has been demonstrated using both 4-hydroxy tamoxifen and thyroid hormone (see, e.g., US Pat. 2016 Dec 16;5(12): 1475-1484; and 2005 Feb;14(2): 523-532 In some embodiments, the post-transcriptional regulatory switch may be selected from any or combination of switches from Table 11.

[00232] F. Other examples of control switches

[00233] Any known regulatory switch, including those triggered by environmental changes, can be used to control the gene expression of a transgene expressed by an scDNA vector in said scDNA vector. Additional examples include, but are not limited to: the BOC method of Suzuki et al., Scientific Reports 8; 10051 (2018); expansion of the genetic code and non-physiological amino acid; radiation or ultrasound controlled positive/negative switches (see, for example, Scott S et al, Gene Ther. 2000 Jul;7(13): 1121-5; US Pat. Nos. 5,612,318; 5,571,797; 5,770,581; a system, for example, as described in US Pat.

[00234] In some embodiments, the regulatory switch contemplated for use in said scDNA vector is a hypoxia-mediated or stress-activated switch, such as those described in WO 1999060142A2, US Pat. Nos. 5,834,306; 6218179; 6709858; US 2015/0322410; Greco et al, (2004) Targeted Cancer Therapies 9, S368, as well as FROG, TOAD and NRSE elements and conditionally inducible silencing elements including hypoxia response elements (HRE), inflammatory response elements (IRE) and shear stress activated elements (SSAE), e.g. cDNA vector after ischemia or in ischemic tissues and/or tumors.

[00235] In some embodiments, the regulatory switch provided for use in said zkDJV vector is an optogenetic (e.g., light-controlled) regulatory switch, such as, for example, one of the switches described in Polesskaya et al., BMC Neurosci. 2018; 19(Suppl 1): 12, which are also contemplated for use in the present invention. In such embodiments, the scDNA vector may contain light-sensitive genetic elements and may regulate transgene expression in response to visible wavelength radiation (eg, blue, near infrared). scDNA vectors containing optogenetic regulatory switches are suitable for use in transgene expression at locations in the body that such light sources can reach, e.g., skin, eyes, muscles, etc., and can also be used in expressing transgenes with scDNA vectors in internal organs and tissues, if the light signal can be provided using a suitable method (e.g., an implantable device as described herein). Such optogenetic regulatory switches include the use of light-responsive elements or a light-inducible transcriptional effector (LITE) (e.g., described in: 2014/0287938), a Light-On system (e.g., described in: Wang et al., Nat Methods. 2012 Feb 12;9(3):266-9; which has been reported to allow control of insulin transgene expression in vivo , the Cry2/CIB1 system (e.g., described in Kennedy et al., Nature Methods; 7, 973 975 (2010); and the FKF1/GIGANTEA system (e.g., described in Yazawa et al., Nat Biotechnol. 2009 Oct;27(10):941-5).

[00236] G. "Emergency Switches"

[00237] Other embodiments of the present invention relate to an scDNA vector containing an "emergency switch". The "kill switch" as described herein allows for the killing of a cell containing the ccDNA vector, or its transition to programmed death, as a method of permanently removing the administered ccDNA vector from the subject's system. One skilled in the art will recognize that the use of "kill switches" in ccDNA vectors according to the present invention generally involves targeting said ccDNA vector to a limited number of cells, the loss of which is acceptable to the subject, or the type of cells whose apoptosis is desired (e.g., cancer cells). In all aspects, the "kill switch" as described herein is designed to provide rapid and sustained killing of a cell containing said scDNA vector in the absence of a survival input or other specific condition. In other words, the "kill switch" encoded by the scDNA vector according to the present invention can limit the survival of the cell containing the scDNA vector to the environment given by specific input signals. Such "kill switches" serve to provide a biological containment function if it is desired to either remove the ccDNA vector from the subject or ensure that the encoded transgene is not expressed. Accordingly, "safety switches" are synthetic biological circuits in said scDNA vector, conjugating environmental signals to the conditional survival of the cell containing said scDNA vector. In some embodiments, different scDNA vectors can be developed containing different "kill switches". This makes it possible to control which transgene-expressing cells are killed when using cocktails of scDNA vectors.

[00238] In some embodiments, the scDNA vector may comprise a "kill switch", which is a modular biocontainment circuit. In some embodiments, a "kill switch" for use in said scDNA vector is disclosed in WO 2017/059245, which describes a switch called a "kill switch" that contains a pattern of at least two mutually inhibitory, repressible sequences such that an environmental signal suppresses the activity of a second molecule in said construct (e.g., a small molecule binding transcription factor is used to provide a "survival" state by re suppression of toxin production). In cells containing the scDNA-vector with an emergency blocker, after the disappearance of the environmental signal, the circuit irreversibly switches to a state of "death" with derepression of the toxin, which leads to its production, which kills the cell. In another embodiment, a synthetic biological circuit, referred to as a "password circuit" or "password kill switch", is provided that utilizes hybrid transcription factors (TFs) to construct complex environmental requirements for cell survival. The safety lock and password kill switch described in WO 2017/059245 are particularly suitable for use in ccDNA vectors because they are modular and configurable with respect to both the environmental conditions that control circuit activation and the output modules that control cell fate. With the right choice of toxins, including, but not limited to, endonucleases, for example, EcoRI, password circuits in the scDNA vector can be used not only for killing (killing, destroying) the host cell containing the scDNA vector, but also for decomposing its genome and accompanying plasmids.

[00239] Other "kill switches" known to those skilled in the art are provided for use in the ccDNA vector as described herein, such as those described in US 2010/0175141; US 2013/0009799; US 2011/0172826; US 2013/0109568, as well as "safety switches" described in the source: Jusiak et al, Reviews in Cell Biology and molecular Medicine; 2014; 1-56; Kobayashi et al, PNAS, 2004; 101; 8419-9; Marchisio et al. Int. Journal of Biochem and Cell Biol, 2011; 43; 310-319; and Reinshagen et al. Science Translational Medicine, 2018, 11.

[00240] Accordingly, in some embodiments, said scDNA vector may comprise a "kill switch" nucleic acid construct that contains a nucleic acid encoding an effector toxin or reporter protein, wherein expression of said effector toxin (e.g., death protein) or reporter protein is controlled by a predetermined condition. For example, a predetermined condition may be represented by the presence of an environmental agent, such as, for example, an exogenous agent, without which the cell will by default express an effector toxin (eg, a death protein) and be killed. In alternative embodiments, the predetermined condition is represented by the presence of two or more environmental agents, e.g., the cell will only survive when given two or more of the required exogenous agents, and without either of these, the cell containing said scDNA vector will be killed.

[00241] In some embodiments, the scDNA vector is modified to include a kill switch to kill cells containing said scDNA vector to effectively terminate in vivo expression of said scDNA vector-expressed transgene (e.g., a therapeutic gene, protein, or peptide, etc.). In particular, said scDNA vector is genetically engineered to further express a switch protein that is not functional in mammalian cells under normal physiological conditions. Only after administration of a drug or provision of environmental conditions specifically targeting said switch protein will the cells expressing said switch protein be killed, thereby terminating the expression of said therapeutic protein or peptide. For example, it has been reported that killing of cells expressing HSV thymidine kinase can occur following administration of drugs such as ganciclovir and cytosine deaminase. See, for example, sources: Dey and Evans, Suicide Gene Therapy by Herpes Simplex Virus-1 Thymidine Kinase (HSV-TK), in Targets in Gene Therapy, ed. You (2011); and Bellinger et al, Proc. Natl. Acad. sci. USA 96(15):8699-8704 (1999). In some embodiments, said scDNA vector may contain an siRNA "kill switch" called DISE (Death Induced by Survival gene Elimination))) (Murmann et al, Oncotarget. 2017; 8:84643-84658. Induction of DISE in ovarian cancer cells in vivo).

[00242] In some aspects, a kill switch is a biological circuit or system that sensitizes a cellular response to a predetermined condition, such as the absence of an agent in the cell's environment, such as an exogenous agent. Such a circuit or system may comprise a nucleic acid construct comprising expression modules that form a predetermined condition responsive blocker regulatory circuit, said construct comprising expression modules that form the regulatory circuit and includes:

i) a first repressor protein expression module, wherein said first repressor protein binds a nucleic acid element binding the first repressor protein and suppresses transcription of a coding sequence comprising said first repressor protein binding element, wherein the repressive activity of the first repressor protein is sensitive to inhibition by the first exogenous agent, the presence or absence of which is a predetermined condition;

ii) a second repressor protein expression module, wherein said second repressor protein binds a second repressor protein-binding element of a nucleic acid and represses transcription of a coding sequence comprising said second repressor protein-binding element, wherein the second repressor protein is different from the first repressor protein; And

iii) an effector expression module comprising a nucleic acid sequence encoding an effector protein operably linked to a genetic element containing a binding element for a second repressor protein such that expression of the second repressor protein causes repression of the effector expression from the effector expression module, wherein the second expression module comprises a nucleic acid element binding the first repressor protein that allows transcription of the second repressor protein to be repressed when said element is bound by the first repressor protein om, and the corresponding modules form a regulatory circuit in such a way that in the absence of the first exogenous agent, the first repressor protein is produced from the first repressor protein expression module and suppresses transcription from the second repressor protein expression module, so that repression of the effector expression by the second repressor protein is canceled, which leads to the expression of the effector protein, however, in the presence of the first exogenous agent, the activity of the first repressor protein is inhibited, which allows the expression of the second repressor protein, which supports expression of the effector protein in the "off" state, thus the first exogenous agent is required for the circuit to keep the expression of the effector protein in the "off" state, and the removal or absence of the first exogenous agent by default results in the expression of the effector protein.

[00243] In some embodiments, said effector is a toxin or protein that induces a cell death program. Any protein that is toxic to the host cell can be used. In some embodiments, said toxin only kills cells in which it is expressed. In other embodiments, said toxin also kills other cells of the same host organism. Any of the many cell death products can be used in an emergency bollard. Particularly suitable for use are agents that inhibit DNA replication, protein translation, or other processes, or, for example, destroy the nucleic acid of the host cell. To identify an effective mechanism for killing host cells after circuit activation, several toxin genes were tested that directly damage the DNA or RNA of the host cell. EcoRI ²¹ endonuclease, ccdB ²² DNA gyrase inhibitor, and mazF ²³ ribonuclease-type toxin were tested because they are well characterized, are native to E. coli, and provide a range of killing mechanisms. To improve loop stability and provide an independent pathway for loop-dependent cell death, the system can be further adapted to express, for example, a targeted protease or nuclease that further interacts with a repressor that keeps the death gene in a “turned off” state. After loss or elimination of the survival signal, repression of the death gene is even more effectively eliminated as a result of, for example, active degradation of the repressor protein or its mRNA. In non-limiting examples, the mƒ-Lon protease has been used not only to destroy LacI, but also to target critical proteins for their degradation. The mƒ-Lon degradation mark pdt#l can be attached to the 3' end of five major genes whose protein products are especially sensitive to mϕ-Lon ²⁰ degradation; cell viability was measured after ATC removal. Of the major target genes tested, the murC peptidoglycan biosynthesis gene provided the most pronounced and rapid cell death phenotype (survival rate <1×10 ^-4 within 6 hours).

[00244] As used herein, the term "predetermined input signal" refers to an agent or condition that affects the activity of a transcription factor polypeptide in a known manner. Typically, such agents can bind to and/or change the conformation of the transcription factor polypeptide, thereby modifying the activity of the transcription factor polypeptide. Examples of predetermined inputs include, but are not limited to, environmental inputs that are not necessary for the survival of a particular host organism (ie, in the absence of a synthetic biological circuit as described herein). Conditions that may provide a predetermined input signal include, for example, temperature, for example, if the activity of one or more factors is temperature sensitive, the presence or absence of light, including light of a certain wavelength spectrum, and the concentration of a gas, salt, metal, or mineral. Environmental inputs include, for example, small molecules, biological agents such as pheromones, hormones, growth factors, metabolites, nutrients, and the like, and the like; chemical concentrations, environmental by-products, metal ions; and other such molecules or agents; lighting levels; temperature; mechanical stress or pressure; or electrical signals such as current and voltage.

[00245] In some embodiments, reporters are used to quantify the strength or activity of a signal received by the modules or programmable synthetic biological circuits of the present invention. In some embodiments, reporters can be fused in-frame with other protein coding sequences to identify the location of said protein in a cell or organism. Luciferases can be used as effector proteins in the various embodiments described herein, for example, to measure low levels of gene expression because cells tend to have little or no background luminescence in the absence of luciferase. In other embodiments, enzymes that produce colored substrates can be quantified using spectrophotometers or other absorbance measuring instruments, including plate readers. Like luciferases, enzymes such as β-galactosidase can be used to measure low levels of gene expression because they tend to amplify weak signals. In some embodiments, the effector protein may be an enzyme that can break down or otherwise destroy a toxin. In some embodiments, the effector protein may be an odor-producing enzyme that converts the substrate into an odorous product. In some embodiments, the effector protein can be an enzyme that phosphorylates or dephosphorylates either small molecules or other proteins, or an enzyme that methylates or demethylates other proteins or DNA.

[00246] In some embodiments, the effector protein may be a receptor, ligand, or lytic protein. Receptors often contain three domains: an extracellular domain for binding ligands such as proteins, peptides, or small molecules, a transmembrane domain, and an intracellular or cytoplasmic domain that can often be involved in some kind of signal transduction event, such as phosphorylation. In some embodiments, transporter, channel, or pump gene sequences are used as effector proteins. Non-limiting examples and sequences of effector proteins for use with "kill switches" as described herein can be found in the Registry of Standard Biological Parts on the Internet at parts.igem.org.

[00247] As used herein, a "modulator protein" is a protein that modulates expression from a target nucleic acid sequence. Modulatory proteins include, for example, transcription factors, transcription activators and repressors, and proteins that bind or modify a transcription factor and affect its activity. In some embodiments, the modulator protein includes, for example, a protease that disrupts a protein factor involved in regulating expression from a target nucleic acid sequence. Preferred modulator proteins include modular proteins in which, for example, DNA binding elements or domains and input binding or responsive elements or domains can be separated and transferred, i.e., for example, fusion of the DNA binding domain of the first modulator protein with the input agent responsive domain of the second protein results in the formation of a new protein that binds a DNA sequence recognized by the first protein, but is sensitive to the input agent to which the second protein normally responds. Accordingly, as used herein, the term "modulator polypeptide" and more specifically the term "repressor polypeptide" includes, along with said polypeptides, e.g., "(repressor) LacI polypeptide", variants or derivatives of such polypeptides that respond to another input agent or input agent variant. Accordingly, in the case of a LacI polypeptide, LacI mutants or variants that bind to agents other than lactose or IPTG are included. A wide range of such agents are known in the art.

[00248] Table 11. Examples of Regulator Switches ^b Positive switch switched to "On" by an effector; not by removing the effector, which provides an "off" state. ^with Negative switch switchable to "off" mode by the effector; not removing an effector that provides an "on" state, ^d Tigand or other physical stimuli (eg, temperature, electromagnetic radiation, electricity) that stabilize the switch in the "on" or "off" state. e Refers to the reference number cited in Kis et al, JR Soc Interface. 12:20141000 (2015), and both this article and the sources cited therein are incorporated herein by reference.

VI. Detailed description of the method for obtaining the scDNA vector

A. Receipt in general

[00249] According to the present disclosure, a scDNA vector can be obtained using a method comprising the following steps: a) incubating a population of host cells (e.g., insect cells) carrying an expression construct polynucleotide template (e.g., scDNA plasmid, scDNA bacmid, and/or scDNA baculovirus) that does not contain viral capsid coding sequences in the presence of a Rep protein under conditions effective for, and for a period of time sufficient to induce the production of said ccDNA vector in host cells, said host cells not containing viral capsid coding sequences; and b) collecting and isolating the scDNA vector from host cells. The presence of the Rep protein induces replication of the ITR-modified vector polynucleotide to produce the ccDNA vector in the host cell. However, viral particles (eg AAV virions) are not expressed. Accordingly, there are no size limitations such as those naturally imposed by AAV or other virus vectors.

[00250] The presence of an scDNA vector isolated from host cells can be confirmed by cleaving DNA isolated from a host cell with a restriction enzyme having a single recognition site on the scDNA vector and analyzing said cleaved DNA material on a non-denaturing gel to confirm the presence of characteristic linear and continuous DNA bands distinct from linear and discontinuous DNA bands.

[00251] According to another aspect of the present invention, the use of host cell lines that have stably integrated an expression vector DNA polynucleotide template (scDNA template) into their own genome to obtain a non-viral DNA vector, for example, as described in the source: Lee, L. et al. (2013) Plos One 8(8): e69879. Preferably, Rep is added to host cells with an MOI of approximately 3. If the host cell line is a mammalian cell line, such as HEK293 cells, said cell lines may contain a stably integrated polynucleotide vector template, and a second vector, such as a herpesvirus vector, may be used to introduce the Rep protein into the cells, allowing excision and amplification of scDNA in the presence of Rep and a helper virus.

[00252] In one embodiment, the host cells used to generate the ccDNA vectors described herein are insect cells, and the baculovirus is used to deliver both a polynucleotide that encodes a Rep protein and a non-viral ccDNA DNA vector polynucleotide expression template construct, e.g., as described in FIG. 4A-4C and Example 1. In some embodiments, said host cell is engineered to express the Rep protein.

[00253] The scDNA vector is then recovered and isolated from the host cells. The timing of extraction and harvesting of the ccDNA vectors described herein from cells can be chosen and optimized to ensure that these ccDNA vectors are highly productive. For example, the extraction time may be selected based on cell viability, cell morphology, cell growth, and the like. In one embodiment, the cells are cultured under sufficient conditions and harvested a sufficient time after baculovirus infection to produce scDNA vectors, but before most cells die due to baculovirus toxicity. These DNA vectors can be isolated using plasmid purification kits such as Endo-Free kits from Qiagen. Other methods developed for isolating plasmids can also be adapted to DNA vectors. Generally, any nucleic acid purification methods can be used.

[00254] DNA vectors can be purified by any DNA purification method known to those skilled in the art. In one embodiment, the scDNA vectors are purified as DNA molecules. In another embodiment, said scDNA vectors are purified as exosomes or microparticles.

[00255] The presence of said scDNA vector can be confirmed by cleaving vector DNA isolated from cells with a restriction enzyme having a single recognition site on the DNA vector, and analyzing both the cleaved and uncleaved DNA material using gel electrophoresis to confirm the presence of characteristic linear and continuous DNA bands that are distinct from linear and discontinuous DNA bands. Fig. 4C and 4E illustrate one implementation for identifying the presence of closed-ended scDNA vectors generated using the methods described herein. For example, in FIG. 5 shows a gel confirming the production of ccDNA from several plasmid constructs using one embodiment to generate vectors as described in the examples.

B. scDNA plasmid

[00256] An scDNA plasmid is a plasmid used for subsequent production of an scDNA vector. In some embodiments, an scDNA plasmid can be constructed using known techniques to provide, as operably linked components, at least the following in the direction of transcription: (1) a 5' ITR sequence; (2) an expression cassette containing a cis regulatory element such as a promoter, an inducible promoter, a regulatory switch, enhancers, and the like; and (3) a 3' ITR sequence, wherein the 3' ITR sequence is asymmetric with respect to the 5' ITR sequence. In some embodiments, said ITR-flanked expression cassette contains a cloning site for introducing an exogenous sequence. This expression cassette replaces the rep and cap coding regions of the AAV genomes.

[00257] In one aspect, a scDNA vector is generated from a plasmid, herein referred to as a "zcDNA plasmid", encoding in this order: an adeno-associated virus (AAV) first inverted terminal repeat (ITR), an expression cassette containing a transgene, and a mutated or modified AAV ITR, wherein said scDNA plasmid does not contain the AAV capsid protein coding sequences. In alternative embodiments, said scDNA plasmid encodes, in this order: a first (or 5') modified or mutated AAV ITR, an expression cassette containing a transgene, and a second (or 3') wild type AAV ITR, wherein said scDNA plasmid does not contain AAV capsid protein coding sequences, and the 5' and 3' ITRs are asymmetric with respect to each other. In alternative embodiments, said scDNA plasmid encodes, in this order: a first (or 5') modified or mutated AAV ITR, an expression cassette containing a transgene, and a second (or 3') mutated or modified AAV ITR, wherein said scDNA plasmid does not contain AAV capsid protein coding sequences, and the modified 5' and 3' ITRs are different and do not contain the same modifications.

[00258] In a further embodiment, said zcDNA plasmid system does not contain the viral capsid protein coding sequences (ie, does not contain the AAV capsid genes as well as the capsid genes of other viruses). In addition, in a specific embodiment, the scDNA plasmid also lacks the coding sequences for the AAV Rep protein. Accordingly, in a preferred embodiment, the scDNA plasmid does not contain the functional cap AAV and rep AAV, GG-3' for AAV2) genes other than the variable palindromic hairpin sequence.

[00259] The ZcDNA plasmid of the present invention can be generated using natural nucleotide sequences from the genomes of any AAV serotypes well known in the art. In one embodiment, the backbone of the ccDNA plasmid is derived from the genome of AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV 12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8. For example, NCBI: NC 002077; NC 001401; NC001729; NC001829; NC006152; NC 006260; NC 006261; from the Kotin and Smith Virus Index (“The Springer Index of Viruses”), available from a link maintained by Springer (web address: oesys.springer.de/viruses/database/mkchapter.asp?virID=42.04.) (Note: Reference or database reference refers to the contents of the linked resource or database as of the effective filing date of this application). In a specific embodiment, the backbone of the ccDNA plasmid is derived from the AAV2 genome. In another specific embodiment, the backbone of the ccDNA plasmid is a synthetic backbone genetically engineered to include at the 5' and 3' ends ITRs derived from one of the indicated AAV genomes.

[00260] The scDNA plasmid may optionally include a selectable or selectable marker for use in the production of scDNA vector-producing cell lines. In one embodiment, said selectable marker may be inserted downstream (ie, in the 3' direction) of the 3' ITR sequence. According to another implementation variant of the specified selective marker can be inserted upstream (ie, in the 5'-direction) 5'-sequence of ITR. Suitable selectable markers include, for example, markers conferring drug resistance. The selectable markers may be, for example, the blasticidin S resistance gene, kanamycin, geneticin, and the like. In a preferred embodiment, said selective drug marker is the blasticidin S resistance gene.

[00261] An example ccDNA (eg, rAAV0) is produced from the rAAV plasmid. The method for producing a rAAV vector may include: (a) delivering an rAAV plasmid to a host cell as described above, wherein both the host cell and the plasmid are devoid of genes encoding the capsid protein, (b) culturing said host cell under conditions allowing the production of an scDNA genome; and (c) extracting the cells and isolating the AAV genome derived from said cells.

C. Examples of a method for producing ccDNA vectors from ccDNA plasmids

[00262] This document also provides methods for producing capsidless scDNA vectors, namely a method that provides a high enough yield to provide enough vector for in vivo experiments.

[00263] In some embodiments, a method for producing a scDNA vector includes the steps of: (1) introducing a nucleic acid construct comprising an expression cassette and two asymmetric ITR sequences into a host cell (e.g., Sf9 cells), (2) optionally, obtaining a clonal cell line, e.g. a baculovirus carrying said gene) into said insect cell; and (4) recovering said cell and purifying the scDNA vector. The nucleic acid construct containing the expression cassette and the two ITR sequences described above to produce a capsidless AAV vector can be a cfAAV plasmid, or a bacmid, or a baculovirus generated with a cfAAV plasmid as described below. Said nucleic acid construct may be introduced into a host cell by transfection, viral transduction, stable integration, or other methods known in the art.

D. Cell lines:

[00264] The host cell lines used to generate the ccDNA vector may include insect cell lines derived from Spodoptera frugiperda, e.g., Sf9 Sf21, or Trichoplusia ni or other invertebrate, vertebrate, or other eukaryotic cell lines, including mammalian cells. Other cell lines known to the person skilled in the art may also be used, such as HEK293, Huh-7, HeLa, HepG2, Hep1A, 911, CHO, COS, MeWo, NIH3T3, A549, HT1 180, monocytes; and mature and immature dendritic cells. Host cell lines can be transfected to stably express the ceDBA plasmid for high production of the scDNA vector.

[00265] ccDNA plasmids can be introduced into Sf9 cells by transient transfection using reagents (eg, liposomal, calcium phosphate) or physical methods (eg, electroporation) known in the art. Alternatively, stable Sf9 cell lines can be obtained that have stably integrated the ccDNA plasmid into the genome. Such stable cell lines can be obtained by incorporating a selectable marker into the scDNA plasmid as described above. If the scDNA plasmid used to transfect the cell line contains a selectable marker, for example for an antibiotic, cells that have been transfected with said ccDNA plasmid and have integrated the scDNA plasmid DNA into the genome can be selected by adding said antibiotic to the cell growth media. Resistant clones of said cells can then be isolated by single cell dilution or colony transfer techniques and propagated.

E. Isolation and purification of ccDNA vectors:

[00266] Examples of the method for obtaining and isolating scDNA vectors are described in FIG. 4A-4E and in the specific examples below. The scDNA vectors as described herein can be obtained from a producer cell expressing the AAV Rep protein or proteins further transformed with an scDNA plasmid, scDNA bacmid, or scDNA baculovirus. Plasmids suitable for the production of ccDNA vectors include the plasmids shown in FIG. 8A (suitable for obtaining Rep BIIC), FIG. 8B (plasmid used to generate the scDNA vector).

[00267] In one aspect, the polynucleotide encodes an AAV Rep protein (Rep 78 or 68) delivered to a producer cell in a plasmid (Rep plasmid), bacmid (Rep bakmida), or baculovirus (Rep baculovirus). Rep plasmid, Rep bacmid and Rep baculovirus can be obtained using the methods described above.

[00268] Methods for producing an scDNA vector, which is an example of an scDNA vector, are described herein. The expression construct used to generate the scDNA vector of the present invention may be a plasmid (eg, scDNA plasmids), bacmid (eg, scDNA bakmida), and/or a baculovirus (eg, scDNA baculovirus). By way of example only, a ccDNA vector can be obtained from cells co-infected with ccDNA baculovirus and Rep baculovirus. Rep proteins produced from Rep baculovirus can replicate the baculovirus ccDNA to produce ccDNA vectors. Alternatively, ccDNA vectors can be obtained from cells stably transfected with a construct containing the sequence encoding the AAV Rep protein (Rep78/52) delivered in Rep plasmids, Rep bacmids, or Rep baculovirus. The ccDNA baculovirus can be transiently transfected into cells, it can replicate by the Rep protein and produce ccDNA vectors.

[00269] Bacmida (e.g., scDNA bacmida) can be transfected into permissive insect cells such as Sf9, Sf21, Tni (Trichoplusia ni), High Five, and can generate scDNA baculovirus, which is a recombinant baculovirus including sequences containing an asymmetric ITR and an expression cassette. The scDNA baculovirus can be re-infected with insect cells to produce a new generation of recombinant baculovirus. Optionally, this step may be repeated once or several times to obtain more recombinant baculovirus.

[00270] The timing of extraction and harvesting of the scDNA vectors described herein from cells can be chosen and optimized to ensure high production of said scDNA vectors. For example, the extraction time may be selected based on cell viability, cell morphology, cell growth, and the like. Typically, cells can be recovered long enough after baculovirus infection to generate scDNA vectors (eg, scDNA vectors), but before most cells begin to die due to viral toxicity. ZcDNA vectors can be isolated from Sf9 cells using plasmid purification kits such as those from Qiagen ENDO-FREE PLASMID®. Other methods developed for isolating plasmids can also be adapted to ccDNA vectors. Generally, any nucleic acid purification methods known in the art, as well as commercially available DNA extraction kits, can be used.

[00271] Alternatively, purification can be accomplished using a process of alkaline cell pellet lysis, centrifugation of the final lysate, and chromatographic separation. In one non-limiting example, this process can be carried out by loading the supernatant onto an ion exchange column (for example, SARTOBIND Q®) that retains nucleic acids, followed by elution (for example, 1.2 M NaCl solution), and additional chromatographic purification on a gel filtration column (for example, 6 Fast Flow GE). The capsidless AAV vector is then isolated, for example, by precipitation.

[00272] In some embodiments, scDNA vectors may also be purified in the form of exosomes or microparticles. It is known in the art that many cell types release not only soluble proteins but also complex protein/nucleic acid cargoes by shedding membrane microvesicles (Cocucci et al, 2009; EP 10306226.1) Such vesicles include microvesicles (also called microparticles) and exosomes (also called nanovesicles); both contain proteins and RNA as cargo. Microvesicles are formed by direct budding from the plasma membrane, and exosomes are released into the extracellular environment after the fusion of multivesicular endosomes with the plasma membrane. Accordingly, microvesicles and/or exosomes containing the scDNA vector can be isolated from cells that have been transduced with the scDNA plasmid, or with the baculovirus produced with the scDNA plasmid.

[00273] Microvesicles can be isolated by filtration or ultracentrifugation of the culture medium at 20,000×g and exosomes at 100,000×g. The optimal duration of ultracentrifugation can be experimentally determined and depends on the specific cell type from which the vesicles are isolated. Preferably, the culture medium is first purified by low speed centrifugation (eg at 2000×g for 5-20 minutes) and subjected to centrifugal concentration using, for example, an AMICON® spin column (Millipore, Watford, UK). Microvesicles and exosomes can be further purified by FACS or MACS using specific antibodies that recognize specific surface antigens present on microvesicles and exosomes. Other methods for purifying microvesicles and exosomes include, but are not limited to, immunoprecipitation, affinity chromatography, filtration, and magnetic beads coated with specific antibodies or aptamers. After cleansing, the vesicles are washed with, for example, phosphate buffered saline. One of the advantages of using microvesicles or exosomes to deliver ccDNA-containing vesicles is that these vesicles can be targeted to different cell types by incorporating proteins into their membranes that are recognized by specific receptors on the respective cell types (see also EP 10306226)

[00274] Another aspect of the invention described herein relates to methods for purifying ccDNA vectors from host cell lines that have stably integrated the ccDNA construct into their own genome. In one embodiment, the scDNA vectors are purified as DNA molecules. In another embodiment, the scDNA vectors are purified as exosomes or microparticles.

[00275] FIG. 5 shows a gel confirming the production of scDNA from several scDNA plasmid constructs using the method described in the examples. The presence of ccDNA confirms the characteristic banding pattern on the gel as described for FIG. 4D in examples. Other characteristics of the scDNA production process and intermediates are summarized in FIG. 6A and 6B, and in FIG. 7A and 7B as described in the examples.

VII. Pharmaceutical compositions

[00276] According to another aspect, pharmaceutical compositions are provided. Said pharmaceutical composition contains an scDNA vector as described herein and a pharmaceutically acceptable carrier or diluent.

[00277] DNA vectors as described herein can be incorporated into pharmaceutical compositions suitable for administration to a subject for in vivo delivery to the cells, tissues, or organs of said subject. Typically, said pharmaceutical composition comprises a ccDNA vector as described herein and a pharmaceutically acceptable carrier. For example, the scDNA vectors described herein can be incorporated into a pharmaceutical composition suitable for the desired route of therapeutic administration (eg, parenteral administration). Also contemplated are passive tissue transduction by intravenous or intra-arterial infusion under high pressure, as well as intracellular injection, such as intranuclear microinjection or intracytoplasmic injection. Pharmaceutical compositions for therapeutic use may be formulated into a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for a high concentration ccDNA vector. Sterile injectable solutions can be prepared by incorporating the scDNA vector compound in the required amount in a suitable buffer, along with one ingredient or combination of ingredients listed above, as needed, followed by sterile filtration.

[00278] Pharmaceutically active compositions containing the scDNA vector can be formulated to deliver the transgene in nucleic acid to recipient cells resulting in therapeutic expression of said transgene. Said composition may also include a pharmaceutically acceptable carrier.

[00279] The zcDNA vector as described herein can be incorporated into a pharmaceutical composition suitable for topical, systemic, intra-amniotic, intrathecal, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, interstitial (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (eg, extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, intrastromal, intracameral, and intravitreal), intracochlear, and mucosal (eg, oral, rectal, nasal) administration. Also contemplated are passive tissue transduction by intravenous or intra-arterial infusion under high pressure, as well as intracellular injection, such as intranuclear microinjection or intracytoplasmic injection.

[00280] Pharmaceutical compositions for therapeutic purposes, as a rule, must be sterile and stable under the conditions of receipt and storage. Said composition may be incorporated into a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for a high concentration scDNA vector. Sterile injectable solutions can be prepared by incorporating the ccDNA vector compound in the required amount in a suitable buffer, together with one ingredient or combination of ingredients listed above, as needed, followed by sterile filtration.

[00281] Various techniques and methods for delivering nucleic acids to cells are known in the art. For example, nucleic acids such as scDNA can be incorporated into lipid nanoparticles (LNPs), lipidoids, liposomes, lipid nanoparticles, lipoplexes, or core/shell nanoparticles. Typically, LNPs are composed of nucleic acid molecules (e.g., scDNA), one or more ionizable or cationic lipids (or salts thereof), one or more nonionic or neutral lipids (e.g., a phospholipid), a molecule that prevents aggregation (e.g., PEG or a PEG-lipid conjugate), and optionally a sterol (e.g., cholesterol).

[00282] Another method for delivering nucleic acids, such as ccDNA, into a cell is by conjugating the nucleic acid to a ligand that the cell internalizes. For example, a ligand can bind a receptor on a cell surface and be internalized by endocytosis. Said ligand may be covalently linked to a nucleotide in a nucleic acid. Examples of conjugates for delivery of nucleic acids into a cell are described, for example, in WO 2015/006740, WO 2014/025805, WO 2012/037254, WO 2009/082606, WO 2009/073809, WO 2009/018332, WO 2006/112 872, WO 2004/090108, WO 2004/091515 and WO 2017/177326.

[00283] Nucleic acids, such as ccDNA, can also be delivered into a cell by transfection. Suitable transfection methods include, but are not limited to, lipid-mediated transfection, cationic polymer-mediated transfection, or calcium phosphate precipitation. Transfection reagents are well known in the art and include, but are not limited to, TurboFect Transfection Reagent (Thermo Fisher Scientific), Pro-Ject Reagent (Thermo Fisher Scientific), TRANSPASS™ P Protein Transfection Reagent (New England Biolabs), CHARIOT™ Protein Delivery Reagent (Active Motif), PROTEOJUICE™ Protein Transfection Reagent (EMD Millipore), 293-fectin , Lipofectamine (LIPOFECTAMINE™ 2000), LIPOFECTAMINE™ 3000 (Thermo Fisher Scientific), LIPOFECTAMTNE™ (Thermo Fisher Scientific), Lipofectin (LIPOFECTIN™, Thermo Fisher Scientific), DMPJE-C, CELLFECTIN™ (Thermo Fisher Scientific), OLIGOFECTAMINE™ (Thermo Fisher Scientific), LIPOFECTACE™, FUGEN E™ (Roche, Basel, Switzerland), FUGENE™ HD (Roche), TRANSFECTAM™ (transfectam, Promega, Madison, WI), TFX-10™ (Promega), TFX-20™ (Promega), TFX-50™ (Promega), transfectin (TRANSFECTIN™, BioRad, Hercules, CA), SILENTFECT™ (Bio-Rad), Effectene™ (Qiagen, Valencia, CA), DC -chol (Avanti Polar Lipids), GENEPORTER™ (Gene Therapy Systems, San Diego, CA), DHARMAFECT 1™ (Dharmacon, Lafayette, CO), DHARMAFECT 2™ (Dharmacon), DHARMAFECT 3™ (Dharmacon), DHARMAFECT 4™ (Dharmacon), ESCORT™ III (Sigma, St. Louis, MO), and ES CORT™ IV (Sigma Chemical Co.). Nucleic acids, such as scDNA, can also be delivered into the cell using microfluidics techniques known to those skilled in the art.

[00284] Methods for non-viral delivery of nucleic acids in vivo or ex vivo include electrosurgery, lipofection (see, for example, US Pat. 04-410 (1995); Blaese et al., Cancer Gene Ther. 2: 291-297 (1995); Behr et al., Bioconjugate Chem. 5: 382-389 (1994); Remy et al., Bioconjugate Chem. 5: 647-654 (1994); Gao et al., Gene Therapy 2: 710-722 (1995), Ahmad et al., Cancer Res. 52: 4817-4820 (1992), US Pat. 946787), immunoliposomes, conjugates of nucleic acids with polycations or lipids, deproteinized DNA, and agent-enhanced DNA uptake. Sonoporation using, for example, the Sonitron 2000 system (Rich-Mar) can also be used to deliver nucleic acids.

[00285] ccDNA vectors as described herein can also be administered directly to an organism for transduction of cells in vivo. Administration is by any of the routes commonly used to bring the molecule into as close contact as possible with blood or tissue cells including, but not limited to, injection, infusion, topical application, and electrosurgery. Suitable routes for administering such nucleic acids are available and well known to those skilled in the art, and while more than one route may be used to administer a particular composition, a particular route can often provide a faster and more efficient response than another route.

[00286] Methods for introducing a nucleic acid vector scDNA-vector as described herein can provide delivery, for example, to hematopoietic stem cells, for example, methods as described in US patent No. 5928638.

[00287] ZcDNA vectors in accordance with the present invention can be added to liposomes for delivery to a cell or target organ of a subject. Liposomes are vesicles having at least one lipid bilayer. Liposomes are commonly used as drug/therapeutic delivery vehicles in the context of pharmaceutical development. They work by fusing with the cell membrane and rearranging its lipid structure to deliver a drug or active pharmaceutical ingredient (API). Liposomal compositions for such delivery consist of phospholipids, in particular compounds containing a phosphatidylcholine group, however, these compositions may also include other lipids.

[00288] According to some aspects of the present invention, a liposomal formulation is provided that includes one or more compounds with a polyethylene glycol (PEG) functional group (so-called "pegylated compounds"), which can reduce the immunogenicity/antigenicity, provide the hydrophilicity and hydrophobicity of the specified compound or compounds, and the frequency of dosing. Alternatively, said liposomal formulation simply includes a polyethylene glycol (PEG) polymer as an additional component. In such aspects, the molecular weight of the PEG or PEG moiety may be from 62 Da to about 5000 Da.

[00289] According to some aspects of the present invention, a liposome formulation is provided that delivers an API with an extended release or controlled release profile over a period of hours to weeks. In some related aspects, said liposomal formulation may comprise aqueous chambers linked by lipid bilayers. In other related aspects, said liposomal formulation encapsulates an API with components that undergo a physical transition at elevated temperature, releasing said API over a period of hours to weeks.

[00290] In some aspects, said liposomal formulation comprises sphingomyelin and one or more lipids as described herein. In some aspects, said liposomal formulation contains optisomes.

[00291] According to certain aspects of the present invention, a liposomal formulation is provided that comprises one or more lipids selected from: N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, (distearoyl-sn-glycero-phosphoethanolamine), MPEG (methoxypolyethylene glycol)-conjugated lipid, HSPC (hydrogenated soy phosphatidylcholine); PEG (polyethylene glycol); DSPE (distearoyl-sn-glycero-phosphoethanolamine); DSPC (distearoylphosphatidylcholine); DOPC (dioleylphosphatidylcholine); DPPG (dipalmitoylphosphatidylglycerol); EPC (egg phosphatidylcholine); DOPS (dioleylphosphatidylserine); POPC (palmitoiloleoylphosphatidylcholine); SM (sphingomyelin); MPEG (methoxypolyethylene glycol); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DSPG (distearoylphosphatidylglycerol); DEPC (dierucoylphosphatidylcholine); DOPE (dioleoli-sn-glycerophosphatidylamine), cholesteryl sulfate (CS), dipalmitoylphosphatidylglycerol (DPPG), DOPC (dioleoli-sn-glycerophosphatidylcholine) or any combination of the above.

[00292] According to some aspects of the present invention, a liposomal formulation is provided comprising a phospholipid, cholesterol, and pegylated lipid in a molar ratio of 56:38:5. In some aspects, the total lipid content of said liposomal formulation is 2-16 mg/mL. According to some aspects of the present invention, a liposomal formulation is provided comprising a lipid comprising a phosphatidylcholine functional group, a lipid comprising an ethanolamine functional group, and a pegylated lipid. In some aspects, the present invention provides a liposomal formulation comprising a phosphatidylcholine functional lipid, an ethanolamine functional lipid, and a pegylated lipid in a molar ratio of 3:0.015:2, respectively. According to some aspects of the present invention, a liposomal formulation is provided comprising a lipid comprising a phosphatidylcholine functional group, cholesterol, and a pegylated lipid. According to some aspects of the present invention, a liposomal formulation is provided comprising a lipid comprising a phosphatidylcholine functional group and cholesterol. In some aspects, said pegylated lipid is PEG-2000-DSPE. In some aspects of the present invention, a liposomal formulation is provided comprising DPPG, soy PC, an MPEG-DSPE lipid conjugate, and cholesterol.

[00293] According to some aspects of the present invention, a liposomal formulation is provided comprising one or more lipids comprising a phosphatidylcholine functional group and one or more lipids comprising an ethanolamine functional group. According to some aspects of the present invention, a liposome formulation is provided comprising one or more of: lipids comprising a phosphatidylcholine functional group, lipids comprising an ethanolamine functional group, and sterols, for example, cholesterol. In some aspects, said liposomal formulation contains DOPC/DEPC; and DOPE.

[00294] According to some aspects of the present invention, a liposomal formulation is provided, further comprising one or more pharmaceutical excipients, for example, sucrose and/or glycine.

[00295] According to some aspects of the present invention, a liposomal formulation with a single layer or multilayer structure is provided. According to some aspects of the present invention, a liposome formulation is provided that contains multivesicular particles and/or foam-based particles. According to some aspects of the present invention, a liposome formulation is provided that has a relative particle size larger than conventional nanoparticles, approximately 150-250 nm. In some aspects, said liposomal formulation is a lyophilized powder.

[00296] According to some aspects of the present invention, a liposomal formulation is provided that is prepared and loaded with ccDNA vectors provided or described herein by adding a weak base to a mixture containing isolated ccDNA outside the liposome. This addition increases the pH outside the liposomes to about 7.3 and directs the API to the liposome. According to some aspects of the present invention, a liposome formulation is provided with an acidic pH within the liposome. In such cases, the pH within the liposome may be 4-6.9, more preferably 6.5. According to other aspects of the present invention, a liposomal formulation is provided using intraliposomal drug stabilization technology. In such cases, polymeric or non-polymeric highly charged anions and intraliposomal capturing agents such as sucrose polyphosphate or octasulfate are used.

[00297] According to other aspects of the present invention, a liposome formulation is provided comprising phospholipids, lecithin, phosphatidylcholine, and phosphatidylethanolamine.

[00298] Delivery reagents such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like can be used to administer the compositions of the present disclosure into suitable host cells. In particular, said nucleic acids may be formulated for delivery in an encapsulated form in a lipid particle, liposome, vesicle, nanosphere, nanoparticle, gold particle, or the like. Such formulations may be preferred for administration of pharmaceutically acceptable nucleic acid formulations as described herein.

[00299] Various delivery methods known in the art, or modifications thereof, can be used to deliver scDNA vectors in vitro or in vivo. For example, in some embodiments, scDNA vectors are delivered by transiently penetrating the cell membrane using mechanical, electrical, ultrasonic, hydrodynamic, or laser energy in a manner that facilitates DNA entry into target cells. For example, the ccDNA vector can be delivered by transient disruption of the cell membrane by pushing the cell through a limited size channel, or by other methods known in the art. In some cases, the ccDNA vector is directly injected as deproteinized DNA directly into skin, thymus, cardiac muscle, skeletal muscle, or liver cells.

[00300] In some cases, the scDNA vector is delivered using a gene gun. Gold or tungsten spherical particles (1-3 µm in diameter) coated with capsidless AAV vectors can be accelerated to high speed by pressurized gas to penetrate the target tissue cells.

[00301] In some embodiments, electroporation is used to deliver scDNA vectors. Electroporation causes a temporary destabilization of cell membranes of the target tissue by introducing a pair of electrodes into the tissue, so that DNA molecules in the environment surrounding the destabilized membrane can penetrate into the cytoplasm and nucleoplasm of the cell. Electroporation has been used in vivo for many tissue types such as skin, lungs and muscles.

[00302] In some cases, the ccDNA vector is delivered by hydrodynamic injection, which is a simple and highly efficient method for direct intracellular delivery of any water-soluble compounds and particles to the internal organs and skeletal muscles of the entire limb.

[00303] In some cases, scDNA vectors are delivered using ultrasound, creating nanoscopic pores in the membrane to facilitate intracellular delivery of DNA particles to visceral or tumor cells, so the size and concentration of plasmid DNA is of great importance for the efficiency of the system. In some cases, scDNA vectors are delivered by magnetofection using magnetic fields to concentrate particles containing the nucleic acid into target cells.

[00304] In some cases, chemical delivery systems may be used, for example, using nanoscale complexes, which involve the compaction of a negatively charged nucleic acid with polycationic other nanosized particles belonging to cationic liposomes/micelles or cationic polymers. Cationic lipids used in this delivery method include, but are not limited to, monovalent cationic lipids, polyvalent cationic lipids, guanidine-containing compounds, cholesterol derivatives, cationic polymers (e.g., poly(ethyleneimine), poly-L-lysine, protamine, other cationic polymers), and lipid-polymer hybrids.

[00305] A. Exosomes:

[00306] In some embodiments, the scDNA vector as described herein is delivered by packaging into an exosome. Exosomes are small membrane-bound vesicles of endocytic origin that are released into the extracellular environment after the fusion of multivesicular bodies from the plasma membrane. Their surface is composed of the lipid bilayer of the donor cell's cell membrane, they contain the cytosol from the cell that produced the exosome, and expose the parent cell's membrane proteins on the surface. Exosomes produce various types of cells, including epithelial cells. B- and T-lymphocytes, mast cells (MC), and dendritic cells (DC). Some implementation options include the use of exosomes with a diameter of 10 nm to 1 μm, 20 nm to 500 nm, 30 nm to 250 nm, 50 nm to 100 nm. Exosomes can be isolated for delivery to target cells either using donor cells or by introducing specific nucleic acids into them. Various methods known in the art can be used to obtain exosomes containing capsidless AAV vectors according to the present invention.

[00307] B. Microparticles/Nanoparticles:

[00308] In some embodiments, the scDNA vector as described herein is delivered via a lipid nanoparticle. Typically, lipid nanoparticles contain an ionizable amino lipid (e.g., heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin-MC3-DMA, phosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol, and coating lipid (polyethylene glycol-di myristolglycerol, PEG-DMG), for example, as described in Tam et al (2013) Advances in Lipid Nanoparticles for siRNA delivery Pharmaceuticals 5(3): 498-507.

[00309] In some embodiments, the lipid nanoparticle has an average diameter of about 10 to about 1000 nm. In some embodiments, the lipid nanoparticle has a diameter of less than 300 nm. In some embodiments, the lipid nanoparticle has a diameter of from about 10 to about 300 nm. In some embodiments, the lipid nanoparticle has a diameter of less than 200 nm. In some embodiments, the lipid nanoparticle has a diameter of from about 25 to about 200 nm. In some embodiments, a lipid nanoparticle formulation (e.g., a composition comprising a pool of lipid nanoparticles) has a particle size distribution where the average size (e.g., diameter) is from about 70 nm to about 200 nm, and more often is about 100 nm or less.

[00310] Various lipid nanoparticles known in the art can be used to deliver the scDNA vector as described herein. For example, various delivery methods using lipid nanoparticles are described in US Pat.

[00311] In some embodiments, the scDNA vector as described herein is delivered using gold nanoparticles. Typically, the nucleic acid can be covalently linked to the gold nanoparticle or non-covalently linked to the gold nanoparticle (eg, charge-linked), for example, as described in Ding et al. (2014). Gold Nanoparticles for Nucleic Acid Delivery. Mol. Ther. 22(6); 1075-1083. In some embodiments, gold nanoparticle-nucleic acid conjugates are prepared using the methods described, for example, in US Pat. No. 6,812,334.

[00312] C. Liposomes

[00313] The formation and use of liposomes is generally known to those skilled in the art. Liposomes have been developed with improved serum stability and increased circulating half-life (US Pat. No. 5,741,516). Additionally, various methods have been described using liposomes and liposome-like formulations as potential drug carriers (US Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868;

[00314] Liposomes have been successfully used in several cell types that are generally resistant to transfection using other procedures. In addition, liposomes do not have the DNA length limitations typical of virus-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into various cultured cell lines and animals. In addition, several successful clinical trials have been completed to investigate the efficacy of liposome-mediated drug delivery.

[00315] Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multilamellar vesicles from concentric bilayers (also called multilamellar vesicles (MLVs). MLVs typically have a diameter of 25 nm to 4 µm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with a diameter ranging from 200 to 500 Å, containing in the core aqueous solution.

[00316] In some embodiments, the liposome contains cationic lipids. The term "cationic lipid" includes lipids and synthetic lipids containing both polar and non-polar domains that are capable of becoming positively charged at or near physiological pH, bind to polyanions such as nucleic acids, and facilitate the delivery of nucleic acids into cells. In some embodiments, cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides, or derivatives thereof. In some embodiments, cationic lipids include straight chain, branched alkyl, alkenyl groups, or any combination of the above. Согласно некоторым вариантам реализации катионные липиды содержат от 1 до приблизительно 25 атомов углерода (например, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 или 25 атомов углерода. Согласно некоторым вариантам реализации катионные липиды содержат более 25 атомов углерода. Согласно некоторым вариантам реализации прямоцепочечные или разветвленные алкильные или алкеновые группы содержат шесть или более атомов углерода. Катионный липид может также содержать, согласно некоторым вариантам реализации, одну или более алициклических групп. Неограничивающие примеры алициклических групп включают холестерин и другие стероидные группы. Согласно некоторым вариантам реализации катионные липиды получают с одним или более противоионами. Примеры противоионов (анионов) включают, не ограничиваясь перечисленными, Cl ^- , Br ^- , I ^- , F ^- , ацетат, трифторацетат, сульфат, нитрит и нитрат.

[00317] Non-limiting examples of cationic lipids include polyethyleneimine, polyamidoamine (PAMAM) starburst hyperbranched dendrimers, lipofectin (a combination of DOTMA and DOPE), lipofectase, Lipofectamine™ (e.g., LIPOFECTAMINE™ 2000), DOPE, cytofectin (Gilead Sciences, Foster City, CA), and eufectins (JBL, San Louis Obispo, California). Examples of cationic liposomes can be prepared from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethyl ammonium chloride (DOTMA), N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC -Chol), 2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide; and dimethyldioctadecylammonium bromide (DDAB). Nucleic acids (eg, CELiD) may also be complexed with, for example, poly(L-lysine), or avidin and lipids, for example, sterile poly(L-lysine), may or may not be included in said mixture.

[00318] In some embodiments, the scDNA vector as described herein is delivered using a cationic lipid as described in US Pat. No. 8,158,601 or a polyamine compound or lipid as described in US Pat. No. 8,034,376.

[00319] D. Conjugates

[00320] In some embodiments, the scDNA vector as described herein is conjugated (eg, covalently linked) to an agent that increases cellular uptake. An "agent that enhances cellular uptake" is a molecule that facilitates the transport of a nucleic acid across a lipid membrane. For example, the nucleic acid can be conjugated to a lipophilic compound (eg, cholesterol, tocopherol, etc.), a cell penetrating peptide (CPP) (eg, penetratin, TAT, Syn1B, etc.), and polyamines (eg, spermine). Additional examples of agents that increase cellular uptake are disclosed, for example, in Winkler (2013). Oligonucleotide conjugates for therapeutic applications. Ther. Deliv. 4(7); 791-809.

[00321] In some embodiments, the scDNA vector as described herein is conjugated to a polymer (eg, a polymer molecule) or a folate molecule (eg, a folic acid molecule). It is generally known in the art to deliver polymer-conjugated nucleic acids, such as as described in WO2000/34343 and WO200 8/0223 09. In some embodiments, the scDNA vector as described herein is conjugated to a poly(amide) polymer, such as as described in US Pat. No. 8,987,377. In some embodiments, the nucleic acid described herein is conjugated to a folate molecule as described in US Pat. No. 8,507,455.

[00322] In some embodiments, the scDNA vector as described herein is conjugated to a carbohydrate, such as as described in US Pat. No. 8,450,467.

[00323] E. Nanocapsule

[00324] Alternatively, scDNA vector nanocapsule formulations as described herein can be used. In general, substances can be encapsulated in nanocapsules in a stable and reproducible manner. To avoid side effects due to excessive intracellular loading of polymers, such ultrafine particles (about 0.1 μm in size) must be developed using polymers capable of degrading in vivo. Provided for use are biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements.

VIII. Delivery methods for ccDNA vectors

[00325] In some embodiments, the scDNA vector can be delivered to the target cell in vitro or in vivo by various suitable methods. ZcDNA vectors can be applied or injected separately. ZcDNA vectors can be delivered into a cell without the aid of a transfection reagent or other physical means. Alternatively, scDNA vectors can be delivered using any transfection reagent known in the art or other physical method known in the art that facilitates entry of DNA into the cell, such as liposomes, alcohols, polylysine-rich compounds, arginine-rich compounds, calcium phosphate, microvesicles, microinjection, electroporation, and the like.

[00326] At the same time, transduction with capsidless AAV vectors as described herein can effectively target cell and tissue types that are difficult to transduce with standard AAV virions using various delivery reagents.

[00327] In another embodiment, the scDNA vector is administered to the CNS (eg, brain or eye). This zcDNA vector can be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (striatum, cerebrum, including occipital, temporal, parietal and frontal lobes, cerebral cortex, basal ganglia, hippocampus, and amygdala), limbic system, neocortex, striatum, cerebrum, and inferior colliculus. Said scDNA vector may also be introduced into different regions of the eye such as the retina, cornea and/or optic nerve. Said scDNA vector can be delivered to the cerebrospinal fluid (eg, by lumbar puncture). Said ccDNA vector may also be intravascularly introduced into the CNS in situations where the blood-brain barrier has been compromised (eg, in a brain tumor or cerebral infarction).

[00328] In some embodiments, said scDNA vector may be introduced into the desired region or regions of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intraocular, intraocular (e.g., intravitreal, subretinal, anterior chamber) and periocular (for example, to the sub-Tenon space) delivery, as well as intramuscular delivery with retrograde delivery to motor neurons.

[00329] In some embodiments, said scDNA vector is administered as a liquid formulation by direct injection (eg, stereotaxic injection) into the desired region or compartment in the CNS. In other embodiments, said scDNA vector may be administered by topical application to the desired area, or by intranasal administration of an aerosol formulation. Administration to the eye can be accomplished by topical application of liquid drops. In a further alternative, the scDNA vector may be administered as a slow release solid formulation (see, for example, US Pat. No. 7,201,898). In other additional embodiments, said scDNA vector can be used for retrograde transport to treat, alleviate, and/or prevent diseases and disorders that involve motor neurons (e.g., amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA), etc.). For example, said scDNA vector can be delivered to muscle tissue from which it can migrate to neurons.

VIII. Additional Uses for scDNA Vectors

[00330] Compositions and scDNA vectors proposed according to the present invention can be used to deliver a transgene for various purposes. In some embodiments, said transgene encodes a protein or functional RNA for use in scientific research, for example, to create a somatic transgenic model in animals carrying said transgene, for example, to study the function of a product of said transgene. According to another example, said transgene encodes a protein or functional RNA for use in creating an animal model of a disease. In some embodiments, said transgene encodes one or more peptides, polypeptides, or proteins that are suitable for treating, preventing, or alleviating disease states or disorders in a mammalian subject. The specified transgene can be transferred to the subject (eg, expressed in the subject) in sufficient quantity to treat a disease associated with reduced expression, lack of expression or dysfunction of the gene. In some embodiments, said transgene can be transferred to a subject (e.g., expressed in a subject) in sufficient quantity to treat a disease associated with increased expression, gene product activity, or inappropriate upregulation of a gene that said transgene represses or otherwise causes the desired reduction in expression.

IX. Methods of application

[00331] The ZcDNA vector of the present invention may also be used in a method for delivering a nucleotide sequence of interest to a target cell. Said method may in particular be a method of delivering a therapeutic gene of interest into a cell of a subject in need thereof. The present invention allows for in vivo expression of a polypeptide, protein or oligonucleotide encoded by a therapeutic exogenous DNA sequence in cells of a subject such that therapeutic levels of said polypeptide, protein or oligonucleotide are expressed. These results are observed in both in vivo and in vitro delivery modes of the scDNA vector.

[00332] A method for delivering a nucleic acid of interest to a cell of a subject may include administering to said subject an scDNA vector of the present invention containing said nucleic acid of interest. In addition, the present invention provides a method for delivering a nucleic acid of interest to a cell in need thereof, comprising multiple injections of an scDNA vector of the present invention containing the nucleic acid of interest. Because the ccDNA vector of the present invention does not induce an immune response, such a multi-administration strategy will not interfere with the response of the host immune system against the ccDNA vector of the present invention, contrary to what is often observed with encapsidated vectors.

[00333] The scDNA vector nucleic acid or acids are administered in sufficient amounts to transfect cells of the desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse events. Standard and pharmaceutically acceptable routes of administration include, but are not limited to, intravenous delivery (eg, in a liposomal formulation), direct delivery to a selected organ (eg, intraportal delivery to the liver), intramuscular delivery, and other parenteral routes of administration. Routes of administration can be combined if desired.

[00334] Delivery of the ccDNA vector is not limited to one kind of ccDNA vector. Accordingly, in another aspect, multiple scDNA vectors containing different exogenous DNA sequences can be delivered simultaneously or sequentially to a target cell, tissue, organ, or body of a subject. Accordingly, this strategy may allow the expression of several genes. Delivery can also be repeated and, importantly for gene therapy in the clinical setting, with subsequent dose increases or decreases, given the absence of host-directed immune response against the capsid due to the absence of the viral capsid. No anti-capsid response is expected because the capsid is absent.

[00335] The present invention also provides a method of treating a disease in a subject, comprising administering to a target cell in need (particularly a muscle cell or tissue) of the subject a therapeutically effective amount of the scDNA vector, optionally with a pharmaceutically acceptable carrier. Although the scDNA vector can be introduced in the presence of a carrier, such a carrier is not necessary. The scDNA vector used contains a nucleotide sequence of interest suitable for the treatment of a disease. In particular, said scDNA vector may contain the desired exogenous DNA sequence operably linked to control elements capable of directing the transcription of the desired polypeptide, protein, or oligonucleotide encoded by said exogenous DNA sequence when administered to a subject. Said scDNA vector may be introduced by any suitable route as described above and elsewhere herein.

X. Methods of treatment

[00336] The technology described herein also demonstrates methods for producing as well as methods for using the proposed ccDNA vectors in a variety of ways, including, for example, ex situ, in vitro, and in vivo uses, techniques, diagnostic procedures, and/or gene therapy regimens.

[00337] The present invention provides a method of treating a disease or disorder in a subject, comprising administering to a target cell in need (e.g., a muscle cell or tissue, or other affected cell type) of the subject, a therapeutically effective amount of an scDNA vector, optionally with a pharmaceutically acceptable carrier. While said scDNA vector may be introduced in the presence of a carrier, such a carrier is not necessary. The scDNA vector used contains a nucleotide sequence of interest suitable for the treatment of a disease. In particular, said scDNA vector may contain the desired exogenous DNA sequence operably linked to control elements capable of directing the transcription of the desired polypeptide, protein, or oligonucleotide encoded by said exogenous DNA sequence when administered to a subject. Said scDNA vector may be introduced by any suitable route as described above and elsewhere herein.

[00338] Any transgene can be delivered using scDNA vectors as described herein. Transgenes of interest include nucleic acids encoding polypeptides or non-coding nucleic acids (eg, RNA interference, siRNA, etc.), preferably therapeutic (eg, for medical, diagnostic, or veterinary use) or immunogenic (eg, for vaccines) polypeptides.

[00339] In some embodiments, transgenes for expression using the scDNA vectors described herein express or encode one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAi, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments; or any combination of the above.

[00340] In particular, said transgene may encode one or more therapeutic agents, including, but not limited to, for example, proteins, polypeptides, peptides, enzymes, antibodies, antigen-binding fragments, as well as their variants and/or active fragments, agonists, antagonists, mimetics for use in the treatment, prevention and/or alleviation of one or more symptoms of a disease, dysfunction, injury and/or disorder. In one aspect, said disease, dysfunction, injury, injury, and/or disorder is a disease, dysfunction, injury, injury, and/or disorder in a human.

[00341] As noted herein, said transgene may encode a therapeutic protein or peptide, or a therapeutic nucleic acid sequence or therapeutic agent, including, but not limited to, one or more agonists, antagonists, anti-apoptotic factors, inhibitors, receptors, cytokines, cytotoxins, erythropoietic agents, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interfer anons, interleukins, interleukin receptors, nerve growth factors, neuroactive peptides, neuroactive peptide receptors, proteases, protease inhibitors, protein decarboxylases, protein kinases, protein kinase inhibitors, protein receptor-binding enzymes, transport proteins or one or more inhibitors thereof, serotonin receptors or one or more uptake inhibitors, serpins, serpin receptors, tumor suppressors , diagnostic molecules, chemotherapeutic agents, cytotoxins; or any combination of the above.

[00342] In some embodiments, a transgene in an expression cassette, expression construct, or scDNA vector described herein can be codon-optimized for the host cell. As used herein, the term "codon-optimized" or "codon-optimization" refers to a method of modifying a nucleic acid sequence to improve expression in cells of a vertebrate animal of interest, e.g., a mouse or a human (e.g., humanized), by replacing at least one, more than one, or a significant number of codons in a natural sequence (e.g., a prokaryotic sequence) with codons commonly or most frequently used in the genes of said vertebrate animal. In different species, there is a particular bias with a preference for certain codons for a particular amino acid. Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be determined using, for example, the codon optimization and personalized gene synthesis platform from Aptagen Gene Forge® (Aptagen, Inc.) or other publicly available database.

[00343] In some embodiments, said scDNA vector expresses a transgene in a host cell subject. In some embodiments, said host cell is a human host cell, including, for example, blood cells, stem cells, hematopoietic cells, CD34 ⁺ cells, liver cells, cancer cells, vascular cells, muscle cells, pancreatic cells, nerve or retinal cells, epithelial or endothelial cells, dendritic cells, fibroblasts, or any other mammalian cell, including, but not limited to, hepatocytes ( i.e., liver cells), lung cells, heart cells, pancreatic cells, gastrointestinal cells, diaphragmatic cells, renal (i.e., kidney) cells, nerve cells, blood cells, bone marrow cells, or cells from any one or more selected tissues of the subject for whom the gene therapy is intended. In one aspect, the subject host cell is a human host cell.

[00344] The present invention provides ccDNA vector compositions and formulations that include one or more ccDNA vectors of the present invention along with one or more pharmaceutically acceptable buffers, diluents, or excipients. Such compositions may be included in one or more diagnostic or therapeutic kits for diagnosing, preventing, treating, or alleviating one or more symptoms of a disease, injury, disorder, injury, or dysfunction. In one aspect, said disease, injury, disorder, injury, or dysfunction is a disease, injury, disorder, injury, or dysfunction in a human.

[00345] According to another aspect of the technology described herein, there is provided a method of providing a subject in need with a diagnostically or therapeutically effective amount of a scDNA vector, said method comprising delivering to a cell, tissue, or organ of the subject in need an amount of a scDNA vector as described herein; for a period of time effective to cause expression of the transgene from said scDNA vector, thereby providing said subject with a diagnostically or therapeutically effective amount of a protein, peptide, nucleic acid expressed by said scDNA vector. In a further aspect, said subject is a human.

[00346] According to another aspect of the technology described herein, a method for diagnosing, preventing, treating, or alleviating at least one or more symptoms of a disease, disorder, dysfunction, injury, abnormal condition, or injury in a subject is provided. In a general and broad sense, said method includes at least the step of administering to a subject in need one or more of the described scDNA vectors, in an amount and for a period of time sufficient to diagnose, prevent, treat, or alleviate one or more symptoms of a disease, disorder, dysfunction, injury, abnormal condition, or injury in the subject. In a further aspect, said subject is a human.

[00347] In another aspect, the use of said scDNA vector is provided as a tool for treating or reducing one or more symptoms of a disease or condition. There are a number of hereditary diseases for which defective genes are known, which generally fall into two classes: deficiencies, typically enzyme deficiencies, which are usually inherited in a recessive manner, and imbalanced conditions, in which regulatory or structural proteins may be involved, and which are usually, but not always, inherited in a dominant manner. In diseases with deficient conditions, ccDNA vectors can be used to deliver transgenes, to introduce a normal gene into diseased tissues for replacement therapy, and also, in some embodiments, to create animal models of the disease using antisense mutations. In the case of imbalanced disease states, scDNA vectors can be used to create a disease state model in a model system, which can then be used as part of an effort to counter said disease state. Accordingly, ccDNA vectors and methods as described herein allow the treatment of genetic diseases. According to the present document, the treatment of a disease state is carried out by partially or completely eliminating the insufficiency or imbalance that causes the disease or makes it more severe.

[00348] In general, an scDNA vector as described herein can be used to deliver any transgene to treat, prevent, or alleviate symptoms associated with any gene expression disorder. Indicative painful states include, not limited to the listed: cystic fiber (and other lung diseases), hemophilia A, hemophilia B, talassemia, anemia and other blood disorders, AIDS, Alzheimer disease, Parkinson disease, Gentington disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, weapons, diabetes. hacker dystrophy (for example, Duchenna, Becker), Gurler's disease, adenosinezaminase deficiency, metabolic defects, degenerative diseases of the retinal (and other eyes of the eyes), mitochondriopathy (for example, hereditary optical neuropathy (Lhon), Leia syndrome and subacute sclerosizing enmulopathy (for example,), myopathy (for example,), myopathy (for example), myopathy (for example , shoulder-lyceum fiber myopathy (FSHD) and cardiomyopathy), diseases of solid organs (for example, brain, liver, kidneys, heart); and so on. In some embodiments, scDNA vectors as described herein may be advantageously used in the treatment of individuals with metabolic disorders (eg, ornithine transcarbamylase deficiency).

[00349] In some embodiments, the scDNA vector described herein can be used to treat, alleviate, and/or prevent a disease or disorder caused by a mutation in a gene or gene product. Examples of diseases or disorders that can be treated using ccDNA vectors include, but are not limited to, metabolic diseases or disorders (eg, Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage disease); diseases or disorders of the urea cycle (eg, ornithine transcarbamylase (OTC) deficiency); lysosomal or storage disorders (eg, metachromatic leukodystrophy (MLD), mucopolysaccharidosis type II (MPSII; Gunther's syndrome)); liver diseases or disorders (eg, progressive familial intrahepatic cholestasis (PSIC); blood diseases or disorders (eg, hemophilia (A and B), thalassemia, and anemia); cancer and tumors; and genetic diseases or disorders (eg, cystic fibrosis).

[00350] According to another additional aspect, a scDNA vector as described herein can be used to deliver a heterologous nucleotide sequence in situations in which regulation of the level of transgene expression (e.g., transgenes encoding hormones or growth factors, as described herein) is desired.

[00351] Accordingly, in some embodiments, the scDNA vector described herein can be used to correct an abnormal level and/or function of a gene product (e.g., a missing or defective protein) that results in a disease or disorder. Said ccDNA vector may produce a functional protein and/or modify levels of said protein to alleviate or reduce symptoms resulting from a particular disease or disorder, or provide benefits for a particular disease or disorder caused by the absence or defect of the protein. For example, treatment of OTC deficiency can be accomplished by producing a functional OTC enzyme; treatment of hemophilia A and B can be done by modifying the levels of factor VIII, factor IX and factor X; PKU can be treated by modifying levels of the enzyme phenylalanine hydroxylase; treatment of Fabry's disease or Gaucher's disease can be carried out by producing a functional alpha-galactosidase or beta-glucocerebrosidase, respectively; treatment of FSHD or MPSII can be accomplished by producing a functional arylsulfatase A or iduronate-2-sulfatase, respectively; treatment of cystic fibrosis can be carried out by producing a functional regulator of transmembrane conductance in cystic fibrosis; treatment of glycogen storage disease can be carried out by restoring the function of the functional enzyme glucose-6-phosphatase; and treatment of PSVH can be carried out by obtaining functional ATP8 B1, ABCB11, ABCB4 or TJP2 genes.

[00352] In alternative embodiments, the scDNA vectors as described herein can be used to provide an antisense nucleic acid to a cell in vitro or in vivo. For example, if the transgene is an RNAi molecule, expression of the antisense nucleic acid or RNAi in the target cell reduces the level of expression of a particular protein by that cell. Accordingly, transgenes, which are RNA interference molecules or antisense nucleic acids, can be introduced to reduce the expression of a particular protein in a subject in need thereof. Antisense nucleic acids can also be introduced into cells in vitro to regulate cell physiology, for example, to optimize cell and tissue culture systems.

[00353] In some embodiments, examples of transgenes encoded by the ccDNA vector include, but are not limited to: X, lysosomal enzymes (e.g., hexosaminidase A associated with Tay-Sachs disease or iduronate sulfatase associated with Gunther syndrome/MPS II), erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, as well as cytokines (eg, interferon, β-interferon, interferon-γ, interleukin-2, interleukin-4, interleukin 12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.), peptide growth factors and hormones (eg, somatotropin, insulin, insulin-like growth factors 1 and 2, platelet-derived growth factor (TRF), epi dermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), neurotrophic factor-3 and 4, brain-derived neurotrophic factor (BDNF), glial growth factor (GDNF), transforming growth factors α and β, etc.), receptors (for example, tumor necrosis factor receptor). In some exemplary embodiments, said transgene encodes a monoclonal antibody that is specific for one or more of the desired targets. In some exemplary embodiments, the scDNA vector encodes more than one transgene. In some exemplary embodiments, said transgene encodes a fusion protein containing two different polypeptides of interest. In some embodiments, said transgene encodes an antibody, including a full-length antibody or antibody fragment, as defined herein. In some embodiments, said antibody is an immunoglobulin antigen-binding domain or variable domain sequence as defined herein. Other illustrative transgenic sequences encode products of suicide genes (thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, and tumor necrosis factor), proteins conferring resistance to a drug used in cancer therapy, and products of tumor suppressor genes.

[00354] In a representative embodiment, a transgene expressed by a scDNA vector can be used to treat muscular dystrophy in a subject in need thereof, said method comprising: administering an effective to treat, alleviate, or prevent amount of the scDNA vector described herein, said scDNA vector containing a heterologous nucleic acid encoding dystrophies, minidystrophin, microdystrophin, myostatin propeptide , follistatin, soluble type II activin receptor, IPFR-1, anti-inflammatory polypeptides such as dominant I-κB mutant, sarcospan, utrophin, microdystrophin, laminin-α2, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, IPFR-1, anti-myostatin or anti-myostatin propeptide antibody or antibody fragment , and/or RNAi to myostatin. In specific embodiments, said scDNA vector can be introduced into skeletal, diaphragmatic, and/or cardiac muscle as described herein.

[00355] In some embodiments, said scDNA vector can be used to deliver a transgene to skeletal, cardiac, or diaphragmatic muscle, to produce a polypeptide (e.g., enzyme) or functional RNA (e.g., RNAi, miRNA, antisense RNA) that normally circulates in the blood, or for systemic delivery to other tissues to treat, alleviate, and/or prevent a disorder (e.g., a metabolic disorder such as diabetes (e.g., insulin delivery). a), hemophilia (eg, VIII), a mucopolysaccharidosis disorder (eg, Sly's syndrome, Hurler's syndrome, Schie's syndrome, Hurler-Scheie's syndrome, Gunter's syndrome, Sanfilippo A, B, C, D syndrome, Morquio's syndrome, Maroteau-Lami's syndrome, etc.) or a lysosomal storage disorder (such as Gaucher's disease [glucocerebrosidase], Pompe [lysosomes] disease acid alpha-glucosidase] or Fabry disease [alpha-galactosidase A]) or glycogen storage disorders (such as Pompe disease [lysosomal acid alpha-glucosidase]). Other suitable proteins for the treatment, alleviation and/or prevention of metabolic disorders are described above.

[00356] In other embodiments, an scDNA vector as described herein may be used to deliver a transgene in a method for treating, alleviating, and/or preventing a metabolic disorder in a subject in need thereof. Exemplary metabolic disorders and transgenes encoding polypeptides are described herein. Optionally, said polypeptide is a secreted polypeptide (eg, a polypeptide that is a secreted polypeptide in its natural state or engineered to be secreted, for example by operably linking it to a secretory signal sequence, as is known in the art).

[00357] Another aspect of the present invention relates to a method for treating, ameliorating and/or preventing congenital heart failure or PAD in a subject in need thereof, said method comprising administering an scDNA vector as described herein to a mammalian subject, said scDNA vector containing a transgene encoding, for example, Ca ²⁺ -ATPase of the sarcoplasmic endoreticulum (SERCA2a), angina genic factor, phosphatase inhibitor I (I-1), RNAi to phospholamban; a phospholamban inhibitory or dominant negative molecule such as phospholamban S16E, a zinc finger protein that regulates the phospholamban gene, β2-adrenergic receptor, beta-2-adrenergic receptor kinase (BARK), PI3 kinase, calsarcan, beta-adrenergic receptor kinase (βARKct) inhibitor, protein phosphatase 1 inhibitor 1, S 100A1, parvalbumin, adenylyl cyclase type 6, a molecule that knocks down G-protein coupled receptor kinase type 2, such as truncated constitutively active βARKct, Pim-1, PGC-1α, SOD-1, SOD-2, EC-SOD, kallikrein, HIF, thymosin-β4, mir-1, mir-133, mir- 206 and/or mir-208.

[00358] The zcDNA vectors as described herein may be administered to the lungs of a subject by any suitable means, optionally by administering an aerosol suspension of respirable particles containing the zcDNA vectors that the subject inhales. Said respirable particles may be liquid or solid. Liquid particle aerosols containing the scDNA vectors can be produced by any suitable method, for example, using a pneumatic aerosol nebulizer or an ultrasonic nebulizer, as is known to those skilled in the art. See, for example, US Pat. No. 4,501,729. Particulate aerosols containing scDNA vectors can similarly be generated with any particulate aerosol generator using techniques known in the pharmaceutical art.

[00359] In some embodiments, said scDNA vectors can be introduced into CNS tissues (eg, brain, eyes). In specific embodiments, the scDNA vectors as described herein may be administered to treat, alleviate, or prevent diseases of the CNS, including genetic disorders, neurodegenerative disorders, psychiatric disorders(disorders), and tumors. Exemplary CNS diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, spinal cord injury. of the brain or head, Tay-Sachs disease, Lesch-Nychen disease, epilepsy, cerebral infarctions, psychiatric disorders, including mood disorders (eg, depression, bipolar affective disorder (disorder), persistent affective disorder, secondary mood disorder), schizophrenia, drug or drug dependence (eg, alcoholism and dependence on other substances), neuroses (eg, anxiety, obsessive disorder, somatic form disorder, dissociative disorder, grief disorder, postpartum depression), psychosis (eg, hallucinations and delusions), dementia, paranoia, attention-deficit disorders, psychosexual disorders, sleep disturbances, pain-related disorders, dietary or weight-related disorders (eg, obesity, cachexia, anorexia nervosa and bulimia), cancer, and CNS tumors (eg, pituitary tumors).

[00360] Ocular disorders that can be treated, alleviated, or prevented using the ccDNA vectors of the present invention include ophthalmic disorders affecting the retina, posterior tract, and optic nerve (e.g., retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma). Many ophthalmic diseases and disorders are associated with one or more of three types of features: (1) angiogenesis, (2) inflammation, and (3) degeneration. In some embodiments, the scDNA vector as described herein may be used to deliver anti-angiogenic factors; anti-inflammatory factors; factors that slow cell degeneration, promote cell preservation or promote cell growth, and combinations of the foregoing. Diabetic retinopathy, for example, is characterized by angiogenesis. Treatment of diabetic retinopathy may be by delivering one or more anti-angiogenic factors either intraocularly (eg, into the vitreous) or periocularly (eg, into the sub-Tenon's space). One or more neurotrophic factors can be delivered together, either intraocularly (eg, intravitreally) or periocularly. Additional eye diseases (ocular diseases) that can be treated or prevented using the ccDNA vectors of the present invention include geographic atrophy, vascular or "wet" macular degeneration, Stargardt's disease, Leber's congenital amaurosis (LCA), Usher's syndrome, pseudoxanthoma elasticum (PXE), X-linked retinitis pigmentosa (XLRP), X-linked retinal dissection (XLRS). ), choroideremia, Leber's congenital optic neuropathy (LHON), achromatopsia, rod-cone dystrophy, Fuchs endothelial corneal dystrophy, diabetic macular edema; cancer and eye tumors.

[00361] In some embodiments, treatment, alleviation, or prevention of inflammatory diseases or ocular disorders (eg, uveitis) can be accomplished using the scDNA vectors of the present invention. One or more anti-inflammatory factors can be expressed by intraocular administration (eg, into the vitreous chamber or anterior chamber) of the scDNA vector as described herein. In other embodiments, the treatment, alleviation, or prevention of diseases or ocular disorders characterized by retinal degeneration (eg, retinitis pigmentosa) can be accomplished using the scDNA vectors of the present invention. Intraocular administration (eg, into the vitreous) of a ccDNA vector as described herein encoding one or more neurotrophic factors can be used to treat such diseases associated with retinal degeneration. In some embodiments, a disease or disorder that involves both angiogenesis and retinal degeneration (eg, age-related macular degeneration) can be treated using the scDNA vectors of the present invention. Age-related macular degeneration can be treated by administering an ccDNA vector as described herein encoding one or more neurotrophic factors intraocularly (e.g. into the vitreous) and/or encoding one or more anti-angiogenic factors intraocularly or periocularly (e.g. into the sub-Tenonian space). Glaucoma is characterized by increased intraocular pressure and loss of retinal ganglion cells. Treatment options for glaucoma include administering one or more neuroprotective agents that protect cells from excitotoxic damage using a scDNA vector as described herein. Accordingly, such agents include N-methyl-D-aspartate (NMDA) antagonists, cytokines, and neurotrophic factors that can be delivered intraocularly, optionally intravitreally, using a scDNA vector as described herein.

[00362] In other embodiments, the scDNA vector as described herein may be used to treat seizures, such as to reduce the occurrence, frequency, or severity of seizures. The effectiveness of the therapeutic treatment of seizures can be assessed based on behavioral signs (eg, trembling, eye or mouth tics) and/or electrographic methods (most seizures are characterized by distinctive electrographic abnormalities). Accordingly, the ccDNA vector as described herein can also be used to treat epilepsy, which is characterized by multiple seizures at different time points. In one representative embodiment, somatostatin (or an active fragment thereof) is administered to the brain using the ccDNA vector a as described herein for the treatment of a pituitary tumor. According to this embodiment, the scDNA vector as described herein encoding somatostatin (or its active fragment) is administered by microinfusion into the pituitary gland. Similarly, such treatment can be used to treat acromegaly (abnormal secretion of growth hormone by the pituitary gland). The nucleic acid (eg, GenBank Accession No. J00306) and amino acid (eg, GenBank Accession No. P01166; contains truncated somatostatin-28 and somatostatin-14 active peptides) sequences of somatostatins are known in the art. In specific embodiments, the scDNA vector may encode a transgene that contains a secretion signal as described in US Pat. No. 7,071,172.

[00363] Another aspect of the present invention relates to the use of a scDNA vector as described herein to generate antisense RNA, RNA interference, or other functional RNA (eg, a ribozyme) for systemic delivery to a subject in vivo. Accordingly, in some embodiments, said scDNA vector may contain a transgene that encodes an antisense nucleic acid, a ribozyme (e.g., as described in US Pat. No. 5,877,022), an RNA that affects spliceosome-mediated trans-splicing (see Puttaraju et al., (1999) Nature Biotech. 17:246; US Pat. No. 6,013,487; US patent No. 6083702), interfering RNA (RNA interference), mediating gene silencing (see Sharp et al., (2000) Science 287:2431), or other non-translated RNA, such as "guide" RNA (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; US patent No. 586 9248 issued by Yuan et al.), etc.

[00364] In some embodiments, said scDNA vector may additionally also contain a transgene that encodes a reporter polypeptide (eg, an enzyme such as green fluorescent protein or alkaline phosphatase). In some embodiments, the transgene that encodes a reporter protein suitable for experimental or diagnostic use is selected from any of the following: β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art. In some aspects, scDNA vectors containing a transgene encoding a reporter polypeptide can be used for diagnostic purposes or as markers of the activity of the scDNA vector in a subject to which it is administered.

[00365] In some embodiments, said scDNA vector may contain a transgene or a heterologous nucleotide sequence that has homology to and recombines with a locus on the host chromosome. This approach can be used to correct a genetic defect in a host cell.

[00366] In some embodiments, said scDNA vector may contain a transgene that can be used to express an immunogenic polypeptide in a subject, such as for vaccination. Said transgene may encode any immunogen of interest known in the art, including, but not limited to, human immunodeficiency virus, influenza virus immunogens, gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like.

XI. Introduction

[00367] In specific embodiments, more than one administration (e.g., two, three, four, or more administrations) at various intervals, such as daily, weekly, monthly, yearly, and the like, may be used to achieve the desired level of gene expression.

[00368] Exemplary routes of administration of the ccDNA vector as described herein include oral, rectal, transmucosal, intranasal, inhalation (e.g., aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intraendothelial, in utero (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular [including injection into the skeletal, diaphragmatic and / or cardiac muscle], intrapleural, intracerebral and intraarticular), topical (for example, on the skin and mucosal surfaces, including the surface of the respiratory tract, and transdermal administration), intralymphatic administration, etc., as well as direct injection into a tissue or organ (for example, into the liver, eyes, skeletal muscles, heart muscle, diaphragmatic muscle, or brain).

[00369] The scDNA vector can be introduced at any site in the subject's body, including, without limitation, a site selected from the group consisting of the brain, skeletal muscle, smooth muscle, heart, diaphragm, respiratory tract epithelium, liver, kidney, spleen, pancreas, skin, and eye. Can also be the introduction of the specified ccDNA vector in the tumor (for example, in the tumor or near the tumor, or in the lymph node). The most appropriate route in any given case depends on the nature and severity of the condition being treated, alleviated and/or prevented, and on the nature of the particular scDNA vector used. In addition, the use of scDNA allows the introduction of more than one transgene in a single vector or multiple scDNA vectors (eg, an scDNA cocktail).

[00370] Administration of an scDNA vector as described herein to skeletal muscle in accordance with the present invention includes, but is not limited to, administration to skeletal muscle in the extremities (e.g., upper arm, forearm, thigh, and/or lower leg), back, neck, head (e.g., tongue), chest, abdomen, pelvis/perineum, and/or fingers. The ccDNA vector as described herein can be delivered to skeletal muscle by intravenous, intra-arterial, intraperitoneal, limb perfusion, (optionally isolated limb, leg, and/or arm perfusion; see e.g., Arruda et al., (2005) Blood 105: 3458-3464), and/or direct intramuscular injection. In specific embodiments, an scDNA vector as described herein is administered to a limb (arm and/or leg) of a subject (e.g., a subject with muscular dystrophy such as DMD) by perfusion of the limb, optionally by perfusion of an isolated limb (e.g., by intravenous or intra-articular injection). In embodiments, the scDNA vector as described herein can be introduced without using "hydrodynamic" techniques.

[00371] Administration of an scDNA vector as described herein to cardiac muscle includes administration to the left atrium, right atrium, left ventricle, right ventricle, and/or septum. The ccDNA vector as described herein can be delivered to the heart muscle by intravenous administration, intra-arterial administration such as intra-aortic administration, direct injection into the heart (e.g., left atrium, right atrium, left ventricle, right ventricle), and/or coronary artery perfusion. The introduction into the diaphragmatic muscle can be carried out by any suitable method, including intravenous administration, intra-arterial administration and/or intraperitoneal administration. Administration to smooth muscle may be by any suitable route, including intravenous administration, intra-arterial administration, and/or intraperitoneal administration. According to one implementation variant, the introduction can be carried out in endothelial cells present in smooth muscles, near and/or on them.

[00372] In some embodiments, the scDNA vector of the present invention is administered to skeletal muscle, diaphragmatic muscle, and/or cardiac muscle (e.g., to treat, alleviate, and/or prevent muscular dystrophy or heart disease (e.g., PAD or congestive heart failure).

[00373] A. Ex vivo treatment

[00374] In some embodiments, cells are removed from the subject, injected with the scDNA vector, and returned to the subject. Methods for extracting cells from a subject's body for ex vivo treatment, followed by introduction back into the subject's body, are known in the art (see, for example, US Pat. No. 5,399,346, the contents of which are fully incorporated herein). Alternatively, the scDNA vector is introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and said cells are administered to a subject in need thereof.

[00375] Cells transduced with the ccDNA vector are preferably administered to a subject in a "therapeutically effective amount" in combination with a pharmaceutical carrier. Those skilled in the art will appreciate that the therapeutic effects need not be complete or provide a cure, as long as the subject receives some benefit.

[00376] In some embodiments, said scDNA vector can encode a transgene (sometimes referred to as a heterologous nucleotide sequence) that is any polypeptide that is desired to be produced in a cell in vitro, ex vivo, or in vivo. For example, in contrast to the use of said ccDNA vectors in a treatment method as described herein, in some embodiments, said ccDNA vectors can be introduced into cultured cells and the expressed gene product isolated from them, for example, to produce antigens or vaccines.

[00377] These ccDNA vectors can be used in both veterinary and human medicine. Suitable subjects for the use of ex vivo gene delivery methods as described above include both birds (e.g., chicken, ducks, geese, quails, turkey, and pheasant) and mammals (e.g., humans, cattle, sheep, goats, horses, felines, canines, and lagomorphs), with mammals being preferred. Human subjects are most preferred. Human subjects include neonates, infants, young adults and adults.

[00378] One aspect of the technology described herein relates to a method for delivering a transgene into a cell. Typically, in in vitro methods, said scDNA vector can be introduced into a cell using methods as described herein, as well as other methods known in the art. ZcDNA vectors as described herein are preferably introduced into the cell in a biologically effective amount. When an scDNA vector is administered to a cell in vivo (eg, a subject), a biologically effective amount of said scDNA vector is an amount sufficient to allow transduction and expression of said transgene in the target cell.

[00379] B. Dose Ranges

[00380] In vivo and/or in vitro assays may optionally be performed to facilitate identification of optimal dosage ranges for use. The precise dosage to be used in the formulation also depends on the route of administration and the severity of the condition, and should be determined based on the judgment of one of skill in the art and in the circumstances of each subject. Effective doses can be extrapolated from dose-response curves obtained in vitro or in test animal models.

[00381] The ZcDNA vector is administered in sufficient amounts to transfect cells of the desired tissue and to ensure sufficient levels of gene transfer and expression without excessive adverse events. Standard and pharmaceutically acceptable routes of administration include, but are not limited to, those described above in the "Introduction" section, such as direct delivery to a selected organ (for example, intraportal delivery to the liver), oral, inhalation delivery (including intranasal and intratracheal), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral delivery; and other routes for parenteral administration. Routes of administration can be combined if desired.

[00382] The dosage amount of the scDNA vector required to achieve a particular "therapeutic effect" varies depending on a number of factors, including, but not limited to: the route of administration of the nucleic acid, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of said gene or genes, RNA product or products, or the resulting expressed protein or proteins. A person skilled in the art can easily determine the dosage range of the zcDNA vector for treating a patient with a particular disease or disorder based on the above factors as well as other factors well known in the art.

[00383] The dosage regimen may be adjusted to provide the optimal therapeutic response. For example, the oligonucleotide may be administered multiple times, eg, multiple doses may be administered daily, or the dose may be proportionally reduced to suit the therapeutic need. One of skill in the art will readily be able to determine suitable doses and administration schedules for the oligonucleotides in question, both for administration to cells and for administration to subjects.

[00384] A "therapeutically effective dose" is a relatively broad range that can be determined in clinical trials and depends on the particular application (neural cells require very small amounts, while systemic injections require significant amounts). For example, for direct in vivo injection into the skeletal or cardiac muscle of a human subject, a therapeutically effective dose would be about 1 μg-100 g of the scDNA vector. Where exosomes or microparticles are used to deliver the ccDNA vector, a therapeutically effective dose can be determined experimentally, but it is expected that 1 μg to about 100 g of vector will be delivered.

[00385] The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of suitable dosing and treatment regimens for the use of the particular compositions described herein in various treatment regimens.

[00386] For in vitro transfection, an effective amount of ccDNA vector to deliver into cells (1 x 10 ⁶ cells) is about 0.1-100 μg ccDNA vector a, preferably 1 to 20 μg, more preferably 1 to 15 μg, or 8 to 10 μg. Larger ccDNA vectors require higher doses. If exosomes or microparticles are used, an effective in vitro dose can be determined experimentally, but should be expected to deliver the same amount of scDNA vector in total.

[00387] Treatment may include the administration of a single dose or multiple doses. In some embodiments, more than one dose may be administered to a subject; in fact, multiple doses may be administered as needed, since the ccDNA vector does not elicit an immune response against the capsid in the host due to the absence of a viral capsid. Accordingly, a person skilled in the art can readily determine the appropriate number of doses. The number of doses administered may, for example, be in the order of 1-100, preferably 2-20 doses.

[00388] Without wishing to be bound by any particular theory, the lack of a typical antiviral immune response elicited by administration of an scDNA vector as described herein (i.e. without capsid components) allows said scDNA vector to be repeatedly administered to a host. In some embodiments, the number of administrations of the heterologous nucleic acid to a subject is 2 to 10 times (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). In some embodiments, the scDNA vector is delivered to a subject more than 10 times.

[00389] In some embodiments, the dose of the scDNA vector is administered to the subject no more than once per calendar day (eg, a 24-hour period). In some embodiments, the dose of the ccDNA vector is administered to the subject no more than once every 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, the dose of the scDNA vector is administered to the subject no more than once per calendar week (eg, 7 calendar days). In some embodiments, the dose of the scDNA vector is administered to the subject no more than once every two weeks (eg, once every two calendar weeks). In some embodiments, the dose of the scDNA vector is administered to the subject no more than once per calendar month (eg, once every 30 calendar days). In some embodiments, a dose of the scDNA vector is administered to a subject no more than once every six calendar months. In some embodiments, a dose of the scDNA vector is administered to a subject no more than once per calendar year (eg, 365 days or 366 days in a leap year).

[00390] C. Unit dosage forms

[00391] In some embodiments, these pharmaceutical compositions may conveniently be presented in unit dosage form. The unit dosage form is typically adapted to one or more specific routes of administration of the pharmaceutical composition. In some embodiments, said unit dosage form is adapted for administration by inhalation. In some embodiments, said unit dosage form is adapted for administration via a vaporizer. In some embodiments, said unit dosage form is adapted for administration via a nebulizer. In some embodiments, said unit dosage form is adapted for administration with an aerosol generator. In some embodiments, said unit dosage form is adapted for oral administration, for buccal administration, or for sublingual administration. In some embodiments, said unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments, said unit dosage form is adapted for intrathecal or intracerebroventricular administration. In some embodiments, said pharmaceutical composition is formulated for topical administration. The amount of active ingredient which can be combined with the carrier material to form a unit dosage form is usually that amount of said compound which provides a therapeutic effect.

XII. Various applications

[00392] The compositions and scDNA vectors of the present invention can be used to deliver a transgene for a variety of purposes as described above. In some embodiments, said transgene encodes a protein or functional RNA for use in scientific research, for example, to create a somatic transgenic animal model carrying said transgene, for example, to study the function of said transgenic product. According to another example, said transgene encodes a protein or functional RNA for use in creating an animal model of a disease.

[00393] In some embodiments, said transgene encodes one or more peptides, polypeptides, or proteins that are suitable for treating, alleviating, or preventing disease states in a mammalian subject. The specified transgene can be transferred into the patient's body (eg, expressed in the patient) in sufficient quantities to treat diseases associated with reduced expression, lack of expression or gene dysfunction.

[00394] In some embodiments, scDNA vectors are provided for use in diagnostic and screening methods by which a transgene is transiently or stably expressed in a cell culture system, or alternatively in a transgenic animal model.

[00395] In another aspect, the technology described herein provides a method for transducing a population of mammalian cells. In a general and broad sense, said method includes at least the step of introducing into one or more cells in a population a composition that contains an effective amount of one or more scDNA as described herein.

[00396] In addition, according to the present invention, compositions, as well as therapeutic and / or diagnostic kits, are provided that include one or more of the described ccDNA vectors or ccDNA compositions, formulated with one or more additional ingredients, or prepared along with one or more instructions for their use.

[00397] The cells for administration of the ccDNA vector as described herein may be of any type, including, but not limited to, nerve cells (including cells of the peripheral and central nervous system, in particular brain cells), lung cells, retinal cells, epithelial cells (for example, epithelial cells of the gastrointestinal tract and respiratory tract), muscle cells, dendritic cells, pancreatic cells (including islet cells), liver cells, cells myocardium, bone cells (eg, bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, sex germ cells, and the like. Alternatively, the cell may be any progenitor cell. In a further embodiment, the cell may be a stem cell (eg, neural stem cell, liver stem cell). In yet another embodiment, the cell may be a cancer or tumor cell. In addition, said cells may be from any species as indicated above.

[00398] In some embodiments, the present invention may be described by any of the following:

1A. scDNA vector containing:

an expression cassette containing a cis regulatory element, wherein said cis regulatory element is selected from the group consisting of a post-transcriptional regulatory element and a BGH poly(A) signal;

A wild-type ITR at the start (5' end) of the expression cassette, wherein said wild-type ITR contains a polynucleotide from SEQ ID NO: 51; And

a modified ITR at the end (3' end) of the expression cassette, wherein said modified ITR contains a polynucleotide from SEQ ID NO: 2,

while the specified DNA vector is devoid of specific prokaryotic methylation and is not enclosed in the AAV capsid protein.

2A. The DNA vector according to 1A, wherein said DNA vector has a linear and continuous structure.

3A. The DNA vector according to any one of items 1A-2A, wherein said post-transcriptional regulatory element comprises a WHP post-transcriptional regulatory element (WPRE).

4A. The DNA vector according to any one of paragraphs 1A-3A, wherein said expression cassette further comprises a cloning site.

5A. The DNA vector according to any one of paragraphs 1A-4A, wherein said expression cassette contains a promoter selected from the group consisting of the CAG promoter, the AAT promoter, the LP1 promoter and the EF1a promoter.

6A. The DNA vector according to item 1A, wherein said expression cassette contains polynucleotides from SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

7A. The DNA vector according to any one of paragraphs 1A-6A, wherein said expression cassette further comprises a cloning site and an exogenous sequence inserted into the cloning site.

8A. DNA vector according to item 7A, where the specified exogenous sequence contains at least 2000 nucleotides.

9A. DNA vector according to item 7A, where the specified exogenous sequence encodes a protein.

10A. DNA vector according to item 7A, where the specified exogenous sequence encodes a reporter protein.

11A. A cell containing a DNA vector according to any one of paragraphs 1A-10A.

12A. A cell according to item 11A further comprising a replication protein selected from the group consisting of: AAV Rep 78, AAV Rep 68, AAV Rep52 and AAV Rep 40.

13A. The cell according to item 12A, characterized in that said replication protein encodes a helper virus.

14A. A cell according to any one of paragraphs 11A-13A, characterized in that said cell does not contain a gene encoding an AAV capsid protein.

15A. A pharmacologically active ingredient containing a DNA vector according to any one of paragraphs 1A-10A and, optionally, an excipient.

16A. A method for delivering an exogenous sequence into a cell, comprising the step of introducing a DNA vector according to any one of paragraphs 1A-10A into said cell.

17A. The method of claim 16A, wherein said step of introducing the vector DNA comprises hydrodynamic injection.

18A. A method for obtaining a DNA vector, including the following steps:

introduction into the cell of a nucleic acid construct or a virus containing (containing):

an expression cassette containing a cis regulatory element, wherein said cis regulatory element is selected from the group consisting of a post-transcriptional regulatory element and a BGH poly(A) signal;

A wild-type ITR at the start (5' end) of the expression cassette, wherein said wild-type ITR contains a polynucleotide from SEQ ID NO: 51; And

a modified ITR at the end (3' end) of the expression cassette, wherein said modified ITR contains a polynucleotide from SEQ ID NO: 2,

wherein said cell does not contain the AAV capsid protein; And

collecting said DNA vector derived from said nucleic acid construct or said virus,

while said DNA vector is devoid of specific prokaryotic methylation.

19A. The method of claim 18A, wherein said DNA vector has a linear and continuous structure.

20A. The method of any one of paragraphs 18A-19A, wherein said post-transcriptional regulatory element comprises a WHP post-transcriptional regulatory element (WPRE).

21A. The method of any one of paragraphs 18A-20A, wherein said expression cassette further comprises a promoter selected from the group consisting of a CAG promoter, an AAT promoter, an LP1 promoter, and an EF1a promoter.

22A. The method of claim 18, wherein said expression cassette contains polynucleotides from SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

23A. The method of any one of items 18A-22A, wherein said expression cassette further comprises an exogenous sequence.

24A. The method according to item 23A, where the specified exogenous sequence contains at least 2000 nucleotides.

25A. The method of claim 23A, wherein said exogenous sequence encodes a protein.

26A. The method of claim 23A, wherein said exogenous sequence encodes a reporter protein.

27A. The method of any one of items 18A-26A, wherein said cell is an insect cell.

28A. DNA vector obtained by the method according to any one of paragraphs 18-27.

29A. A cell for obtaining a DNA vector, containing:

the first polynucleotide containing:

an expression cassette containing a cis regulatory element, wherein said cis regulatory element is selected from the group consisting of a post-transcriptional regulatory element and a BGH poly(A) signal;

A wild-type ITR at the start (5' end) of the expression cassette, wherein said wild-type ITR contains a polynucleotide from SEQ ID NO: 51; And

a modified ITR at the end (3' end) of the expression cassette, wherein said modified ITR contains a polynucleotide from SEQ ID NO: 2; And

a second polynucleotide encoding a replication protein selected from the group consisting of AAV78, AAV52, AAV Rep68 and AAV Rep 40,

wherein said cell does not contain the gene encoding the AAV capsid protein.

30A. The cell according to item 29A, characterized in that said post-transcriptional regulatory element contains a post-transcriptional regulatory element WHP (WPRE).

31A. A cell according to any one of paragraphs 29A-30A, characterized in that said cell is an insect cell.

32A. A DNA vector obtained from a cell according to any one of paragraphs 29A-31A by replication of the specified first polynucleotide.

33A. A polynucleotide for obtaining a DNA vector, containing:

an expression cassette containing a cis regulatory element, wherein said cis regulatory element is selected from the group consisting of a post-transcriptional regulatory element and a BGH poly(A) signal;

A wild-type ITR at the start (5' end) of the expression cassette, wherein said wild-type ITR contains a polynucleotide from SEQ ID NO: 51; And

a modified ITR at the end (3' end) of the expression cassette, wherein said modified ITR contains a polynucleotide from SEQ ID NO: 2.

34A. The polynucleotide of item 33A, wherein said post-transcriptional regulatory element comprises a WHP post-transcriptional regulatory element (WPRE).

35A. A polynucleotide according to any one of paragraphs 33A-34A, wherein said polynucleotide is in a plasmid, in a bacmid, or in a baculovirus.

36A. A polynucleotide according to any one of paragraphs 33A-35A, further comprising an exogenous sequence.

37A. The polynucleotide according to item 36A, where the specified exogenous sequence contains at least 2000 nucleotides.

38A. The polynucleotide of item 36A, wherein said exogenous sequence encodes a protein.

39A. The polynucleotide of item 36A, wherein said exogenous sequence encodes a reporter

40A. A DNA vector obtained by replicating a polynucleotide according to any one of paragraphs 33A-39A with a replication protein selected from the group consisting of AAV78, AAV52, AAV Rep68 and AAV Rep 40, where said DNA vector has a linear and continuous structure, is devoid of specific prokaryotic methylation and is not enclosed in an AAV capsid protein.

41 A. DNA vector containing: expression cassette;

modified ITR at the beginning (at the 5' end)

expression cassette, where said modified

ITR contains a polynucleotide from SEQ ID NO: 52; And

A wild-type ITR at the end (3' end) of the expression cassette, wherein said wild-type ITR contains a polynucleotide from SEQ ID NO:1,

at the same time, the DNA vector is devoid of specific prokaryotic methylation and is not enclosed in the AAV capsid protein.

42A. The DNA vector of item 41A, wherein said expression cassette contains a cis regulatory element, wherein said cis regulatory element is selected from the group consisting of a post-transcriptional response element and a poly(A) signal.

43A. The DNA vector of item 42A, wherein said post-transcriptional response element comprises a WHP post-transcriptional response element (WPRE).

44A. The DNA vector of any one of paragraphs 42A-43A, wherein said poly(A) signal comprises a BGH poly(A) signal.

45A. The DNA vector of any one of paragraphs 41A-44A, wherein said expression cassette contains a promoter selected from the group consisting of a CAG promoter, an AAT promoter, an LP1 promoter, and an EF1a promoter.

46A. The DNA vector of item 41A, wherein said expression cassette contains polynucleotides from SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

47A. The DNA vector according to any one of paragraphs 41A-46A, where the specified expression cassette contains an exogenous sequence.

48A. DNA vector according to item 47A, where the specified exogenous sequence contains at least 2000 nucleotides.

49A. DNA vector according to item 47A, where the specified exogenous sequence encodes a protein.

50A. DNA vector according to item 47A, where the specified exogenous sequence encodes a reporter protein.

51A. A cell containing a DNA vector according to any one of items 41A-50A.

52A. The cell of item 52A further comprising a replication protein selected from the group consisting of: AAV Rep 78, AAV Rep 68, AAV Rep52 and AAV Rep 40.

53A. The cell according to item 51A, characterized in that said replication protein encodes a helper virus.

54A. A cell according to any one of paragraphs 51-53, characterized in that said cell does not contain a gene encoding an AAV capsid protein.

55A. A pharmacologically active ingredient comprising: a DNA vector according to any one of paragraphs 1A-10A; and optionally an excipient.

56A. A method for delivering an exogenous sequence into a cell, comprising the step of: introducing said non-encased DNA vector according to any one of paragraphs 1A-10A into said cell.

57A. The method of claim 16A, wherein said step of introducing the vector DNA comprises hydrodynamic injection.

58A. A method for obtaining a DNA vector, including the following steps:

introduction into a cell of a nucleic acid or virus construct containing or containing:

expression cassette;

a modified ITR at the beginning (at the 5' end) of the expression cassette, wherein said modified ITR contains a polynucleotide from SEQ ID NO: 52; And

ITR wild-type at the end (at the 3' end)

expression cassette, where the specified

The wild-type ITR contains a polynucleotide from SEQ ID NO: 1,

wherein said cell does not contain the AAV capsid protein; And

collecting said DNA vector derived from said nucleic acid construct or said virus,

while said DNA vector is devoid of specific prokaryotic methylation and is not enclosed in the AAV capsid protein.

59A. The method of claim 18A, wherein said expression cassette contains a cis regulatory element, wherein said cis regulatory element is selected from the group consisting of a post-transcriptional response element and a poly(A) signal.

60A. The method of claim 59A, wherein said post-transcriptional response element comprises a WHP post-transcriptional response element (WPRE).

61A. The method of any one of 59A-60A, wherein said poly(A) signal comprises a BGH poly(A) signal.

62A. The method of any one of paragraphs 18A-61A, wherein said expression cassette contains a promoter selected from the group consisting of a CAG promoter, an AAT promoter, an LP1 promoter, and an EF1a promoter.

63A. The method of claim 18A, wherein said expression cassette contains polynucleotides from SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

64A. The method of any one of items 18A-63A, wherein said expression cassette further comprises an exogenous sequence.

65A. The method of claim 23A, wherein said exogenous sequence contains at least 2000 nucleotides.

66A. The method of claim 23A, wherein said exogenous sequence encodes a protein.

67A. The method of claim 23A, wherein said exogenous sequence encodes a reporter protein.

68A. The method of any one of items 18A-26A, wherein said cell is an insect cell.

69A. A DNA vector obtained by the method of any one of paragraphs 18A-27A.

70A. The DNA vector of item 69A, wherein said DNA vector has a linear and continuous structure.

71 A. A cell for obtaining a DNA vector, containing: the first polynucleotide containing: an expression cassette;

modified ITR at the beginning (at the 5' end)

expression cassette, where said modified

ITR contains a polynucleotide from SEQ ID NO: 52; And

A wild-type ITR at the end (3' end) of the expression cassette, wherein said wild-type ITR contains a polynucleotide from SEQ ID NO: 1; And

a second polynucleotide encoding a replication protein selected from the group consisting of AAV78, AAV52, AAV Rep68 and AAV Rep 40,

wherein said cell does not contain the gene encoding the AAV capsid protein.

72A. A cell according to claim 71A, characterized in that said cell is an insect cell.

73A. A vector DNA obtained from a cell according to any one of paragraphs 29A-50A by replicating said first polynucleotide.

74A. A polynucleotide for obtaining a DNA vector, containing:

expression cassette;

modified ITR at the beginning (at the 5' end)

expression cassette, where said modified

ITR contains a polynucleotide from SEQ ID NO: 52; And

A wild-type ITR at the end (3' end) of the expression cassette, wherein said wild-type ITR contains a polynucleotide from SEQ ID NO: 1.

75 A. The polynucleotide of 74A, wherein said polynucleotide is in a plasmid, bacmid, or baculovirus.

76A. A polynucleotide according to any one of paragraphs 74A-75A, further comprising an exogenous sequence.

77A. The polynucleotide according to item 74A, where the specified exogenous sequence contains at least 2000 nucleotides.

78A. The polynucleotide of item 74A, wherein said exogenous sequence encodes a protein.

79A. The polynucleotide of item 74A, wherein said exogenous sequence encodes a reporter

80A. A DNA vector obtained by replicating a polynucleotide according to any one of paragraphs 33-39 with a replication protein selected from the group consisting of AAV78, AAV52, AAV Rep68 and AAV Rep 40, wherein said DNA vector lacks specific prokaryotic methylation and is not enclosed in an AAV capsid protein.

81A. The DNA vector of item 80A, wherein said DNA vector is produced in an insect cell.

82A. scDNA vector derived from a plasmid containing the mutated AAV ITR sequence from any of Tables 2-6 or Tables 7-10A or 10B.

In some embodiments, the present invention may be described by any of the following:

1 B. DNA vector obtained using a method including:

(a) transfection of the first population of insect cells with the first recombinant bacmid obtained by transposing the first DNA plasmid construct into a baculovirus expression vector, wherein the first DNA plasmid construct contains sequentially in the following order in the same direction: the first site of the replicative protein (RPS-1), a promoter operably linked to the reporter sequence of ORF polynucleotides, a post-translation and termination signal; and a second replicative protein site (RPS-2), wherein RPS-1 and RPS-2 independently contain intramolecular duplexes and are covalently linked, and RPS-1 contains one or more deletions, substitutions, or one or more polynucleotide base pair truncations relative to RPS-2; and then extracting the first population of insect cells injected with baculovirus (BIIC-1);

(b) transfecting a second population of insect cells with a second recombinant bacmid obtained by transposing a second DNA plasmid construct into a second baculovirus expression vector, the second DNA plasmid construct encoding a protein that binds to at least one of RPS-1 and RPS-2, and then recovering a second population of insect cells with injected baculovirus (BIIC-2);

(c) combining BIIC-1 and BIIC-2 with a third population of insect cells in relative amounts to provide substantially the same multiplicity of infection (MOI), followed by incubation of said third population of insect cells, BIIC-1 and BIIC-2 under cell growth conditions such that the protein encoded by the second DNA plasmid construct reacts with the RPS-1 and/or RPS-2 sites to induce replication of the DNA vector encoded by the first DNA plasmas idoy, until the insect cell reaches an observed diameter of approximately 15-20 micrometers and cell viability of approximately 50-80%, followed by extraction of DNA from insect cell pellets by centrifugation;

(d) extracting DNA from insect cell pellets to obtain produced DNA from insect cell pellets;

(e) identifying DNA derived from insect cell pellets on a native gel in both the primary lane and the secondary lane at a relative distance apart indicative of material in the second lane with approximately twice the mass of the primary lane material;

(f) confirming the presence of said vector DNA in insect cell pellet-derived DNA on a denaturing gel by shifting both the primary and secondary bands up towards the lower mass region on the gel after restriction endonuclease digestion of the DNA; And

(g) isolating the remaining DNA vector obtained from insect cell pellets to obtain a highly stable DNA vector.

2B. The DNA vector of item 1B, wherein RPS-1 and RPS-2 are distinct and independently selected from the sequence of DNA polynucleotide pairs of SEQ ID NO: SEQ ID NO: 1 and SEQ ID NO: 52; or SEQ ID NO: 2 and SEQ ID NO: 51; or SEQ ID NO: 101 and SEQ ID NO: 102; or SEQ ID NO: 103 and SEQ ID NO: 104; or SEQ ID NO: 105 and SEQ ID NO: 106; or SEQ ID NO: 107 and SEQ ID NO: 108; or SEQ ID NO: 109 and SEQ ID NO: 110; or SEQ ID NO: 111 and SEQ ID NO: 112; or SEQ ID NO: 113 or SEQ ID NO: 114; and SEQ ID NO: 115 or SEQ ID NO: 116.

In some embodiments, the present invention may be described by any of the following:

1C. A capsidless AAV vector (cfAAV) containing:

an expression cassette containing a cis-regulatory element, a promoter and an exogenous sequence; And

two self-complementary sequences flanking said expression cassette,

wherein said cfAAV vector is not associated with a capsid protein and is devoid of specific prokaryotic methylation.

2C. CfAAV vector according to 1C, wherein said cis-regulatory elements are selected from the group consisting of a ribo switch, an insulator, an siRNA-regulated element, and a post-transcriptional regulatory element.

3C. A CfAAV vector according to any one of 1C-2C, wherein said DNA vector is CELiD.

4C. A CfAAV vector according to any one of 1C-3C, wherein said two self-complementary sequences are AAV2 ITR sequences.

5C. A CfAAV vector according to any one of 1C-4C, wherein said exogenous sequence contains an internal ribosome entry site (IRES) and a 2A element.

6C. The CfAAV vector of item 5C, wherein said expression cassette contains a sequence encoding more than one protein.

7C. A CfAAV vector according to any one of 1C-6C, wherein said expression cassette contains more than 4,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides.

8C. Pharmacological composition containing the cfAAV vector according to any one of paragraphs 1C-7C.

9C. Expression construct containing:

an expression cassette containing a cis-regulatory element, a promoter and an exogenous sequence; and two inverted terminal repeat (ITR) sequences flanking said expression cassette,

wherein said expression construct does not contain an open reading frame encoding a capsid protein.

10C. The expression construct of claim 9C, wherein said cis-regulatory elements are selected from the group consisting of a riboswitch, an insulator, an siRNA-regulated element, and a post-transcriptional regulatory element.

11C. An expression cassette according to any one of paragraphs 9C-10C, characterized in that said exogenous sequence contains an internal ribosome entry site (IRES) and a 2A element.

12C. An expression cassette according to claim 11, characterized in that said expression cassette contains a sequence encoding more than one protein.

13C. An expression construct according to any one of paragraphs 9C-12C, characterized in that said expression construct is in a plasmid, in a bacmid, or in a baculovirus.

14C. A method for obtaining a cfAAV vector, including:

introducing an expression construct according to any one of paragraphs 9C-13C into a cell; And

collecting said cfAAV vector obtained by replicating the expression construct.

15C. The method of claim 14C, further comprising the step of repeatedly replicating the expression construct prior to introducing said expression construct into the cell.

16C. The method of claim 15C, wherein said expression construct is in a plasmid and the replication step is carried out in E. coli.

17C. The method of claim 16C, further comprising the step of transferring the expression construct from the plasmid to the bacmid prior to introducing said expression construct into the cell.

18C. The method of claim 17C, further comprising the step of transferring the expression construct from bacmid to baculovirus prior to introducing said expression construct into the cell.

19C. The method of any one of items 14C-18C, further comprising the step of introducing the Rep protein into the cell.

20C. The method of any one of 14C-19C, wherein said cell is an insect cell.

21C. A CfAAV vector obtained using the method of any one of 14C-20C.

22C.CfAAV vector according to 21C, wherein said DNA vector is CELiD.

In some embodiments, the present invention may be described by any of the following:

1D. A capsid non-viral DNA vector derived from vector polynucleotide a, wherein said vector polynucleotide encodes a heterologous nucleic acid functionally located between the DNA polynucleotide sequences of the first and second inverted terminal repeats (ITRs) of AAV2, wherein at least one of the ITRs contains at least one polynucleotide division, insertion, or substitution relative to the corresponding wild-type AAV2 ITR of SEQ ID NO: 1 or SEQ ID NO: 51 to induce replication of said DNA vector in an insect cell in the presence of Rep protein, wherein said vector DNA can be obtained by a method comprising the following steps:

a. incubating a population of insect cells carrying said vector polynucleotide that does not contain viral capsid coding sequences in the presence of a Rep protein under conditions effective to and for a time sufficient to induce the production of capsid-free non-viral DNA in the insect cells, said insect cells not containing viral capsid coding sequences; And

b. collection and isolation of non-capsid non-viral DNA from insect cells;

moreover, the presence of non-capsid non-viral DNA isolated from insect cells can be confirmed by cleaving DNA isolated from insect cells with a restriction enzyme having a single recognition site on a DNA vector, and analyzing said cleaved DNA material on a non-denaturing gel to confirm the presence of characteristic linear and continuous DNA bands that differ from linear and discontinuous DNA bands.

2D. Capsid-free non-viral DNA vector according to item 1, wherein said mutated ITR is selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO: 52.

3D. Capsid non-viral DNA vector according to paragraph 1, where the specified vector polynucleotide contains a pair of ITR selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 52; and SEQ ID NO: 2 and SEQ ID NO: 51.

4D. A non-capsid non-viral DNA vector derived from a vector polynucleotide, wherein said vector polynucleotide encodes a heterologous nucleic acid functionally located between two different inverted terminal repeat (ITR) sequences, wherein at least one of the ITRs is a functional ITR containing a functional terminal resolution site and a Rep binding site, and one of the ITRs contains a deletion, insertion, or substitution relative to said functional ITR; the presence of the Rep protein induces replication of said vector polynucleotide and production of a vector DNA in the insect cell, said vector DNA can be obtained by a method comprising the following steps:

a. incubating a population of insect cells carrying said vector polynucleotide that does not contain viral capsid coding sequences in the presence of a Rep protein under conditions effective to, and for a time sufficient to induce, production of a capsid-free, non-viral DNA vector in insect cells, characterized in that the capsid-free, non-viral DNA produced is not contained in said insect cells; And

b. collection and isolation of non-capsid non-viral DNA from insect cells;

moreover, the presence of non-capsid non-viral DNA isolated from insect cells can be confirmed by cleaving DNA isolated from insect cells with a restriction enzyme having a single recognition site on a DNA vector, and analyzing said cleaved DNA material on a non-denaturing gel to confirm the presence of characteristic linear and continuous DNA bands that differ from linear and discontinuous DNA bands.

In some embodiments, the present invention may be described by any of the following:

1E. A vector containing (i) a non-viral non-capsid DNA vector with covalently closed ends (ccDNA vector), where said ccDNA vector contains a heterologous nucleic acid sequence encoding a transgene functionally located between two different AAV inverted terminal repeat (ITR) sequences, wherein one of the ITRs contains a functional AAV terminal resolution site and a Rep binding site, and one of the ITRs contains a deletion, an insertion or a substitution relative to another ITR (modified ITR), wherein said vector is not enclosed in a viral capsid.

2E. The zcDNA vector according to claim 1, wherein said zcDNA vector, when cleaved with a restriction enzyme having a single recognition site on said zcDNA vector, exhibits the presence of characteristic linear and continuous DNA bands that differ from linear and discontinuous control DNA bands when analyzed on a non-denaturing gel.

3E. The zcDNA vector of item 1E, wherein said modified ITR is from a different serotype.

4E. The zcDNA vector of item 3E, wherein said modified ITR is not a wild-type ITR.

5E. The zcDNA vector of item 1E, wherein the deletion, insertion, or replacement occurs in at least one of the ITRs selected from the group consisting of A, A', B, B', C, C, and D.

6E. The 3cDNA vector of item 5E, wherein the deletion, insertion, or substitution results in the deletion of one of the loops formed by regions A, A', B, B'C, or C.

7E. The zcDNA vector of item 1E, wherein said modification corresponds to a change in the modified AAV2 ITR selected from the group consisting of SEQ ID NOs: 101-498 and 499 or 545-547.

8E. The zcDNA vector of item 1E, wherein said modification corresponds to a change in the modified AAV2 ITR selected from the group consisting of SEQ ID NO. 2, 52, 63 and 64.

9E. ZcDNA vector according to items 1E-8E, where the specified vector is in the nanocarrier.

10E. The ZcDNA vector according to item 9E, wherein said nanocarrier contains a lipid nanoparticle (LNP).

11E. A method including:

obtaining non-viral non-capsid DNA with covalently closed ends (ccDNA) using vector polynucleotide a, where said vector polynucleotide encodes a heterologous nucleic acid functionally located between two different sequences of inverted terminal repeats (ITRs), wherein at least one of the ITRs contains a functional terminal resolution site and a Rep binding site, and one of the ITRs contains a deletion, insertion, or substitution relative to another ITR; the presence of the Rep protein induces replication of the vector polynucleotide and production of a vector DNA in the insect cell, said vector DNA can be obtained by a method comprising the following steps:

A. incubating a population of insect cells carrying said vector polynucleotide that does not contain viral capsid coding sequences in the presence of a Rep protein under conditions effective to, and for a time sufficient to induce, production of a capsid-free, non-viral DNA vector in insect cells, characterized in that the capsid-free, non-viral DNA produced is not contained in said insect cells; And

b. collection and isolation of non-capsid non-viral DNA from insect cells;

moreover, the presence of non-capsid non-viral DNA isolated from insect cells can be confirmed by cleaving DNA isolated from insect cells with a restriction enzyme having a single recognition site on a DNA vector, and analyzing said cleaved DNA material on a non-denaturing gel to confirm the presence of characteristic linear and continuous DNA bands that differ from linear and discontinuous DNA bands.

12E. A capsid non-viral DNA vector derived from a vector polynucleotide, wherein said vector encodes a heterologous gene operably located between DNA polynucleotide sequences of the first and second inverted terminal repeats (ITRs) of AAV2, wherein at least one of the ITRs contains at least one polynucleotide deletion, insertion, or substitution relative to the corresponding wild-type AAV2 ITR from SEQ ID NO: 1 or SEQ ID NO: 51 to induce replication of said A DNA vector in an insect cell in the presence of a Rep protein, said DNA vector being obtained by a method comprising the following steps:

(a) incubating a population of insect cells carrying said vector polynucleotide, which does not contain viral capsid coding sequences, in the presence of a Rep protein under conditions effective to and for a time sufficient to induce the production of capsidless non-viral DNA in the insect cells, said insect cells not containing viral capsid coding sequences; And

(b) collecting and isolating capsid-free non-viral DNA from insect cells;

moreover, the presence of non-capsid non-viral DNA isolated from insect cells can be confirmed by cleaving DNA isolated from insect cells with a restriction enzyme having a single recognition site on a DNA vector, and analyzing said cleaved DNA material on a non-denaturing gel to confirm the presence of characteristic linear and continuous DNA bands that differ from linear and discontinuous DNA bands.

13E. The non-capsid non-viral DNA vector according to item 12E, wherein said modified ITR is selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO: 52.

14E. Capsid non-viral DNA vector according to paragraph 12E, where the specified vector polynucleotide contains a pair of ITR selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 52; SEQ ID NO: 2 and SEQ ID NO: 51.

15E. A capsid non-viral DNA vector derived from vector polynucleotide a, wherein said vector polynucleotide encodes a heterologous gene operably located between two different inverted terminal repeat (ITR) sequences, wherein at least one of the ITRs contains a functional terminal resolution site and a Rep binding site, and one of the ITRs contains a deletion, insertion, or substitution, and the presence of the Rep protein induces replication of the vector polynucleotide and production of the vector DNA in insect cell, wherein said DNA vector can be obtained by a method comprising the following steps:

(a) incubating a population of insect cells carrying said vector polynucleotide that does not contain viral capsid coding sequences in the presence of a Rep protein under conditions effective to, and for a time sufficient to induce, production of a capsid-free, non-viral DNA vector in insect cells, characterized in that the capsid-free, non-viral DNA produced is not contained in said insect cells; And

(b) collecting and isolating capsid-free non-viral DNA from insect cells;

moreover, the presence of non-capsid non-viral DNA isolated from insect cells can be confirmed by cleaving DNA isolated from insect cells with a restriction enzyme having a single recognition site on a DNA vector, and analyzing said cleaved DNA material on a non-denaturing gel to confirm the presence of characteristic linear and continuous DNA bands that differ from linear and discontinuous DNA bands.

17E. A capsid non-viral DNA vector according to item 15E, wherein one ITR is the wild-type AAV ITR.

18E. A method of treating a disease in a subject, said method comprising:

co-administering to a subject in need thereof a composition comprising (i) a non-viral capsidless DNA vector with covalently closed ends (ccDNA vector), wherein said ccDNA vector contains a heterologous nucleic acid sequence encoding a transgene operably located between two different AAV inverted terminal repeat (ITR) sequences, wherein one of the ITRs contains a functional AAV terminal clearance site and a Rep binding site, and one of the ITRs contains a division, insertion, or substitution relative to the other ITR.

19E. The method of claim 17E, wherein said scDNA vector is administered in combination with a pharmaceutically acceptable carrier.

20E. A method for delivering a therapeutic agent to a subject, said method comprising:

administering to the subject a composition containing the scDNA vector according to items 1E-10E and 12E-16E.

EXAMPLES

[00399] The following examples are intended to be illustrative and not restrictive.

EXAMPLE 1 Construction of ccDNA Vectors

[00400] The production of scDNA vectors using a polynucleotide template construct is described. For example, the polynucleotide template construct used to generate the scDNA vectors of the present invention may be a scDNA plasmid, scDNA bacmid, and/or scDNA baculovirus. Without being limited by theory, in a permissive host cell in the presence of, for example, Rep, a polynucleotide template construct with two ITRs and an expression construct, with at least one of the ITRs modified, replicates to produce scDNA vectors. Obtaining the scDNA vector involves two steps: first, excision (“rescue”) of the template from the matrix backbone (e.g., zcDNA plasmids, scDNA bacmids, scDNA baculovirus genome, etc.) via Rep proteins, and second, Rep-mediated replication of the excised scDNA vector.

[00401] An example of a method for obtaining scDNA vectors is provided by the preparation of an scDNA plasmid as described herein. 1A and 1B, the polynucleotide template construct of each of the zcDNA plasmids includes both a left ITR and a right mutated ITR, between which are: (i) an enhancer/promoter; (ii) a cloning site for the transgene; (iii) a post-transcriptional response element (eg, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)); and (iv) a polyadenylation signal (e.g., the bovine growth hormone (BGHpA) gene. Unique restriction endonuclease recognition sites (R1-R6) (shown in FIGS. 1A and 1B) were also introduced between all components to facilitate the introduction of new genetic components at specific sites in the construct. Sites for R3 (PmeI) GTTTAAAC (SEQ ID NO: 7) and R4 (PacI) TTAAT enzymes TAA (SEQ ID NO: 542) was inserted into the cloning site to introduce the open reading frame of the transgene These sequences were cloned into pFastBac HT B plasmid obtained from ThermoFisher Scientific.

[00402] Briefly, a series of ccDNA vectors were generated from the ccDNA plasmid constructs shown in Table 12 using the method shown in FIG. 4A-AC. Table 12 lists the corresponding polynucleotide sequence numbers for each component, including sequences with protein replication site (RPS) activity (eg, Rep binding site) at either end of the promoter operably linked to the transgene. The numbers in Table 12 refer to SEQ ID NOs herein corresponding to the sequences of each component.

[00403]

[00404] In some embodiments, a construct for producing ccDNA vectors contains a promoter that is a regulatory switch as described herein, such as an inducible promoter. Other constructs have also been used to generate ccDNA vectors, such as constructs 10, 11, 12, and 13 (see, for example, Table 14A), which contain the MND or HLCR promoter operably linked to the luciferase transgene.

[00405] Obtaining scDNA bacmid:

[00406] As shown in FIG. 4A, DH10Bac competent cells (MAX EFFICIENCY®

DH10Bac™ Competent Cells, Thermo Fisher) were transformed with either test or control plasmids following the protocol according to the manufacturer's instructions. Recombination was induced between the plasmid and the baculovirus shuttle vector in DH10Bac cells to produce recombinant scDNA bacmids. Recombinant bacmids were selected by a positive selection screen based on a blue-white screen in E. coli (marker F80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG with antibiotics to select transformants and maintain bacmids and transposase plasmids . White colonies resulting from a transposition that destroys the β-galactoside indicator gene were collected and cultured in 10 ml of medium.

[00407] Recombinant scDNA bacmids were isolated from E. coli and transfected into Sf9 or Sf21 insect cells using FugeneHD to generate infectious baculovirus. Adhesive insect cells Sf9 or Sf21 were cultured in 50 ml of medium in T25 flasks at 25°C. After 4 days, the culture medium (containing the P0 virus) was separated from the cells, filtered through a filter with a pore size of 0.45 μm, separating infectious baculovirus particles from cells or cell debris.

[00408] Optionally, the first batch of baculovirus (P0) obtained was amplified by infecting intact insect cells with Sf9 or Sf21 in medium volumes of 50 to 500 ml. Cells were maintained in suspension cultures in an incubator with an orbital shaker at 130 rpm at 25°C, monitoring the diameter and cell viability until the cells reached a diameter of 18-19 nm (from the diameter of intact cells 14-15 nm) and a density of ~4.0E+6 cells/ml. 3-8 days after infection, P1 baculovirus particles in the medium were collected after centrifugation to remove cells and debris, followed by filtration through a filter with a pore size of 0.45 μm.

[00409] The scDNA baculovirus containing test constructs was harvested and the infectivity, or titer, of the baculovirus was determined. In particular, 4×20 ml of cultures of Sf9 cells with a density of 2.5E+6 cells/ml were treated with baculovirus P1 in the following dilutions: 1/1000, 1/10,000, 1/50,000, 1/100,000, and incubated at 25-27°C. Infectivity was determined daily for 45 days in terms of cell diameter increase and cell cycle arrest, as well as changes in cell viability.

[00410] As shown in FIG. 4A, the "Rep plasmid" according to FIG. 8A was produced in the pFASTBAC™-Dual expression vector (ThermoFisher) containing both Rep78 (SEQ ID NO: 13) or Rep68 (SEQ ID NO: 12) and Rep52 (SEQ ID NO: 14) or Rep40 (SEQ ID NO: 11).

[00411] Competent DH10Bac cells (MAX EFFICIENCY® DH10Bac™ Competent Cells (Thermo Fisher)) were transformed with the indicated Rep plasmid following the protocol suggested by the manufacturer. Recombination between the Rep plasmid and the baculovirus shuttle vector was induced in DH10Bac cells to obtain recombinant bacmids ("Rep-bacmid"). ant bacmids were selected using positive selection, which included a blue-white screen for E. coli (marker F80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG. (cin, tetracycline in LB broth) Recombinant bacmids (Rep bacmids) were isolated from E. coli and these Rep bacmids were transfected into Sf9 or Sf21 insect cells to produce infectious baculovirus.

[00412] Sf9 or Sf21 insect cells were cultured in 50 ml of medium for 4 days, and infectious recombinant baculovirus ("Rep-baculovirus") was isolated from the culture. Optionally, first generation Rep-baculovirus (P0) was amplified by infecting intact insect cells with Sf9 or Sf21 and cultured in 50-500 ml of medium. 3-8 days after infection, P1 baculovirus particles in the medium were collected by separating the cells by centrifugation or filtration, or using another fractionation method. Collected Rep-baculovirus and determined the infectious activity of the baculovirus. Specifically, 4×20 ml of Sf9 cell cultures at a density of 2.5×10 ⁶ cells/ml were treated with P1 baculovirus at the following dilutions: 1/1000, 1/10000, 1/50000, 1/100000, and incubated. Infectivity was determined daily for 4-5 days in terms of cell diameter increase and cell cycle arrest, as well as changes in cell viability.

[00413] Obtaining and characterizing the scDNA vector

[00414] As shown in FIG. 4B, then a Sf9 insect cell culture medium containing either (1) a sample containing scDNA bacmid or scDNA baculovirus, or (2) Rep baculovirus, as described above, was added to fresh Sf9 cell culture (2.5E+6 cells/ml, 20 ml) at a ratio of 1:1000 and 1:10000, respectively. The cells were then cultured at 130 rpm at 25°C. 4-5 days after coinfection, the diameter and viability of the cells are detected. When the cell diameter reached 18–20 nm with a viability of ~70–80%, cell cultures were centrifuged, the medium was removed, and cell pellets were collected. The cell pellets were first resuspended in an appropriate volume of aqueous medium, either water or buffer. The ccDNA vector was isolated and purified from cells using the purification protocol from Qiagen MIDI PLUS™ (Qiagen, treatment with 0.2 mg cell pellet mass per column).

[00415] Yields for scDNA vectors produced and purified from Sf9 insect cells were initially determined based on UV absorbance at 260 nm. Yields for various scDNA vectors based on UV absorption are shown in Table 13 below.

[00416]

[00417] Evaluation of ccDNA vectors can be performed by identification using agarose gel electrophoresis under native or denaturing conditions, as illustrated in FIG. 4D, where (a) the presence of characteristic bands migrating twice as far on denaturing gels or on native gels after restriction endonuclease digestion and gel electrophoretic analysis, and (b) the presence of monomer and dimer bands (2×) on denaturing gels for undigested material are indicative of the presence of the scDNA vector.

[00418] The structures of the isolated scDNA vectors were further analyzed by digesting DNA derived from co-infected Sf9 cells (as described herein) with restriction endonucleases selected based on a) the presence of only one cut site in the scDNA vectors, and b) generating final fragments large enough to be clearly visible when fractionated on a 0.8% denaturing agarose gel ( >800 bp). As shown in FIG. 4E, the linear scatter DNA vectors and the scDNA linear and contiguous vector can be distinguished by the size of their reaction products. and 2 kb, while the non-capsid-embedded contiguous vector is expected to produce fragments of 2 kb. and 4 kb.

[00419], 1000 pp and 2000 pp). After cleavage and electrophoresis on a denaturing gel (separating two complementary strands of DNA), linear covalently open DNA will be separated into 1000 bp fragments. and 2000 bp, while a covalently closed DNA (i.e., a 3cDNA vector) will split into fragments twice as large (2000 bp and 4000 bp) because the two strands of DNA are connected, and in these circumstances are unfolded and are twice as long (although they are single stranded). In addition, when monomeric, dimeric and n-meric forms of DNA vectors are cleaved, they will all be divided into fragments of the same size due to the binding of the ends of the multimeric DNA vectors (see Fig. 4D).

[00420] FIG. 5 shows an example of a denaturing gel image with the following scDNA vectors: construct-1, construct-2, construct-3, construct-4, construct-5, construct-6, construct-7, and construct-8 (all described in Table 12 above), with (+) or without (-) endonuclease cleavage. Each scDNA vector from constructs 1-8 produced two bands (*) after reaction with endonuclease. The sizes of their fragments in two bands, determined by the size marker, are shown at the bottom of the image. Band sizes confirm that each of the scDNA vectors produced from plasmids containing construct 18 has a contiguous structure.

[00421] As used herein, "assay for identifying DNA vectors by agarose gel electrophoresis under native and denaturing gel conditions" refers to an assay for evaluating the presence of closed ends of ccDNA by performing restriction endonuclease digestion followed by electrophoretic evaluation of cleavage products. One example of such an assay is provided below, although one of ordinary skill in the art will appreciate that many variations of this example known in the art are possible. The restriction endonuclease is chosen to be an enzyme that cuts the scDNA vector of interest at one site, resulting in products approximately 1/3 x and 2/3 x the length of the DNA vector. This ensures band separation on both native and denaturing gels. Before denaturing, it is important to remove the buffer from the sample. The Qiagen PCR PCR purification kit or desalting "spin columns" such as GE HEALTHCARE ILUSTRA™ MICROSPIN™ G-25 columns are some known endonuclease cleavages in the art. Said analysis includes, for example, i) digestion of the DNA with an appropriate restriction endonuclease (or endonucleases), 2) use of, for example, a Qiagen PNR purification kit, eluting with distilled water, iii) addition of 10x denaturing solution (10x=0.5 M NaOH, 10 mM EDTA), addition of 10x dye, unbuffered, and analysis together with DNA ladders obtained by adding 10X denaturing solution in 4×, 0.8-1.0% gel previously incubated with 1 mM EDTA and 200 mM NaOH to ensure uniformity of NaOH concentration in the gel and gel chamber, and running the gel in the presence of 1× denaturing solution (50 mM NaOH, 1 mM EDTA). A person skilled in the art will understand what voltage to use for electrophoresis, depending on the size and the required time to obtain results. After electrophoresis, the gels are dried and neutralized in 1× TBE or TAE, transferred to distilled water or 1× TBE/TAE with 1× SYBR Gold. The bands can then be visualized using, for example, a Thermo Fisher stain, SYBR® Gold Nucleic Acid Gel Stain (10,000X concentrate in DMSO) and epifluorescent light (blue) or UV (312 nm).

[00422] Evaluation of the purity of the resulting scDNA vector can be carried out using any method known in the art. According to one non-limiting example of the method, the contribution of the ccDNA plasmid to the total UV uptake of the sample can be calculated by comparing the fluorescence intensity of the ccDNA vector and the standard. For example, if, based on UV absorbance, 4 μg of the ccDNA vector was loaded onto the gel and the fluorescence intensity of the ccDNA vector is equivalent to a 2 kb band. with a known mass of 1 µg, then there is 1 µg of the scDNA vector, and the scDNA vector makes up 25% of the total UV absorbing material. The intensity of the band on the gel is then plotted as a function of the calculated amount injected to which the band corresponds - for example, if the length of the entire scDNA vector is 8 kb and the excised reference band corresponds to 2 kb, the intensity of the band on the graph will be 25% of the total amount injected, which in this case will be 0.25 μg for an injected 1.0 μg. Using a dilution of the scDNA vector-carrying plasmid to build a standard curve, a regression line equation is obtained, which is then used to calculate the amount of the specified scDNA vector in the corresponding band, which can then be used to determine a percentage of the total amount of scDNA vector injected, or percent purity.

EXAMPLE 2 Production of Viral DNA in ccDNA Cells

[00423] Also, scDNA vectors were generated from constructs 11, 12, 13, and 14 shown in Table 14A. ZcDNA plasmids containing constructs 11-14 were prepared using molecular cloning techniques well known in the art. The plasmids in Table 14A were constructed with the WPRE sequence comprising SEQ ID NO: 8 followed by BGHpA comprising SEQ ID NO: 9 in the untranslated 3' region between the transgene and the right ITR.

[00424] The vector backbone for constructs 11-14 contains: (i) asymmetric ITR-MND-luciferase-wPPE-BCH-poly(A)-ITR in pFB-HTb (construct 11), (ii) ITR-MND-luciferase-wPPE-BCH-poly(A)-asymmetric ITR in pFB-HTb (construct 12), (iii) asymmetric iTR-HLCR-AAT-luc-wPRE(O BGH-poly(A-ITR in pFB-HTb (Construct 13); and ITP-HLCR-AAT-1uc-wPRE(O-BGH-poly(A)-asymmetric ITR in pFB-HTb (Construct 14); each construct contains at least one ITR that is asymmetric with respect to another ITR. These constructs also contain one or more of the following sequences: sequence w PREO (SEQ ID NO: 72) and BGH-poly(A) (SEQ ID NO: 73), or sequences at least 85%, or at least 90%, or at least 95% identical to those indicated.

[00425] Next, the production of the scDNA vector was performed according to the procedure in FIG. 4A-4C, for example, (a) obtaining recombinant scDNA bacmid DNA and transfecting an insect cell with recombinant scDNA bacmid DNA; (b) preparation of P1 stock (low titer), P2 stock (high titer), and detection of virus titer using quantitative PCR to obtain 5 ml of delivery solution containing >1E+7 plaque-forming or infectious units “PFU” per ml of BV stock, BV Stock COA. pair infection: Rep bacmid as described herein and at least one of the following constructs: construct 11, construct 12, construct 13, and construct 14. Isolation of the scDNA vector was performed using the QIAGEN Plasmid Midi Kit to obtain purified DNA material for further analysis. Table 14B and Table 14C show the yields (based on OD detection) of the scDNA vector produced from constructs 11-14.

[00426]

[00427]

[00428] Table 14C lists the amount of DNA material obtained (based on OD detection) using constructs 12 and 14 of Table 14C. The yield of total DNA material was acceptable compared to typical yields of approximately 3 mg/l of DNA material in the method of Example 1 (Table 13) above.

EXAMPLE 3 ccDNA vectors express the luciferase transgene in vitro

[00429] Constructs were generated by introducing an open reading frame encoding a luciferase reporter gene into the cloning site of the scDNA plasmid constructs: construct-1, construct-3, construct-5, and construct-7. The 3cDNA plasmids (see Table 12 above) comprising the luciferase coding sequence are referred to as the 1-Luc plasmid construct, the 3-Luc plasmid construct, the 5-Luc plasmid construct, and the 7-Luc plasmid construct, respectively.

[00430] HEK293 cells were cultured and transfected with 100 ng, 200 ng or 400 ng of plasmid constructs 1, 3, 5 and 7 using FUGENE® (Promega Corp.) as the transfection agent. The expression of luciferase with each of the plasmids was determined based on the activity of luciferase in each cell culture; the results are shown in FIG. 6A. Luciferase activity was not detected in untreated control cells ("untreated") or cells treated with Fugene alone ("Fugene"), confirming that luciferase activity is the result of gene expression from plasmids. As shown in FIG. 6A and FIG. 6B, stable expression of luciferase from constructs 1 and 7 was detected. a dose-dependent increase in luciferase activity was detected.

[00431] Growth and viability of cells transfected with each of the plasmids were also determined and are shown in FIG. 7A and FIG. 7B. Growth and viability of transfected cells did not differ significantly between groups of cells treated with different constructs.

[00432] Accordingly, luciferase activity measured in each group and normalized for cell growth and viability did not differ from unnormalized luciferase activity. The zcDNA plasmid with the 1-Luc construct showed the most consistent expression of luciferase, with or without normalization.

[00433] Accordingly, the data presented in FIG. 6A, 6B, 7A and 7B demonstrate that construct 1, containing in the 5' to 3' direction WT-ITR (SEQ ID NO: 51), CAG promoter (SEQ ID NO: 3), R3/R4 cloning site (SEQ ID NO: 7), WPRE (SEQ ID NO: 8), BGHpA (SEQ ID NO: 9) and modified ITR (SEQ ID NO: 2), effectively produces an scDNA vector that can express the protein of the transgene included in said scDNA vector.

EXAMPLE 4 Expression of transgenic luciferase protein from ccDNA vectors in vivo.

[00434] The expression of the transgenic protein produced from the constructs 1-8 described above is evaluated from scDNA vectors in vivo in mice. The ZKDNK-Vector obtained from the ZKDNK-Plasmid structure 1 (according to the description in table 12) was tested and prolonged and persistent transgenic expression of lucifers in the mice in the mice after a hydrodynamic anti-structure without liposa, re-dosage (up to 42 days) of the ZKDNK of the exogenous lucifer was demonstrated. Lyachkov. In other experiments, luciferase expression from selected scDNA vectors was assessed in vivo, the scDNA vectors containing the luciferase transgene and at least one modified ITR selected from any of those listed in Tables 10A-10B, or an ITR containing at least one of the sequences shown in FIG. 26A-26B.

[00435] In vivo luciferase expression: Male CD-1 IGS mice 5-7 weeks old (Charles River Laboratories) are administered 0.35 mg/kg of luciferase-expressing ccDNA vector in a volume of 1.2 ml via tail vein hydrodynamic injection on day 0. Luciferase expression is assessed by IVIS imaging at 3, 4, 7 , 14, 21, 28, 31, 35 and 42 days. Briefly, mice are intraperitoneally injected with 150 mg/kg of luciferin substrate and then whole body luminescence is assessed using IVIS® imaging.

[00436] IVIS imaging is performed on day 3, day 4, day 7, day 14, day 21, day 28, day 31, day 35, and day 42, and harvested organs are imaged ex vivo after sacrifice at day 42.

[00437] Throughout the study, animals are weighed daily and monitored for general health and well-being. At sacrifice, the blood of each animal is collected by terminal cardiac puncture, divided into two parts and processed to obtain 1) plasma and 2) serum; the plasma is flash frozen and the serum is used for a liver enzyme panel and then flash frozen. In addition, the liver, spleen, kidney and inguinal lymph nodes (LN) are harvested and visualized ex vivo using IVIS.

[00438] Hepatic luciferase expression is assessed using the MAXDISCOVERY® luciferase ELISA kit (BIOO Scientific/PerkmElmer), qPCR for luciferase in liver samples, histopathological analysis of liver samples, and/or a panel of liver enzymes in serum (VetScanVS2; Abaxis Preventative Care Profile Plus).

EXAMPLE 5 Run-through screening for mutant ITRs

[00439] Conducted additional analyzes of the relationship of the ITR structure and the formation of ccDNA. A number of mutants were designed to evaluate the effect of specific structural changes on the formation of scDNA and the ability to express the transgene encoded in scDNA. The construction of mutants, analysis of the formation of ccDNA and evaluation of transgenic expression of ccDNA in human cell culture are described in more detail below.

A. Construction of mutant ITRs

[00440] A library of 31 AAV type II unique asymmetric ITR mutant cassette plasmids was developed in silico and then evaluated in Sf9 insect cells and human embryonic kidney (HEK293) cells. Each ITR cassette contained either a luciferase (LUC) or a green fluorescent protein (GFP) reporter gene driven by a p10 promoter sequence for expression in insect cells and a CAG promoter sequence for expression in mammalian cells. Mutations were created in the ITR sequence in either the right or left ITR region. This library contained 15 right handed (RS) and 16 left handed (LS) mutants shown in Tables 10A and 10B and in FIG. 26A and 26B herein.

[00441] Suspension cultures of Sf9 were maintained on Sf900 III medium (Gibco) in 200 ml vented tissue culture flasks. Cultures were subcultured every 48 hours and cell numbers and growth metrics were determined prior to each subculture using a ViCell counter (Beckman Coulter). The cultures were maintained with shaking (circle 1'', 130 rpm) at 27°C. Adherent cultures of HEK293 cells were maintained on GlutiMax DMEM (Dulbecco's Modified Eagle's Medium, Gibco) with 1% fetal bovine serum and 1% PenStrep in 250 ml culture flasks at 37° C. and 5% CO ₂ . Cultures were trypsinized and subcultured every 96 hours. For each passage, a 1:10 dilution was used from a 90-100% confluent flask.

[00442] ZcDNA vectors were generated and constructed as described in Example 1 above. Briefly, as seen in FIG. 4B, Sf9 cells transduced with plasmid constructs were allowed to adhere to growth for 24 hours under stationary conditions at 27°C. After 24 hours, the transfected Sf9 cells were infected with a Rep vector from baculovirus-infected insect cells (BIIC). Previously, the infectivity of BIICs had been characterized and they were used at a final dilution of 1:2000. BIIC diluted 1:100 in Sf900 insect cell medium was added to each well with previously transfected cells. BIIC without Rep vector was added to a subset of wells as a negative control. The contents of the tablets were mixed by gentle shaking on a plate shaker for 2 minutes. The cells were then cultured for an additional 48 hours at 27° C. under stationary conditions. All experimental constructs and controls were analyzed in triplicate.

[00443] After 48 hours, the 96-well plate was removed from the incubator, left for a short time to equilibrate to room temperature, and analyzed for luciferase expression (OneGlo luciferase assay (Promega Corporation)). Total luminescence was measured using a SpectraMax M Series microplate reader. The results for replicates were averaged. The results are shown in FIG. 27. As expected, the three negative controls (medium only, mock transfection without donor DNA; and a sample that was treated in the absence of Rep-containing baculovirus cells) did not show significant luciferase expression. Consistent luciferase expression was observed in each of the mutant samples, indicating successful transfection and expression of the ccDNA-encoded transgene for each sample, regardless of mutation.

B. Analysis of ccDNA generation

[00444] In order to confirm that the scDNA obtained from the previous study has the expected closed-end structure, experiments were performed to obtain sufficient amounts of each scDNA, which could then be tested for the presence of the proper structure. Briefly, Sf9 suspension cultures transfected DNA from a single plasmid with mutant ITRs from the library. Cultures were seeded at a density of 1.25×10 ⁶ cells/ml in Erlenmeyer culture flasks with limited gas exchange. DNA: lipid complexes for transfection were prepared using the fuGene transfection reagent according to the manufacturer's instructions. Complex mixtures were prepared and incubated as previously described for the luciferase plate assay, with volumes increasing in proportion to the number of transfected cells, as in the reporter gene assay, a ratio of 4.5:1 (reagent volume/DNA mass) was used. In parallel with the experimental cultures, mock-transfected (transfection reagents only) and untreated control cultures were prepared. After adding the transfection reagents, the cultures were left to reconstitute for 10-15 minutes at room temperature with gentle agitation, after which they were transferred to a 27°C shaking incubator. After 24 hours of incubation with shaking, all flasks (experimental and control) were determined by the number of cells and growth metrics using a ViCell counter (Beckman Coulter). All flasks (except growth control) were infected with BIIC containing the Rep vector at a final dilution of 1:5000. A positive control was also obtained using the established BIIC double infection procedure for obtaining ccDNA. The dual infection culture was seeded using a cell number equal to the average number of viable cells in all experimental cultures. Dual infection controls were infected with Rep and BIIC reporter gene carriers at a final dilution of 1:5000 for each construct, respectively. After infection, the cultures were returned to the incubator under previously described conditions with shaking. Cell counts, growth and viability metrics were determined daily in all flasks for 3 days post-infection. Measurements at the time point T=0 were performed after reconstitution of freshly infected cultures for ~2 hours in a shaking incubator. After 3 days, cells were harvested by centrifugation for 15 minutes. The supernatant was discarded, the weight of the precipitate was recorded, and the precipitate was frozen to -80°C until DNA extraction.

[00445] Putative crude ccDNA was extracted from all flasks (experimental and control) using the Qiagen Plasmid Plus Midi Purification kit (Qiagen) according to the manufacturer's "high throughput" protocol. The eluates were quantified based on the optical density measured on NanoDrop OneC (ThermoFisher). The resulting scDNA extracts were stored at 4°C.

[00446] The above ccDNA extracts were run on a native agarose (1% agarose, 1x TAE buffer) gel containing a 1:10,000 dilution of SYBR Safe Gel Stain (ThermoFisher Scientific) along with a Tracldt 1 Kb Plus DNA Ladder. The gel was then visualized using a Gbox Mini Imager under UV/blue illumination. As previously described, two primary bands are expected for scDNA samples run through native gels: a ~5,500 bp band corresponding to the monomeric form and a ~11,000 bp band corresponding to the dimeric form. All mutant samples were tested and all showed the expected monomer and dimer bands on native agarose gels. Results for a representative sample of mutants are shown in FIG. 28. Putative crude ITR mutant scDNA and control extracts from small scale production were further analyzed using conjugated restriction enzyme digests and a denaturing agarose gel to confirm the double-stranded DNA structure that is distinctive of scDNA. Each ccDNA mutant is expected to have a single EcoR1 restriction site, thus, if formed correctly, it will produce two characteristic fragments upon EcoR1 cleavage. EcoR1 high fidelity restriction endonuclease (New England Biolabs) was used to cleave the putative scDNA extract according to the manufacturer's instructions. Mock transfected and growth control extracts were not analyzed because spectrophotometric quantitation using NanoDrop (ThermoFisher) and native agarose gel analysis showed no detectable scDNA/plasmid-like product in the eluates. The digested material was purified using the Qiagen PCR Clean-up Kit (Qiagen) according to the manufacturer's instructions, except that the purified digested material was eluted in nuclease-free water instead of Qiagen's elution buffer. Alkaline agarose gel (0.8% alkaline agarose) was equilibrated in equilibration buffer (1 mm EDHA, 200 mm NaOH) overnight at 4°C. 10x denaturing solution (50 mM NaOH, 1 mM EDHA) was added to samples of purified digested scDNA and corresponding undigested scDNA (1 μg total) and the samples were heated at 65° C. for 10 minutes. 10× loading dye (bromophenol blue, 50% glycerol) was added to each denatured sample and mixed. A Tracldt 1kb Plus DNA Ladder (ThermoFisher Scientific) was also loaded onto the gel as a reference sample. The gel was run for ~18 hours at 4°C and constant voltage (25 V), followed by washing with deionized H ₂ O and neutralization in 1×TAE buffer (Tris-acetate, EDTA), pH 7.6, for 20 minutes with gentle shaking. Then the gel was transferred into a solution of 1× TAE/1× SYBR Gold and left for ~1 hour with gentle shaking. The gel was then visualized using a Gbox Mini Imager (Syngene) under UV/blue illumination. Uncut denatured samples were expected to migrate as ~11,000 bp fragments, and EcoR1-treated samples were expected to yield two bands, one for ~4,000 bp and the other for ~6,000 bp.

[00447] All mutant samples gave similar results in the specified experiment. Two significant bands were seen in each sample lane with EcoR1-treated samples migrating on the denaturing gel as fragments of the expected size, dramatically different from the uncleaved mutant samples, which migrated as fragments of the expected ~11,000 bp size. In FIG. 27 shows the results for a representative sample of mutants, where for each digested mutant sample, two bands above background values are visible, in contrast to the single band visible in the case of non-cleaved mutant samples. Thus, the mutant samples appear to correctly form ccDNA.

C. Functional expression in mammalian cell culture

[00448] To evaluate the functionality of scDNA with mutant ITRs resulting from a small scale manufacturing process, HEK293 cells were transfected with several representative samples of mutated scDNA. Actively dividing HEK293 cells were seeded in 96-well microtiter plates at a density of 3×10 ⁶ cells per well (80% confluence) and incubated for 24 hours under the previously described conditions for adherent HEK293 cultures. 24 hours later, 200 ng of total crude ccDNA from small scale production was transfected using Lipofectamine (Invitrogen, TheromoFisher Scientific). Transfection complexes were prepared according to the manufacturer's instructions; a total volume of 10 μl of the transfection complex was used to transfect previously seeded HEK293 cells. All experimental constructs and controls were analyzed in triplicate. Transfected cells were incubated under previously described conditions for 72 hours. After 72 hours, the 96-well plate was removed from the incubator and left for a short time to equilibrate to room temperature. A OneGlo luciferase assay was performed. After 10 minutes on an orbital shaker, total luminescence was measured using a SpectraMax M Series microplate reader. The results for replicates were averaged. The results are shown in FIG. 30. Each of the tested mutant samples expressed luciferase in human cell culture, which indicates the correct formation and expression of ccDNA for each sample in the context of human cells.

[00449] sources.

[00450] All sources listed and disclosed in the present description and examples, including patents, patent applications, international patent applications and publications, are incorporated herein in their entirety by reference.

--->

SEQUENCE LIST

<110> GENERATION BIO INC.

<120> MODIFIED CLOSED-ENDED DNA (CEDNA)/

MODIFIED CLOSED-ENDED DNA (CMDNA)

<130> 080170-090580WOPT

<140>

<141>

<150> 62/556335

<151> 2017-09-08

<150> 62/556281

<151> 2017-09-08

<150> 62/556319

<151> 2017-09-08

<150> 62/556324

<151> 2017-09-08

<150> 62/556329

<151> 2017-09-08

<150> 62/556331

<151> 2017-09-08

<160> 557

<170> PatentIn version 3.5

<210> 1

<211> 141

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 1

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120

gagcgcgcag ctgcctgcag g 141

<210> 2

<211> 130

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 2

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc 120

tgcctgcagg 130

<210> 3

<211> 1923

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 3

tcaatattgg ccattagcca tattattcat tggttata gcataaatca atattggcta 60

ttggccattg catacgttgt atctatatca taatatgtac attttattg gctcatgtcc 120

aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180

gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240

gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300

agtaacgcca atagggactt tccattgacg tcaatggggtg gagtatttac ggtaaactgc 360

ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420

cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480

gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg 540

cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta 600

attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg 660

gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg 720

cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg 780

aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg ccccgtgccc cgctccgccg 840

ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc 900

gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt 960

ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc gggggggagc 1020

ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc 1080

ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg 1140

aggggagcgc ggccgggggc ggtgccccgc ggtgcgggggg gggctgcgag gggaacaaag 1200

1260

tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg 1320

gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg 1380

ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc 1440

ggccccggga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg 1500

taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct 1560

gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg 1620

aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc 1680

cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg 1740

ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tttagccttc 1800

1860

acagaattcc tcgaagatcc gaaggggttc aagcttggca ttccggtact gttggtaaag 1920

cca 1923

<210> 4

<211> 1272

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 4

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctggggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg gtccgggttc aaaaccactt gctgggtggg gagtcgtcag 360

taagtggcta tgccccgacc ccgaagcctg tttccccatc tgtacaatgg aaatgataaa 420

gacgcccatc tgatagggtt tttgtggcaa ataaacattt ggtttttttg ttttgttttg 480

ttttgttttt tgagatggag gtttgctctg tcgcccaggc tggagtgcag tgacacaatc 540

tcatctcacc acaaccttcc cctgcctcag cctcccaagt agctgggatt acaagcatgt 600

gccaccacac ctggctaatt ttctattttt agtagagacg ggtttctcca tgttggtcag 660

cctcagcctc ccaagtaact gggattacag gcctgtgcca cccacacccgg ctaatttttt 720

ctatttttga cagggacggg gtttcaccat gttggtcagg ctggtctaga ggtaccggat 780

cttgctacca gtggaacagc cactaaggat tctgcagtga gagcagaggg ccagctaagt 840

ggtactctcc cagagactgt ctgactcacg ccaccccctc caccttggac acaggacgct 900

gtggtttctg agccaggtac aatgactcct ttcggtaagt gcagtggaag ctgtacactg 960

cccaggcaaa gcgtccgggc agcgtaggcg ggcgactcag atcccagcca gtggacttag 1020

cccctgtttg ctcctccgat aactggggtg accttggtta atattcacca gcagcctccc 1080

ccgttgcccc tctggatcca ctgcttaaat acggacgagg acagggccct gtctcctcag 1140

cttcaggcac caccactgac ctgggacagt gaatccggac tctaaggtaa atataaaatt 1200

tttaagtgta taatgtgtta aactactgat tctaattgtt tctctctttt agattccaac 1260

ctttggaact ga 1272

<210> 5

<211> 547

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 5

ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc 60

tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac ctccaacatc 120

cactcgaccc cttggaattt ttcggtggag aggagcagag gttgtcctgg cgtggtttag 180

gtagtgtgag aggggaatga ctcctttcgg taagtgcagt ggaagctgta cactgcccag 240

gcaaagcgtc cgggcagcgt aggcgggcga ctcagatccc agccagtgga cttagcccct 300

360

gcccctctgg atccactgct taaatacggga cgaggacagg gccctgtctc ctcagcttca 420

ggcaccacca ctgacctggg acagtgaatc cggactctaa ggtaaatata aaatttttaa 480

gtgtataatg tgttaaacta ctgattctaa ttgtttctct cttttagatt ccaacctttg 540

gaactga 547

<210> 6

<211> 1179

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 6

ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60

ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120

gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180

gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240

gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300

acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg 360

gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg 420

cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct 480

ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg 540

caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc 600

gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga 660

gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct 720

ggtctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca 780

gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg 840

acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg 900

tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat 960

1020

1080 gagtttcccc acactgagtg ggtggagact

ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca 1140

gtggttcaaa gtttttttct tccatttcag gtgtcgtga 1179

<210> 7

<211> 8

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 7

gtttaaac 8

<210> 8

<211> 581

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 8

gagcatctta ccgccattta ttcccatatt tgttctgttt ttcttgattt gggtatacat 60

ttaaatgtta ataaaacaaa atggtggggc aatcatttac atttttaggg atatgtaatt 120

actagttcag gtgtattgcc acaagacaaa catgttaaga aactttcccg ttatttacgc 180

tctgttcctg ttaatcaacc tctggattac aaaatttgtg aaagattgac tgatattctt 240

aactatgttg ctccttttac gctgtgtgga tatgctgctt tatagcctct gtatctagct 300

attgcttccc gtacggcttt cgttttctcc tccttgtata aatcctggtt gctgtctctt 360

ttagaggagt tgtggcccgt tgtccgtcaa cgtggcgtgg tgtgctctgt gtttgctgac 420

gcaaccccca ctggctgggg cattgccacc acctgtcaac tcctttctgg gactttcgct 480

ttccccctcc cgatcgccac ggcagaactc atcgccgcct gccttgcccg ctgctggaca 540

ggggctaggt tgctgggcac tgataattcc gtggtgttgt c 581

<210> 9

<211> 225

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 9

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 60

ggaaggtgcc actcccactg tccttttccta ataaaatgag gaaattgcat cgcattgtct 120

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 180

ggaagacaat agcaggcatg ctggggatgc ggtggggctct atggc 225

<210> 10

<211> 213

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 10

taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 60

tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 120

180

tttaaagcaa gtaaaacctc tacaaatgtg gta 213

<210> 11

<211> 1260

<212> DNA

<213> Adeno-associated virus - 2

<400> 11

atggagctgg tcgggtggct cgtggacaag gggattacct cggagaagca gtggatccag 60

gaggaccagg cctcatacat ctccttcaat gcggcctcca actcgcggtc ccaaatcaag 120

gctgccttgg acaatgcggg aaagattatg agcctgacta aaaccgcccc cgactacctg 180

240

aacgggtacg atccccaata tgcggcttcc gtctttctgg gatgggccac gaaaaagttc 300

ggcaagagga acaccatctg gctgtttggg cctgcaacta ccgggaagac caacatcgcg 360

gaggccatag ccccacactgt gcccttctac gggtgcgtaa actggaccaa tgagaacttt 420

cccttcaacg actgtgtcga caagatggtg atctggtgg aggaggggaa gatgaccgcc 480

aaggtcgtgg agtcggccaa agccattctc ggaggaagca aggtgcgcgt ggaccagaaa 540

tgcaagtcct cggcccagat agacccgact cccgtgatcg tcacctccaa caccaacatg 600

tgcgccgtga ttgacgggaa ctcaacgacc ttcgaacacc agcagccgtt gcaagaccgg 660

atgttcaaat ttgaactcac ccgccgtctg gatcatgact ttgggaaggt caccaagcag 720

gaagtcaaag actttttccg gtgggcaaag gatcacgtgg ttgaggtgga gcatgaattc 780

tacgtcaaaa agggtggagc caagaaaaga cccgccccca gtgacgcaga tataagtgag 840

cccaaacggg tgcgcgagtc agttgcgcag ccatcgacgt cagacgcgga agcttcgatc 900

aactacgcag acaggtacca aaacaaatgt tctcgtcacg tgggcatgaa tctgatgctg 960

tttccctgca gacaatgcga gagaatgaat cagaattcaa atatctgctt cactcacgga 1020

1080

aaggcgtatc agaaactgtg ctacattcat catatcatgg gaaaggtgcc agacgcttgc 1140

actgcctgcg atctggtcaa tgtggatttg gatgactgca tctttgaaca ataaatgatt 1200

taaatcaggt atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga 1260

<210> 12

<211> 1932

<212> DNA

<213> Adeno-associated virus - 2

<400> 12

atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga gcatctgccc 60

ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt gccgccagat 120

tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag 180

240 cgcgactttc tgacggaatg gcgccgtgtg

caatttgaga agggagag ctacttccac atgcacgtgc tcgtggaaac caccggggtg 300

360

taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac cagaaatggc 420

gccgggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt gctccccaaa 480

acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag cgcctgtttg 540

aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag 600

gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag atcaaaaact 660

tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac ctcggagaag 720

cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc caactcgcgg 780

tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac taaaaccgcc 840

ccgactacc tggtggggcca gcagcccgtg gaggacattt ccagcaatcg gattatataaa 900

attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct gggatggggcc 960

acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac taccgggaag 1020

accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt aaactggacc 1080

aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg ggaggagggg 1140

aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag caaggtgcgc 1200

gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat cgtcacctcc 1260

1320

ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga ctttgggaag 1380

1440

gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc cagtgacgca 1500

gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg 1560

gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg 1620

aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc aaatatctgc 1680

ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc tcaacccgtt 1740

tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg 1800

ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa 1860

caataaatga tttaaatcag gtatggctgc cgatggttat cttccagatt ggctcgagga 1920

cactctctct ga 1932

<210> 13

<211> 1876

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 13

cgcagccacc atggcggggt tttacgagat tgtgattaag gtccccagcg accttgacgg 60

gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt 120

gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga 180

gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct 240

tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac 300

caccggggtg aaatccatgg ttttgggacg ttttcctgagt cagattcgcg aaaaactgat 360

tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac 420

cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt 480

gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag 540

cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc 600

gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag 660

atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac 720

ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc 780

caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac 840

taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacattt cgcaatcg 900

gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct 960

gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac 1020

taccgggaag acccaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt 1080

aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg 1140

ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag 1200

1260

cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca 1320

ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga 1380

ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt 1440

ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc 1500

1560

gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca 1620

cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc 1680

aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc 1740

tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat 1800

gggaaaggtg ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg 1860

catctttgaa catataa 1876

<210> 14

<211> 1194

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 14

atggagctgg tcgggtggct cgtggacaag gggattacct cggagaagca gtggatccag 60

gaggaccagg cctcatacat ctccttcaat gcggcctcca actcgcggtc ccaaatcaag 120

gctgccttgg acaatgcggg aaagattatg agcctgacta aaaccgcccc cgactacctg 180

240

aacgggtacg atccccaata tgcggcttcc gtctttctgg gatgggccac gaaaaagttc 300

ggcaagagga acaccatctg gctgtttggg cctgcaacta ccgggaagac caacatcgcg 360

gaggccatag ccccacactgt gcccttctac gggtgcgtaa actggaccaa tgagaacttt 420

cccttcaacg actgtgtcga caagatggtg atctggtgg aggaggggaa gatgaccgcc 480

aaggtcgtgg agtcggccaa agccattctc ggaggaagca aggtgcgcgt ggaccagaaa 540

tgcaagtcct cggcccagat agacccgact cccgtgatcg tcacctccaa caccaacatg 600

tgcgccgtga ttgacgggaa ctcaacgacc ttcgaacacc agcagccgtt gcaagaccgg 660

atgttcaaat ttgaactcac ccgccgtctg gatcatgact ttgggaaggt caccaagcag 720

gaagtcaaag actttttccg gtgggcaaag gatcacgtgg ttgaggtgga gcatgaattc 780

tacgtcaaaa agggtggagc caagaaaaga cccgccccca gtgacgcaga tataagtgag 840

cccaaacggg tgcgcgagtc agttgcgcag ccatcgacgt cagacgcgga agcttcgatc 900

aactacgcag accgctacca aaacaaatgt tctcgtcacg tgggcatgaa tctgatgctg 960

tttccctgca gacaatgcga gagaatgaat cagaattcaa atatctgctt cactcacgga 1020

1080

aaggcgtatc agaaactgtg ctacattcat catatcatgg gaaaggtgcc agacgcttgc 1140

actgcctgcg atctggtcaa tgtggatttg gatgactgca tctttgaaca ataa 1194

<210> 15

<211> 141

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 15

aataaacgat aacgccgttg gtggcgtgag gcatgtaaaa ggttacatca ttatcttgtt 60

cgccatccgg ttggtataaa tagacgttca tgttggtttt tgtttcagtt gcaagttggc 120

tgcggcgcgc gcagcacctt t 141

<210> 16

<211> 556

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 16

ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc 60

tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac ctccaacatc 120

cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg tggtttaggt 180

agtgtgagag gggaatgact cctttcggta agtgcagtgg aagctgtaca ctgcccaggc 240

aaagcgtccg ggcagcgtag gcgggcgact cagatcccag ccagtggact tagcccctgt 300

ttgctcctcc gtaactggg gtgaccttgg ttaatattca ccagcagcct cccccgttgc 360

ccctctggat ccactgctta aatacggacg aggacactcg agggccctgt ctcctcagct 420

tcaggcacca ccactgacct gggacagtga atccggacat cgattctaag gtaaatataa 480

aatttttaag tgtataattt gttaaactac tgattctaat tgtttctctc ttttagattc 540

caacctttgg aactga 556

<210> 17

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 17

gcgcgctcgc tcgctcactg aggccggggcg accaaaggtc gcccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 18

<211> 241

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 18

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

c 241

<210> 19

<211> 215

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 19

gaacgctgac gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa 60

cacccagcgc gcgtgcgcc tggcaggaag atggctgtga gggacagggg agtggcgccc 120

tgcaatattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg 180

gatttgggaa tcgtataaga actgtatgag accac 215

<210> 20

<211> 150

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 20

ataaacgata acgccgttgg tggcgtgagg catgtaaaag gttacatcat tatcttgttc 60

gccatccggt tggtataaat agacgttcat gttggttttt gtttcagttg caagttggct 120

gcggcgcgcg cagcaccttt gcggccatct 150

<210> 21

<211> 546

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 21

ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc 60

tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac ctccaacatc 120

cactcgaccc cttggaattt ttcggtggag aggagcagag gttgtcctgg cgtggtttag 180

gtagtgtgag aggggaatga ctcctttcgg taagtgcagt ggaagctgta cactgcccag 240

gcaaagcgtc cgggcagcgt aggcgggcga ctcagatccc agccagtgga cttagcccct 300

360

gcccctctgg atccactgct taaatacggga cgaggacagg gccctgtctc ctcagcttca 420

ggcaccacca ctgacctggg acagtgaatc cggactctaa ggtaaatata aaatttttaa 480

gtgtataatg tgttaaacta ctgattctaa ttgtttctct cttttagatt ccaacctttg 540

gaactg 546

<210> 22

<211> 317

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 22

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 60

agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 120

tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 180

ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 240

aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag 300

gcctaggctt ttgcaaa 317

<210> 23

<211> 576

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 23

tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa 60

cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata 120

atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag 180

tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc 240

cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta 300

tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg 360

cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt 420

ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca 480

aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag 540

gtctatataa gcagagctgg tttagtgaac cgtcag 576

<210> 24

<211> 1313

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 24

ggagccgaga gtaattcata caaaaggagg gatcgccttc gcaaggggag agcccaggga 60

ccgtccctaa attctcacag acccaaatcc ctgtagccgc cccacgacag cgcgaggagc 120

atgcgcccag ggctgagcgc gggtagatca gagcacacaa gctcacagtc cccggcggtg 180

gggggagggg cgcgctgagc gggggccagg gagctggcgc ggggcaaact gggaaagtgg 240

tgtcgtgtgc tggctccgcc ctcttcccga gggtggggga gaacggtata taagtgcggt 300

agtcgccttg gacgttcttt ttcgcaacgg gttgccgtc agaacgcagg tgagtggcgg 360

gtgtggcttc cgcgggcccc ggagctggag ccctgctctg agcgggccgg gctgatatgc 420

gagtgtcgtc cgcagggttt agctgtgagc attcccactt cgagtggcgg gcggtgcggg 480

ggtgagagtg cgaggcctag cggcaacccc gtagcctcgc ctcgtgtccg gcttgaggcc 540

tagcgtggtg tccgccgccg cgtgccactc cggccgcact atgcgtttt tgtccttgct 600

gccctcgatt gccttccagc agcatgggct aacaaaggga gggtgtgggg ctcactctta 660

aggagcccat gaagcttacg ttggatagga atggaagggc aggaggggcg actggggccc 720

gcccgccttc ggagcacatg tccgacgcca cctggatggg gcgaggcctg tggctttccg 780

aagcaatcgg gcgtgagttt agcctacctg ggccatgtgg ccctagcact gggcacggtc 840

tggcctggcg gtgccgcgtt cccttgcctc ccaacaaggg tgaggccgtc ccgcccggca 900

ccagttgctt gcgcggaaag atggccgctc ccggggccct gttgcaagga gctcaaaatg 960

gaggacgcgg cagcccggtg gagcgggcgg gtgagtcacc cacacaaagg aagagggcct 1020

tgcccctcgc cggccgctgc ttcctgtgac cccgtggtct atcggccgca tagtcacctc 1080

gggcttctct tgagcaccgc tcgtcgcggc ggggggaggg gatctaatgg cgttggagtt 1140

tgttcacatt tggtgggtgg agactagtca ggccagcctg gcgctggaag tcattcttgg 1200

aatttgcccc gttgagtttg gagcgaggct aattctcaag cctcttagcg gttcaaaggt 1260

attttctaaa cccgtttcca ggtgttgtga aagccaccgc taattcaaag caa 1313

<210> 25

<211> 19

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 25

Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly

1 5 10 15

Ala His Ser

<210> 26

<211> 19

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 26

Met Leu Pro Ser Gln Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala

1 5 10 15

Ser Arg Gly

<210> 27

<211> 7

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 27

Pro Lys Lys Lys Arg Lys Val

15

<210> 28

<400> 28

000

<210> 29

<400> 29

000

<210> 30

<400> 30

000

<210> 31

<400> 31

000

<210> 32

<400> 32

000

<210> 33

<400> 33

000

<210> 34

<400> 34

000

<210> 35

<400> 35

000

<210> 36

<400> 36

000

<210> 37

<400> 37

000

<210> 38

<400> 38

000

<210> 39

<400> 39

000

<210> 40

<400> 40

000

<210> 41

<400> 41

000

<210> 42

<400> 42

000

<210> 43

<400> 43

000

<210> 44

<400> 44

000

<210> 45

<211> 6

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 45

ggttga 6

<210> 46

<211> 4

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 46

agtt 4

<210> 47

<211> 6

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 47

ggttgg 6

<210> 48

<211> 6

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 48

agttgg 6

<210> 49

<211> 6

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 49

agttga 6

<210> 50

<211> 6

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 50

rrttrr 6

<210> 51

<211> 141

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 51

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc cggggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc t 141

<210> 52

<211> 130

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 52

60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct 130

<210> 53

<211> 3123

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 53

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggaaacc cgggcgtgcc 60

cgggcgcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg 120

gttcctgaac agagaaacag gagaatatgg gccaaacagg atatctgtgg taagcagttc 180

ctgccccggc tcagggccaa gaacagttgg aacagcagaa tatgggccaa acaggatatc 240

tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 300

ccgccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 360

atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 420

ttctgctccc cgagctctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga 480

gacgccatcc acgctgtttt gacttccata gaaggccgcc accatggaag acgccaaaaa 540

cataaagaaa ggcccggcgc cattctatcc gctggaagat ggaaccgctg gagagcaact 600

gcataaggct atgaagagat acgccctggt tcctggaaca attgctttta cagatgcaca 660

tatcgaggtg gacatcactt acgctgagta cttcgaaatg tccgttcggt tggcagaagc 720

tatgaaacga tatgggctga atacaaatca cagaatcgtc gtatgcagtg aaaactctct 780

tcaattcttt atgccggtgt tgggcgcgtt atttatcgga gttgcagttg cgcccgcgaa 840

cgacatttat aatgaacgtg aattgctcaa cagtatgggc atttcgcagc ctaccgtggt 900

gttcgtttcc aaaaaggggt tgcaaaaaat tttgaacgtg caaaaaaagc tcccaatcat 960

ccaaaaaatt attatcatgg attctaaaac ggattaccag ggatttcagt cgatgtacac 1020

gttcgtcaca tctcatctac ctcccggttt taatgaatac gattttgtgc cagagtcctt 1080

cgatagggac aagacaattg cactgatcat gaactcctct ggatctactg gtctgcctaa 1140

aggtgtcgct ctgcctcata gaactgcctg cgtgagattc tcgcatgcca gagatcctat 1200

ttttggcaat caaatcattc cggatactgc gattttaagt gttgttccat tccatcacgg 1260

ttttggaatg tttactacac tcggatattt gatatgtgga tttcgagtcg tcttaatgta 1320

tagatttgaa gaagagctgt ttctgaggag ccttcaggat tacaagattc aaagtgcgct 1380

gctggtgcca accctattct ccttcttcgc caaaagcact ctgattgaca aatacgattt 1440

atctaattta cacgaaattg cttctggtgg cgctcccctc tctaaggaag tcggggaagc 1500

ggttgccaag aggttccatc tgccaggtat caggcaagga tatgggctca ctgagactac 1560

atcagctatt ctgattacac ccgaggggga tgataaaccg ggcgcggtcg gtaaagttgt 1620

tccatttttt gaagcgaagg ttgtggatct ggataccggg aaaacgctgg gcgttaatca 1680

aagaggcgaa ctgtgtgtga gaggtcctat gattatgtcc ggttatgtaa acaatccgga 1740

agcgaccaac gccttgattg acaaggatgg atggctacat tctggagaca tagcttactg 1800

ggacgaagac gaacacttct tcatcgttga ccgcctgaag tctctgatta agtacaaagg 1860

ctatcaggtg gctcccgctg aattggaatc catcttgctc caacacccca acatcttcga 1920

cgcaggtgtc gcaggtcttc ccgacgatga cgccggtgaa cttcccgccg ccgttgttgt 1980

tttggagcac ggaaagacga tgacggaaaa agagatcgtg gattacgtcg ccagtcaagt 2040

aacaaccgcg aaaaagttgc gcggaggagt tgtgtttgtg gacgaagtac cgaaaggtct 2100

2160

aaagatcgcc gtgtaagagc atcttaccgc catttattcc catatttgtt ctgtttttct 2220

tgatttgggt atacatttaa atgttaataa aacaaaatgg tggggcaatc atttacattt 2280

ttagggatat gtaattacta gttcaggtgt attgccacaa gacaaacatg ttaagaaact 2340

ttcccgttat ttacgctctg ttcctgttaa tcaacctctg gattacaaaa tttgtgaaag 2400

attgactgat attcttaact atgttgctcc ttttacgctg tgtggatatg ctgcttttata 2460

gcctctgtat ctagctattg cttcccgtac ggctttcgtt ttctcctcct tgtataaatc 2520

ctggttgctg tctcttttag aggagttgtg gcccgttgtc cgtcaacgtg gcgtggtgtg 2580

ctctgtgttt gctgacgcaa cccccactgg ctggggcatt gccaccacct gtcaactcct 2640

ttctgggact ttcgctttcc ccctcccgat cgccacggca gaactcatcg ccgcctgcct 2700

tgcccgctgc tggacagggg ctaggttgct gggcactgat aattccgtgg tgttgtctgt 2760

gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 2820

aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 2880

taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 2940

agacaatagc aggcatgctg gggatgcggt gggctctatg gcaggaaccc ctagtgatgg 3000

agttggccac tccctctctg cgcgctcgct cgctcactga ggccggggcga ccaaaggtcg 3060

cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc 3120

agg 3123

<210> 54

<211> 3117

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 54

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc cggggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tgaacagaga aacaggagaa tatgggccaa acaggatatc 180

tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gttggaacag cagaatatgg 240

gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg 300

tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt ttccagggtg 360

ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg 420

cttctgttcg cgcgcttctg ctccccgagc tctatataag cagagctcgt ttagtgaacc 480

gtcagatcgc ctggagacgc catccacgct gttttgactt ccatagaagg ccgccaccat 540

ggaagacgcc aaaaacataa agaaaggccc ggcgccattc tatccgctgg aagatggaac 600

cgctggagag caactgcata aggctatgaa gagatacgcc ctggttcctg gaacaattgc 660

ttttacagat gcacatatcg aggtggacat cacttacgct gagtacttcg aaatgtccgt 720

tcggttggca gaagctatga aacgatatgg gctgaataca aatcacagaa tcgtcgtatg 780

cagtgaaaac tctcttcaat tctttatgcc ggtgttgggc gcgttattta tcggagttgc 840

agttgcgccc gcgaacgaca tttataatga acgtgaattg ctcaacagta tgggcatttc 900

gcagcctacc gtggtgttcg tttccaaaaa ggggttgcaa aaaattttga acgtgcaaaa 960

aaagctccca atcatccaaa aaattattat catggattct aaaacggatt accagggatt 1020

tcagtcgatg tacacgttcg tcacatctca tctacctccc ggttttaatg aatacgattt 1080

tgtgccagag tccttcgata gggacaagac aattgcactg atcatgaact cctctggatc 1140

tactggtctg cctaaaggtg tcgctctgcc tcatagaact gcctgcgtga gattctcgca 1200

1260

1320

agtcgtctta atgtatagat ttgaagaaga gctgtttctg aggagccttc aggattacaa 1380

gattcaaagt gcgctgctgg tgccaaccct attctccttc ttcgccaaaa gcactctgat 1440

tgacaaatac gatttatcta atttacacga aattgcttct ggtggcgctc ccctctctaa 1500

ggaagtcggg gaagcggttg ccaagaggtt ccatctgcca ggtatcaggc aaggatatgg 1560

gctcactgag actacatcag ctattctgat tacacccgag ggggatgata aaccgggcgc 1620

ggtcggtaaa gttgttccat tttttgaagc gaaggttgtg gatctggata ccgggaaaac 1680

gctgggcgtt aatcaaagag gcgaactgtg tgtgagaggt cctatgatta tgtccggtta 1740

1800

agacatagct tactgggacg aagacgaaca cttcttcatc gttgaccgcc tgaagtctct 1860

gattaagtac aaaggctatc aggtggctcc cgctgaattg gaatccatct tgctccaaca 1920

ccccaacatc ttcgacgcag gtgtcgcagg tcttcccgac gatgacgccg gtgaacttcc 1980

cgccgccgtt gttgttttgg agcacggaaa gacgatgacg gaaaaagaga tcgtggatta 2040

2100

agtaccgaaa ggtcttaccg gaaaactcga cgcaagaaaa atcagagaga tcctcataaa 2160

ggccaagaag ggcggaaaga tcgccgtgta agagcatctt accgccattt attcccatat 2220

ttgttctgtt tttcttgatt tgggtataca tttaaatgtt aataaaacaa aatggtgggg 2280

caatcattta catttttagg gatatgtaat tactagttca ggtgtattgc cacaagacaa 2340

acatgttaag aaactttccc gttatttacg ctctgttcct gttaatcaac ctctggatta 2400

caaaatttgt gaaagattga ctgatattct taactatgtt gctcctttta cgctgtgtgg 2460

atatgctgct ttatagcctc tgtatctagc tattgcttcc cgtacggctt tcgttttctc 2520

ctccttgtat aaatcctggt tgctgtctct tttgaggag ttgtggcccg ttgtccgtca 2580

acgtggcgtg gtgtgctctg tgtttgctga cgcaaccccc actggctggg gcattgccac 2640

cacctgtcaa ctcctttctg ggactttcgc ttttccccctc ccgatcgcca cggcagaact 2700

catcgccgcc tgccttgccc gctgctggac aggggctagg ttgctgggca ctgataattc 2760

cgtggtgttg tctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 2820

ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc 2880

atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa 2940

gggggaggat tgggaagaca atagcaggca tgctggggat gcggtggggct ctatggcagg 3000

aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg 3060

3117

<210> 55

<400> 55

000

<210> 56

<400> 56

000

<210> 57

<211> 3912

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 57

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggaaacc cgggcgtgcc 60

cgggcgcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg 120

gttcctggct cagaggctca gaggcacaca ggagtttctg ggctcaccct gcccccttcc 180

aacccctcag ttcccatcct ccagcagctg tttgtgtgct gcctctgaag tccacactga 240

acaaacttca gcctactcat gtccctaaaa tgggcaaaca ttgcaagcag caaacagcaa 300

acacacagcc ctccctgcct gctgaccttg gagctggggc agaggtcaga gacctctctg 360

ggcccatgcc acctccaaca tccactcgac cccttggaat ttcggtggag aggagcagag 420

gttgtcctgg cgtggtttag gtagtgtgag agggtccggg ttcaaaacca cttgctggggt 480

ggggagtcgt cagtaagtgg ctatgccccg accccgaagc ctgtttcccc atctgtacaa 540

tggaaatgat aaagacgccc atctgatagg gtttttgtgg caaataaaca tttggttttt 600

ttgttttgtt ttgttttgtt ttttgagatg gaggtttgct ctgtcgccca ggctggagtg 660

cagtgacaca atctcatctc accacaacct tcccctgcct cagcctccca agtagctggg 720

attacaagca tgtgccacca cacctggcta attttctatt tttagtagag acgggtttct 780

ccatgttggt cagcctcagc ctcccaagta actgggatta caggcctgtg ccaccacacc 840

cggctaattt tttctatttt tgacagggac ggggtttcac catgttggtc aggctggtct 900

agaggtaccg gatcttgcta ccagtggaac agccactaag gattctgcag tgagagcaga 960

gggccagcta agtggtactc tcccagagac tgtctgactc acgccacccc ctccaccttg 1020

gacacaggac gctgtggttt ctgagccagg tacaatgact cctttcggta agtgcagtgg 1080

aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag gcgggcgact cagatcccag 1140

ccagtggact tagcccctgt ttgctcctcc gataactggg gtgaccttgg ttaatattca 1200

ccagcagcct cccccgttgc ccctctggat ccactgctta aatacggacg aggacagggc 1260

cctgtctcct cagcttcagg caccaccact gacctgggac agtgccgcca ccatggaaga 1320

cgccaaaaac ataaagaaag gcccggcgcc attctatccg ctggaagatg gaaccgctgg 1380

agagcaactg cataaggcta tgaagagata cgccctggtt cctggaacaa ttgcttttac 1440

agatgcacat atcgaggtgg acatcactta cgctgagtac ttcgaaatgt ccgttcggtt 1500

atgaaacgat atgggctgaa tacaaatcac agaatcgtcg tatgcagtga 1560

1620

1680

taccgtggtg ttcgtttcca aaaaggggtt gcaaaaaatt ttgaacgtgc aaaaaaagct 1740

cccaatcatc caaaaaatta ttatcatgga ttctaaaacg gattaccagg gatttcagtc 1800

gatgtacacg ttcgtcacat ctcatctacc tcccggtttt aatgaatacg attttgtgcc 1860

agagtccttc gatagggaca agacaattgc actgatcatg aactcctctg gatctactgg 1920

tctgcctaaa ggtgtcgctc tgcctcatag aactgcctgc gtgagattct cgcatgccag 1980

agatcctatt tttggcaatc aaatcattcc ggatactgcg attttaagtg ttgttccatt 2040

ccatcacggt tttggaatgt ttactacact cggatatttg atatgtggat ttcgagtcgt 2100

2160

aagtgcgctg ctggtgccaa ccctattctc cttcttcgcc aaaagcactc tgattgacaa 2220

atacgattta tctaatttac acgaaattgc ttctggtggc gctcccctct ctaaggaagt 2280

cggggaagcg gttgccaaga ggttccatct gccaggtatc aggcaaggat atgggctcac 2340

tgagactaca tcagctattc tgattacacc cgagggggat gataaaccgg gcgcggtcgg 2400

taaagttgtt ccattttttg aagcgaaggt tgtggatctg gataccggga aaacgctgggg 2460

2520

2580

agcttactgg gacgaagacg aacacttctt catcgttgac cgcctgaagt ctctgattaa 2640

gtacaaaggc tatcaggtgg ctcccgctga attggaatcc atcttgctcc aacaccccaa 2700

catcttcgac gcaggtgtcg caggtcttcc cgacgatgac gccggtgaac ttcccgccgc 2760

2820

cagtcaagta acaaccgcga aaaagttgcg cggaggagtt gtgtttgtgg acgaagtacc 2880

gaaaggtctt accggaaaac tcgacgcaag aaaaatcaga gagatcctca taaaggccaa 2940

gaagggcgga aagatcgccg tgtaagagca tcttaccgcc atttattccc atatttgttc 3000

3060

3120

taagaaactt tcccgttatt tacgctctgt tcctgttaat caacctctgg attacaaaat 3180

ttgtgaaaga ttgactgata ttcttaacta tgttgctcct tttacgctgt gtggatatgc 3240

tgctttatag cctctgtatc tagctattgc ttcccgtacg gctttcgttt tctcctcctt 3300

gtataaatcc tggttgctgt ctcttttaga ggagttgtgg cccgttgtcc gtcaacgtgg 3360

cgtggtgtgc tctgtgtttg ctgacgcaac ccccactggc tggggcattg ccaccacctg 3420

tcaactcctt tctgggactt tcgctttccc cctcccgatc gccacggcag aactcatcgc 3480

cgcctgcctt gcccgctgct ggacaggggc taggttgctg ggcactgata attccgtggt 3540

gttgtctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 3600

gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 3660

ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 3720

ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg caggaacccc 3780

tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac 3840

caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 3900

gctgcctgca gg 3912

<210> 58

<211> 3906

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 58

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc cggggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tggctcagag gctcagaggc acacaggagt ttctgggctc 180

accctgcccc cttccaaccc ctcagttccc atcctccagc agctgtttgt gtgctgcctc 240

tgaagtccac actgaacaaa cttcagccta ctcatgtccc taaaatgggc aaacattgca 300

agcagcaaac agcaaacaca cagccctccc tgcctgctga ccttggagct ggggcagagg 360

tcagagacct ctctgggccc atgccacctc caacatccac tcgacccctt ggaatttcgg 420

tggagaggag cagaggttgt cctggcgtgg tttaggtagt gtgagagggt cggggttcaa 480

aaccacttgc tgggtgggga gtcgtcagta agtggctatg ccccgacccc gaagcctgtt 540

tccccatctg tacaatggaa atgataaaga cgcccatctg atagggtttt tgtggcaaat 600

660

gcccaggctg gagtgcagtg acacaatctc atctcaccac aaccttcccc tgcctcagcc 720

tcccaagtag ctgggattac aagcatgtgc caccacacct ggctaatttt ctatttttag 780

tagagacggg tttctccatg ttggtcagcc tcagcctccc aagtaactgg gattacaggc 840

ctgtgccacc acacccggct aatttttttct atttttgaca gggacggggt ttcaccatgt 900

tggtcaggct ggtctagagg taccggatct tgctaccagt ggaacagcca ctaaggattc 960

tgcagtgaga gcagagggcc agctaagtgg tactctccca gagactgtct gactcacgcc 1020

accccctcca ccttggacac aggacgctgt ggtttctgag ccaggtacaa tgactccttt 1080

1140

cgactcagat cccagccagt ggacttagcc cctgtttgct cctccgataa ctggggtgac 1200

cttggttaat attcaccagc agcctccccc gttgcccctc tggatccact gcttaaatac 1260

ggacgaggac agggccctgt ctcctcagct tcaggcacca ccactgacct gggacagtgc 1320

cgccaccatg gaagacgcca aaaacataaa gaaaggcccg gcgccattct atccgctgga 1380

agatggaacc gctggagagc aactgcataa ggctatgaag agatacgccc tggttcctgg 1440

aacaattgct tttacagatg cacatatcga ggtggacatc acttacgctg agtacttcga 1500

aatgtccgtt cggttggcag aagctatgaa acgatatgggg ctgaatacaa atcacagaat 1560

1620

cggagttgca gttgcgccg cgaacgacat ttataatgaa cgtgaattgc tcaacagtat 1680

gggcatttcg cagcctaccg tggtgttcgt ttccaaaaag gggttgcaaa aaattttgaa 1740

cgtgcaaaaa aagctcccaa tcatccaaaa aattattatc atggattcta aaacggatta 1800

ccagggattt cagtcgatgt acacgttcgt cacatctcat ctacctcccg gttttaatga 1860

atacgatttt gtgccagagt ccttcgatag ggacaagaca attgcactga tcatgaactc 1920

ctctggatct actggtctgc ctaaaggtgt cgctctgcct catagaactg cctgcgtgag 1980

attctcgcat gccagagatc ctatttttgg caatcaaatc attccggata ctgcgatttt 2040

aagtgttgtt ccattccatc acggttttgg aatgtttact acactcggat atttgatatg 2100

tggatttcga gtcgtcttaa tgtatagatt tgaagaagag ctgtttctga ggagccttca 2160

ggattacaag attcaaagtg cgctgctggt gccaacccta ttctccttct tcgccaaaag 2220

cactctgatt gacaaatacg atttatctaa tttacacgaa attgcttctg gtggcgctcc 2280

cctctctaag gaagtcgggg aagcggttgc caagaggttc catctgccag gtatcaggca 2340

aggatatggg ctcactgaga ctacatcagc tattctgatt acacccgagg gggatgataa 2400

accgggcgcg gtcggtaaag ttgttccatt ttttgaagcg aaggttgtgg atctggatac 2460

cgggaaaacg ctgggcgtta atcaaagagg cgaactgtgt gtgagaggtc ctatgattat 2520

gtccggttat gtaaacaatc cggaagcgac caacgccttg attgacaagg atggatggct 2580

acattctgga gacatagctt actgggacga agacgaacac ttcttcatcg ttgaccgcct 2640

gaagtctctg attaagtaca aaggctatca ggtggctccc gctgaattgg aatccatctt 2700

gctccaacac cccaacatct tcgacgcagg tgtcgcaggt cttcccgacg atgacgccgg 2760

tgaacttccc gccgccgttg ttgttttgga gcacggaaag acgatgacgg aaaaagagat 2820

cgtggattac gtcgccagtc aagtaacaac cgcgaaaaag ttgcgcggag gagttgtgtt 2880

tgtggacgaa gtaccgaaag gtcttaccgg aaaactcgac gcaagaaaaa tcagagagat 2940

cctcataaag gccaagaagg gcggaaagat cgccgtgtaa gagcatctta ccgccattta 3000

3060

atggtggggc aatcatttac atttttaggg atatgtaatt actagttcag gtgtattgcc 3120

acaagacaaa catgttaaga aactttcccg ttatttacgc tctgttcctg ttaatcaacc 3180

tctggattac aaaatttgtg aaagattgac tgatattctt aactatgttg ctccttttac 3240

gctgtgtgga tatgctgctt tatagcctct gtatctagct attgcttccc gtacggcttt 3300

3360

tgtccgtcaa cgtggcgtgg tgtgctctgt gtttgctgac gcaaccccca ctggctgggg 3420

cattgccacc acctgtcaac tcctttctgg gactttcgct ttccccctcc cgatcgccac 3480

ggcagaactc atcgccgcct gccttgcccg ctgctggaca ggggctaggt tgctgggcac 3540

tgataattcc gtggtgttgt ctgtgccttc tagttgccag ccatctgttg tttgcccctc 3600

ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 3660

ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 3720

ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc 3780

tatggcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 3840

ctgaggccgc ccgggaaacc cgggcgtgcg cctcagtgag cgagcgagcg cgcagctgcc 3900

tgcagg 3906

<210> 59

<400> 59

000

<210> 60

<400> 60

000

<210> 61

<400> 61

000

<210> 62

<400> 62

000

<210> 63

<211> 126

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 63

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggaaacc cgggcgtgcc 60

cgggcgcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg 120

gttcct 126

<210> 64

<211> 120

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 64

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

120

<210> 65

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 65

gcgcgctcgc tcgctcactg aggccgcccg ggcgtcggggc gacctttggt cgcccggcct 60

cagtgagcga gcgagcgcgc 80

<210> 66

<211> 141

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 66

aataaacgat aacgccgttg gtggcgtgag gcatgtaaaa ggttacatca ttatcttgtt 60

cgccatccgg ttggtataaa tagacgttca tgttggtttt tgtttcagtt gcaagttggc 120

tgcggcgcgc gcagcacctt t 141

<210> 67

<211> 1876

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 67

cgcagccacc atggcggggt tttacgagat tgtgattaag gtccccagcg accttgacga 60

gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt 120

gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga 180

gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct 240

tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac 300

caccggggtg aaatccatgg ttttgggacg ttttcctgagt cagattcgcg aaaaactgat 360

tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac 420

cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt 480

gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag 540

cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc 600

gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag 660

atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac 720

ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc 780

caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac 840

taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacattt cgcaatcg 900

gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct 960

gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac 1020

taccgggaag acccaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt 1080

aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg 1140

ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag 1200

1260

cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca 1320

ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga 1380

ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt 1440

ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc 1500

1560

gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca 1620

cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc 1680

aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc 1740

tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat 1800

gggaaaggtg ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg 1860

catctttgaa catataa 1876

<210> 68

<211> 129

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 68

atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc 60

gtaacagttt tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca 120

tcgggcgcg 129

<210> 69

<211> 1203

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 69

gccgccacca tggagttggt gggctggctc gtggacaaag gcattacttc ggaaaagcag 60

tggattcagg aggatcaggc atcttacatc tcattcaacg ctgccagtaa ctcgaggtcc 120

cagatcaagg cagcgctgga caacgcggga aagattatga gtctgaccaa aactgctcca 180

gactacctcg ttggtcagca accggtggaa gatatctcca gcaacaggat ctacaagatt 240

ctggagctca acggctacga ccctcaatac gctgcctcag tgttcttggg ttgggccacc 300

aagaaattcg gcaagagaaa cactatctgg ctgttcggcc ccgctaccac tggaaagaca 360

aacatcgcag aagcgattgc tcacacggtg ccattctacg gctgcgtcaa ctggacaaac 420

gagaacttcc cgttcaacga ctgtgtcgat aagatggtta tctggtggga ggaaggaaag 480

atgacggcca aagtggtcga aagcgccaag gcaattctgg gtggctctaa agtgcgcgtc 540

gaccagaagt gcaaatcttc agctcaaatc gatcctaccc ccgttattgt gacatcaaac 600

acgaacatgt gtgccgtgat cgacggaaac agtacaacgt tcgaacacca gcaacctctc 660

caggatcgta tgttcaagtt cgagctcacc cgccgtttgg accatgattt cggcaaggtc 720

actaaacaag aggttaagga cttcttccgc tgggctaaag atcacgttgt ggaggttgaa 780

catgagttct acgtcaagaa aggaggtgct aagaaacgtc cagccccgtc ggacgcagat 840

atctccgaac ctaagagggt gagagagtcg gtcgcacagc caagcacttc tgacgcagaa 900

gcttccatta actacgcaga taggtaccaa aacaagtgca gcagacacgt gggtatgaac 960

ttgatgctgt tcccatgccg ccagtgtgag cgtatgaacc aaaactctaa catctgtttc 1020

acacatggcc agaaggactg cctcgaatgt ttccctgtgt cagagagtca gcccgtctca 1080

gtcgttaaga aagcttacca aaagttgtgc tacatccacc atattatggg taaagtccct 1140

gatgcctgta ccgcttgtga tctggtcaac gtggatttgg acgactgtat tttcgagcaa 1200

taa 1203

<210> 70

<211> 388

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 70

gaacagagaa acaggagaat atgggccaaa caggatatct gtggtaagca gttcctgccc 60

cggctcaggg ccaagaacag ttggaacagc agaatatgggg ccaaacagga tatctgtggt 120

aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc ggtcccgccc 180

tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc 240

ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc 300

tccccgagct ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc 360

atccacgctg ttttgacttc catagaag 388

<210> 71

<211> 1662

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 71

gccgccacca tggaagacgc caaaaacata aagaaaggcc cggcgccatt ctatccgctg 60

gaagatggaa ccgctggaga gcaactgcat aaggctatga agagatacgc cctggttcct 120

ggaacaattg cttttacaga tgcacatatc gaggtggaca tcacttacgc tgagtacttc 180

gaaatgtccg ttcggttggc agaagctatg aaacgatatg ggctgaatac aaatcacaga 240

atcgtcgtat gcagtgaaaa ctctcttcaa ttctttatgc cggtgttggg cgcgttattt 300

atcggagttg cagttgcgcc cgcgaacgac atttataatg aacgtgaatt gctcaacagt 360

atgggcattt cgcagcctac cgtggtgttc gtttccaaaa aggggttgca aaaaattttg 420

aacgtgcaaa aaaagctccc aatcatccaa aaaattatta tcatggattc taaaacggat 480

taccagggat ttcagtcgat gtacacgttc gtcacatctc atctacctcc cggttttaat 540

gaatacgatt ttgtgccaga gtccttcgat agggacaaga caattgcact gatcatgaac 600

tcctctggat ctactggtct gcctaaaggt gtcgctctgc ctcatagaac tgcctgcgtg 660

agattctcgc atgccagaga tcctattttt ggcaatcaaa tcattccgga tactgcgatt 720

ttaagtgttg ttccattcca tcacggtttt ggaatgttta ctacactcgg atatttgata 780

tgtggatttc gagtcgtctt aatgtataga tttgaagaag agctgtttct gaggagcctt 840

caggattaca agattcaaag tgcgctgctg gtgccaaccc tattctcctt cttcgccaaa 900

agcactctga ttgacaaata cgatttatct aatttacacg aaattgcttc tggtggcgct 960

cccctctcta aggaagtcgg ggaagcggtt gccaagaggt tccatctgcc aggtatcagg 1020

caaggatatg ggctcactga gactacatca gctattctga ttacacccga gggggatgat 1080

aaaccgggcg cggtcggtaa agttgttcca ttttttgaag cgaaggttgt ggatctggat 1140

accgggaaaa cgctgggcgt taatcaaaga ggcgaactgt gtgtgagagg tcctatgatt 1200

1260

ctacattctg gagacatagc ttactgggac gaagacgaac acttcttcat cgttgaccgc 1320

ctgaagtctc tgattaagta caaaggctat caggtggctc ccgctgaatt ggaatccatc 1380

ttgctccaac accccaacat cttcgacgca ggtgtcgcag gtcttcccga cgatgacgcc 1440

ggtgaacttc ccgccgccgt tgttgttttg gagcacggaa agacgatgac ggaaaaagag 1500

atcgtggatt acgtcgccag tcaagtaaca accgcgaaaa agttgcgcgg aggagttgtg 1560

tttgtggacg aagtaccgaa aggtcttacc ggaaaactcg acgcaagaaa aatcagagag 1620

atcctcataa aggccaagaa gggcggaaag atcgccgtgt aa 1662

<210> 72

<211> 581

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 72

gagcatctta ccgccattta ttcccatatt tgttctgttt ttcttgattt gggtatacat 60

ttaaatgtta ataaaacaaa atggtggggc aatcatttac atttttaggg atatgtaatt 120

actagttcag gtgtattgcc acaagacaaa catgttaaga aactttcccg ttatttacgc 180

tctgttcctg ttaatcaacc tctggattac aaaatttgtg aaagattgac tgatattctt 240

aactatgttg ctccttttac gctgtgtgga tatgctgctt tatagcctct gtatctagct 300

attgcttccc gtacggcttt cgttttctcc tccttgtata aatcctggtt gctgtctctt 360

ttagaggagt tgtggcccgt tgtccgtcaa cgtggcgtgg tgtgctctgt gtttgctgac 420

gcaaccccca ctggctgggg cattgccacc acctgtcaac tcctttctgg gactttcgct 480

ttccccctcc cgatcgccac ggcagaactc atcgccgcct gccttgcccg ctgctggaca 540

ggggctaggt tgctgggcac tgataattcc gtggtgttgt c 581

<210> 73

<211> 225

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 73

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 60

ggaaggtgcc actcccactg tccttttccta ataaaatgag gaaattgcat cgcattgtct 120

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 180

ggaagacaat agcaggcatg ctggggatgc ggtggggctct atggc 225

<210> 74

<211> 1177

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 74

ggctcagagg ctcagaggca cacaggagtt tctgggctca ccctgccccc ttccaacccc 60

tcagttccca tcctccagca gctgtttgtg tgctgcctct gaagtccaca ctgaacaaac 120

ttcagcctac tcatgtccct aaaatgggca aacattgcaa gcagcaaaca gcaaacacac 180

agccctccct gcctgctgac cttggagctg gggcagaggt cagagacctc tctgggccca 240

tgccacctcc aacatccact cgaccccttg gaatttcggt ggagaggagc agaggttgtc 300

ctggcgtggt ttaggtagtg tgagagggtc cgggttcaaa accacttgct gggtggggag 360

tcgtcagtaa gtggctatgc cccgaccccg aagcctgttt ccccatctgt acaatggaaa 420

tgataaagac gcccatctga tagggttttt gtggcaaata aacatttggt ttttttgttt 480

tgttttgttt tgttttttga gatggaggtt tgctctgtcg cccaggctgg agtgcagtga 540

cacaatctca tctcaccaca accttcccct gcctcagcct cccaagtagc tgggattaca 600

agcatgtgcc accacacctg gctaattttc tatttttagt agagacgggt ttctccatgt 660

tggtcagcct cagcctccca agtaactggg attacaggcc tgtgccacca cacccggcta 720

attttttcta tttttgacag ggacggggtt tcaccatgtt ggtcaggctg gtctagaggt 780

accggatctt gctaccagtg gaacagccac taaggattct gcagtgagag cagaggggcca 840

gctaagtggt actctcccag agactgtctg actcacgcca ccccctccac cttggacaca 900

ggacgctgtg gtttctgagc caggtacaat gactcctttc ggtaagtgca gtggaagctg 960

1020

gacttagccc ctgttgctc ctccgataac tggggtgacc ttggttaata ttcaccagca 1080

1140

tcctcagctt caggcaccac cactgacctg ggacagt 1177

<210> 75

<400> 75

000

<210> 76

<400> 76

000

<210> 77

<400> 77

000

<210> 78

<400> 78

000

<210> 79

<400> 79

000

<210> 80

<400> 80

000

<210> 81

<400> 81

000

<210> 82

<400> 82

000

<210> 83

<400> 83

000

<210> 84

<400> 84

000

<210> 85

<400> 85

000

<210> 86

<400> 86

000

<210> 87

<400> 87

000

<210> 88

<400> 88

000

<210> 89

<400> 89

000

<210> 90

<400> 90

000

<210> 91

<400> 91

000

<210> 92

<400> 92

000

<210> 93

<400> 93

000

<210> 94

<400> 94

000

<210> 95

<400> 95

000

<210> 96

<400> 96

000

<210> 97

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 97

gcgcgctcgc tcgctcactg aggccggggcg accaaaggtc gcccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 98

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 98

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacggc ctcagtgagc 60

gagcgagcgc gc 72

<210> 99

<211> 122

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 99

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacggcctc agtgagcgag cgagcgcgca gctgcctgca 120

gg 122

<210> 100

<211> 130

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 100

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc 120

tgcctgcagg 130

<210> 101

<211> 70

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 101

gcgcgctcgc tcgctcactg aggccgcccg ggaaacccgg gcgtgcgcct cagtgagcga 60

gcgagcgcgc 70

<210> 102

<211> 70

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 102

gcgcgctcgc tcgctcactg aggcgcacgc ccgggtttcc cgggcggcct cagtgagcga 60

gcgagcgcgc 70

<210> 103

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 103

gcgcgctcgc tcgctcactg aggccgtcgg gcgacctttg gtcgcccggc ctcagtgagc 60

gagcgagcgc gc 72

<210> 104

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 104

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacggc ctcagtgagc 60

gagcgagcgc gc 72

<210> 105

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 105

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagcc gggcgtcggc ctcagtgagc 60

gagcgagcgc gc 72

<210> 106

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 106

gcgcgctcgc tcgctcactg aggccgacgc ccgggctttg cccgggcggc ctcagtgagc 60

gagcgagcgc gc 72

<210> 107

<211> 83

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 107

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg ctttgccgg 60

cctcagtgag cgagcgagcg cgc 83

<210> 108

<211> 83

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 108

60

cctcagtgag cgagcgagcg cgc 83

<210> 109

<211> 77

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 109

gcgcgctcgc tcgctcactg aggccgaaac gtcgggcgac ctttggtcgc ccggcctcag 60

tgagcgagcg agcgcgc 77

<210> 110

<211> 77

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 110

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgtt tcggcctcag 60

tgagcgagcg agcgcgc 77

<210> 111

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 111

gcgcgctcgc tcgctcactg aggcaaagcc tcagtgagcg agcgagcgcg c 51

<210> 112

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 112

gcgcgctcgc tcgctcactg aggctttgcc tcagtgagcg agcgagcgcg c 51

<210> 113

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 113

gcgcgctcgc tcgctcactg aggccgcccg ggcgtcggggc gacctttggt cgcccggcct 60

cagtgagcga gcgagcgcgc 80

<210> 114

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 114

gcgcgctcgc tcgctcactg aggccggggcg accaaaggtc gcccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 115

<211> 77

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 115

gcgcgctcgc tcgctcactg aggcgcccgg gcgtcgggcg acctttggtc gcccggcctc 60

agtgagcgag cgagcgc 77

<210> 116

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 116

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcgcctc 60

agtgagcgag cgagcgcgc 79

<210> 117

<211> 89

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 117

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgactttgtc 60

gcccggcctc agtgagcgag cgagcgcgc 89

<210> 118

<211> 89

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 118

gcgcgctcgc tcgctcactg aggccgggcg acaaagtcgc ccgacgcccg ggctttgccc 60

gggcggcctc agtgagcgag cgagcgcgc 89

<210> 119

<211> 87

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 119

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagcc gggcgtcggg cgattttcgc 60

ccggcctcag tgagcgagcg agcgcgc 87

<210> 120

<211> 87

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 120

60

gcggcctcag tgagcgagcg agcgcgc 87

<210> 121

<211> 85

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 121

60

ggcctcagtg agcgagcgag cgcgc 85

<210> 122

<211> 85

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 122

60

ggcctcagtg agcgagcgag cgcgc 85

<210> 123

<211> 89

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 123

gcgcgctcgc tcgctcactg aggccgcccg ggaaacccgg gcgtcgggcg acctttggtc 60

gcccggcctc agtgagcgag cgagcgcgc 89

<210> 124

<211> 89

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 124

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggtttccc 60

gggcggcctc agtgagcgag cgagcgcgc 89

<210> 125

<211> 87

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 125

gcgcgctcgc tcgctcactg aggccgcccg gaaaccgggc gtcgggcgac ctttggtcgc 60

ccggcctcag tgagcgagcg agcgcgc 87

<210> 126

<211> 87

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 126

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cggtttccgg 60

gcggcctcag tgagcgagcg agcgcgc 87

<210> 127

<211> 85

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 127

gcgcgctcgc tcgctcactg aggccgcccg aaacgggcgt cgggcgacct ttggtcgccc 60

ggcctcagtg agcgagcgag cgcgc 85

<210> 128

<211> 85

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 128

60

ggcctcagtg agcgagcgag cgcgc 85

<210> 129

<211> 83

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 129

gcgcgctcgc tcgctcactg aggccgccca aagggcgtcg ggcgaccttt ggtcgcccgg 60

cctcagtgag cgagcgagcg cgc 83

<210> 130

<211> 83

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 130

60

cctcagtgag cgagcgagcg cgc 83

<210> 131

<211> 81

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 131

gcgcgctcgc tcgctcactg aggccgccaa aggcgtcggg cgacctttgg tcgcccggcc 60

tcagtgagcg agcgagcgcg c 81

<210> 132

<211> 81

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 132

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc tttggcggcc 60

tcagtgagcg agcgagcgcg c 81

<210> 133

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 133

gcgcgctcgc tcgctcactg aggccgcaaa gcgtcgggcg acctttggtc gcccggcctc 60

agtgagcgag cgagcgcgc 79

<210> 134

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 134

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgct ttgcggcctc 60

agtgagcgag cgagcgcgc 79

<210> 135

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 135

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagcc cgggctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 136

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 136

gcgcgctcgc tcgctcactg aggccggggcg accaaaggtc gcccgacacc cgggtggcct 60

cagtgagcga gcgagcgcgc 80

<210> 137

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 137

gcgcgctcgc tcgctcactg aggccggggcg accaaaggtc gcccgacgac cggtcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 138

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 138

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgca cgtgcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 139

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 139

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc atggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 140

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 140

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagac cggtctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 141

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 141

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacaca cgtgtggcct 60

cagtgagcga gcgagcgcgc 80

<210> 142

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 142

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgac atgtcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 143

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 143

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagca cgtgctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 144

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 144

gcgcgctcgc tcgctcactg aggccggggcg accaaaggtc gcccgaaacc cgggttgcct 60

cagtgagcga gcgagcgcgc 80

<210> 145

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 145

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagcc atggctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 146

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 146

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacaac cggttggcct 60

cagtgagcga gcgagcgcgc 80

<210> 147

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 147

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacacc atggtggcct 60

cagtgagcga gcgagcgcgc 80

<210> 148

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 148

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgaa cgttcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 149

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 149

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgca attgcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 150

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 150

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaaaac cggtttgcct 60

cagtgagcga gcgagcgcgc 80

<210> 151

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 151

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagaa cgttctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 152

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 152

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagca attgctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 153

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 153

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacaaa cgtttggcct 60

cagtgagcga gcgagcgcgc 80

<210> 154

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 154

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgaa atttcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 155

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 155

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagca attgctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 156

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 156

gcgcgctcgc tcgctcactg aggccggggcg accaaaggtc gcccgaaacc atggttgcct 60

cagtgagcga gcgagcgcgc 80

<210> 157

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 157

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacaac atgttggcct 60

cagtgagcga gcgagcgcgc 80

<210> 158

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 158

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagac atgtctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 159

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 159

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacaca attgtggcct 60

cagtgagcga gcgagcgcgc 80

<210> 160

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 160

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaaaaa cgttttgcct 60

cagtgagcga gcgagcgcgc 80

<210> 161

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 161

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaaaac atgtttgcct 60

cagtgagcga gcgagcgcgc 80

<210> 162

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 162

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaaaca attgttgcct 60

cagtgagcga gcgagcgcgc 80

<210> 163

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 163

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagaa atttctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 164

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 164

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacaaa attttggcct 60

cagtgagcga gcgagcgcgc 80

<210> 165

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 165

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaaaaa atttttgcct 60

cagtgagcga gcgagcgcgc 80

<210> 166

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 166

gcgcgctcgc tcgctcgctg aggccggggcg accaaaggtc gcccgacgcc cgggcggcct 60

cagcgagcga gcgagcgcgc 80

<210> 167

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 167

gcgcgctcgc tcgctcaatg aggccggggcg accaaaggtc gcccgacgcc cgggcggcct 60

cattgagcga gcgagcgcgc 80

<210> 168

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 168

gcgcgctcgc tcgctcaccg aggccggggcg accaaaggtc gcccgacgcc cgggcggcct 60

cggtgagcga gcgagcgcgc 80

<210> 169

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 169

gcgcgctcgc tcgctcacta aggccggggcg accaaaggtc gcccgacgcc cgggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 170

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 170

gcgcgctcgc tcgctcactg gggccgggcg accaaaggtc gcccgacgcc cgggcggccc 60

cagtgagcga gcgagcgcgc 80

<210> 171

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 171

gcgcgctcgc tcgctcactg aagccgggcg accaaaggtc gcccgacgcc cgggcggctt 60

cagtgagcga gcgagcgcgc 80

<210> 172

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 172

gcgcgctcgc tcgctcactg agaccgggcg accaaaggtc gcccgacgcc cgggcggtct 60

cagtgagcga gcgagcgcgc 80

<210> 173

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 173

gcgcgctcgc tcgctcgatg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 174

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 174

60

ccgcgagcga gcgagcgcgc 80

<210> 175

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 175

gcgcgctcgc tcgctcgcta aggccgggcg accaaaggtc gcccgacgcc cgggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 176

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 176

60

cagcgagcga gcgagcgcgc 80

<210> 177

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 177

gcgcgctcgc tcgctcgctg aagccgggcg accaaaggtc gcccgacgcc cgggcggctt 60

cagcgagcga gcgagcgcgc 80

<210> 178

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 178

60

cagcgagcga gcgagcgcgc 80

<210> 179

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 179

gcgcgctcgc tcgctcgagg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct 60

cctcgagcga gcgagcgcgc 80

<210> 180

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 180

gcgcgctcgc tcgctcgata aggccggggcg accaaaggtc gcccgacgcc cgggcggcct 60

tatcgagcga gcgagcgcgc 80

<210> 181

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 181

gcgcgctcgc tcgctcgatg gggccggggcg accaaaggtc gcccgacgcc cgggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 182

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 182

gcgcgctcgc tcgctcgatg aagccgggcg accaaaggtc gcccgacgcc cgggcggctt 60

catcgagcga gcgagcgcgc 80

<210> 183

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 183

60

catcgagcga gcgagcgcgc 80

<210> 184

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 184

gcgcgctcgc tcgctcgaga aggccggggcg accaaaggtc gcccgacgcc cgggcggcct 60

tctcgagcga gcgagcgcgc 80

<210> 185

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 185

gcgcgctcgc tcgctcgagg gggccgggcg accaaaggtc gcccgacgcc cgggcggccc 60

cctcgagcga gcgagcgcgc 80

<210> 186

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 186

gcgcgctcgc tcgctcgagg aagccgggcg accaaaggtc gcccgacgcc cgggcggctt 60

cctcgagcga gcgagcgcgc 80

<210> 187

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 187

gcgcgctcgc tcgctcgagg gggccgggcg accaaaggtc gcccgacgcc cgggcggccc 60

cctcgagcga gcgagcgcgc 80

<210> 188

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 188

gcgcgctcgc tcgctcgagg aagccgggcg accaaaggtc gcccgacgcc cgggcggctt 60

cctcgagcga gcgagcgcgc 80

<210> 189

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 189

gcgcgctcgc tcgctcgagg agaccgggcg accaaaggtc gcccgacgcc cgggcggtct 60

cctcgagcga gcgagcgcgc 80

<210> 190

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 190

gcgcgctcgc tcgctcgaga gggccgggcg accaaaggtc gcccgacgcc cgggcggccc 60

tctcgagcga gcgagcgcgc 80

<210> 191

<400> 191

000

<210> 192

<400> 192

000

<210> 193

<400> 193

000

<210> 194

<400> 194

000

<210> 195

<400> 195

000

<210> 196

<400> 196

000

<210> 197

<400> 197

000

<210> 198

<400> 198

000

<210> 199

<400> 199

000

<210> 200

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 200

gcgcgctcgc tcgctcgaga aagccgggcg accaaaggtc gcccgacgcc cgggcggctt 60

tctcgagcga gcgagcgcgc 80

<210> 201

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 201

60

tctcgagcga gcgagcgcgc 80

<210> 202

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 202

gcgcgctcgc tcgctcgaga gagccgggcg accaaaggtc gcccgacgcc cgggcggctc 60

tctcgagcga gcgagcgcgc 80

<210> 203

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 203

gcgcgctcgc tcgctcgaga ggaccgggcg accaaaggtc gcccgacgcc cgggcggtcc 60

tctcgagcga gcgagcgcgc 80

<210> 204

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 204

60

tctcgagcga gcgagcgcgc 80

<210> 205

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 205

gcgcgctcgc tcgctcaaga gaaccgggcg accaaaggtc gcccgacgcc cgggcggttc 60

tcttgagcga gcgagcgcgc 80

<210> 206

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 206

gcgcgctcgc tcgctcacga gaaccgggcg accaaaggtc gcccgacgcc cgggcggttc 60

tcgtgagcga gcgagcgcgc 80

<210> 207

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 207

gcgcgctcgc tcgctcacta gaaccgggcg accaaaggtc gcccgacgcc cgggcggttc 60

tagtgagcga gcgagcgcgc 80

<210> 208

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 208

gcgcgctcgc tcgctcactg gaaccgggcg accaaaggtc gccgacgcc cgggcggttc 60

cagtgagcga gcgagcgcgc 80

<210> 209

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 209

60

cagtgagcga gcgagcgcgc 80

<210> 210

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 210

gcgcgctcgc tcgctcgcgg gggccgggcg accaaaggtc gcccgacgcc cgggcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 211

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 211

60

tattgagcga gcgagcgcgc 80

<210> 212

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 212

gcgcgctcgc tcgctcactg aggccaggcg accaaaggtc gcctgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 213

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 213

gcgcgctcgc tcgctcactg aggccgagcg accaaaggtc gctcgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 214

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 214

gcgcgctcgc tcgctcactg aggccggacg accaaaggtc gtccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 215

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 215

gcgcgctcgc tcgctcactg aggccgggag accaaaggtc tcccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 216

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 216

gcgcgctcgc tcgctcactg aggccggggca accaaaggtt gcccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 217

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 217

gcgcgctcgc tcgctcactg aggccggggcg gccaaaggcc gcccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 218

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 218

gcgcgctcgc tcgctcactg aggccgggcg aacaaagttc gcccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 219

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 219

gcgcgctcgc tcgctcactg aggccgggcg acaaaatgtc gcccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 220

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 220

gcgcgctcgc tcgctcactg aggccaagcg accaaaggtc gcttgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 221

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 221

gcgcgctcgc tcgctcactg aggccaaacg accaaaggtc gtttgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 222

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 222

gcgcgctcgc tcgctcactg aggccaaaag accaaaggtc ttttgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 223

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 223

gcgcgctcgc tcgctcactg aggccaaaaa accaaaggtt ttttgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 224

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 224

gcgcgctcgc tcgctcactg aggccaaaaa gccaaaggct ttttgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 225

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 225

gcgcgctcgc tcgctcactg aggccaaaaa gacaaagtct ttttgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 226

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 226

gcgcgctcgc tcgctcactg aggccaaaaa gaaaaattct ttttgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 227

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 227

gcgcgctcgc tcgctcactg aggccaaaaa aaaaaattttt ttttgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 228

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 228

gcgcgctcgc tcgctcactg aggccgaaaa aaaaaattttt tttcgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 229

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 229

gcgcgctcgc tcgctcactg aggccggaaa gaaaaattct ttccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 230

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 230

gcgcgctcgc tcgctcactg aggccgggaa gaaaaattct tcccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 231

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 231

gcgcgctcgc tcgctcactg aggccggggca gaaaaattct gcccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 232

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 232

gcgcgctcgc tcgctcactg aggccggggcg gaaaaattcc gcccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 233

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 233

gcgcgctcgc tcgctcactg aggccgggcg aaaaaatttc gcccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 234

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 234

gcgcgctcgc tcgctcactg aggccaggcg accaaaggtc gcctgacacc cgggtggcct 60

cagtgagcga gcgagcgcgc 80

<210> 235

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 235

gcgcgctcgc tcgctcactg aggccaggcg accaaaggtc gcctgacgcc atggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 236

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 236

gcgcgctcgc tcgctcactg aggccaggcg accaaaggtc gcctgacgac atgtcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 237

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 237

gcgcgctcgc tcgctcactg aggccaggcg accaaaggtc gcctgacgaa cgttcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 238

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 238

gcgcgctcgc tcgctcactg aggccaggcg accaaaggtc gcctgaagca attgctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 239

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 239

gcgcgctcgc tcgctcactg aggccggggcg gccaaaggcc gcccgacacc cgggtggcct 60

cagtgagcga gcgagcgcgc 80

<210> 240

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 240

gcgcgctcgc tcgctcactg aggccggggcg gccaaaggcc gcccgacgcc atggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 241

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 241

gcgcgctcgc tcgctcactg aggccggggcg gccaaaggcc gcccgacgac atgtcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 242

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 242

gcgcgctcgc tcgctcactg aggccgggcg gccaaaggcc gcccgacgaa cgttcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 243

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 243

gcgcgctcgc tcgctcactg aggccgggcg gccaaaggcc gcccgaagca attgctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 244

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 244

gcgcgctcgc tcgctcactg aggccgggcg acaaaatgtc gcccgacacc cgggtggcct 60

cagtgagcga gcgagcgcgc 80

<210> 245

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 245

gcgcgctcgc tcgctcactg aggccgggcg acaaaatgtc gcccgacgcc atggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 246

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 246

gcgcgctcgc tcgctcactg aggccgggcg acaaaatgtc gcccgacgac atgtcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 247

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 247

gcgcgctcgc tcgctcactg aggccgggcg acaaaatgtc gcccgacgaa cgttcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 248

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 248

gcgcgctcgc tcgctcactg aggccgggcg acaaaatgtc gcccgaagca attgctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 249

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 249

gcgcgctcgc tcgctcactg aggccaaaag accaaaggtc ttttgacacc cgggtggcct 60

cagtgagcga gcgagcgcgc 80

<210> 250

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 250

gcgcgctcgc tcgctcactg aggccaaaag accaaaggtc ttttgacgcc atggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 251

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 251

gcgcgctcgc tcgctcactg aggccaaaag accaaaggtc ttttgacgac atgtcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 252

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 252

gcgcgctcgc tcgctcactg aggccaaaag accaaaggtc ttttgacgaa cgttcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 253

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 253

gcgcgctcgc tcgctcactg aggccaaaag accaaaggtc ttttgaagca attgctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 254

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 254

gcgcgctcgc tcgctcactg aggccaaaaa aaaaaattttt ttttgacacc cgggtggcct 60

cagtgagcga gcgagcgcgc 80

<210> 255

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 255

gcgcgctcgc tcgctcactg aggccaaaaa aaaaaattttt ttttgacgcc atggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 256

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 256

gcgcgctcgc tcgctcactg aggccaaaaa aaaaaattttt ttttgacgac atgtcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 257

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 257

gcgcgctcgc tcgctcactg aggccaaaaa aaaaaattttt ttttgacgaa cgttcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 258

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 258

gcgcgctcgc tcgctcactg aggccaaaaa aaaaaattttt ttttgaagca attgctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 259

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 259

gcgcgctcgc tcgctcactg aggccggggca gaaaaattct gcccgacacc cgggtggcct 60

cagtgagcga gcgagcgcgc 80

<210> 260

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 260

gcgcgctcgc tcgctcactg aggccggggca gaaaaattct gcccgacgcc atggcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 261

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 261

gcgcgctcgc tcgctcactg aggccggggca gaaaaattct gcccgacgac atgtcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 262

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 262

gcgcgctcgc tcgctcactg aggccggggca gaaaaattct gcccgacgaa cgttcggcct 60

cagtgagcga gcgagcgcgc 80

<210> 263

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 263

gcgcgctcgc tcgctcactg aggccggggca gaaaaattct gcccgaagca attgctgcct 60

cagtgagcga gcgagcgcgc 80

<210> 264

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 264

gcgcgctcgc tcgctcacta aggccaggcg accaaaggtc gcctgacgcc cgggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 265

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 265

gcgcgctcgc tcgctcacta aggccggggcg gccaaaggcc gcccgacgcc cgggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 266

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 266

gcgcgctcgc tcgctcacta aggccgggcg acaaaatgtc gcccgacgcc cgggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 267

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 267

gcgcgctcgc tcgctcacta aggccaaaag accaaaggtc ttttgacgcc cgggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 268

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 268

gcgcgctcgc tcgctcacta aggccaaaaa aaaaaattttt ttttgacgcc cgggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 269

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 269

gcgcgctcgc tcgctcacta aggccggggca gaaaaattct gcccgacgcc cgggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 270

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 270

gcgcgctcgc tcgctcgatg gggccaggcg accaaaggtc gcctgacgcc cgggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 271

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 271

gcgcgctcgc tcgctcgatg gggccggggcg gccaaaggcc gcccgacgcc cgggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 272

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 272

gcgcgctcgc tcgctcgatg gggccgggcg acaaaatgtc gcccgacgcc cgggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 273

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 273

gcgcgctcgc tcgctcgatg gggccaaaag accaaaggtc ttttgacgcc cgggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 274

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 274

gcgcgctcgc tcgctcgatg gggccaaaaa aaaaaattttt ttttgacgcc cgggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 275

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 275

gcgcgctcgc tcgctcgatg gggccggggca gaaaaattct gcccgacgcc cgggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 276

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 276

gcgcgctcgc tcgctcgaga gaaccaggcg accaaaggtc gcctgacgcc cgggcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 277

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 277

gcgcgctcgc tcgctcgaga gaaccgggcg gccaaaggcc gcccgacgcc cgggcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 278

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 278

gcgcgctcgc tcgctcgaga gaaccgggcg acaaaatgtc gcccgacgcc cgggcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 279

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 279

gcgcgctcgc tcgctcgaga gaaccaaaag accaaaggtc ttttgacgcc cgggcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 280

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 280

gcgcgctcgc tcgctcgaga gaaccaaaaa aaaaaattttt ttttgacgcc cgggcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 281

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 281

gcgcgctcgc tcgctcgaga gaaccgggca gaaaaattct gcccgacgcc cgggcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 282

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 282

gcgcgctcgc tcgctcgcgg gggccaggcg accaaaggtc gcctgacgcc cgggcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 283

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 283

gcgcgctcgc tcgctcgcgg gggccgggcg gccaaaggcc gcccgacgcc cgggcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 284

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 284

60

ccgcgagcga gcgagcgcgc 80

<210> 285

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 285

gcgcgctcgc tcgctcgcgg gggccaaaag accaaaggtc ttttgacgcc cgggcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 286

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 286

gcgcgctcgc tcgctcgcgg gggccaaaaa aaaaaatttt ttttgacgcc cgggcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 287

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 287

gcgcgctcgc tcgctcgcgg gggccggggca gaaaaattct gcccgacgcc cgggcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 288

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 288

gcgcgctcgc tcgctcaata aaaccaggcg accaaaggtc gcctgacgcc cgggcggttt 60

tattgagcga gcgagcgcgc 80

<210> 289

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 289

60

tattgagcga gcgagcgcgc 80

<210> 290

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 290

60

tattgagcga gcgagcgcgc 80

<210> 291

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 291

gcgcgctcgc tcgctcaata aaaccaaaag accaaaggtc ttttgacgcc cgggcggttt 60

tattgagcga gcgagcgcgc 80

<210> 292

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 292

60

tattgagcga gcgagcgcgc 80

<210> 293

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 293

60

tattgagcga gcgagcgcgc 80

<210> 294

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 294

gcgcgctcgc tcgctcacta aggccggggcg accaaaggtc gcccgacacc cgggtggcct 60

tagtgagcga gcgagcgcgc 80

<210> 295

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 295

gcgcgctcgc tcgctcacta aggccgggcg accaaaggtc gcccgacgcc atggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 296

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 296

gcgcgctcgc tcgctcacta aggccgggcg accaaaggtc gcccgacgac atgtcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 297

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 297

gcgcgctcgc tcgctcacta aggccggggcg accaaaggtc gcccgacgaa cgttcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 298

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 298

gcgcgctcgc tcgctcacta aggccgggcg accaaaggtc gcccgaagca attgctgcct 60

tagtgagcga gcgagcgcgc 80

<210> 299

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 299

gcgcgctcgc tcgctcgatg gggccgggcg accaaaggtc gcccgacacc cgggtggcct 60

catcgagcga gcgagcgcgc 80

<210> 300

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 300

gcgcgctcgc tcgctcgatg gggccgggcg accaaaggtc gcccgacgcc atggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 301

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 301

gcgcgctcgc tcgctcgatg gggccgggcg accaaaggtc gcccgacgac atgtcggcct 60

catcgagcga gcgagcgcgc 80

<210> 302

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 302

gcgcgctcgc tcgctcgatg gggccgggcg accaaaggtc gcccgacgaa cgttcggcct 60

catcgagcga gcgagcgcgc 80

<210> 303

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 303

gcgcgctcgc tcgctcgatg gggccgggcg accaaaggtc gcccgaagca attgctgcct 60

catcgagcga gcgagcgcgc 80

<210> 304

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 304

gcgcgctcgc tcgctcgaga gaaccgggcg accaaaggtc gcccgacacc cgggtggttc 60

tctcgagcga gcgagcgcgc 80

<210> 305

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 305

gcgcgctcgc tcgctcgaga gaaccgggcg accaaaggtc gcccgacgcc atggcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 306

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 306

gcgcgctcgc tcgctcgaga gaaccgggcg accaaaggtc gcccgacgac atgtcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 307

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 307

gcgcgctcgc tcgctcgaga gaaccgggcg accaaaggtc gcccgacgaa cgttcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 308

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 308

gcgcgctcgc tcgctcgaga gaaccgggcg accaaaggtc gcccgaagca attgctgttc 60

tctcgagcga gcgagcgcgc 80

<210> 309

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 309

gcgcgctcgc tcgctcgcgg gggccgggcg accaaaggtc gcccgacacc cgggtggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 310

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 310

gcgcgctcgc tcgctcgcgg gggccgggcg accaaaggtc gcccgacgcc atggcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 311

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 311

gcgcgctcgc tcgctcgcgg gggccgggcg accaaaggtc gcccgacgac atgtcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 312

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 312

gcgcgctcgc tcgctcgcgg gggccgggcg accaaaggtc gcccgacgaa cgttcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 313

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 313

gcgcgctcgc tcgctcgcgg gggccgggcg accaaaggtc gcccgaagca attgctgccc 60

ccgcgagcga gcgagcgcgc 80

<210> 314

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 314

60

tattgagcga gcgagcgcgc 80

<210> 315

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 315

60

tattgagcga gcgagcgcgc 80

<210> 316

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 316

60

tattgagcga gcgagcgcgc 80

<210> 317

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 317

60

tattgagcga gcgagcgcgc 80

<210> 318

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 318

gcgcgctcgc tcgctcaata aaaccgggcg accaaaggtc gcccgaagca attgctgttt 60

tattgagcga gcgagcgcgc 80

<210> 319

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 319

gcgcgctcgc tcgctcacta aggccaggcg accaaaggtc gcctgacacc cgggtggcct 60

tagtgagcga gcgagcgcgc 80

<210> 320

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 320

gcgcgctcgc tcgctcacta aggccaggcg accaaaggtc gcctgacgcc atggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 321

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 321

gcgcgctcgc tcgctcacta aggccaggcg accaaaggtc gcctgacgac atgtcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 322

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 322

gcgcgctcgc tcgctcacta aggccaggcg accaaaggtc gcctgacgaa cgttcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 323

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 323

gcgcgctcgc tcgctcacta aggccaggcg accaaaggtc gcctgaagca attgctgcct 60

tagtgagcga gcgagcgcgc 80

<210> 324

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 324

gcgcgctcgc tcgctcacta aggccggggcg gccaaaggcc gcccgacacc cgggtggcct 60

tagtgagcga gcgagcgcgc 80

<210> 325

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 325

gcgcgctcgc tcgctcacta aggccgggcg gccaaaggcc gcccgacgcc atggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 326

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 326

gcgcgctcgc tcgctcacta aggccgggcg gccaaaggcc gcccgacgac atgtcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 327

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 327

gcgcgctcgc tcgctcacta aggccgggcg gccaaaggcc gcccgacgaa cgttcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 328

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 328

gcgcgctcgc tcgctcacta aggccgggcg gccaaaggcc gcccgaagca attgctgcct 60

tagtgagcga gcgagcgcgc 80

<210> 329

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 329

gcgcgctcgc tcgctcacta aggccgggcg acaaaatgtc gcccgacacc cgggtggcct 60

tagtgagcga gcgagcgcgc 80

<210> 330

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 330

gcgcgctcgc tcgctcacta aggccgggcg acaaaatgtc gcccgacgcc atggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 331

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 331

gcgcgctcgc tcgctcacta aggccgggcg acaaaatgtc gcccgacgac atgtcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 332

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 332

gcgcgctcgc tcgctcacta aggccgggcg acaaaatgtc gcccgacgaa cgttcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 333

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 333

gcgcgctcgc tcgctcacta aggccgggcg acaaaatgtc gcccgaagca attgctgcct 60

tagtgagcga gcgagcgcgc 80

<210> 334

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 334

gcgcgctcgc tcgctcacta aggccaaaag accaaaggtc ttttgacacc cgggtggcct 60

tagtgagcga gcgagcgcgc 80

<210> 335

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 335

gcgcgctcgc tcgctcacta aggccaaaag accaaaggtc ttttgacgcc atggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 336

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 336

gcgcgctcgc tcgctcacta aggccaaaag accaaaggtc ttttgacgac atgtcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 337

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 337

gcgcgctcgc tcgctcacta aggccaaaag accaaaggtc ttttgacgaa cgttcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 338

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 338

gcgcgctcgc tcgctcacta aggccaaaag accaaaggtc ttttgaagca attgctgcct 60

tagtgagcga gcgagcgcgc 80

<210> 339

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 339

gcgcgctcgc tcgctcacta aggccaaaaa aaaaaattttt ttttgacacc cgggtggcct 60

tagtgagcga gcgagcgcgc 80

<210> 340

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 340

gcgcgctcgc tcgctcacta aggccaaaaa aaaaaattttt ttttgacgcc atggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 341

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 341

gcgcgctcgc tcgctcacta aggccaaaaa aaaaaattttt ttttgacgac atgtcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 342

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 342

gcgcgctcgc tcgctcacta aggccaaaaa aaaaaattttt ttttgacgaa cgttcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 343

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 343

gcgcgctcgc tcgctcacta aggccaaaaa aaaaaattttt ttttgaagca attgctgcct 60

tagtgagcga gcgagcgcgc 80

<210> 344

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 344

gcgcgctcgc tcgctcacta aggccggggca gaaaaattct gcccgacacc cgggtggcct 60

tagtgagcga gcgagcgcgc 80

<210> 345

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 345

gcgcgctcgc tcgctcacta aggccggggca gaaaaattct gcccgacgcc atggcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 346

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 346

gcgcgctcgc tcgctcacta aggccggggca gaaaaattct gcccgacgac atgtcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 347

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 347

gcgcgctcgc tcgctcacta aggccggggca gaaaaattct gcccgacgaa cgttcggcct 60

tagtgagcga gcgagcgcgc 80

<210> 348

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 348

gcgcgctcgc tcgctcacta aggccgggca gaaaaattct gcccgaagca attgctgcct 60

tagtgagcga gcgagcgcgc 80

<210> 349

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 349

gcgcgctcgc tcgctcgatg gggccaggcg accaaaggtc gcctgacacc cgggtggcct 60

catcgagcga gcgagcgcgc 80

<210> 350

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 350

gcgcgctcgc tcgctcgatg gggccaggcg accaaaggtc gcctgacgcc atggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 351

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 351

gcgcgctcgc tcgctcgatg gggccaggcg accaaaggtc gcctgacgac atgtcggcct 60

catcgagcga gcgagcgcgc 80

<210> 352

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 352

gcgcgctcgc tcgctcgatg gggccaggcg accaaaggtc gcctgacgaa cgttcggcct 60

catcgagcga gcgagcgcgc 80

<210> 353

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 353

gcgcgctcgc tcgctcgatg gggccaggcg accaaaggtc gcctgaagca attgctgcct 60

catcgagcga gcgagcgcgc 80

<210> 354

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 354

gcgcgctcgc tcgctcgatg gggccggggcg gccaaaggcc gcccgacacc cgggtggcct 60

catcgagcga gcgagcgcgc 80

<210> 355

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 355

gcgcgctcgc tcgctcgatg gggccgggcg gccaaaggcc gcccgacgcc atggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 356

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 356

gcgcgctcgc tcgctcgatg gggccggggcg gccaaaggcc gcccgacgac atgtcggcct 60

catcgagcga gcgagcgcgc 80

<210> 357

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 357

gcgcgctcgc tcgctcgatg gggccgggcg gccaaaggcc gcccgacgaa cgttcggcct 60

catcgagcga gcgagcgcgc 80

<210> 358

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 358

gcgcgctcgc tcgctcgatg gggccgggcg gccaaaggcc gcccgaagca attgctgcct 60

catcgagcga gcgagcgcgc 80

<210> 359

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 359

gcgcgctcgc tcgctcgatg gggccgggcg acaaaatgtc gcccgacacc cgggtggcct 60

catcgagcga gcgagcgcgc 80

<210> 360

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 360

gcgcgctcgc tcgctcgatg gggccgggcg acaaaatgtc gcccgacgcc atggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 361

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 361

gcgcgctcgc tcgctcgatg gggccgggcg acaaaatgtc gcccgacgac atgtcggcct 60

catcgagcga gcgagcgcgc 80

<210> 362

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 362

gcgcgctcgc tcgctcgatg gggccgggcg acaaaatgtc gcccgacgaa cgttcggcct 60

catcgagcga gcgagcgcgc 80

<210> 363

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 363

gcgcgctcgc tcgctcgatg gggccgggcg acaaaatgtc gcccgaagca attgctgcct 60

catcgagcga gcgagcgcgc 80

<210> 364

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 364

gcgcgctcgc tcgctcgatg gggccaaaag accaaaggtc ttttgacacc cgggtggcct 60

catcgagcga gcgagcgcgc 80

<210> 365

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 365

gcgcgctcgc tcgctcgatg gggccaaaag accaaaggtc ttttgacgcc atggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 366

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 366

gcgcgctcgc tcgctcgatg gggccaaaag accaaaggtc ttttgacgac atgtcggcct 60

catcgagcga gcgagcgcgc 80

<210> 367

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 367

gcgcgctcgc tcgctcgatg gggccaaaag accaaaggtc ttttgacgaa cgttcggcct 60

catcgagcga gcgagcgcgc 80

<210> 368

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 368

gcgcgctcgc tcgctcgatg gggccaaaag accaaaggtc ttttgaagca attgctgcct 60

catcgagcga gcgagcgcgc 80

<210> 369

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 369

gcgcgctcgc tcgctcgatg gggccaaaaa aaaaaattttt ttttgacacc cgggtggcct 60

catcgagcga gcgagcgcgc 80

<210> 370

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 370

gcgcgctcgc tcgctcgatg gggccaaaaa aaaaaattttt ttttgacgcc atggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 371

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 371

gcgcgctcgc tcgctcgatg gggccaaaaa aaaaaattttt ttttgacgac atgtcggcct 60

catcgagcga gcgagcgcgc 80

<210> 372

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 372

gcgcgctcgc tcgctcgatg gggccaaaaa aaaaaattttt ttttgacgaa cgttcggcct 60

catcgagcga gcgagcgcgc 80

<210> 373

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 373

gcgcgctcgc tcgctcgatg gggccaaaaa aaaaaattttt ttttgaagca attgctgcct 60

catcgagcga gcgagcgcgc 80

<210> 374

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 374

gcgcgctcgc tcgctcgatg gggccggggca gaaaaattct gcccgacacc cgggtggcct 60

catcgagcga gcgagcgcgc 80

<210> 375

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 375

gcgcgctcgc tcgctcgatg gggccggggca gaaaaattct gcccgacgcc atggcggcct 60

catcgagcga gcgagcgcgc 80

<210> 376

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 376

gcgcgctcgc tcgctcgatg gggccgggca gaaaaattct gcccgacgac atgtcggcct 60

catcgagcga gcgagcgcgc 80

<210> 377

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 377

gcgcgctcgc tcgctcgatg gggccgggca gaaaaattct gcccgacgaa cgttcggcct 60

catcgagcga gcgagcgcgc 80

<210> 378

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 378

gcgcgctcgc tcgctcgatg gggccgggca gaaaaattct gcccgaagca attgctgcct 60

catcgagcga gcgagcgcgc 80

<210> 379

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 379

gcgcgctcgc tcgctcgaga gaaccaggcg accaaaggtc gcctgacacc cgggtggttc 60

tctcgagcga gcgagcgcgc 80

<210> 380

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 380

gcgcgctcgc tcgctcgaga gaaccaggcg accaaaggtc gcctgacgcc atggcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 381

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 381

gcgcgctcgc tcgctcgaga gaaccaggcg accaaaggtc gcctgacgac atgtcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 382

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 382

gcgcgctcgc tcgctcgaga gaaccaggcg accaaaggtc gcctgacgaa cgttcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 383

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 383

gcgcgctcgc tcgctcgaga gaaccaggcg accaaaggtc gcctgaagca attgctgttc 60

tctcgagcga gcgagcgcgc 80

<210> 384

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 384

gcgcgctcgc tcgctcgaga gaaccgggcg gccaaaggcc gcccgacacc cgggtggttc 60

tctcgagcga gcgagcgcgc 80

<210> 385

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 385

gcgcgctcgc tcgctcgaga gaaccgggcg gccaaaggcc gcccgacgcc atggcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 386

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 386

gcgcgctcgc tcgctcgaga gaaccgggcg gccaaaggcc gcccgacgac atgtcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 387

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 387

gcgcgctcgc tcgctcgaga gaaccgggcg gccaaaggcc gcccgacgaa cgttcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 388

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 388

gcgcgctcgc tcgctcgaga gaaccgggcg gccaaaggcc gcccgaagca attgctgttc 60

tctcgagcga gcgagcgcgc 80

<210> 389

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 389

gcgcgctcgc tcgctcgaga gaaccgggcg acaaaatgtc gcccgacacc cgggtggttc 60

tctcgagcga gcgagcgcgc 80

<210> 390

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 390

gcgcgctcgc tcgctcgaga gaaccgggcg acaaaatgtc gcccgacgcc atggcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 391

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 391

gcgcgctcgc tcgctcgaga gaaccgggcg acaaaatgtc gcccgacgac atgtcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 392

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 392

gcgcgctcgc tcgctcgaga gaaccgggcg acaaaatgtc gcccgacgaa cgttcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 393

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 393

gcgcgctcgc tcgctcgaga gaaccgggcg acaaaatgtc gcccgaagca attgctgttc 60

tctcgagcga gcgagcgcgc 80

<210> 394

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 394

gcgcgctcgc tcgctcgaga gaaccaaaag accaaaggtc ttttgacacc cgggtggttc 60

tctcgagcga gcgagcgcgc 80

<210> 395

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 395

gcgcgctcgc tcgctcgaga gaaccaaaag accaaaggtc ttttgacgcc atggcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 396

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 396

gcgcgctcgc tcgctcgaga gaaccaaaag accaaaggtc ttttgacgac atgtcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 397

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 397

gcgcgctcgc tcgctcgaga gaaccaaaag accaaaggtc ttttgacgaa cgttcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 398

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 398

gcgcgctcgc tcgctcgaga gaaccaaaag accaaaggtc ttttgaagca attgctgttc 60

tctcgagcga gcgagcgcgc 80

<210> 399

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 399

gcgcgctcgc tcgctcgaga gaaccaaaaa aaaaaattttt ttttgacacc cgggtggttc 60

tctcgagcga gcgagcgcgc 80

<210> 400

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 400

gcgcgctcgc tcgctcgaga gaaccaaaaa aaaaaattttt ttttgacgcc atggcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 401

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 401

gcgcgctcgc tcgctcgaga gaaccaaaaa aaaaaattttt ttttgacgac atgtcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 402

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 402

gcgcgctcgc tcgctcgaga gaaccaaaaa aaaaaattttt ttttgacgaa cgttcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 403

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 403

gcgcgctcgc tcgctcgaga gaaccaaaaa aaaaaattttt ttttgaagca attgctgttc 60

tctcgagcga gcgagcgcgc 80

<210> 404

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 404

gcgcgctcgc tcgctcgaga gaaccgggca gaaaaattct gcccgacacc cgggtggttc 60

tctcgagcga gcgagcgcgc 80

<210> 405

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 405

gcgcgctcgc tcgctcgaga gaaccgggca gaaaaattct gcccgacgcc atggcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 406

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 406

gcgcgctcgc tcgctcgaga gaaccgggca gaaaaattct gcccgacgac atgtcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 407

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 407

gcgcgctcgc tcgctcgaga gaaccgggca gaaaaattct gcccgacgaa cgttcggttc 60

tctcgagcga gcgagcgcgc 80

<210> 408

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 408

gcgcgctcgc tcgctcgaga gaaccgggca gaaaaattct gcccgaagca attgctgttc 60

tctcgagcga gcgagcgcgc 80

<210> 409

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 409

gcgcgctcgc tcgctcgcgg gggccaggcg accaaaggtc gcctgacacc cgggtggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 410

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 410

gcgcgctcgc tcgctcgcgg gggccaggcg accaaaggtc gcctgacgcc atggcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 411

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 411

gcgcgctcgc tcgctcgcgg gggccaggcg accaaaggtc gcctgacgac atgtcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 412

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 412

gcgcgctcgc tcgctcgcgg gggccaggcg accaaaggtc gcctgacgaa cgttcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 413

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 413

gcgcgctcgc tcgctcgcgg gggccaggcg accaaaggtc gcctgaagca attgctgccc 60

ccgcgagcga gcgagcgcgc 80

<210> 414

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 414

gcgcgctcgc tcgctcgcgg gggccgggcg gccaaaggcc gcccgacacc cgggtggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 415

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 415

gcgcgctcgc tcgctcgcgg gggccgggcg gccaaaggcc gcccgacgcc atggcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 416

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 416

gcgcgctcgc tcgctcgcgg gggccgggcg gccaaaggcc gcccgacgac atgtcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 417

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 417

gcgcgctcgc tcgctcgcgg gggccgggcg gccaaaggcc gcccgacgaa cgttcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 418

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 418

gcgcgctcgc tcgctcgcgg gggccgggcg gccaaaggcc gcccgaagca attgctgccc 60

ccgcgagcga gcgagcgcgc 80

<210> 419

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 419

gcgcgctcgc tcgctcgcgg gggccgggcg acaaaatgtc gcccgacacc cgggtggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 420

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 420

gcgcgctcgc tcgctcgcgg gggccgggcg acaaaatgtc gcccgacgcc atggcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 421

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 421

gcgcgctcgc tcgctcgcgg gggccgggcg acaaaatgtc gcccgacgac atgtcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 422

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 422

gcgcgctcgc tcgctcgcgg gggccgggcg acaaaatgtc gcccgacgaa cgttcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 423

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 423

gcgcgctcgc tcgctcgcgg gggccgggcg acaaaatgtc gcccgaagca attgctgccc 60

ccgcgagcga gcgagcgcgc 80

<210> 424

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 424

gcgcgctcgc tcgctcgcgg gggccaaaag accaaaggtc ttttgacacc cgggtggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 425

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 425

gcgcgctcgc tcgctcgcgg gggccaaaag accaaaggtc ttttgacgcc atggcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 426

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 426

gcgcgctcgc tcgctcgcgg gggccaaaag accaaaggtc ttttgacgac atgtcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 427

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 427

gcgcgctcgc tcgctcgcgg gggccaaaag accaaaggtc ttttgacgaa cgttcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 428

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 428

gcgcgctcgc tcgctcgcgg gggccaaaag accaaaggtc ttttgaagca attgctgccc 60

ccgcgagcga gcgagcgcgc 80

<210> 429

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 429

gcgcgctcgc tcgctcgcgg gggccaaaaa aaaaaattttt ttttgacacc cgggtggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 430

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 430

gcgcgctcgc tcgctcgcgg gggccaaaaa aaaaaatttt ttttgacgcc atggcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 431

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 431

gcgcgctcgc tcgctcgcgg gggccaaaaa aaaaaatttt ttttgacgac atgtcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 432

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 432

gcgcgctcgc tcgctcgcgg gggccaaaaa aaaaaattttt ttttgacgaa cgttcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 433

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 433

gcgcgctcgc tcgctcgcgg gggccaaaaa aaaaaatttt ttttgaagca attgctgccc 60

ccgcgagcga gcgagcgcgc 80

<210> 434

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 434

gcgcgctcgc tcgctcgcgg gggccggggca gaaaaattct gcccgacacc cgggtggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 435

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 435

gcgcgctcgc tcgctcgcgg gggccggggca gaaaaattct gcccgacgcc atggcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 436

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 436

gcgcgctcgc tcgctcgcgg gggccggggca gaaaaattct gcccgacgac atgtcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 437

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 437

gcgcgctcgc tcgctcgcgg gggccggggca gaaaaattct gcccgacgaa cgttcggccc 60

ccgcgagcga gcgagcgcgc 80

<210> 438

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 438

gcgcgctcgc tcgctcgcgg gggccgggca gaaaaattct gcccgaagca attgctgccc 60

ccgcgagcga gcgagcgcgc 80

<210> 439

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 439

gcgcgctcgc tcgctcaata aaaccaggcg accaaaggtc gcctgacacc cgggtggttt 60

tattgagcga gcgagcgcgc 80

<210> 440

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 440

gcgcgctcgc tcgctcaata aaaccaggcg accaaaggtc gcctgacgcc atggcggttt 60

tattgagcga gcgagcgcgc 80

<210> 441

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 441

gcgcgctcgc tcgctcaata aaaccaggcg accaaaggtc gcctgacgac atgtcggttt 60

tattgagcga gcgagcgcgc 80

<210> 442

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 442

gcgcgctcgc tcgctcaata aaaccaggcg accaaaggtc gcctgacgaa cgttcggttt 60

tattgagcga gcgagcgcgc 80

<210> 443

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 443

gcgcgctcgc tcgctcaata aaaccaggcg accaaaggtc gcctgaagca attgctgttt 60

tattgagcga gcgagcgcgc 80

<210> 444

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 444

60

tattgagcga gcgagcgcgc 80

<210> 445

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 445

gcgcgctcgc tcgctcaata aaaccgggcg gccaaaggcc gcccgacgcc atggcggttt 60

tattgagcga gcgagcgcgc 80

<210> 446

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 446

gcgcgctcgc tcgctcaata aaaccgggcg gccaaaggcc gcccgacgac atgtcggttt 60

tattgagcga gcgagcgcgc 80

<210> 447

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 447

60

tattgagcga gcgagcgcgc 80

<210> 448

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 448

gcgcgctcgc tcgctcaata aaaccgggcg gccaaaggcc gcccgaagca attgctgttt 60

tattgagcga gcgagcgcgc 80

<210> 449

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 449

60

tattgagcga gcgagcgcgc 80

<210> 450

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 450

60

tattgagcga gcgagcgcgc 80

<210> 451

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 451

60

tattgagcga gcgagcgcgc 80

<210> 452

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 452

60

tattgagcga gcgagcgcgc 80

<210> 453

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 453

60

tattgagcga gcgagcgcgc 80

<210> 454

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 454

gcgcgctcgc tcgctcaata aaaccaaaag accaaaggtc ttttgacacc cgggtggttt 60

tattgagcga gcgagcgcgc 80

<210> 455

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 455

gcgcgctcgc tcgctcaata aaaccaaaag accaaaggtc ttttgacgcc atggcggttt 60

tattgagcga gcgagcgcgc 80

<210> 456

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 456

gcgcgctcgc tcgctcaata aaaccaaaag accaaaggtc ttttgacgac atgtcggttt 60

tattgagcga gcgagcgcgc 80

<210> 457

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 457

gcgcgctcgc tcgctcaata aaaccaaaag accaaaggtc ttttgacgaa cgttcggttt 60

tattgagcga gcgagcgcgc 80

<210> 458

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 458

gcgcgctcgc tcgctcaata aaaccaaaag accaaaggtc ttttgaagca attgctgttt 60

tattgagcga gcgagcgcgc 80

<210> 459

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 459

gcgcgctcgc tcgctcaata aaaccaaaaa 60

tattgagcga gcgagcgcgc 80

<210> 460

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 460

60 gcgcgctcgc tcgctcaata aaaccaaaaa

tattgagcga gcgagcgcgc 80

<210> 461

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 461

60 gcgcgctcgc tcgctcaata aaaccaaaaa

tattgagcga gcgagcgcgc 80

<210> 462

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 462

60 gcgcgctcgc tcgctcaata aaaccaaaaa

tattgagcga gcgagcgcgc 80

<210> 463

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 463

gcgcgctcgc tcgctcaata aaaccaaaaa 60

tattgagcga gcgagcgcgc 80

<210> 464

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 464

60

tattgagcga gcgagcgcgc 80

<210> 465

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 465

60

tattgagcga gcgagcgcgc 80

<210> 466

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 466

60

tattgagcga gcgagcgcgc 80

<210> 467

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 467

60

tattgagcga gcgagcgcgc 80

<210> 468

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 468

gcgcgctcgc tcgctcaata aaaccgggca gaaaaattct gcccgaagca attgctgttt 60

tattgagcga gcgagcgcgc 80

<210> 469

<211> 120

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 469

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

120

<210> 470

<211> 122

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 470

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca 120

gg 122

<210> 471

<211> 129

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 471

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gcgcctcagt gagcgagcga gcgcgcagct 120

gcctgcagg 129

<210> 472

<211> 101

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 472

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ctttgcctca gtgagcgagc gagcgcgcag ctgcctgcag g 101

<210> 473

<211> 139

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 473

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgaca aagtcgcccg acgcccggggc tttgcccggg cggcctcagt gagcgagcga 120

gcgcgcagct gcctgcagg 139

<210> 474

<211> 137

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 474

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgaaa atcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc 120

gcgcagctgc ctgcagg 137

<210> 475

<211> 135

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 475

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgaaa cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc 120

gcagctgcct gcagg 135

<210> 476

<211> 133

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 476

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcaaag cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 120

agctgcctgc agg 133

<210> 477

<211> 139

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 477

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gtttcccggg cggcctcagt gagcgagcga 120

gcgcgcagct gcctgcagg 139

<210> 478

<211> 137

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 478

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg tttccgggcg gcctcagtga gcgagcgagc 120

gcgcagctgc ctgcagg 137

<210> 479

<211> 135

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 479

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgt ttcgggcggc ctcagtgagc gagcgagcgc 120

gcagctgcct gcagg 135

<210> 480

<211> 133

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 480

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgccctt tgggcggcct cagtgagcga gcgagcgcgc 120

agctgcctgc agg 133

<210> 481

<211> 131

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 481

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgccttt ggcggcctca gtgagcgagc gagcgcgcag 120

ctgcctgcag g 131

<210> 482

<211> 129

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 482

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgctttg cggcctcagt gagcgagcga gcgcgcagct 120

gcctgcagg 129

<210> 483

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 483

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgtttcg gcctcagtga gcgagcgagc gcgcagctgc 120

ctgcagg 127

<210> 484

<211> 120

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 484

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggaaacc cgggcgtgcg 60

cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct 120

<210> 485

<211> 122

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 485

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgt cgggcgacct ttggtcgccc 60

ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc 120

ct 122

<210> 486

<211> 122

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 486

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc cggggcaaag cccgggcgtc 60

ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc 120

ct 122

<210> 487

<211> 129

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 487

cctgcaggca gctgcgcgct cgctcgctca ctgaggcgcc cgggcgtcgg gcgacctttg 60

gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta 120

ggggttcct 129

<210> 488

<211> 101

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 488

cctgcaggca gctgcgcgct cgctcgctca ctgaggcaaa gcctcagtga gcgagcgagc 60

gcgcagagag ggagtggcca actccatcac taggggttcc t 101

<210> 489

<211> 139

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 489

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc cggggcaaag cccgggcgtc 60

gggcgacttt gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac 120

tccatcacta ggggttcct 139

<210> 490

<211> 137

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 490

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc cggggcaaag cccgggcgtc 60

gggcgatttt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc 120

catcactagg ggttcct 137

<210> 491

<211> 135

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 491

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc cggggcaaag cccgggcgtc 60

gggcgtttcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca 120

tcactagggg ttcct 135

<210> 492

<211> 133

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 492

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc cggggcaaag cccgggcgtc 60

gggctttgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc 120

actaggggtt cct 133

<210> 493

<211> 139

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 493

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggaaacc cgggcgtcgg 60

gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac 120

tccatcacta ggggttcct 139

<210> 494

<211> 137

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 494

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc cgggaaaccg ggcgtcgggc 60

gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc 120

catcactagg ggttcct 137

<210> 495

<211> 135

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 495

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgaaacggg cgtcgggcga 60

cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca 120

tcactagggg ttcct 135

<210> 496

<211> 133

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 496

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccaaagggcg tcgggcgacc 60

tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc 120

actaggggtt cct 133

<210> 497

<211> 131

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 497

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc caaaggcgtc gggcgacctt 60

tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac 120

tagggttcc t 131

<210> 498

<211> 129

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 498

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc aaagcgtcgg gcgacctttg 60

gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta 120

ggggttcct 129

<210> 499

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 499

cctgcaggca gctgcgcgct cgctcgctca ctgaggccga aacgtcgggc gacctttggt 60

cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg 120

ggttcct 127

<210> 500

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 500

gcccgctggt ttccagcggg ctgcgggccc gaaacgggcc cgc 43

<210> 501

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 501

cgggcccgtg cgggcccaaa gggcccgc 28

<210> 502

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 502

gcccgggcac gcccgggttt ccggggcg 28

<210> 503

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 503

cgtgcgggcc caaagggccc gc 22

<210> 504

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 504

cgggcgacca aaggtcgccc g 21

<210> 505

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 505

cgcccggggct ttgcccgggc 20

<210> 506

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 506

cgggcgacca aaggtcgcc gacgcccgggg ctttgcccgg gc 42

<210> 507

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 507

cgggcgacca aaggtcgccc g 21

<210> 508

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 508

cgcccggggct ttgcccgggc 20

<210> 509

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 509

cgggcgacca aaggtcgccc gacgcccgggg cggc 34

<210> 510

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 510

cgggcgacca aaggtcgccc g 21

<210> 511

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 511

cgcccggggct ttgcccgggc 20

<210> 512

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 512

cggggcccga cgcccggggct ttgcccggggc 30

<210> 513

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 513

cgggcgacca aaggtcgccc g 21

<210> 514

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 514

cgcccggggct ttgcccgggc 20

<210> 515

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 515

cgggccgac gcccggggctt tgcccggggc 29

<210> 516

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 516

cgggcgacca aaggtcgccc g 21

<210> 517

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 517

cgcccggggct ttgcccgggc 20

<210> 518

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 518

gcccgggcaa agcccgggcg 20

<210> 519

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 519

cgggcgacct ttggtcgccc g 21

<210> 520

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 520

gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cg 42

<210> 521

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 521

gcccgggcaa agcccgggcg 20

<210> 522

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 522

gcccgggcgt cgggcgacct ttggtcgccc g 31

<210> 523

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 523

gcccgggcaa agcccgggcg 20

<210> 524

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 524

cgggcgacct ttggtcgccc g 21

<210> 525

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 525

gccgcccggg cgacgggcga cctttggtcg cccg 34

<210> 526

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 526

gcccgggcaa agcccgggcg 20

<210> 527

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 527

cgggcgacct ttggtcgccc g 21

<210> 528

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 528

gcccgggcgt cgggcgacct ttggtcgccc g 31

<210> 529

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 529

cgggcgacct ttggtcgccc g 21

<210> 530

<211> 1653

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 530

atgccgccac ccggaccgg ccgaggcctt ctctggctgg gtctggttct gagctccgtc 60

tgcgtcgccc tcggatccga aacgcaggcc aactcgacca cagatgctct gaacgttctt 120

ctcatcatcg tggatgacct gcgcccctcc ctgggctgtt atggggataa gctggtgagg 180

tccccaaata ttgaccaact ggcatcccac agcctcctct tccagaatgc ctttgcgcag 240

caagcagtgt gcgccccgag ccgcgtttct ttcctcactg gcaggagacc tgacaccacc 300

cgcctgtacg acttcaactc ctactggagg gtgcacgctg gaaacttctc caccatcccc 360

cagtacttca aggagaatgg ctatgtgacc atgtcggtgg gaaaagtctt tcaccctggg 420

atatcttcta accataccga tgattctccg tatagctggt cttttccacc ttatcatcct 480

tcctctgaga agtatgaaaa cactaagaca tgtcgaggggc cagatggaga actccatgcc 540

aacctgcttt gccctgtgga tgtgctggat gttcccgagg gcaccttgcc tgacaaacag 600

agcactgagc aagccataca gttgttggaa aagatgaaaa cgtcagccag tcctttcttc 660

ctggccgttg ggtatcataa gccacacatc cccttcagat accccaagga atttcagaag 720

ttgtatccct tggagaacat caccctggcc cccgatcccg aggtccctga tggcctaccc 780

cctgtggcct acaacccctg gatggacatc aggcaacggg aagacgtcca agccttaaac 840

atcagtgtgc cgtatggtcc aattcctgtg gactttcagc ggaaaatccg ccagagctac 900

tttgcctctg tgtcatattt ggatacacag gtcggccgcc tcttgagtgc tttggacgat 960

cttcagctgg ccaacagcac catcattgca tttacctcgg atcatgggtg ggctctaggt 1020

gaacatggag aatgggccaa atacagcaat tttgatgttg ctacccatgt tcccctgata 1080

ttctatgttc ctggaaggac ggcttcactt ccggaggcag gcgagaagct tttcccttac 1140

ctcgaccctt ttgattccgc ctcacagttg atggagccag gcaggcaatc catggacctt 1200

gtggaacttg tgtctctttt tcccacgctg gctggacttg caggactgca ggttccacct 1260

cgctgccccg ttccttcatt tcacgttgag ctgtgcagag aaggcaagaa ccttctgaag 1320

cattttcgat tccgtgactt ggaagaggat ccgtacctcc ctggtaatcc ccgtgaactg 1380

attgcctata gccagtatcc ccggccttca gacatccctc agtggaattc tgacaagccg 1440

agtttaaaag atataaagat catgggctat tccatacgca ccatagacta taggtatact 1500

gtgtgggttg gcttcaatcc tgatgaattt ctagctaact tttctgacat ccatgcaggg 1560

gaactgtatt ttgtggattc tgacccattg caggatcaca atatgtataa tgattcccaa 1620

ggtggagatc ttttccagtt gttgatgcct tga 1653

<210> 531

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 531

gcgcgctcgc tcgctc 16

<210> 532

<211> 8

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 532

actgaggc 8

<210> 533

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 533

cgggcgacca aaggtcgccc ga 22

<210> 534

<211> 10

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 534

cgcccgggcg 10

<210> 535

<211> 8

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 535

gcctcagt 8

<210> 536

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 536

gagcgagcga gcgcgc 16

<210> 537

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 537

gcccgggcaa agcccgggcg 20

<210> 538

<211> 165

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 538

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc 120

gagcgcgcag agagggagtg gccaactcca tcactagggg ttcct 165

<210> 539

<211> 140

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 539

cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccgcc 60

120

cgcagagaga tcactagggg 140

<210> 540

<211> 91

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 540

60

tcgcccggcc tcagtgagcg agcgagcgcg c 91

<210> 541

<211> 91

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 541

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc 60

ccgggcggcc tcagtgagcg agcgagcgcg c 91

<210> 542

<211> 8

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 542

ttaattaa 8

<210> 543

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 543

gcgcgctcgc tcgctcactg aggccgcccg ggcgtcggggc gacctttggt cgcccggcct 60

cagtgagcga gcgagcgcgc 80

<210> 544

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 544

gcgcgctcgc tcgctcactg aggcctttgc ctcagtgagc gagcgagcgc gc 52

<210> 545

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 545

gcgcgctcgc tcgctcactg aggcgcccgg gcgtcgggcg acctttggtc gcccggcctc 60

agtgagcgag cgagcgcgc 79

<210> 546

<211> 81

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 546

ctgcgcgctc gctcgctcac tgaggccgggg cgaccaaagg tcgcccgacg tttcggcctc 60

agtgagcgag cgagcgcgca g 81

<210> 547

<211> 81

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 547

ctgcgcgctc gctcgctcac tgaggccgaa acgtcgggcg acctttggtc gcccggcctc 60

agtgagcgag cgagcgcgca g 81

<210> 548

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 548

cgggcgacca aaggtcgcc gacgcccgggg ctttgcccgg gc 42

<210> 549

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 549

gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cg 42

<210> 550

<211> 144

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 550

aggaacccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc 60

cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg

agcgcgcaga gagggagtgg ccaa 144

<210> 551

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 551

gcccgctggt ttccagcggg ctgcgggccc gaaacgggcc cgc 43

<210> 552

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 552

cgggcccgtg cgggcccaaa gggcccgc 28

<210> 553

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 553

gcccgggcac gcccgggttt ccggggcg 28

<210> 554

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 554

cgtgcgggcc caaagggccc gc 22

<210> 555

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 555

gcgggccgga aacgggccg ctgcccgctg gtttccagcg ggc 43

<210> 556

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 556

cgcccgggaa acccggggcgt gcccggggc 28

<210> 557

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 557

gggccgccg ggaaacccgg gcgtgccc 28

<---

Claims (78)

1. Non-viral non-capsid DNA vector with covalently closed ends (scDNA vector) for nucleic acid delivery, characterized in that said scDNA vector contains at least one nucleotide sequence located between asymmetric inverted terminal repeats (asymmetric ITRs), where one of the ITRs contains a deletion in at least one of the ITR regions selected from the group consisting of A, A', B, B', C, C', D and D' ITR regions, wherein at least one of the asymmetric ITRs contains a functional terminal clearance site and a Rep binding site. 2. The zcDNA vector according to claim 1, wherein one of the ITRs contains a deletion in at least one structure selected from the group consisting of (i) a stem structure that corresponds to regions A and A', (ii) a first stem loop structure that corresponds to regions B and B', (iii) a second stem loop structure that corresponds to regions C and C', and (iv) a structure containing at least transcription factor binding sites , which corresponds to the regions D and D'. 3. ZcDNA vector according to any one of paragraphs. 1 or 2, where one of the ITRs contains a deletion of all ITR regions A, A', B, B', C, C', D and D'. 4. ZcDNA vector according to any one of paragraphs. 1-3, wherein said scDNA vector exhibits characteristic linear and continuous DNA bands compared to linear and discontinuous DNA controls when digested with a restriction enzyme having a single recognition site on said scDNA vector analyzed using both native and denaturing gel electrophoresis. 5. ZcDNA vector according to paragraphs. 1-3 or 4, wherein one or more of the asymmetric ITRs are from a virus selected from the group consisting of parvovirus, dependdovirus, and adeno-associated virus (AAV). 6. ZcDNA vector according to claim 5, wherein said asymmetric ITRs are from different viral serotypes. 7. The zcDNA vector of claim 6, wherein said asymmetric ITRs are from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. 8. ZcDNA vector according to any one of paragraphs. 1-7, wherein one or more of the asymmetric ITRs are synthetic. 9. ZcDNA vector according to any one of paragraphs. 1-8, where one or both of the asymmetric ITRs contain a single stem loop structure in a region that in a wild-type ITR would contain a first stem loop structure formed by regions B and B' and a second stem loop structure formed by regions C and C'. 10. The zcDNA vector of claim 9, wherein one or both of the asymmetric ITRs comprise one stem and two loops in a region that in a wild-type ITR would contain a first stem-loop structure formed by regions B and B' and a second stem-loop structure formed by regions C and C'. 11. The zcDNA vector according to claim 9 or 10, wherein one or both of the asymmetric ITRs contain one stem and one loop in a region that, in a wild-type ITR, would contain a first stem-loop structure formed by regions B and B' and a second stem-loop structure formed by regions C and C'. 12. ZcDNA vector according to claim 1, wherein at least one asymmetric ITR is a modified AAV2 ITR containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 101-499 and 545-547, and an ITR having a sequence at least 95% identical to SEQ ID NOs: 101-499 and 545-547. 13. ZcDNA vector according to claim 1, where at least one asymmetric ITR is a modified AAV2 ITR containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 52, 63, or 64, or a nucleotide sequence at least 95% identical to SEQ ID NO: 2, 52, 63, or 64. 14. The zcDNA vector according to claim 1, wherein the 5'-ITR is a wild-type AAV ITR and the 3'-ITR contains a sequence selected from the group consisting of SEQ ID NOs: 2, 64, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 469-483 and 546 and sequences at least 95% identical to any of the above sequences. 15. The zcDNA vector of claim 1, wherein the 3'-ITR is a wild-type AAV ITR and the 5'-ITR contains a sequence selected from SEQ ID NOs: 52, 63, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 484-499, 545 and 547 and sequences at least 95% identical to any of the above sequences. 16. ZcDNA vector according to claim 1, where the 5'-ITR contains a sequence selected from the group consisting of SEQ ID NO: 52, 63, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 12 9, 131, 133, 484-499, 545 and 547 and sequences at least 95% identical to any of the above sequences; and the 3'-ITR contains a sequence selected from the group consisting of SEQ ID NOs: 2, 64, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 469- 483 and 546 and sequences at least 95% identical to any of the above sequences. 17. ZcDNA vector according to claim 1, containing at least two asymmetric ITRs selected from the group consisting of: a. SEQ ID NO: 1 and SEQ ID NO: 52; And b. SEQ ID NO: 2 and SEQ ID NO: 51. 18. ZcDNA vector according to claim 1, containing a pair of asymmetric ITRs selected from the group consisting of: a. SEQ ID NO: 1 and SEQ ID NO: 52; And b. SEQ ID NO: 2 and SEQ ID NO: 51. 19. ZcDNA vector according to any one of paragraphs. 1-18, wherein one or both of the asymmetric ITRs contain a sequence other than SEQ ID NOs: 2, 52, 63, 64, 113, 114 and 557. 20. ZcDNA vector according to any one of paragraphs. 1-19, where the scDNA vector contains at least one regulatory switch. 21. ZkDNA vector according to claim 20, where at least one regulatory switch is selected from the group consisting of: ABA; AIR; ART; BEARON, BEAROFF; BirA-tTA; BIT; Cry2-CIB1; STA, CTS; cTA, rcTA; ecdysone; EcR:RXR; electrogenetic switch; ER-p65-ZF; E.REX; EthR; GAL4-ER; GAL4-hPR; GAL4-Raps; GAL4-TR; GyrB; HEA-3; intramer; Lacl; LAD; Lighton; NICE; PPAR*; PEACE; P.I.T.; REDOX; QuoRex; ST-TA; TIGR; TraR; TET-OFF, TET-ON; TRT; UREX; VAC; ZF-ER, ZF-RXR/EcR; ZF Raps; ZF switches; ZF(TF); RNAi aptamer; aptamer RNAi MiRNA; aptamer splicing; aptazim; replicon CytTS; shRNA TET-OFF, shRNA TET-ON; aptamer with theophylline; aptazim 3' UTR; aptazim 5' UTR; aptamer with Hoechst dye; aptamer H23; aptamer L7Ae; aptamer MS2; AID; ERDD; FM; halo tag; HDV-aptazim; PROTAC; Shield DD; Shield LID and TMP DD. 22. ZcDNA vector according to any one of paragraphs. 1-21, where the specified vector is in the nanocarrier. 23. ZcDNA vector according to claim 22, wherein said nanocarrier contains a lipid nanoparticle (LNP). 24. ZcDNA vector according to any one of paragraphs. 1-23, where said scDNA vector includes an aptamer. 25. ZcDNA vector according to any one of paragraphs. 1-23, wherein at least one of the asymmetric ITRs includes an aptamer. 26. ZcDNA vector according to any one of paragraphs. 24 or 25, where at least one nucleotide sequence contains a sequence that encodes a therapeutic protein. 27. ZcDNA vector according to claim 26, wherein the therapeutic protein is selected from the group consisting of Factor VIII, Factor IX, Factor X, and phenylalanine hydroxylase. 28. Non-viral capsidless DNA vector with covalently closed ends (scDNA vector) according to any one of paragraphs. 1-23, which is obtained by a method including the following steps: A. incubating a population of insect cells bearing the scDNA expression construct in the presence of at least one Rep protein, said scDNA expression construct encoding the scDNA vector, under conditions effective to and for a time sufficient to induce production of said scDNA vector in the insect cells; And b. isolating said scDNA vector from insect cells. 29. The scDNA vector of claim 28, wherein said scDNA expression construct is selected from the group consisting of scDNA plasmid, scDNA bacmid, and scDNA baculovirus. 30. ZcDNA vector according to claim 28 or claim 29, wherein said insect cell expresses at least one Rep protein. 31. The zcDNA vector of claim 30, wherein at least one Rep protein is from a virus selected from the group consisting of parvovirus, dependdovirus, and adeno-associated virus (AAV). 32. The zcDNA vector of claim 31 wherein at least one Rep protein is from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. 33. Expression design zkDNA for the production of zkDNA vector according to any one of paragraphs. 1-21. 34. The scDNA expression construct of claim 33, which is selected from the group consisting of scDNA plasmid, scDNA bacmid, and scDNA baculovirus. 35. A host cell producing an ccDNA expression construct according to claim 33 or 34. 36. A host cell according to claim 35 that expresses at least one Rep protein. 37. A host cell according to claim 36, wherein at least one Rep protein is from a virus selected from the group consisting of parvovirus, dependdovirus, and adeno-associated virus (AAV). 38. The host cell according to claim 37, characterized in that at least one Rep protein is taken from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. 39. The host cell according to any one of paragraphs. 35-38, which is an insect cell. 40. The host cell according to claim 39, which is an Sf9 cell. 41. A method for obtaining a scDNA vector, including: a. incubation of the host cell according to any one of paragraphs. 35-40 under conditions effective to, and for a time sufficient to induce production of the scDNA vector; And b. isolating said scDNA vector from host cells. 42. The use of the scDNA vector according to any one of paragraphs. 1-21 for the manufacture of a medicament for the treatment, prevention, alleviation, monitoring, or diagnosis of a disease or disorder caused by a mutation in a gene or gene product in a subject, wherein at least one nucleotide sequence is selected to treat, prevent, alleviate, diagnose, or monitor said disease or disorder caused by a mutation in the gene or gene product, wherein the disease or disorder caused by the mutation in the gene or gene product is selected from the group consisting of: metabolic diseases or disorders , diseases or disorders of the urea cycle, lysosomal diseases or storage disorders, diseases or disorders of the liver, diseases or disorders of the blood, cancer and tumors, and genetic diseases or disorders. 43. Application according to paragraph 42, where: the metabolic disease is Fabry's disease, Gaucher's disease, phenylketonuria (PKU), or glycogen storage disease; the disease or disorder of the urea cycle is ornithine transcarbamylase (OTC) deficiency; the lysosomal disease or storage disorder is metachromatic leukodystrophy (MLD) or mucopolysaccharidosis type II (MPSII; Gunther's syndrome); the liver disease or disorder is progressive familial intrahepatic cholestasis (FIC); the blood disease or disorder is hemophilia A, hemophilia B, thalassemia, or anemia; or the genetic disease or disorder is cystic fibrosis. 44. Use according to claim 42 or 43, wherein said at least one nucleotide sequence, when transcribed or translated, modifies an abnormal amount of endogenous protein in said subject. 45. Use according to claim 42 or 43, wherein said at least one nucleotide sequence, when transcribed or translated, modifies abnormal function or activity of an endogenous protein or pathway in said subject. 46. Application according to any one of paragraphs. 42-45, wherein said at least one nucleotide sequence encodes or contains a nucleotide molecule selected from the group consisting of RNAi, siRNA, miRNA, dncRNA, aptamer, peptide nucleic acid, and antisense oligo or polynucleotide. 47. Application according to any one of paragraphs. 42-45, wherein said at least one nucleotide sequence encodes a protein. 48. Use according to claim 42, wherein said at least one nucleotide sequence encodes a marker protein. 49. Application according to any one of paragraphs. 42-47, wherein said at least one nucleotide sequence encodes an agonist or antagonist of an endogenous protein or pathway associated with said disease or disorder. 50. Application according to any one of paragraphs. 42-47, wherein said at least one nucleotide sequence encodes an antibody. 51. Application according to paragraphs. 42-50, wherein said scDNA vector is administered in combination with a pharmaceutically acceptable carrier. 52. The use of a composition containing the scDNA vector according to any one of paragraphs. 1-21 to obtain a drug for delivering a therapeutic protein to a subject to correct an abnormal level and/or function of a gene product, wherein said at least one nucleotide sequence encodes said therapeutic protein. 53. Use according to claim 52, wherein said therapeutic protein is a therapeutic antibody. 54. Use according to claim 52 wherein the therapeutic protein is selected from the group consisting of Factor VIII, Factor IX, Factor X and phenylalanine hydroxylase. 55. Set for the delivery of nucleic acid containing scDNA vector according to any one of paragraphs. 1-21 and nanocarrier packaged in a container with a package insert. 56. Set for obtaining zkDNA vector according to any one of paragraphs. 1-21 containing: a. an expression construct comprising at least one restriction site for insertion of at least one nucleotide sequence, or a regulatory switch, or both, wherein said at least one restriction site is located between asymmetric inverted terminal repeats (asymmetric ITRs), and at least one of the asymmetric ITRs contains a functional terminal clearance site and a Rep binding site, and b. instructions for use for obtaining the scDNA vector. 57. The kit of claim 56, wherein one of the ITRs contains a deletion in at least one of the ITRs selected from the group consisting of A, A', B, B', C, C', D, and D' of the ITRs. 58. The kit of claim 57, wherein one of the ITRs contains a deletion in at least one structure selected from the group consisting of (i) a stem structure that corresponds to regions A and A', (ii) a first stem loop structure that corresponds to regions B and B', (iii) a second stem loop structure that corresponds to regions C and C', and (iv) a structure containing at least transcription factor binding sites that corresponds to regions D and D'. 59. Set according to any one of paragraphs. 56, 57, or 58, wherein one of the ITRs contains a deletion of all ITR regions A, A', B, B', C, C', D, and D'. 60. Set according to any one of paragraphs. 56-58 or 59, which is suitable for obtaining scDNA vector according to any one of paragraphs. 1-22. 61. Set according to any one of paragraphs. 56-59 or 60, further comprising a population of insect cells lacking viral capsid coding sequences, where the production of the scDNA vector can be induced in the presence of the Rep protein. 62. Set according to any one of paragraphs. 56-61 further comprising a vector containing a polynucleotide sequence that encodes at least one Rep protein, wherein said vector is suitable for expressing said at least one Rep protein in an insect cell.

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