HK40062724A - Optimized human clotting factor viii gene expression cassettes and their use - Google Patents

Description

Optimized human coagulation factor VIII gene expression cassette and application thereof

The present application is a divisional application of chinese patent application No.201680020293.7 entitled "optimized human blood coagulation factor VIII gene expression cassette and use thereof" filed on 5/2/2016.

Priority declaration

This application claims the benefit of U.S. provisional application serial No. 62/112,901 filed on 6/2/2015, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to synthetic liver-specific promoters and expression constructs for producing polypeptides and functional nucleic acids in the liver of a subject. The invention also relates to factor VIII proteins containing modifications in the amino acid sequence of the factor VIII protein, as well as nucleic acid constructs encoding the factor VIII protein and methods of using these compositions to treat bleeding disorders.

Background

Factor viii (factor viii) (fviii) plays a key role in the coagulation cascade by accelerating the conversion of factor X to factor Xa. FVIII activity deficiency is the cause of the bleeding disorder hemophilia a. The current treatment for hemophilia a is intravenous infusion of plasma-derived or recombinant FVIII proteins. Although this treatment is effective in controlling bleeding episodes, the requirement for frequent infusions makes FVIII inherently costly due to its short half-life (8-12 hours). Gene therapy has become an attractive strategy for the ultimate cure of this disease. However, the progress in FVIII gene delivery using adeno-associated virus (AAV), one of the most promising viral vectors, has lagged behind the progress in coagulation factor IX delivery, as the large size of the FVIII coding sequence approaches the packaging capacity of AAV.

The present invention overcomes the disadvantages of the art by providing short synthetic liver-specific promoters and expression constructs suitable for use in AAV vectors. The invention further provides FVIII proteins comprising additional glycosylation sites with amino acid sequence modifications and methods for their use in treating bleeding disorders.

Disclosure of Invention

The present invention is based in part on the development of synthetic liver-specific promoters that are only about 200 base pairs in length. The promoters may be used to produce polypeptides and functional nucleic acids in a liver-specific manner, particularly using AAV vectors, which have stringent length limitations and may benefit from the availability of short but strong promoters.

The present invention is also based in part on the development of modified FVIII proteins comprising additional glycosylation sites in the heavy chain. The modified proteins provide long-term and high levels of activity relative to FVIII proteins without the modifications described herein.

In one aspect, the invention relates to a polynucleotide comprising a synthetic liver-specific promoter, wherein said promoter comprises the nucleotide sequence of SEQ ID No. 1 or a sequence having at least 90% identity thereto.

In another aspect, the invention relates to vectors, cells and/or transgenic animals comprising a polynucleotide of the invention.

In yet another aspect, the invention relates to a method of producing a polypeptide or functional nucleic acid in the liver of a subject, comprising delivering a polynucleotide, vector and/or transformed cell of the invention to the subject, thereby producing the polypeptide or functional nucleic acid in the liver of the subject.

In an additional aspect, the present invention relates to a method of treating hemophilia a in a subject, comprising delivering to the subject a therapeutically effective amount of a polynucleotide, vector and/or transformed cell of the invention, thereby treating hemophilia a in the subject.

In another aspect, the present invention relates to a method of increasing the bioavailability of a factor VIII polypeptide in a subject, comprising delivering to the subject an effective amount of a polynucleotide, vector and/or transformed cell of the present invention, thereby increasing the bioavailability of the factor VIII polypeptide in the subject.

In a further aspect, the present invention relates to a modified human factor VIII polypeptide, wherein the amino acid residues in the heavy chain are modified to create one or more glycosylation sites.

In additional aspects, the invention relates to a polynucleotide encoding the modified human factor VIII polypeptide of the invention and vectors, cells, and/or transgenic animals comprising the polynucleotide.

In another aspect, the present invention relates to a method of producing factor VIII in the liver of a subject, comprising delivering to the subject a polynucleotide encoding a modified human factor VIII polypeptide of the invention or a vector and/or transformed cell comprising said polynucleotide, thereby producing factor VIII in the liver of the subject.

In another aspect, the invention relates to a method of treating hemophilia a in a subject, comprising delivering to the subject a therapeutically effective amount of a modified human factor VIII polypeptide, polynucleotide, vector, and/or transformed cell of the invention, thereby treating hemophilia a in the subject.

In an additional aspect, the present invention relates to a method of increasing the bioavailability of a factor VIII polypeptide in a subject, comprising delivering to the subject an effective amount of a polynucleotide encoding a modified human factor VIII polypeptide of the invention or a vector and/or transformed cell comprising the polynucleotide, thereby increasing the bioavailability of the factor VIII polypeptide in the subject.

These and other aspects of the invention are set forth in more detail in the description of the invention below.

Drawings

FIG. 1 shows the sequence of the LXP3.3 promoter (SEQ ID NO: 1). Putative liver and housekeeping (house-keeping) transcription factor binding sites are highlighted by underlining.

FIG. 2A shows a comparison of LacZ expression in liver and heart following intravenous injection of AAV9-TBG-LacZ or AAV9-Lxp3.3-LacZ into mice. Double X-gal and H & E staining of thin sections of liver and heart are shown.

FIG. 2B shows the use of 1x10¹¹Quantitative comparison of LacZ enzyme activity in various tissues of mice treated with the vector genome (v.g.) of AAV9-LacZ vector,the AAV9-LacZ vector contains a non-specific CMV promoter, a liver-specific TBG promoter or an LXP3.3 promoter, respectively (as shown in FIG. 1).

FIG. 2C shows the use of AAV9-LacZ vector as shown in FIG. 2B but at lower doses (2X 10)¹⁰Individual vector genome (v.g.)) treated mice in the liver, quantitative comparison of LacZ enzyme activity.

Figure 3 shows factor VIII activity in the supernatant of Huh7 cells transfected with different AAV vector plasmids containing the NBP promoter (177bp) driving the human BDD factor VIII gene without the intron or containing the VH4 intron or the chimeric CIN intron.

FIG. 4 shows the AAV-Lxp3.3i-BDD-F8 construct and the sequence of the Lxp3.3i promoter-intron (SEQ ID NO: 2). ITR represents 145bp inverted terminal repeat of AAV.

FIG. 5 shows FVIII expression of two constructs containing either a non-specific CMV promoter or a liver-specific LXP3.3 promoter in human hepatoma Huh7 cells and mouse Hepa1-6 cells.

Figure 6 shows FVIII activity in Huh7 cells transfected with different BDD factor VIII (synthetic opti-F8 or wild type wtF8) plasmids containing the promoter LXP3.3 or the weak promoter TkPro, respectively.

FIG. 7A shows high dose injection at IV (2X 10)¹¹V.g./mouse) or low dose (4x 10)¹⁰V.g./mouse), AAV9-lxp 3.3-F8-mediated long-term human FVIII gene expression and FVIII activity in FVIII knockout mice. Factor VIII activity (as a percentage of normal human levels) was measured at 2,6, 10 and 18 weeks post-injection, respectively.

FIG. 7B shows high dose injection at IV (2X 10)¹¹V.g./mouse) or low dose (4x 10)¹⁰V.g./mouse), AAV9-lxp 3.3-F8-mediated long-term human FVIII gene expression and human FVIII protein concentration in FVIII knockout mice. Factor VIII protein (as a percentage of normal human levels) was measured by ELISA at 2,6, 10 and 18 weeks post injection, respectively.

FIG. 8 shows the amino acid sequence of the modified BDD FVIII protein (SEQ ID NOS: 31-34). SQ represents serine 743(Ser 743) and glutamine (Gln 1638) at the heavy and light chain junction of BDD factor VIII (numbering based on SEQ ID NO: 5). The underlined letters highlight the mutated amino acids in the heavy chain.

Figure 9A shows human FVIII activity in Huh7 cells transfected with expression plasmids containing BDD factor VIII or mutants F8X1 and F8X2 (see figure 8 for details).

Figure 9B shows AAV8 vector-mediated long-term expression of mutant human BDD FVIII gene in FVIII knockout mice. Human factor VIII activity (as a percentage of normal human levels) was measured at 5X10 by i.v. injection¹⁰Measurements were made at various time points after the vector genome of AAV8-LXP3.3i-F8X1 or AAV8-LXP3.3i-F8X 2.

Detailed Description

The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Furthermore, the present invention also contemplates that, in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted. For purposes of illustration, if the specification states that the compound comprises components A, B and C, it is specifically intended that either one or a combination of A, B or C may be omitted and disclaimed, either alone or in any combination.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Unless otherwise specifically indicated, nucleotide sequences are presented herein only as single strands from left to right in the 5 'to 3' direction. Nucleotides and amino acids are referred to herein by the one-letter code or the three-letter code, in the manner recommended by the IUPAC-IUB biochemical nomenclature commission, or (for amino acids), both of which conform to 37c.f.r. § 1.822 and established usage.

Unless otherwise indicated, standard methods known to those skilled in the art can be used to clone genes, amplify and detect nucleic acids, and the like. These techniques are known to those skilled in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 nd edition (Cold Spring Harbor, NY, 1989); current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

Definition of

As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Further, as used herein, "and/or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

The term "about" as used herein is intended to encompass variations of a specified quantity by ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5% or even ± 0.1% when referring to a measurable value such as the amount of a polypeptide, dose, time, temperature, enzymatic activity or other biological activity, etc.

The transitional phrase "consisting essentially of means that the scope of the claims should be interpreted to include the specified materials or steps," and not to includeSubstantially in the nature ofAffecting the claimed inventionBasicAndnovelThose of a feature ". See Inre Herz,537F.2d 549,551-52,190USPQ 461,463(CCPA1976) (with emphasis on original); see also MPEP § 2111.03.

The term "consisting essentially of" (and grammatical variants) as applied to a polynucleotide or polypeptide sequence of the present invention refers to a polynucleotide or polypeptide consisting of the sequence (e.g., SEQ ID NO) and a total of ten or fewer (e.g., 1, 2,3, 4,5, 6,7, 8, 9, or 10) additional nucleotides or amino acids at the 5 'and/or 3' or N-and/or C-terminus of the sequence, such that the function of the polynucleotide or polypeptide is not substantially altered. A total of ten or fewer additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids added at both ends. The term "substantially altered" as applied to a polynucleotide of the present invention refers to an increase or decrease in the ability to express an encoded polypeptide by at least about 50% or more as compared to the expression level of a polynucleotide consisting of the sequence. The term "substantially altered" as applied to a polypeptide of the present invention refers to an increase or decrease in coagulation stimulating activity of at least about 50% or more as compared to the activity of a polypeptide consisting of the sequence.

The term "enhance" or "increase" or grammatical variations thereof, as used herein, means an increase in a specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, or even 15-fold.

As used herein, the term "inhibit" or "reduce" or grammatical variations thereof means a reduction or decrease in a specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, inhibition or reduction results in little or substantially no detectable activity (at most an insignificant amount, e.g., less than about 10% or even 5%).

An "effective" amount, as used herein, is an amount that provides the desired effect.

As used herein, a "therapeutically effective" amount is an amount that provides some improvement or benefit to the subject. Alternatively, a "therapeutically effective" amount is an amount that will provide some relief, alleviation, or reduction in at least one clinical symptom in a subject. One skilled in the art will appreciate that the therapeutic effect need not be complete or curative, so long as some benefit is provided to the subject.

As used herein, a "prophylactically effective" amount is an amount sufficient to prevent (as defined herein) a disease, disorder, and/or clinical symptom in a patient. One skilled in the art will appreciate that the level of prophylaxis need not be complete as long as some benefit is provided to the subject.

As is well known to those skilled in the art, the efficacy of treatment of a bleeding disorder by the methods of the present invention can be determined by detecting a clinical improvement indicated by a change in a subject's symptoms and/or clinical parameters.

The terms "treat", "treating" or "treated" are intended to reduce or at least partially ameliorate or change the severity of the condition in a subject and to achieve some alleviation, reduction or reduction of at least one clinical symptom.

The terms "prevent", "preventing" and "prevented" (and grammatical variations thereof) refer to a reduction or delay in the extent or severity of a disease, disorder and/or clinical symptom after onset relative to the situation that would occur without the method of the invention prior to the onset of the disease, disorder and/or clinical symptom. In the context of hemophilia a, "prevention" refers to a reduction in the number and/or severity of bleeding episodes that occur as compared to the number and/or severity of bleeding episodes that occur in the absence of prophylactic treatment.

As used herein, "nucleic acid," "nucleotide sequence," and "polynucleotide" are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA, and chimeras of RNA and DNA. The term polynucleotide, nucleotide sequence or nucleic acid refers to a strand of nucleotides regardless of strand length. The nucleic acid may be double-stranded or single-stranded. When single-stranded, the nucleic acid may be the sense strand or the antisense strand. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids with altered base-pairing abilities or increased nuclease resistance. The invention also provides a nucleic acid which is a complement (which may be a full complement or a partial complement) of a nucleic acid, nucleotide sequence or polynucleotide of the invention.

An "isolated polynucleotide" is a nucleotide sequence (e.g., DNA or RNA) that is not directly contiguous with its immediately contiguous (one at the 5 'end and one at the 3' end) nucleotide sequence in the naturally-occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequence immediately adjacent to the coding sequence. Thus, the term includes, for example, recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or present as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), which is independent of other sequences. It also includes recombinant DNA that is part of a hybrid nucleic acid encoding additional polypeptide or peptide sequences. An isolated polynucleotide comprising a gene, not a fragment of a chromosome comprising such a gene, but rather a coding region and a regulatory region associated with the gene, but without additional genes naturally occurring on the chromosome.

The term "fragment" as applied to a polynucleotide will be understood to refer to, consist essentially of, and/or consist of a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence, and comprising, consisting of, or consisting of contiguous nucleotides that are identical or nearly identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such nucleic acid fragments according to the invention may, where appropriate, be included in the larger polynucleotides of which they are a constituent. In some embodiments, such fragments may comprise, consist essentially of, and/or consist of oligonucleotides having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more contiguous nucleotides of a nucleic acid or nucleotide sequence according to the present invention.

The term "isolated" may refer to a nucleic acid, nucleotide sequence, or polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Furthermore, an "isolated fragment" is a fragment of a nucleic acid, nucleotide sequence, or polypeptide that is not a naturally occurring fragment and is not found in nature. "isolated" does not mean that the preparation is technically pure (homogeneous), but that it is sufficiently pure to provide the polypeptide or nucleic acid in a form that can be used for its intended purpose.

The term "fragment" as applied to a polypeptide will be understood to refer to, consist essentially of, and/or consist of an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence, and comprising, consisting essentially of, and/or consisting of contiguous amino acids that are identical or nearly identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such polypeptide fragments according to the invention may, where appropriate, be comprised in the larger polypeptide of which it is a constituent. In some embodiments, such fragments may comprise, consist essentially of, and/or consist of a peptide having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more contiguous amino acids of a polypeptide or amino acid sequence according to the present invention.

A "vector" is any nucleic acid molecule used for the cloning and/or transfer of nucleic acids into cells. The vector may be a replicon to which another nucleotide sequence may be attached to allow the attached nucleotide sequence to replicate. A "replicon" can be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) that is an autonomous unit of nucleic acid replication in vivo (i.e., capable of replication under its own control). The term "vector" includes viral and non-viral (e.g., plasmid) nucleic acid molecules that introduce nucleic acid into cells in vitro, ex vivo, and/or in vivo. Numerous vectors known in the art can be used to manipulate nucleic acids, incorporate response elements and promoters into genes, and the like. For example, insertion of nucleic acid fragments corresponding to the response element and promoter into an appropriate vector can be accomplished by ligating the appropriate nucleic acid fragments into a selected vector having complementary binding ends. Alternatively, the ends of the nucleic acid molecule may be enzymatically modified, or any site may be created by linking a nucleotide sequence (linker) to the nucleic acid ends. Such vectors may be engineered to contain a sequence encoding a selectable marker that facilitates selection of cells containing the vector and/or cells that have incorporated the nucleic acid of the vector into the genome of the cell. Such markers allow the identification and/or selection of host cells that are combined and express the protein encoded by the marker. By "recombinant" vector is meant a viral or non-viral vector comprising one or more heterologous nucleotide sequences (i.e., transgenes), e.g., comprising two, three, four, five or more heterologous nucleotide sequences.

Viral vectors have been used in a variety of gene delivery applications in cells as well as in living animal subjects. Viral vectors that may be used include, but are not limited to, retroviral, lentiviral, adeno-associated, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, epstein-barr virus, and adenoviral vectors. Non-viral vectors include plasmids, liposomes, charged lipids (cytofectins), nucleic acid-protein complexes, and biopolymers. In addition to the nucleic acid of interest, the vector may also comprise one or more regulatory regions and/or selectable markers that can be used to select, measure, and monitor the results of nucleic acid transfer (delivery to a particular tissue, duration of expression, etc.).

Vectors can be introduced into the desired cells by methods known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosomal fusion), use of a gene gun or nucleic acid vector transporter (see, e.g., Wu et al., J.biol.chem.267:963 (1992); Wu et al., J.biol.chem.263:14621 (1988); and Canadian patent application No.2,012,311 filed 3/15 of Hartmut et al.1990). In various embodiments, other molecules may be used to facilitate delivery of nucleic acids in vivo, such as cationic oligopeptides (e.g., WO95/21931), peptides derived from nucleic acid binding proteins (e.g., WO96/25508), and/or cationic polymers (e.g., WO 95/21931). Vectors that are naked nucleic acids may also be introduced in vivo (see U.S. Pat. nos. 5,693,622, 5,589,466, and 5,580,859). Receptor-mediated nucleic acid delivery methods may also be used (Curiel et al, hum. Gene Ther.3:147 (1992); Wu et al, J.biol. chem.262:4429 (1987)).

The terms "protein" and "polypeptide" as used herein are used interchangeably and include peptides and proteins unless otherwise indicated.

A "fusion protein" is a polypeptide that is produced when two heterologous nucleotide sequences encoding two (or more) different polypeptides, or fragments thereof, that are fused together are not found in nature, and are fused together in the correct translational reading frame. Illustrative fusion polypeptides include fusions of a polypeptide of the invention (or fragment thereof) with all or part of glutathione-S-transferase, maltose binding protein or a reporter protein (e.g., green fluorescent protein, β -glucuronidase, β -galactosidase, luciferase, etc.), hemagglutinin, c-myc, FLAG epitopes, and the like.

As used herein, a "functional" polypeptide or "functional fragment" is a substance that substantially retains at least one biological activity (e.g., angiogenic activity, protein binding, ligand or receptor binding) normally associated with the polypeptide. In particular embodiments, a "functional" polypeptide or "functional fragment" retains substantially all of the activity possessed by the unmodified peptide. By "substantially retains" biological activity, it is meant that the polypeptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 99% or more of the biological activity of the native polypeptide (and may even have a higher level of activity than the native polypeptide). A "non-functional" polypeptide is a polypeptide that exhibits little or substantially no detectable biological activity normally associated with the polypeptide (e.g., at most only an insignificant amount, e.g., less than about 10% or even 5%). Biological activities such as protein binding and angiogenic activity can be measured using assays well known in the art and as described herein.

The term "express" or "expresses" a polynucleotide coding sequence means that the sequence is transcribed and optionally translated. Generally, according to the invention, expression of a coding sequence of the invention will result in the production of a polypeptide of the invention. The whole expressed polypeptide or fragment can also function in intact cells without the need for purification.

In the context of the present invention, the term "adeno-associated virus" (AAV) includes, but is not limited to, this AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV, as well as any other AAV now known or later discovered. See, e.g., BERNARD n. fields et al, VIROLOGY, volume 2, chapter 69 (4 th edition, Lippincott-Raven Publishers). Several additional AAV serotypes and clades (clades) have been identified (see, e.g., Gao et al, (2004) j. virol.78: 6381-.

The genomic sequences of various AAV and autonomous parvoviruses, as well as the sequences of ITRs, Rep proteins, and capsid subunits are known in the art. These sequences can be found in the literature or public databases, such asA database. See, e.g.Accession numbers NC 002077, NC 001401, NC 001729, NC 001863, NC 001829, NC 001862, NC 000883, NC 001701, NC 001510, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC 001358, NC 001540, AF513851, AF513852, AY530579, AY631965, AY 631966; the disclosure of which is incorporated herein in its entirety. See also, e.g., Srivistava et al, (1983) j.virol.45: 555; chiorini et al, (1998) J.Virol.71: 6823; chiorini et al, (1999) J.Virol.73: 1309; Bantel-Schaal et al, (1999) J.Virol.73: 939; xiao et al, (1999) j.virol.73: 3994; muramatsu et al, (1996) Virology 221: 208; shade et al, (1986) j.virol.58: 921; gao et al, (2002) proc.nat.acad.sci.usa 99: 11854; international patent publications WO 00/28061, WO 99/61601, WO 98/11244; U.S. patent nos. 6,156,303; the disclosures of which are incorporated herein in their entirety. See also table 1. Xiao, X., (1996), "Characterisation of amplified viruses (AAV) DNA replication and integration," Ph.D.DisservationThe University of Pittsburgh, PA (incorporated herein in its entirety) provides an early description of the AAV1, AAV2, and AAV3 terminal repeats.

TABLE 1

A "recombinant AAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) that comprises at least one inverted terminal repeat (e.g., one, two, or three inverted terminal repeats) and one or more heterologous nucleotide sequences. rAAV vectors typically retain 145 base-Terminal Repeats (TR) in cis (cis) to produce virus; however, modified AAV TR and non-AAV TR (including partially or fully synthetic sequences) may also be used for this purpose. All other viral sequences are not necessary and may be provided in trans (trans) (Muzyczka, (1992) curr. topics Microbiol. Immunol.158: 97). The rAAV vector optionally comprises two TRs (e.g., AAV TRs), which will typically be 5 'and 3' to the heterologous nucleotide sequence, but need not be contiguous therewith. The TRs may be the same as or different from each other. The vector genome may also contain a single ITR at its 3 'or 5' end.

The term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and serves as an inverted terminal repeat (i.e., mediates a desired function such as replication, viral packaging, integration, and/or proviral rescue, etc.). The TR may be an AAV TR or a non-AAV TR. For example, non-AAV TR sequences, such as those of other parvoviruses (e.g., Canine Parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or the SV40 hairpin sequence that is the origin of replication of SV40, can be used as the TR, which can be further modified by truncation, substitution, deletion, insertion, and/or addition. In addition, TR may be partially or fully synthetic, such as the "double D sequence" described in U.S. patent No. 5,478,745 to Samulski et al.

The "AAV terminal repeats" or "AAV TRs" may be from any AAV, including but not limited to serotypes 1, 2,3, 4,5, 6,7, 8, 9, 10, or 11 or any other AAV now known or later discovered (see, e.g., table 1). AAV terminal repeats need not have native terminal repeats (e.g., the native AAV TR sequence can be altered by insertion, deletion, truncation, and/or missense mutation), so long as the terminal repeats mediate the desired functions, e.g., replication, viral packaging, integration, and/or proviral rescue, etc.

The terms "rAAV particle" and "rAAV viral particle" are used interchangeably herein. A "rAAV particle" or "rAAV viral particle" comprises a rAAV vector genome packaged within an AAV capsid.

AAV capsid structures are described in more detail in BERNARD n. fields et al, VIROLOGY, volume 2, chapters 69&70 (4 th edition, Lippincott-Raven Publishers).

The term "pharmacokinetic properties" has the usual and customary meaning and refers to the absorption, distribution, metabolism and excretion of FVIII proteins.

By "bioavailability" is meant, generally and conventionally, the fraction or amount of a dose of the biologically active agent administered that reaches the systemic circulation. In the context of embodiments of the present invention, the term "bioavailability" includes the usual and customary meaning, but is also to be considered to have a broader meaning to include the degree of biological activity of the FVIII protein. In the case of FVIII, for example, one measure of "bioavailability" is the procoagulant activity of the FVIII protein obtained in the post-infusion cycle.

"post-translational modifications" have the usual and customary meaning and include, but are not limited to, removal of leader sequences, gamma-carboxylation of glutamic acid residues, beta-hydroxylation of aspartic acid residues, N-linked glycosylation of asparagine residues, O-linked glycosylation of serine and/or threonine residues, sulfation of tyrosine residues, phosphorylation of serine residues and any combination thereof.

As used herein, "biological activity" is determined with reference to a standard, e.g., from human plasma. For FVIII, the standard may be(CSL Behring). The biological activity of the standard was taken as 100%.

The term "factor VIII protein" or "FVIII protein" as used herein includes wild-type FVIII proteins as well as naturally occurring or artificial proteins (e.g. B domain deleted proteins). FVIII proteins of the invention may further comprise mutated forms of FVIII known in the literature. FVIII proteins of the invention also include any other naturally occurring human FVIII proteins or artificial human FVIII proteins now known or later identified, as well as derivatives and active fragments/active domains thereof known in the art.

The amino acid sequence of FVIII from various mammalian species is available from sequence databases such as GenBank. Examples of FVIII sequences are shown in the table below.

Species (II)	GenBank accession number
		Human being (Homo sapiens)	AAA52484.1
Little mouse (Mus musculus)	NP_032003.2
		Wild boar (Sus scrofa)	AAB06705.1
Cattle (Bos Taurus)	NP_001138980.1
		Domestic dog (Canis lupus familiaris)	NP-001003212.1
Brown rat (Rattus norvegicus)	ADU79112.1

FVIII proteins of the invention also include pharmacologically active forms of FVIII, which are molecules from which the signal peptide has been removed and which have been engineered (enginer) from the protein by action of proteases to cleave the B domain (or by removal thereof at the nucleic acid level) resulting in two discrete polypeptide chains (light and heavy) of FVIII folded into a functional FVIII coagulation factor. Multiple B-domain deleted versions of human FVIII are known, including the frequently used SQ version, in which residues between S743 and Q1638 are deleted. In particular, modified FVIII proteins with an increased degree of glycosylation are specifically included in a broad sense.

The amino acid sequence of the human FVIII protein is well known in the art and can be found in GenBank accession No. AAA 52484. The human FVIII protein is 2351 amino acids in length and consists of a signal peptide (residues 1-19), heavy chain (residues 20-759), B domain (residues 760-1332) and light chain (residue 1668-2351). The amino acid sequence without the signal peptide (SEQ ID NO:5) is disclosed below.

The term "half-life (half life)" is a broad term that includes the usual and customary meanings as well as those found in the FVIII scientific literature. Specifically included in this definition is the measurement of a parameter associated with FVIII, which defines the time after infusion taken to decrease from the initial value measured at the time of infusion to half the initial value. In some embodiments, antibodies to FVIII can be used in various immunoassays to measure the half-life of FVIII in blood and/or blood components, as is well known in the art and described herein. Alternatively, half-life can be measured as a reduction in FVIII activity using functional assays including standard coagulation assays, as is well known in the art and as described herein.

The term "recovery" as used herein includes the amount of FVIII measured by any acceptable method, including but not limited to the level of FVIII antigen or FVIII protease or coagulation activity detected, in a recipient animal or human subject (e.g. in circulation) at the earliest practical time when a biological sample (e.g. blood or blood product sample) is taken, for the purpose of measuring the level of FVIII after its infusion, injection, delivery or other administration. Using existing methods, the earliest biological sampling times for measuring FVIII recovery are typically within the first 15 minutes after infusion, injection, or other delivery/administration of FVIII, but as scientific and/or clinical techniques improve, faster sampling times are expected to be reasonable. Essentially, the recovery value of FVIII herein represents the maximum fraction of FVIII infused, injected, or otherwise delivered/administered that can be measured in the recipient (e.g., in the circulation) at the earliest possible time point following infusion, injection, or otherwise delivery to the recipient animal or patient.

The term "glycosylation site" is a broad term having its usual and customary meaning. In the context of the present application, the term applies to both sites that potentially can accept a carbohydrate moiety as well as to sites within a protein, in particular FVIII, which has in fact attached a carbohydrate moiety and includes any amino acid sequence that can act as a receptor for oligosaccharides and/or carbohydrates.

As used herein, a "transformed" cell is a cell that has been transformed, transduced and/or transfected with a nucleic acid molecule encoding a FVIII protein of the invention, including but not limited to FVIII protein vectors constructed using recombinant DNA techniques.

As used herein, the term "bleeding disorder" reflects any defect, congenital, acquired or inducibility of cellular, physiological or molecular origin exhibited by bleeding. Examples are deficiencies of blood coagulation factors (e.g. deficiencies of haemophilia a and B or of blood coagulation factors XI, VII, VIII or IX), inhibitors of blood coagulation factors, platelet insufficiency, thrombocytopenia, von Willebrand's disease, or bleeding caused by surgery or trauma.

Excessive bleeding also occurs in subjects with a normally functioning blood coagulation cascade (no coagulation factor deficiency or inhibitors against any coagulation factor) and may be caused by platelet insufficiency, thrombocytopenia, or von willebrand disease. In this case, bleeding may be similar to that caused by hemophilia, as the hemostatic system (as in hemophilia) lacks or has abnormal essential clotting "compounds" (such as platelets or von willebrand factor proteins) resulting in significant bleeding. In subjects experiencing extensive tissue damage associated with surgery or trauma, the normal hemostatic mechanisms may be overwhelmed by the need for immediate hemostasis, and thus bleeding may still occur despite the normal hemostatic mechanisms. Surgical hemostasis has limited potential to achieve satisfactory hemostasis when bleeding in organs such as the brain, inner ear area, and eyes, and is also a problem. The same problem may occur during biopsies in various organs (liver, lung, tumor tissue, gastrointestinal tract) as well as during laparoscopic surgery. Common to all these cases is the difficulty in providing hemostasis by surgical techniques (sutures, clips, etc.), also when bleeding is diffuse (hemorrhagic gastritis and profuse uterine bleeding). Acute and massive hemorrhage may also occur in subjects with anticoagulation therapy, in which defective hemostasis is induced by the administered therapy. In cases where anticoagulation must be promptly counteracted, these subjects may require surgical intervention. Radical pubic prostatectomy (radial retropubic prostatectomy) is a routine procedure for subjects with localized prostate cancer. Surgery is often complicated by significant and sometimes massive blood loss. Considerable blood loss during prostatectomy is primarily associated with complex anatomical conditions, with various densely vascularized sites where surgical hemostasis is not readily available, and can lead to extensive diffuse bleeding. In addition, intracerebral hemorrhage is the least treatable form of stroke and is associated with high mortality and hematoma growth in the first hours following intracerebral hemorrhage. Another situation that may cause problems in cases of poor hemostasis is when subjects with normal hemostasis mechanisms are on anticoagulation therapy to prevent thromboembolic disease. Such treatments may include heparin, other forms of proteoglycans, warfarin or other forms of vitamin K antagonists as well as aspirin and other platelet aggregation inhibitors.

In one embodiment of the invention, bleeding is associated with hemophilia. In another embodiment, bleeding is associated with hemophilia as an acquired inhibitor. In another embodiment, the bleeding is associated with thrombocytopenia. In another embodiment, bleeding is associated with von Willebrand's disease. In another embodiment, bleeding is associated with severe tissue damage. In another embodiment, the bleeding is associated with severe trauma. In another embodiment, the bleeding is associated with surgery. In another embodiment, the bleeding is associated with laparoscopic surgery. In another embodiment, the bleeding is associated with hemorrhagic gastritis. In another embodiment, the bleeding is a massive uterine bleeding. In another embodiment, bleeding occurs in an organ with limited mechanical hemostatic potential. In another embodiment, the bleeding occurs in the brain, inner ear area, or eye. In another embodiment, the bleeding is associated with a biopsy procedure. In another embodiment, the bleeding is associated with anticoagulation therapy.

A "subject" of the invention includes any animal that is suffering from or susceptible to a bleeding disorder or bleeding condition for which control of bleeding is needed and/or desired, which can be treated, ameliorated or prevented by administration of FVIII to the subject (e.g., hemophilia a and acquired FVIII deficiency (e.g., due to autoantibodies against FVIII or a hematologic malignancy)). Such subjects are typically mammalian subjects (e.g., laboratory animals such as rats, mice, guinea pigs, rabbits, primates, etc.), farm or commercial animals (e.g., cows, horses, goats, donkeys, sheep, etc.), or livestock (e.g., cats, dogs, ferrets, etc.). In particular embodiments, the subject is a primate subject, a non-human primate subject (e.g., a chimpanzee, baboon, monkey, gorilla, etc.), or a human. The subject of the invention may be a subject known or believed to be at risk of a bleeding disorder or bleeding condition that needs and/or desires to control. Alternatively, a subject according to the present invention may also include a subject previously unknown or suspected of having a risk of a bleeding disorder or bleeding condition in need or desire of control. As another option, the subject can be a laboratory animal and/or an animal model of disease.

Subjects include males and/or females of any age, including neonatal, juvenile, adult and geriatric subjects. With respect to human subjects, in representative embodiments, the subject can be an infant (e.g., an age of less than about 12 months, 10 months, 9 months, 8 months, 7 months, 6 months, or less), a toddler (e.g., at least about 12, 18, or 24 months and/or less than about 36, 30, or 24 months), or a child (e.g., at least about 1, 2,3, 4, or 5 years and/or less than about 14, 12, 10, 8,7, 6,5, or 4 years old). In embodiments of the invention, the subject is a human subject of about 0 to 3,4, 5,6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 3 to 6,9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 6 to 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 9 to 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 12 to 18, 24, 36, 48 or 60 months of age, a human subject of about 18 to 24, 30, 36, 48 or 60 months of age, or a human subject of about 24 to 30, 36, 48 or 60 months of age.

Promoter and expression cassette (expression cassette)

One aspect of the invention relates to a polynucleotide comprising a synthetic liver-specific promoter, wherein the promoter comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID No. 1 or a sequence having at least about 90% identity thereto. In some embodiments, the nucleotide sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID No. 1. The promoter is a short (about 200 base pairs) and strong liver-specific promoter, which is ideal for liver-specific expression of the polynucleotide of interest, and is particularly suitable for use in AAV vectors due to its short length and limited ability of AAV vectors. The promoter is designed to contain conserved basal promoter elements and a transcription start site. The basal promoter is linked at its 5' end to a number of liver-specific transcription factor binding sites for liver-specific expression (fig. 1). The promoter showed high activity as preliminarily identified in vitro by using luciferase reporter gene and transfection experiments in human hepatoma cell line Huh7, and then confirmed in vivo in mice.

In some embodiments, the promoter is part of an expression cassette in which it is operably linked to an intron, e.g., on the 3' end of the promoter. This may be done to increase the expression level of the polynucleotide of interest linked to the promoter. Any suitable intron may be used, for example the chimeric intron cin (promega). In some embodiments, the intron is from VH 4. In some embodiments, the intron can further comprise a short non-native exon junction sequence. In one embodiment, the promoter and intron together comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO.2 or a sequence that is at least 90% identical thereto (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO. 1).

The promoter may be operably linked to the polynucleotide of interest. In some embodiments, the polynucleotide of interest encodes a polypeptide or a functional nucleic acid. In certain embodiments, the polynucleotide of interest encodes a coagulation factor, e.g., FVIII, e.g., B-domain deleted FVIII. The B domain deleted FVIII may be encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO. 3 or a sequence having at least 90% identity thereto (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO. 3). In one embodiment, the expression cassette comprising the promoter, intron, and polynucleotide of interest encoding a B-domain deleted FVIII comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID No. 4 or a sequence having at least 90% identity thereto (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID No. 4).

Another aspect of the invention is a vector, e.g., an expression vector, comprising a polynucleotide of the invention. The vector may be any type of vector known in the art, including but not limited to plasmid vectors and viral vectors. In some embodiments, the viral vector is a retroviral or lentiviral vector. In some embodiments, the viral vector is an AAV vector from any known AAV serotype, including but not limited to AAV type 1, AAV type 2, AAV type 3 (including type 3A and type 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV, as well as any other AAV now known or later discovered. In some embodiments, the AAV vector is AAV8 or AAV 9.

Another aspect of the invention relates to cells (e.g., isolated cells, transformed cells, recombinant cells, etc.) comprising a polynucleotide and/or vector of the invention. Accordingly, various embodiments of the present invention relate to recombinant host cells containing vectors (e.g., expression cassettes). Such cells may be isolated and/or present in a transgenic animal. Transformation of the cells is described further below.

Another aspect of the invention relates to transgenic animals comprising the polynucleotides, vectors and/or transformed cells of the invention. Transgenic animals are described further below.

The polynucleotides, vectors and/or cells of the invention may be included in a pharmaceutical composition. Some embodiments relate to kits comprising a polynucleotide, vector, and/or cell of the invention, and/or reagents and/or instructions for using the kit, e.g., to perform a method of the invention.

Modified factor VIII proteins

One aspect of the invention relates to modified mammalian factor VIII polypeptides (e.g., human FVIII polypeptides) in which amino acid residues in the heavy chain are modified to create one or more additional glycosylation sites. In certain embodiments, the one or more additional glycosylation sites are located in the C-terminus of the heavy chain, e.g., the last 100 amino acid residues of the heavy chain, e.g., the last 50, 40, 30, 20, 10, 9, 8,7, 6, or 5 residues. In some embodiments, the polypeptide is modified to produce at least 2 glycosylation sites, e.g., 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 or more glycosylation sites. Modifications may include amino acid substitutions, additions, deletions, or any combination thereof. These modifications are introduced into the amino acid sequence of the FVIII protein to produce FVIII proteins with increased activity after in vivo expression.

By "additional" glycosylation sites is meant that the number of glycosylation sites in the FVIII protein is greater than the number of glycosylation sites typically present in an unmodified (e.g., wild-type) FVIII protein (e.g., SEQ ID NO: 5).

The invention also relates to FVIII proteins comprising one or more (e.g. 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15 etc.) additional sugar side chains. Such additional sugar side chains may be present at one or more glycosylation sites in the FVIII protein of the invention. Alternatively, the additional sugar side chain may be present at a site on the FVIII protein as a result of chemical and/or enzymatic methods of introducing such sugar chains into the FVIII molecule, as is well known to those skilled in the art. By "additional" or "new" carbohydrate chains is meant that the number of carbohydrate chains in the FVIII protein is greater than the number of carbohydrate chains normally present in "wild type" form FVIII. In various embodiments, about 1 to about 50 additional sugar side chains (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) may be added.

The glycosylation sites can be N-linked glycosylation sites, O-linked glycosylation sites, and combinations of N-linked glycosylation sites and O-linked glycosylation sites. In some embodiments, the added glycosylation sites include N-linked glycosylation sites, and the consensus sequence is NXT/S, with the proviso that X is not proline. In other embodiments, the glycosylation site comprises an O-linked glycosylation site comprising a consensus sequence selected from the group consisting of CXXGGT/S-C (SEQ ID NO:24), NSTE/DA (SEQ ID NO:25), NITQS (SEQ ID NO:26), QSTQS (SEQ ID NO:27), D/E-FT-R/K-V (SEQ ID NO:28), C-E/D-SN (SEQ ID NO:29), GGSC-K/R (SEQ ID NO:30), and any combination thereof.

In some embodiments, about 1 to about 15 glycosylation sites can be added to the amino acid sequence of a FVIII protein of the invention. In various embodiments, about 1 to about 50 glycosylation sites (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) can be added.

As used herein, "glycosylation attachment site" or "glycosylation site" can refer to a sugar attachment consensus sequence (i.e., a series of amino acids that serve as a consensus sequence for attaching a sugar (mono-, oligo-or polysaccharide) to an amino acid sequence) or it can refer to the actual amino acid residues to which a sugar moiety is covalently attached. The saccharide moiety may be a monosaccharide (simple sugar molecule), an oligosaccharide or a polysaccharide.

In particular embodiments, additional amino acids may be inserted between and/or substituted for any of the amino acid residues comprising the heavy chain. Furthermore, the same insert of the invention can be introduced multiple times at the same and/or different positions of the amino acid sequence of the FVIII protein. Furthermore, different inserts and/or the same insert may be introduced one or more times at the same and/or different positions between the amino acid residues of the entire amino acid sequence of the FVIII protein.

Some proteins can support a large number of sugar side chains, and the distance between N-linked glycosylation sites can be as little as three, four, five, or six amino acids (see, e.g., Lundin et al, FEBS Lett.581:5601 (2007); Apweiler et al, Biochim. Biophys. acta 1473:4(1991), the entire contents of which are incorporated herein by reference).

In some embodiments, amino acid residues 736 and 737 of the wild-type human sequence (SEQ ID NO:5) are replaced with amino acid residue XX, wherein X is S or T. Thus, residues 736 and 737 may be SS, ST, TS, or TT.

In some embodiments, amino acid residue 736-742 of the wild-type human sequence (SEQ ID NO:5) is replaced with amino acid residue XXYVNRXL (SEQ ID NO:6), wherein X is S or T. Thus, residues 736-742 can be as follows.

Residue 736-742	SEQ ID NO
		TTYVNRSL	7
TTYVNRTL	8
		TSYVNRSL	9
TSYVNRTL	10
		STYVNRSL	11
STYVNRTL	12
		SSYVNRSL	13
SSYVNRTL	14

In some embodiments, amino acid residue 736-742 in the wild-type human sequence (SEQ ID NO:5) is replaced with amino acid residue XXNNX (SEQ ID NO:15), wherein X is S or T. Thus, residue 736-740 can be as follows.

In some embodiments, the modified human factor VIII polypeptide is a polypeptide in which the B domain is deleted, e.g., the SQ deletion from S743 to Gln1638 (numbering as in SEQ ID NO: 5).

FVIII proteins of the invention with additional glycosylation sites can be produced by recombinant methods such as site-directed mutagenesis using PCR. Alternatively, FVIII proteins of the invention may be chemically synthesized to produce FVIII proteins having one or more (e.g., 1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) additional glycosylation sites.

Modifying any amino acid residue or residues in the mature FVIII amino acid sequence according to methods well known in the art and as taught herein, and testing any resulting FVIII protein for activity, stability, recovery, half-life, etc. according to well known methods and as described herein, are within the skill of the person of skill in the art and within the scope of the present invention (see e.g., Elliott et al.

Embodiments of the present invention relate to recombinant FVIII proteins (e.g. X0, X1, X2) wherein glycosylation sites have been added to improve FVIII activity and/or recovery and/or half-life and/or stability. FVIII proteins of the invention comprise modifications that allow for increased bioavailability of FVIII proteins to subjects to whom FVIII proteins of the invention have been administered. In some embodiments, increased bioavailability refers to the standard current thinking in hematology that the concentration of FVIII in plasma is a relevant concentration. In some embodiments of the invention, increased bioavailability refers to the ability of FVIII protein to stay longer in the circulation of a subject. Thus, in some embodiments of the invention, the FVIII protein described herein is modified to produce a FVIII protein having increased activity following expression in vivo, and in some embodiments, the invention provides a method of increasing the hemostatic effectiveness of a FVIII protein in a subject comprising administering to said subject an effective amount of a FVIII protein of the invention, a polynucleotide of the invention, a vector of the invention, and/or a cell of the invention, wherein the FVIII protein administered to said subject in any of these embodiments is a FVIII protein of the invention having increased activity.

FVIII proteins according to the invention are produced and characterized by methods well known in the art and described herein. As is well known in the art, these methods include measuring clotting time (partial thromboplastin time (PPT) assay)) and administering FVIII protein to test animals to determine recovery, half-life and bioavailability by appropriate immunoassay and/or activity assay.

Another aspect of the invention provides isolated polynucleotides encoding FVIII proteins of the invention and expression cassettes for use in producing FVIII proteins.

Another aspect of the invention is a vector, e.g., an expression vector, comprising a polynucleotide of the invention. The vector may be any type of vector known in the art, including but not limited to plasmid vectors and viral vectors. In some embodiments, the viral vector is an AAV vector from any known AAV serotype, including but not limited to this AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV, as well as any other AAV now known or later discovered. In some embodiments, the AAV vector is AAV8 or AAV 9.

Another aspect of the invention relates to cells (e.g., isolated cells, transformed cells, recombinant cells, etc.) comprising a polynucleotide and/or vector of the invention. Accordingly, various embodiments of the present invention relate to recombinant host cells containing such vectors (e.g., expression cassettes). Such cells may be isolated and/or present in a transgenic animal. Transformation of the cells is described further below.

Another aspect of the invention relates to transgenic animals comprising the polynucleotides, vectors and/or transformed cells of the invention. Transgenic animals are described further below.

FVIII proteins, polynucleotides, vectors and/or cells of the invention may be included in pharmaceutical compositions. Some embodiments relate to kits comprising a FVIII protein, polynucleotide, vector and/or cell of the invention, and/or reagents and/or instructions for using the kit, e.g., to perform a method of the invention.

Method of the invention

Another aspect of the invention relates to the use of the promoters and expression cassettes of the invention for producing polypeptides or functional nucleic acids, for example in a liver-specific manner. Accordingly, one aspect relates to a method of producing a polypeptide or functional nucleic acid in the liver of a subject, comprising delivering to the subject a polynucleotide, vector and/or transformed cell of the invention, thereby producing the polypeptide or functional nucleic acid in the liver of the subject. Delivering the polynucleotide, vector and/or transformed cell under conditions in which expression of the polynucleotide of interest occurs to produce a polypeptide or functional nucleic acid. These conditions are well known in the art and are further described below.

Another aspect of the invention relates to methods of treating hemophilia a or acquired factor VIII deficiency in a subject using the promoters and expression cassettes of the invention, comprising delivering to the subject a therapeutically effective amount of a polynucleotide, vector and/or transformed cell of the invention, thereby treating hemophilia a in the subject. In some embodiments, the polynucleotide of interest encodes a FVIII polypeptide as described above.

Another aspect of the invention relates to methods of increasing the bioavailability of a FVIII polypeptide in a subject using the promoters and expression cassettes of the invention comprising delivering to the subject an effective amount of a polynucleotide, vector and/or transformed cell of the invention, thereby increasing the bioavailability of the FVIII polypeptide in the subject. In this aspect, the polynucleotide of interest encodes a FVIII polypeptide as described above.

The modified FVIII proteins of the invention may be used in a method of treating a bleeding disorder by administering an effective amount of the FVIII protein to a subject in need thereof (e.g., a human patient). Accordingly, the present invention also provides a method of treating a bleeding disorder comprising administering to a subject in need thereof an effective amount of a FVIII protein, polynucleotide, vector and/or cell of the invention.

One aspect of the invention relates to a method of producing factor VIII in the liver of a subject, comprising delivering to the subject a polynucleotide, vector and/or transformed cell of the invention encoding a modified human factor VIII polypeptide, thereby producing factor VIII in the liver of the subject.

Another aspect of the invention relates to a method of treating hemophilia a or acquired factor VIII deficiency in a subject, comprising delivering to the subject a therapeutically effective amount of a modified human factor VIII polypeptide, polynucleotide, vector, and/or transformed cell of the invention, thereby treating hemophilia a or acquired factor VIII deficiency in the subject.

Another aspect of the invention relates to a method of increasing the bioavailability of a factor VIII polypeptide in a subject, comprising delivering to the subject an effective amount of a polynucleotide, vector and/or transformed cell of the invention encoding a modified human factor VIII polypeptide, thereby increasing the bioavailability of the factor VIII polypeptide in the subject.

Bleeding disorders that can be treated according to the methods of the present invention include any disorder that can be treated with FVIII, such as hemophilia a and acquired FVIII deficiency. Such treatment protocols and dosing regimens for administering or delivering a FVIII protein of the invention and/or a FVIII protein-encoding polynucleotide of the invention to a subject (e.g., a subject in need thereof) are well known in the art.

In embodiments of the invention, the dosage of a vector (e.g., a viral vector or other nucleic acid vector) encoding a FVIII protein of the invention may be an amount such that a therapeutic plasma concentration of the FVIII protein is achieved. The therapeutic concentration of FVIII protein is considered to be above the normal level of 1% of healthy individuals, measured on average 100%, and is therefore one International Unit (IU) of FVIII in 1mL of normal human plasma. One skilled in the art will be able to determine the optimal dosage for a given subject and a given condition.

For treatments associated with deliberate intervention, FVIII proteins of the invention are typically administered within about 24 hours prior to the intervention, and for up to 7 days or more thereafter. Administration as a coagulant may be by a variety of routes as described herein.

The pharmaceutical compositions are primarily intended for parenteral administration for prophylactic and/or therapeutic treatment. Preferably, the pharmaceutical composition is administered parenterally, i.e. intravenously, subcutaneously or intramuscularly, or by continuous or pulsed infusion. Alternatively, the pharmaceutical composition may be formulated for administration in a variety of ways, including but not limited to oral, subcutaneous, intravenous, intracerebral, intranasal, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, intraocular, or any other acceptable manner.

Compositions for parenteral administration comprise a FVIII protein of the invention in association with (e.g. dissolved in) a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous carriers can be used, such as water, buffered water, 0.4% saline, 0.3% glycine and the like. FVIII proteins of the invention may also be formulated using extended stability and storage compositions (e.g. methionine and sucrose). FVIII proteins of the invention may also be formulated in liposomal formulations for delivery or targeting to the site of injury. Liposome formulations are generally described in U.S. Pat. nos. 4,837,028, 4,501,728, and 4,975,282. The composition may be sterilized by conventional well-known sterilization techniques. The resulting aqueous solution may be packaged for use, or filtered under sterile conditions and lyophilized, the lyophilized formulation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and the like. The composition may also contain preservatives, isotonicity agents, nonionic surfactants or detergents, antioxidants, and/or other various additives.

The concentration of FVIII protein in these formulations can vary widely, i.e., from less than about 0.5 wt% (typically, or at least about 1 wt%) up to about 15 wt% or 20 wt%, and will be selected primarily by fluid volume, viscosity, etc., depending upon the particular mode of administration selected. Thus, as a non-limiting example, a typical pharmaceutical composition for intravenous infusion may be formulated to contain 250ml of sterile ringer's solution and 10mg of FVIII protein. The actual methods for preparing parenterally administrable compositions are known or obvious to those skilled in the art and are described, for exampleRemington's Pharmaceutical SciencesMore detailed description is given in 21 st edition, Mack Publishing Company, Easton, Pa. (2005).

Compositions comprising a FVIII protein according to the invention and/or a nucleic acid molecule encoding a FVIII protein according to the invention may be administered for prophylactic and/or therapeutic treatment. In therapeutic applications, the compositions are administered to a subject already suffering from a disease as described above, in an amount sufficient to cure, alleviate or partially arrest the disease and its complications. An amount sufficient to achieve this goal is defined as a "therapeutically effective amount". As will be appreciated by those skilled in the art, the amount effective for this purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject.

In prophylactic applications, a composition containing a FVIII polypeptide of the invention is administered to a subject susceptible to or otherwise at risk of a disease state or injury, in order to enhance the subject's own coagulation ability. Such an amount is defined as a "prophylactically effective dose". In prophylactic applications, the precise amount again depends on the health and weight of the subject.

Single or multiple administrations of the composition can be carried out with dose levels and patterns selected by the treating physician. For ambulatory subjects (ambulant subjects) that require daily maintenance levels, FVIII protein may be administered by continuous infusion using, for example, a portable pump system.

FVIII proteins of the invention may also be formulated as sustained or extended release formulations. Methods of formulating sustained or extended release compositions are known in the art and include, but are not limited to, semipermeable matrices of solid hydrophobic particles containing the polypeptide.

Local delivery, e.g. topical administration, of FVIII proteins of the invention may be carried out e.g. by spraying, perfusion, double balloon catheters, stents, incorporation into vascular grafts or stents, hydrogels for coating balloon catheters or other established methods. In any event, the pharmaceutical composition should provide an amount of FVIII protein sufficient to effectively treat the subject.

In some embodiments, an AAV vector is used to deliver a polynucleotide of interest (e.g., a FVIII protein) to a subject. Thus, the invention also provides AAV viral particles (i.e., virosomes) comprising a polynucleotide of interest, wherein the viral particle packages (i.e., encapsulates) a vector genome, optionally an AAV vector genome.

In particular embodiments, a virosome (virion) is a recombinant vector comprising a heterologous polynucleotide of interest (e.g., for delivery to a cell). Thus, the invention can be used to deliver polynucleotides to cells in vitro, ex vivo, and in vivo. In representative embodiments, the recombinant vectors of the invention can be advantageously used to deliver or transfer polynucleotides into animal (e.g., mammalian) cells.

Any heterologous nucleotide sequence can be delivered by the viral vectors of the invention. Polynucleotides of interest include polynucleotides encoding polypeptides, optionally therapeutic (e.g., for medical or veterinary use) and/or immunogenic (e.g., vaccine) polypeptides.

Therapeutic polypeptides include, but are not limited to, Cystic Fibrosis Transmembrane Regulator (CFTR), dystrophin (including protein products of the dystrophin minigene (mini-gene) or minigene (micro-gene), see, e.g., Vincent et al, (1993) Nature Genetics 5: 130; U.S. patent application No. 2003017131; Wang et al, (2000) Proc. Natl. Acad. Sci. USA97:13714-9[ Mini-dystrophin (min-dystrophin)](ii) a Harper et al, (2002) Nature Med.8:253-61[ micro-dystrophin)]) (ii) a Mini-agrin (min-agrin), laminin-alpha 2, troponin (α, β, γ or δ), Fukutin-related proteins, myostatin pro peptide, follistatin, dominant negative myostatin (dominant negative myostatin), angiogenic factors (e.g., VEGF, angiopoietin-1 or 2), inhibitors of anti-apoptotic factors (e.g., heme oxygenase-1, TGF- β, pro-apoptotic signal) such as caspase, protease, kinase, death receptor [ e.g., CD-095)]Modulators of cytochrome C release, inhibitors of mitochondrial pore opening and swelling); activin type II soluble receptors, anti-inflammatory polypeptides such as Ikappa B dominant mutants, sarcospan, dystrophin-related protein (utrophin), mini-dystrophin-related protein (mini-utrophin), antibodies or antibody fragments directed against myostatin or myostatin pro peptide, cell cycle regulators, Rho kinase modulators such as Cethrin, which are modified bacterial C3 extracellular enzymes [ available from BioAxone Therapeutics, Inc., Saint-Lauren, Quebec, Canada]BCL-xL, BCL2, XIAP, FLICEc-s, dominant negative caspase (caspase) -8, dominant negative caspase-9, SPI-6 (see, e.g., U.S. patent application No. 20070026076), transcription factors PGC-alpha 1, Pinch gene, ILK gene, and thymosin 4 gene), coagulation factors (e.g., factor VIII, factor IX, factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, intracellular and @Or extracellular superoxide dismutase, leptin, LDL receptor, enkephalinase (neprilysin), lipoprotein lipase, ornithine transcarbamylase, beta-globulin, alpha-globulin, spectrin, alpha-globulin₁Antitrypsin, methylcytosine binding protein 2, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, β -glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase A, branched chain keto acid dehydrogenase, RP65 protein, cytokines (e.g., α -interferon, α 1-interferon, interferon- γ, interleukin-1 to-14, granulocyte-macrophage colony stimulating factor, lymphotoxin, etc.), peptide growth factors, neurotrophic factors and hormones (e.g., growth hormone (somatotropin), insulin-like growth factors including IGF-1 and IGF-2, GLP-1, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factors-3 and-4, brain derived neurotrophic factors, glial derived growth factor, transforming growth factors-alpha 0 and-beta, etc.), bone morphogenic proteins (including RANKL and VEGF), lysosomal proteins, glutamate receptors, lymphokines, soluble CD4, Fc receptors, T cell receptors, ApoE, ApoC, inhibitor 1 of protein phosphatase inhibitor 1(I-1), phospholamban (phospholambban), serca2a, lysosomal acid alpha-glucosidase, alpha-galactosidase A, Barkct, beta 2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), calsaccin, receptors (e.g., tumor necrosis growth factor-alpha soluble receptor), anti-inflammatory factors such as AP IRs, Pim-1, PGC-1 alpha, SOD-1, SOD-2, ECF-SOD, ECF-1, SOD-2, and VEGF), Kallikrein, thymosin-beta 4, hypoxia inducible transcription factor [ HIF]Angiogenesis factor, S100a1, parvalbumin (parvalbumin), adenylyl cyclase type 6, molecules affecting the knockout of the G protein-coupled receptor kinase type 2 (e.g. truncated constitutively active bsarkct); phospholamban inhibitory or dominant negative molecules such as phospholamban S16E, monoclonal antibodies (including single chain monoclonal antibodies) or suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor such as TNF-a), and any other polypeptide having a therapeutic effect in a subject in need thereof.

Heterologous nucleotide sequences encoding polypeptides include those encoding reporter polypeptides (e.g., enzymes). Reporter polypeptides are known in the art and include, but are not limited to, fluorescent proteins (e.g., EGFP, GFP, RFP, BFP, YFP, or dsRED2), enzymes that produce detectable products, such as luciferase (e.g., from Gaussia, Renilla, or Photinus), β -galactosidase, β -glucuronidase, alkaline phosphatase, and chloramphenicol acetyl transferase genes, or directly detectable proteins. Almost any protein can be detected directly by using, for example, an antibody specific for the protein. Other markers (and related antibiotics) suitable for positive or negative selection of eukaryotic cells are disclosed in Sambrook and Russell (2001), Molecular Cloning, 3 rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Ausubel et al (1992), Current Protocols in Molecular Biology, John Wiley & Sons (including periodic updates).

Alternatively, the heterologous nucleic acid may encode functional RNA, such as antisense oligonucleotides, ribozymes (e.g., as described in U.S. Pat. No. 5,877,022), RNAs that affect spliceosome-mediated trans-splicing (see Puttajaju et al, (1999) Nature Biotech.17: 246; U.S. Pat. No. 6,013,487; U.S. Pat. No. 6,083,702), interfering RNAs (RNAi), including small interfering RNAs (siRNAs) that mediate gene silencing (see Sharp et al, (2000) Science287:2431), microRNAs, or other non-translated "functional" RNAs such as "guide" RNAs (Gorman et al, (1998) Proc.Nat.Acad.Sci.USA 95: 4929; Yuan et al, U.S. Pat. No. 5,869,248), and the like. Exemplary untranslated RNAs include RNAi or antisense RNA to a multidrug resistance (MDR) gene product (e.g., for treating tumors and/or administering to the heart to prevent damage from chemotherapy), RNAi or antisense RNA to myostatin (Duchenne or Becker muscular dystrophy), RNAi or antisense RNA to VEGF or tumor immunogens (including but not limited to those tumor immunogens specifically described herein) (for treating tumors), RNAi or antisense oligonucleotides to mutated dystrophin (Duchenne or Becker muscular dystrophy), RNAi or antisense RNA to a hepatitis b surface antigen gene (to prevent and/or treat hepatitis b infection), RNAi or antisense RNA to an HIV tat and/or rev gene (to prevent and/or treat HIV), and/or RNAi or antisense RNA to any other gene product from a pathogen (to protect a subject from a pathogen) or a defective gene product (to prevent or treat disease) RNAi or antisense RNA of (a). RNAi or antisense RNA directed against the above target or any other target may also be used as research reagents.

As known in the art, antisense nucleic acid (e.g., DNA or RNA) and inhibitory RNA (e.g., microrna and RNAi such as siRNA or shRNA) sequences can be used to induce "exon skipping" in patients with muscular dystrophy resulting from a dystrophin gene deficiency. Thus, the heterologous nucleic acid may encode an antisense nucleic acid or inhibitory RNA that induces appropriate exon skipping. One skilled in the art will appreciate that the specific method of exon skipping depends on the nature of the underlying defect of the dystrophin gene, and that many such strategies are known in the art. Exemplary antisense nucleic acids and inhibitory RNA sequences target upstream branch points and/or downstream donor splice sites and/or internal splice enhancer sequences of one or more dystrophin exons (e.g., exon 19 or 23). For example, in particular embodiments, the heterologous nucleic acid encodes an antisense nucleic acid or inhibitory RNA directed to a branch point upstream and a splice donor site downstream of exon 19 or 23 of the dystrophin gene. Such sequences can be incorporated into AAV vectors that deliver modified U7 snRNA and antisense or inhibitory RNA (see, e.g., Goyenvalle et al, (2004) Science306: 1796-. As another strategy, the modified U1 snRNA can be incorporated into AAV vectors with siRNA, microrna or antisense RNA complementary to splice sites upstream and downstream of dystrophin exons (e.g., exon 19 or 23) (see, e.g., Denti et al, (2006) proc.nat.acad.sci.usa 103: 3758-. In addition, antisense nucleic acids and inhibitory RNA can target splice enhancer sequences within exons 19, 43, 45, or 53 (see, e.g., U.S. Pat. No. 6,653,467; U.S. Pat. No. 6,727,355; and U.S. Pat. No. 6,653,466).

Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific manner. Ribozymes have specific catalytic domains with endonuclease activity (Kim et al, (1987) Proc. Natl. Acad. Sci. USA 84: 8788; Gerlach et al, (1987) Nature328: 802; Forster and Symons, (1987) Cell 49: 211). For example, a large number of ribozymes accelerate phosphotransesterification reactions with a high degree of specificity, typically cleaving only one of a number of phosphoesters in an oligonucleotide substrate (Michel and Westhof, (1990) J.mol.biol.216: 585; Reinhold-Hurek and Shub, (1992) Nature 357: 173). This specificity has been attributed to the following requirements: prior to the chemical reaction, the substrate binds to the internal guide sequence ("IGS") of the ribozyme via specific base-pairing interactions.

Ribozyme catalysis has been observed primarily as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, (1989) Nature 338: 217). For example, U.S. patent No. 5,354,855 reports that certain ribozymes can act as endonucleases with sequence specificities greater than that of known ribozymes and approaching that of DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of nucleic acid expression may be particularly useful for therapeutic applications (Scanlon et al, (1991) Proc. Natl. Acad. Sci. USA 88: 10591; Sarver et al, (1990) Science 247: 1222; Sioud et al, (1992) J.mol.biol.223: 831).

Micro RNA (mir) is a native cellular RNA molecule that can regulate the expression of a variety of genes by controlling the stability of mRNA. Overexpression or reduction of specific micrornas, useful in the treatment of dysfunction, has been shown to be effective in a number of animal models of disease states and diseases (see, e.g., Couzin, (2008) Science 319: 1782-4). Chimeric AAVs are useful for the delivery of micrornas into cells, tissues and subjects, for the treatment of genetic and acquired diseases, or for enhancing the functionality and growth promotion of certain tissues. For example, mir-1, mir-133, mir-206, and/or mir-208 can be used to treat cardiac and skeletal muscle diseases (see, e.g., Chen et al, (2006) Genet.38: 228-33; van Rooij et al, (2008) Trends Genet.24: 159-66). microRNAs can also be used to modulate the immune system following gene delivery (Brown et al, (2007) Blood 110: 4144-52).

The term "antisense oligonucleotide" (including "antisense RNA") as used herein refers to a nucleic acid that is complementary to, and specifically hybridizes to, a specified DNA or RNA sequence. Antisense oligonucleotides and nucleic acids encoding the antisense oligonucleotides can be prepared according to conventional techniques. See, for example, U.S. Pat. nos. 5,023,243 to Tullis; U.S. Pat. No. 5,149,797 to Pederson et al.

One skilled in the art will appreciate that the antisense oligonucleotide need not be fully complementary to the target sequence, so long as the degree of sequence similarity is sufficient to allow the antisense nucleotide sequence to specifically hybridize to its target (as defined above) and reduce production of protein products (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more).

To determine the specificity of hybridization, hybridization of such oligonucleotides to target sequences can be performed under reduced stringency, moderate stringency, or even stringent conditions. Suitable conditions for achieving reduced, moderate, and stringent hybridization conditions are described herein.

Alternatively, in particular embodiments, the antisense oligonucleotides of the invention have at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or more sequence identity to the complement of the target sequence and reduce production of the protein product (as defined above). In some embodiments, the antisense sequence contains 1, 2,3, 4,5, 6,7, 8, 9, or 10 mismatches compared to the target sequence.

Methods of determining percent nucleic acid sequence identity are described in more detail elsewhere herein.

The length of the antisense oligonucleotide is not critical as long as it specifically hybridizes to the predetermined target and reduces production of the protein product (as defined above) and can be determined according to conventional procedures. Typically, the antisense oligonucleotide is at least about 8, 10 or 12 or 15 nucleotides in length and/or less than about 20, 30, 40, 50, 60, 70, 80, 100 or 150 nucleotides in length.

RNA interference (RNAi) is another useful method to reduce the production of protein products (e.g., shRNA or siRNA). RNAi is a mechanism of post-transcriptional gene silencing in which double-stranded rna (dsrna) corresponding to a target sequence of interest is introduced into a cell or organism, resulting in degradation of the corresponding mRNA. Sharp et al, (2001) Genes Dev 15: 485-; and Hammond et al, (2001) Nature Rev.Gen.2:110-119) reviewed the mechanism by which RNAi effects gene silencing. The RNAi effect persists through multiple cell divisions before gene expression is regained. Therefore, RNAi is a powerful method for targeted knock-outs or "knockdowns" at the RNA level. RNAi has proven successful in human cells including human embryonic kidney and HeLa cells (see, e.g., Elbashir et al, Nature (2001)411: 494-8).

Initial attempts to use RNAi in mammalian cells have resulted in antiviral defense mechanisms involving PKR in response to dsRNA molecules (see, e.g., Gil et al, (2000) Apoptosis5: 107). It has been demonstrated that short synthetic dsRNAs of about 21 nucleotides, termed "short interfering RNAs" (siRNAs), can mediate silencing in mammalian cells without eliciting an antiviral response (see, e.g., Elbashir et al, Nature (2001)411: 494-8; Caplen et al, (2001) Proc. Nat. Acad. Sci. USA 98: 9742).

RNAi molecules, including siRNA molecules, can be short hairpin RNAs (shRNAs; see Paddison et al, (2002), Proc. nat. Acad. Sci. USA 99:1443-1448), which are thought to be siRNA molecules processed into 20-25 mer in cells by the action of the RNase III-like enzyme Dicer. shrnas typically have a stem-loop structure in which two inverted repeats are separated by a short spacer sequence that loops out. Shrnas having loops of 3 to 23 nucleotides in length have been reported. The loop sequence is generally not critical. Exemplary loop sequences include the following motifs: AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC and UUCAAGAGA.

The RNAi may further comprise a circular molecule comprising sense and antisense regions flanked on one of its sides by two loop regions so as to form a "dumbbell" shaped structure when the dsRNA is formed between said sense and antisense regions. The molecule can be treated in vitro or in vivo to release dsRNA moieties, such as siRNA.

International patent publication WO 01/77350 describes a vector for bidirectional transcription to produce sense and antisense transcripts of heterologous sequences in eukaryotic cells. This technique can be used to generate RNAi for use according to the present invention.

Shinagawa et al, (2003) Genes Dev.17:1340 report methods for expressing long dsRNA from the CMV promoter (pol II promoter), which are also applicable to tissue-specific pol II promoters. Likewise, the method of Xia et al, (2002) Nature Biotech.20:1006 avoids the poly (A) tailing and can be used in conjunction with tissue-specific promoters.

Methods for producing RNAi include chemical synthesis, in vitro transcription, digestion of long dsRNA by Dicer (in vitro or in vivo), expression from delivery vectors in vivo, and expression from PCR-derived RNAi expression cassettes (see, e.g., TechNotes 10 (3)' Five Ways to product siRNAs, "from Ambion, Inc., Austin TX; available at www.ambion.com).

Guidelines for designing siRNA molecules are available (see, e.g., literature from Ambion, Inc., Austin TX; available at www.ambion.com). In a specific embodiment, the siRNA sequence has a G/C content of about 30-50%. Furthermore, long stretches of more than four T or a residues are typically avoided if RNA polymerase III is used to transcribe RNA. An online siRNA target finder (target finder) may be obtained, for example, from Ambion, inc. (www.ambion.com), by the wye black biomedical Research institute (www.jura.wi.mit.edu), or from Dharmacon Research, inc. (www.dharmacon.com).

The antisense region of the RNAi molecule can be fully complementary to the target sequence, but need not be fully complementary so long as it specifically hybridizes to the target sequence (as defined above) and reduces production of the protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments, hybridization of such oligonucleotides to a target sequence can be performed under reduced stringency, moderate stringency, or even stringent conditions as defined above.

In other embodiments, the antisense region of the RNAi has at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or more sequence identity to the complement of the target sequence and reduces production of the protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments, the antisense region contains 1, 2,3, 4,5, 6,7, 8, 9, or 10 mismatches compared to the target sequence. Mismatches are generally better tolerated at the ends of the dsRNA than in the central portion.

In a specific embodiment, the RNAi is formed by intermolecular complexation between two separate sense and antisense molecules. RNAi comprises a ds region formed by intermolecular base pairing between two separate strands. In other embodiments, the RNAi comprises a ds region, typically an inverted repeat (e.g., shRNA or other stem-loop structure, or a circular RNAi molecule), formed by intramolecular base pairing within a single nucleic acid molecule comprising the sense and antisense regions. The RNAi can further comprise a spacer region between the sense region and the antisense region.

Generally, RNAi molecules are highly selective. If desired, one skilled in the art can readily exclude candidate RNAi that may interfere with the expression of nucleic acids other than the target by, for example, using BLAST (available on www.ncbi.nlm.nih.gov/BLAST), searching relevant databases to identify RNAi sequences that do not have substantial sequence homology to other known sequences.

Kits for producing RNAi are commercially available, for example, from New England Biolabs, Inc.

The recombinant viral vector may also comprise a heterologous nucleotide sequence that shares homology with and recombines with a site on the host chromosome. The method can be used to correct a genetic defect in a host cell.

The invention also provides recombinant viral vectors expressing immunogenic polypeptides, e.g., for vaccination. The heterologous nucleic acid can encode any immunogen of interest known in the art, including but not limited to immunogens from human immunodeficiency virus, influenza virus, gag protein, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like. Alternatively, the immunogen may be present in (e.g., incorporated into) or linked to (e.g., by covalent modification) the viral capsid.

The use of parvoviruses as vaccines is known in the art (see, e.g., Miyamura et al, (1994) Proc. Nat. Acad. Sci. USA 91: 8507; U.S. Pat. No. 5,916,563 to Young et al, U.S. Pat. No. 5,905,040 to Mazzara et al, U.S. Pat. No. 5,882,652, U.S. Pat. No. 5,863,541 to Samulski et al; the disclosures of which are incorporated herein by reference in their entirety). The antigen may be present in the viral capsid. Alternatively, the antigen may be expressed from a heterologous nucleic acid introduced into the recombinant vector genome.

The immunogenic polypeptide or immunogen may be any polypeptide suitable for protecting a subject against a disease including, but not limited to, microbial, bacterial, protozoan, parasitic, fungal, and viral diseases. For example, the immunogen may be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as an influenza virus Hemagglutinin (HA) surface protein or an influenza virus nucleoprotein gene, or an equine influenza virus immunogen), or a lentiviral immunogen (e.g., an equine infectious anemia virus immunogen, a Simian Immunodeficiency Virus (SIV) immunogen, or a Human Immunodeficiency Virus (HIV) immunogen, such as an HIV or SIV envelope GP160 protein, an HIV or SIV matrix/capsid protein, and an HIV or SIV gag, pol, and env gene product). The immunogen can also be an arenavirus immunogen (e.g., a lassa fever virus immunogen, such as the lassa fever virus nucleocapsid protein gene and the lassa fever envelope glycoprotein gene), a poxvirus immunogen (e.g., a vaccinia, such as the vaccinia L1 or L8 gene), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an ebola virus immunogen or a marburg virus immunogen, such as NP and GP genes), a bunyavirus immunogen (e.g., RVFV, CCHF, and SFS viruses), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, such as a human coronavirus envelope glycoprotein gene, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen, or a Severe Acute Respiratory Syndrome (SARS) immunogen, such as S [ S1 or S2], M, E, or an N protein, or an immunogenic fragment thereof). The immunogen may further be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogen), a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis a, hepatitis b, or hepatitis c) immunogen, or any other vaccine immunogen known in the art.

Alternatively, the immunogen may be any tumor or cancer cell antigen. Alternatively, a tumor or cancer antigen is expressed on the surface of a cancer cell. Exemplary cancer and tumor cell antigens are described in s.a. rosenberg, (1999) Immunity 10: 281). Illustrative cancer and tumor antigens include, but are not limited to: BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1/2, BAGE, RAGE, NY-ESO-1, CDK-4, β -catenin, MUM-1, caspase (caspase) -8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigen (Kawakami et al., (1994) Proc.Natl.Acad.Sci.USA 91: 3515; Kawakami et al., (1994) J.exp.Med.,180: 347; Kawakami et al., (1994) Cancer Res.54:3124) including MART-1(Coulie et al., (1991) J.exp.180: 35), gp100 (gp 100 et al., (1988) Cuthiol.254.1994), MAGE-1, MAGE-1643, MAGE-1, TRP-1, MAGE, TRP, TME, TM; TRP-2; p-15 and tyrosinase (Brichard et al, (1993) J.Exp.Med.178: 489); the HER-2/neu gene product (U.S. Pat. No. 4,968,603); CA 125; HE 4; LK 26; FB5 (endosialin); TAG 72; AFP; CA 19-9; NSE; DU-PAN-2; CA 50; span-1; CA 72-4; HCG; STN (sialyltn antigen); c-erbB-2 protein; PSA; L-CanAg; an estrogen receptor; (ii) milk fat globulin; p53 tumor suppressor protein (Levine, (1993) Ann. Rev. biochem.62: 623); mucin antigens (international patent publication WO 90/05142); a telomerase; nuclear matrix protein; prostatic acid phosphatase; papillomavirus antigens; and antigens associated with: melanoma, adenocarcinoma, thymoma, sarcoma, lung cancer, liver cancer, colorectal cancer, non-hodgkin's lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, etc. (see, e.g., Rosenberg, (1996) annu.rev.med.47: 481-91).

Alternatively, the heterologous nucleotide sequence may encode any polypeptide which is desired to be produced in a cell in vitro, ex vivo or in vivo. For example, viral vectors can be introduced into cultured cells and the expressed protein product isolated therefrom.

One skilled in the art will appreciate that the heterologous polynucleotide of interest can be operably associated with appropriate control sequences. For example, a heterologous nucleic acid can be operably associated with an expression control element, such as a transcription/translation control signal, an origin of replication, a polyadenylation signal, an Internal Ribosome Entry Site (IRES), a promoter, an enhancer, and the like.

One skilled in the art will further appreciate that various promoter/enhancer elements may be used depending on the level and tissue-specific expression desired. Promoters/enhancers can be constitutive or inducible, depending on the desired expression pattern. Promoters/enhancers may be natural or exogenous, and may be natural or synthetic sequences. "exogenous (for) means that the transcriptional initiation region is not found in the wild-type host into which it is introduced.

The promoter/enhancer element may be native to the target cell or subject to be treated, and/or native to the heterologous nucleic acid sequence. The promoter/enhancer element is typically selected to function in the target cell of interest. In representative embodiments, the promoter/enhancer element is a mammalian promoter/enhancer element. The promoter/enhancer element may be constitutive or inducible.

Inducible expression control elements are commonly used in those applications where it is desirable to provide for the regulation of expression of a heterologous nucleic acid sequence. Inducible promoter/enhancer elements for gene delivery can be tissue specific or tissue preferred promoter/enhancer elements and include muscle specific or preferred (including cardiac, skeletal and/or smooth muscle), neural tissue specific or preferred (including brain specific), eye (including retina specific and cornea specific), liver specific or preferred, bone marrow specific or preferred, pancreas specific or preferred, spleen specific or preferred, and lung specific or preferred promoter/enhancer elements. Other inducible promoter/enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoter/enhancer elements include, but are not limited to, a Tet on/off element, a RU486 inducible promoter, an ecdysone inducible promoter, a rapamycin inducible promoter, and a metallothionein promoter.

In embodiments in which the heterologous nucleic acid sequence is transcribed and translated in the target cell, a specific initiation signal is typically used for efficient translation of the inserted protein-coding sequence. These exogenous translational control sequences, which may include the ATG initiation codon and adjacent sequences, may be of various origins (both natural and synthetic).

The invention also provides methods of producing the viral vectors of the invention. In representative embodiments, the invention provides a method of producing a recombinant viral vector, the method comprising providing to a cell in vitro (a) a template comprising (i) a polynucleotide of interest and (ii) a packaging signal sequence (e.g., one or more (e.g., two) terminal repeats, such as an AAV terminal repeat) sufficient to encapsulate an AAV template into a viral particle, and (b) an AAV sequence (e.g., an AAV rep and AAV cap sequence) sufficient to allow replication of the template and incorporation of the template into the viral particle. The template and AAV replication and capsid sequences are provided under conditions such that a recombinant viral particle comprising the template packaged within a capsid is produced in a cell. The method may further comprise the step of collecting viral particles from said cells. The virus particles can be collected from the culture medium and/or by lysing the cells.

In one illustrative embodiment, the invention provides a method of producing a rAAV particle comprising an AAV capsid, the method comprising: providing to a cell in vitro nucleic acid encoding an AAV capsid, an AAV rep coding sequence, an AAV vector genome comprising a polynucleotide of interest, and helper (helper) functions for generating productive AAV infection; and allowing assembly of an AAV particle comprising an AAV capsid and encapsidating the AAV vector genome.

The cell is typically one that allows replication of the AAV virus. Any suitable cell known in the art, such as a mammalian cell, may be used. Also suitable are trans-complementing packaging cell lines (trans-complementing cell lines) which provide a function deleted from the replication-defective helper virus, such as 293 cells or other E1a trans-complementing cells (trans-complementing cells).

AAV replication and capsid sequences can be provided by any method known in the art. Current protocols typically express the AAV rep/cap gene on a single plasmid. AAV replication and packaging sequences need not be provided together, although this may be convenient. The AAV rep and/or cap sequences may be provided by any viral or non-viral vector. For example, the rep/cap sequence may be provided by a hybrid adenovirus or herpes virus vector (e.g., inserted into the E1a or E3 region of a deleted adenovirus vector). The AAV cap and rep genes can also be expressed using EBV vectors. One advantage of this approach is that the EBV vector is episomal (episomal), but will maintain a high copy number throughout consecutive cell divisions (i.e., stably integrated into cells as an extrachromosomal element, designated as EBV-based nuclear episome (episome)).

As a further alternative, the rep/cap sequence (episomal or integrated) may be stably carried within the cell.

Typically, the AAV rep/cap sequences are not flanked by AAV packaging sequences (e.g., AAV ITRs) to prevent rescue (rescue) and/or packaging of these sequences.

The template (e.g., rAAV vector genome) can be provided to the cell using any method known in the art. For example, the template may be provided by a non-viral (e.g., plasmid) or viral vector. In particular embodiments, the template is provided by a herpesvirus or an adenoviral vector (e.g., inserted into the E1a or E3 region of a deleted adenovirus). As another illustration, Palombo et al, (1998) J.Virol.72:5025 describe baculovirus vectors carrying reporter genes flanked by AAV ITRs. EBV vectors can also be used to deliver templates, as described above for the rep/cap gene.

In another representative embodiment, the template is provided by a replicating rAAV virus. In yet other embodiments, the AAV provirus is stably integrated into the chromosome of the cell.

To achieve maximum viral titers, the cells are typically provided with helper viral functions (e.g., adenovirus or herpes virus) important for productive AAV infection. Helper viral sequences required for AAV replication are known in the art. Typically, these sequences are provided by a helper adenovirus or herpes virus vector. Alternatively, the adenoviral or herpesvirus sequences may be provided by another non-viral or viral vector, for example as a non-infectious adenovirus mini-plasmid (minilasmid) carrying all the helper genes required for efficient AAV production, as described in Ferrari et al, (1997) Nature Med.3:1295 and U.S. Pat. Nos. 6,040,183 and 6,093,570.

Furthermore, helper virus function (helper virus function) can be provided by a packaging cell having a helper gene (helper gene) integrated in the chromosome or maintained as a stable extrachromosomal element. In representative embodiments, the helper viral sequences cannot be packaged in an AAV virion, e.g., are not flanked by AAV ITRs.

Those skilled in the art will appreciate that it may be advantageous to provide AAV replication and capsid sequences as well as helper viral sequences (e.g., adenoviral sequences) on a single helper construct. The helper construct may be a non-viral or viral construct, but may alternatively be a hybrid adenovirus or a hybrid herpesvirus comprising an AAV rep/cap gene.

In a specific embodiment, the AAV rep/cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector. The vector also contains a rAAV template. AAV rep/cap sequences and/or rAAV templates may be inserted into the deleted region of the adenovirus (e.g., the E1a or E3 region).

In another embodiment, the AAV rep/cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector. The rAAV template is provided as a plasmid template.

In another illustrative embodiment, the AAV rep/cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector, and the rAAV template is integrated into the cell as a provirus. Alternatively, the rAAV template is provided by an EBV vector that is maintained intracellularly as an extrachromosomal element (e.g. as an "EBV-based nuclear episome", see Margolski, (1992) curr. top. microbiol. immun.158: 67).

In another exemplary embodiment, the AAV rep/cap sequences and adenoviral helper sequences are provided by a single adenoviral helper (single adenoviral helper). The rAAV template is provided as a separate replicating viral vector. For example, the rAAV template may be provided by a rAAV particle or a second recombinant adenovirus particle.

In accordance with the methods described above, hybrid adenoviral vectors typically comprise sufficient 5 'and 3' cis sequences (i.e., adenoviral terminal repeats and PAC sequences) of adenovirus for replication and packaging of the adenovirus. The AAV rep/cap sequences and, if present, rAAV template are embedded in the adenoviral backbone and flanked by the 5 'and 3' cis sequences so that these sequences can be packaged into an adenoviral capsid. As described above, in representative embodiments, the adenoviral helper sequence (helper sequence) and AAV rep/cap sequences are not flanked by AAV packaging sequences (e.g., AAV ITRs) such that these sequences are not packaged into AAV virions.

Herpes viruses may also be used as helper viruses in AAV packaging methods. Hybrid herpesviruses encoding AAV rep proteins may advantageously facilitate a more scalable AAV vector production scheme. Hybrid herpes simplex virus type I (HSV-1) vectors have been described which express the AAV-2rep and cap genes (Conway et al, (1999) Gene Therapy 6:986 and WO 00/17377, the disclosures of which are incorporated herein in their entirety).

As another alternative, the viral vectors of the invention can be produced in insect cells using baculovirus vectors to deliver rep/cap genes and rAAV templates as described in Urabe et al, (2002) Human Gene Therapy 13: 1935-43.

Other methods of producing AAV use stably transformed packaging cells (see, e.g., U.S. patent No. 5,658,785).

A stock (stock) of AAV vectors free of contaminating helper virus can be obtained by any method known in the art. For example, AAV and helper virus can be easily distinguished by size. AAV can also be isolated from helper viruses based on affinity for heparin substrates (Zolotukhin et al, (1999) Gene Therapy 6: 973). In representative embodiments, a deleted replication-defective helper virus is used, such that any contaminating helper virus is not replication-competent. As a further alternative, adenoviral helpers lacking late gene expression can be used, as only adenoviral early gene expression is required to mediate packaging of AAV viruses. Adenoviral mutants deficient in late gene expression are known in the art (e.g., ts100K and ts149 adenoviral mutants).

The packaging method of the invention can be used to produce a high titer viral particle stock. In particular embodiments, the viral stock (stock) has a titer of at least about 10⁵A transduction unit (tu)/ml of at least about 10⁶tu/ml, at least about 10⁷tu/ml, at least about 10⁸tu/ml, at least about 10⁹tu/ml or at least about 10¹⁰tu/ml。

In a specific embodiment, the present invention provides a pharmaceutical composition comprising the viral vector of the present invention and optionally other therapeutic agents, stabilizers, buffers, carriers, adjuvants, diluents and the like in a pharmaceutically acceptable carrier. For injection, the carrier is typically a liquid. For other modes of administration, the carrier may be a solid or a liquid. For administration by inhalation, the carrier will be respirable and will preferably be in the form of solid or liquid particles.

The term "pharmaceutically acceptable" refers to a substance that is not toxic or otherwise undesirable, i.e., the substance can be administered to a subject without causing any undesirable biological effects.

One aspect of the invention is a method of transferring a polynucleotide of interest to a cell in vitro. Viral vectors can be introduced into cells at an appropriate multiplicity of infections according to standard transduction methods appropriate for the particular target cell. The titer of the viral vector or capsid for administration can vary depending on the type and number of target cells and the particular viral vector or capsid, and can be determined by one skilled in the art without undue experimentation. In particular embodiments, at least about 10 is introduced into the cell³An infectious unit, more preferably at least about 10⁵And (4) infectious units.

The cells into which the viral vector can be introduced can be of any type, including, but not limited to, neural cells (including cells of the peripheral and central nervous systems, in particular, brain cells such as neurons, oligodendrocytes, glial cells, astrocytes), lung cells, eye cells (including retinal cells, retinal pigment epithelium, and corneal cells), epithelial cells (e.g., intestinal and respiratory epithelial cells), skeletal muscle cells (including myoblasts, myotubes, and muscle fibers), diaphragm muscle cells, dendritic cells, pancreatic cells (including islet cells), liver cells, gastrointestinal tract cells (including smooth muscle cells, epithelial cells), heart cells (including cardiac muscle cells), bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, joint cells (including, for example, cartilage, meniscus, synovium, and bone marrow), germ cells, and the like. Alternatively, the cell may be any progenitor cell. Alternatively, the cell may be a stem cell (e.g., neural stem cell, hepatic stem cell). As another alternative, the cell may be a cancer or tumor cell (such as the cancers and tumors described above). Furthermore, as noted above, the cells may be from any source species.

The viral vector may be introduced into the cell in vitro with the purpose of administering the modified cell to a subject. In particular embodiments, the cells have been removed from the subject, the viral vector introduced therein, and then the cells replaced back into the subject. Methods of removing cells from a subject for in vitro therapy and then introducing back into the subject are known in the art (see, e.g., U.S. Pat. No. 5,399,346). Alternatively, the recombinant viral vector is introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof.

Suitable cells for ex vivo gene therapy are described above. The dosage of cells administered to a subject will vary depending on the age, condition and species of the subject, the cell type, the nucleic acid expressed by the cell, the mode of administration, and the like. Typically, will be at least about 10 per dose in a pharmaceutically acceptable carrier²To about 10⁸Or about 10³To about 10⁶The administration is carried out on individual cells. In a specific embodiment, cells transduced with a viral vector are administered in an effective amount in combination with a pharmaceutical carrierA subject.

In some embodiments, cells that have been transduced with a viral vector can be administered to elicit an immunogenic response (e.g., expressed as a transgene or in the capsid) against the delivered polypeptide. Typically, an amount of cells expressing an effective amount of the polypeptide is administered in combination with a pharmaceutically acceptable carrier. Optionally, the dose is sufficient to generate a protective immune response (as defined above). The degree of protection conferred need not be complete or permanent, so long as the benefit of administering the immunogenic polypeptide outweighs any disadvantage thereof.

Another aspect of the invention is a method of administering a viral vector of the invention to a subject. In particular embodiments, the method comprises a method of delivering a polynucleotide of interest to an animal subject, the method comprising: an effective amount of a viral vector according to the invention is administered to an animal subject. The viral vectors of the invention can be administered to a human subject or animal in need thereof by any method known in the art. Optionally, the viral vector (vector) is delivered in an effective dose in a pharmaceutically acceptable carrier (carrier).

The viral vectors of the invention may further be administered to a subject to elicit an immunogenic response (e.g., as a vaccine). Typically, the vaccines of the present invention comprise an effective amount of the virus in combination with a pharmaceutically acceptable carrier. Optionally, the dose is sufficient to generate a protective immune response (as defined above). The degree of protection conferred need not be complete or permanent, so long as the benefit of administering the immunogenic polypeptide outweighs any of its disadvantages. The subject and immunogen are as described above.

The dose of the viral vector to be administered to a subject will depend on the mode of administration, the disease or disorder to be treated, the condition of the individual subject, the particular viral vector and the nucleic acid to be delivered, and can be determined in a conventional manner. An exemplary dose for achieving a therapeutic effect is at least about 10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²、10³、10¹⁴、10¹⁵Viruses of one transduction unit or higherTitre, preferably about 10⁷Or 10⁸、10⁹、10¹⁰、10¹¹、10¹²、10¹³Or 10¹⁴A transduction unit, even more preferably about 10¹²And (4) transduction units.

In particular embodiments, more than one administration (e.g., two, three, four, or more administrations) can be used to achieve a desired level of gene expression over various spaced time periods (e.g., daily, weekly, monthly, yearly, etc.).

Exemplary modes of administration include oral, rectal, transmucosal, topical, intranasal, inhalation (e.g., by aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [ including to bone, septum and/or myocardium ], intradermal, intrapleural, intracerebral and intraarticular), topical (e.g., skin and mucosal surfaces, including airway surfaces and transdermal administration), intralymphatic, and the like, and direct tissue or organ injection (e.g., liver, skeletal muscle, cardiac muscle, septum muscle or brain). Administration to a tumor (e.g., in or near a tumor or lymph node) is also possible. The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular carrier being used.

Delivery to any of these tissues may also be achieved by delivering a depot (depot) containing the viral vector, which may be implanted in the tissue or which may be in contact with a membrane or other matrix containing the viral vector. Examples of such implantable matrices or substrates are described in U.S. Pat. No. 7,201,898.

The invention is useful for treating a condition of a tissue or organ. Alternatively, the invention can be practiced to deliver nucleic acids to tissues or organs that are used as a platform for producing protein products (e.g., enzymes) or untranslated RNAs (e.g., RNAi, micrornas, antisense RNAs) that circulate in the blood or are systemically delivered to other tissues to treat disorders (e.g., metabolic disorders, such as diabetes (e.g., insulin), hemophilia (e.g., factor IX or factor VIII), or lysosomal storage disorders (e.g., gaucher's disease [ glucocerebrosidase ], pompe disease [ lysosomal acid α -glucosidase ], or glycogen storage diseases (e.g., lysosomal acid α -glucosidase ]).other suitable proteins for treating metabolic disorders are described above.

Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in a liquid prior to injection, or as emulsions. Alternatively, the viral vector may be administered locally rather than systemically, for example in the form of a depot or sustained release formulation. In addition, viral vectors can be delivered dried into surgically implantable matrices, such as bone graft substitutes, sutures, stents, and the like (e.g., as described in U.S. Pat. No. 7,201,898).

Pharmaceutical compositions suitable for oral administration may be presented as discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of a composition of the present invention; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as a water-in-oil or water-in-oil emulsion. Oral delivery can be carried out by complexing the viral vectors of the invention with a vector that is resistant to degradation by digestive enzymes in the intestinal tract of the animal. Examples of such carriers include plastic capsules or tablets as known in the art. These formulations are prepared by any suitable pharmaceutical method, including the step of bringing into association the composition with a suitable carrier (carrier), which may contain one or more accessory ingredients as described above. Generally, the pharmaceutical compositions according to embodiments of the present invention are prepared by uniformly and intimately admixing the composition with liquid or finely divided solid carriers or both, and then, if necessary, shaping the resulting mixture. For example, tablets may be prepared by compressing or molding a powder or granules containing the composition and optionally one or more accessory ingredients. Compressed tablets are prepared by compressing in a suitable machine the composition in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent and/or surfactant/dispersant. Molded tablets are prepared by molding in a suitable machine a powdered compound moistened with an inert liquid binder.

Pharmaceutical compositions suitable for buccal (sublingual) administration include lozenges comprising the composition of the invention in a flavoured base (base), typically sucrose and acacia or tragacanth; and pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia.

Pharmaceutical compositions suitable for parenteral administration may comprise sterile aqueous and non-aqueous injection solutions of the compositions of the present invention, which preparations are optionally isotonic with the blood of the intended recipient. These formulations may contain antioxidants, buffers, bacteriostats and solutes that render the composition isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions, solutions and emulsions may include suspending agents and thickening agents. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles (vehicles) include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution or fixed oils (fixed oils). Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

The compositions may be presented in unit/dose or multi-dose containers, for example, in sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier (carrier), for example, saline or water for injection, immediately prior to use.

Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind described above. For example, the injectable stable sterile compositions of the invention may be provided in unit dosage form in a sealed container. The composition may be provided in the form of a lyophilizate which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject. The unit dosage form can be from about 1 μ g to about 10g of the composition of the invention. When the composition is substantially water-insoluble, a sufficient amount of a physiologically acceptable emulsifier can be included in the aqueous carrier in a sufficient amount to emulsify the composition. One such useful emulsifier is phosphatidylcholine.

Pharmaceutical compositions suitable for rectal administration may be presented as unit dose suppositories. These can be prepared by mixing the composition with one or more conventional solid carriers (e.g., cocoa butter) and then shaping the resulting mixture.

The pharmaceutical compositions of the present invention, which are suitable for topical application to the skin, may take the form of ointments, creams, lotions, pastes, gels, sprays, aerosols or oils. Carriers (carriers) that may be used include, but are not limited to, petrolatum, lanolin, polyethylene glycols, alcohols, dermal penetration enhancers, and combinations of two or more thereof. In some embodiments, for example, topical delivery may be carried out by mixing a pharmaceutical composition of the invention with a lipophilic agent capable of entering the skin (e.g., DMSO).

Pharmaceutical compositions suitable for transdermal administration may be in the form of discrete patches adapted to remain in intimate contact with the epidermis of the subject for an extended period of time. Compositions suitable for transdermal administration may also be delivered by iontophoresis (see, e.g., pharm. res.3:318(1986)) and are typically in the form of an aqueous solution of an optionally buffered composition of the invention. Suitable formulations may comprise citrate or bis \ tris buffer (pH6) or ethanol/water and may contain 0.1 to 0.2M of active ingredient.

The viral vectors disclosed herein may be administered to the lungs of a subject by any suitable means, for example by administering an aerosol suspension of respirable particles comprised of the viral vector, which the subject inhales. The inhalable particles may be liquid or solid. As known to those skilled in the art, aerosols of liquid particles comprising viral vectors may be generated by any suitable means, for example with a pressure-driven nebulizer or ultrasonic atomizer. See, for example, U.S. Pat. No. 4,501,729. Aerosols of solid particles comprising viral vectors may likewise be generated by any solid particulate pharmaceutical aerosol generator by techniques known in the pharmaceutical arts.

Production of factor VIII proteins of the invention

Many expression vectors are available for creating genetically engineered cells. Some expression vectors are designed to express large amounts of recombinant protein following expansion of transfected cells under a variety of conditions that favor selected highly expressing cells. Some expression vectors are designed to express large quantities of recombinant proteins without the need for amplification under selective pressure. The present invention encompasses the production of genetically engineered cells according to standard methods in the art and is not dependent on the use of any specific expression vector or expression system.

To produce genetically engineered cells to produce large quantities of FVIII protein, cells are transfected with expression vectors containing polynucleotides (e.g., cDNA) encoding the protein. In some embodiments, the FVIII protein is expressed using a selected co-transfected enzyme that results in appropriate post-translational modification of the FVIII protein in a given cell system.

The cells can be from a variety of sources, but in other aspects can be cells transfected with an expression vector containing a nucleic acid molecule (e.g., cDNA) encoding a FVIII protein.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. These techniques are well described in the literature. See, e.g., Sambrook, et al,Molecular Cloning；A Laboratory Manual2 nd edition (1989);DNA Cloningvolumes I and II (D.N Glover, eds., 1985);Oligonucleotide Synthesis(m.j.gait editors, 1984);Nucleic Acid Hybridization(B.D.Hames&s.j. higgins editions, 1984);Transcription andTranslation(B.D.Hames&s.j. higgins editions, 1984);Animal Cell Culture(r.i. freshney, editors, 1986);Immobilized Cells and Enzymes(IRL Press,1986)；B.Perbal,APractical Guide to Molecular Cloning(1984)；the series,Methods in Enzymology(Academic Press, Inc.), particularly volumes 154 and 155 (Wu and G, respectively)rossman and Wu edits);Gene Transfer Vectors for Mammalian Cells(edited by J.H.Miller and M.P.Calos, 1987, Cold Spring Harbor Laboratory);Immunochemical Methods in Cell and Molecular Biologyedited by Mayer and Walker (Academic Press, London, 1987); the scope of the crops is shown in the specification,Protein Purification: Principles and Practice2 nd edition, 1987(Springer-Verlag, n.y.); andHandbook of Experimental Immunologyvolumes I-IV (D.M.Weir and C.C.Blackwell, eds., 1986). All patents, patent applications, and publications cited in this specification are herein incorporated by reference in their entirety.

Genetic engineering technology

The production of cloned genes, recombinant DNA, vectors, transformed cells, proteins and protein fragments by genetic engineering is well known. See, e.g., U.S. Pat. No. 4,761,371 at column 6, line 3 to column 9, line 65 of Bell et al; clark et al, U.S. patent No. 4,877,729, column 4, line 38 to line 7, line 6; schilling, U.S. patent No. 4,912,038, column 3, line 26 to column 14, line 12; and Wallner, U.S. patent No. 4,879,224, column 6, line 8 to column 8, line 59.

The vector (vector) is a replicable DNA construct. Vectors are used herein to amplify and/or express FVIII protein-encoding nucleic acids. Expression vectors are replicable nucleic acid constructs in which a nucleotide sequence encoding a FVIII protein is operably linked to appropriate control sequences capable of effecting expression of the nucleotide sequence in a suitable host cell to produce the FVIII protein. The need for such control sequences will vary depending on the host cell chosen and the transformation method chosen. Typically, control sequences include a transcription promoter, an optional operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences which control termination of transcription and translation.

Vectors comprise plasmids, viruses (e.g., AAV, adenovirus, cytomegalovirus), bacteriophages, and integratable DNA fragments (i.e., fragments that integrate into the genome of a host cell by recombination). The vector may replicate and function independently of the host cell genome (e.g., by transient expression), or may integrate into the host cell genome itself (e.g., stable integration). Expression vectors can contain promoters and RNA binding sites that are operably linked to the nucleic acid molecule to be expressed and operable in a host cell and/or organism.

DNA regions or nucleotide sequences are operably linked or operably associated when they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if the promoter controls the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation of the coding sequence.

Suitable host cells include prokaryotes, yeast or higher eukaryotic cells such as mammalian cells and insect cells. Cells derived from multicellular organisms are particularly suitable hosts for recombinant FVIII protein synthesis, and mammalian cells are particularly preferred. The proliferation of such cells in cell culture has become a routine procedure (Tissue CultureAcademic Press, Kruse and Patterson, eds (1973)). Examples of useful host cell lines are VERO and HeLa cells, Chinese Hamster Ovary (CHO) cell lines and WI138, HEK 293, BHK, COS-7, CV and MDCK cell lines. Expression vectors for such cells typically include, if desired, an origin of replication, a promoter upstream of and operably associated with the nucleotide sequence encoding the FVIII protein to be expressed, as well as a ribosome binding site, an RNA splice site (if intron-containing genomic DNA is used), a polyadenylation site, and a transcription termination sequence. In one embodiment, expression may be performed in Chinese Hamster Ovary (CHO) cells using the expression system of U.S. patent No. 5,888,809, which is incorporated herein by reference in its entirety.

Transcriptional and translational control sequences in expression vectors used in transforming vertebrate cells are typically provided by viral sources. Non-limiting examples include promoters derived from polyoma virus, adenovirus 2, and simian virus 40(SV 40). See, for example, U.S. patent No. 4,599,308.

The origin of replication may be provided by vector construction to include exogenous sources, such as may be derived from SV40 or other viral (e.g., polyoma, adenovirus, VSV or BPV) sources, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the host cell chromosome is usually sufficient.

In addition to using vectors containing viral origins of replication, mammalian cells can be transformed by methods using cotransformation of a selectable marker and a nucleic acid molecule encoding a FVIII protein. Non-limiting examples of suitable selectable markers are dihydrofolate reductase (DHFR) or thymidine kinase. This method is further described in U.S. Pat. No. 4,399,216, which is incorporated herein by reference in its entirety.

Other methods suitable for adaptation to synthesize FVIII protein in recombinant vertebrate cell culture include Gething et al nature 293:620 (1981); mantei et al. Nature 281: 40; and Levinson et al, EPO application nos. 117,060a and 117,058A, each of which is incorporated herein by reference in its entirety.

Host cells such as insect cells (e.g., cultured Spodoptera frugiperda cells) and expression vectors such as baculovirus expression vectors (e.g., vectors derived from Autographa californica MNPV, Trichoplusia ni MNPV, Chardonnax arenaria (Rachiplusia ou) MNPV or Galleria ou MNPV) can be used to practice the present invention, as described in U.S. Pat. Nos. 4,745,051 and 4,879,236 to Smith et al. In general, a baculovirus expression vector comprises a baculovirus genome containing a nucleotide sequence to be expressed, which is inserted into a polyhedrin gene at a position ranging from a polyhedrin transcription initiation signal to an ATG initiation site and under the transcriptional control of a baculovirus polyhedrin promoter.

Prokaryotic host cells include gram-negative or gram-positive organisms, such as e.coli (e.coli) or bacilli, respectively. Higher eukaryotic cells include established cell lines of mammalian origin as described herein. Exemplary bacterial host cells are E.coli W3110(ATCC 27,325), E.coli B, E.coli X1776(ATCC 31,537), and E.coli 294(ATCC 31,446). A wide variety of suitable prokaryotic and microbial vectors can be used. Coli is generally transformed using pBR 322. The most commonly used promoters in recombinant microbial expression vectors include the β -lactamase (penicillinase) and lactose promoter systems (Chang et al Nature275:615 (1978); and Goeddel et al Nature 281:544(1979)), the tryptophan (trp) promoter system (Goeddel et al nucleic Acids Res.8:4057(1980) and EPO application publication No. 36,776), and the tac promoter (De Boer et al Proc. Natl.Acad. Sci. USA 80:21 (1983)). The promoter and Shine-Dalgarno sequence (for prokaryotic host expression) are operably linked to nucleic acid encoding FVIII protein, i.e., they are positioned to facilitate transcription of FVIII messenger RNA from DNA.

Eukaryotic microorganisms such as yeast cultures can also be transformed with protein-encoding vectors (see, e.g., U.S. patent No. 4,745,057). Saccharomyces cerevisiae is the most commonly used in lower eukaryotic host microorganisms, although many other strains are commonly available. The yeast vector may contain an origin of replication or Autonomously Replicating Sequence (ARS) from a2 micron yeast plasmid, a promoter, a nucleic acid encoding a FVIII protein, polyadenylation and transcription termination sequences, and a selection gene. An exemplary plasmid is YRp7(Stinchcomb et al Nature 282:39 (1979); Kingsman et al Gene 7:141 (1979); Tschemper et al Gene 10:157 (1980)). Suitable promoter sequences in yeast vectors include the promoter of metallothionein, the promoter of 3-phosphoglycerate kinase (Hitzeman et al J. biol. chem.255:2073(1980) or the promoters of other glycolytic enzymes (Hess et al J. adv. enzyme Reg.7:149 (1968); and Holland et al biochemistry17:4900(1978)) R.Hitzeman et al, EPO publication No. 73,657 further describe vectors and promoters suitable for yeast expression.

The cloned coding sequences of the invention may encode FVIII from any species, including mouse, rat, dog, possum, rabbit, cat, pig, horse, sheep, cow, guinea pig, possum, platypus, and human, but preferably encodes human FVIII protein. FVIII encoding nucleic acids that hybridize to protein encoding nucleic acids disclosed herein are also included. Hybridization of such sequences to a nucleic acid encoding a FVIII protein disclosed herein can be performed in a standard in situ hybridization assay under reduced stringency conditions or even under stringent conditions (e.g., stringent conditions represented by a wash stringency of 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS at 60 ℃ or even 70 ℃). See, e.g., Sambrook et al,Molecular Cloning,A Laboratory Manual(2 nd edition 1989) Cold Spring Harbor Laboratory).

FVIII proteins produced according to the invention can be expressed in transgenic animals by known methods. See, e.g., U.S. patent No. 6,344,596, which is incorporated herein by reference in its entirety. Briefly, transgenic animals may include, but are not limited to, farm animals (e.g., pigs, goats, sheep, cows, horses, rabbits, etc.), rodents (e.g., mice, rats, and guinea pigs), and domestic pets (e.g., cats and dogs). In some embodiments, livestock such as pigs, sheep, goats, and cows are particularly preferred.

The transgenic animals of the invention are produced by introducing into a single cell embryo a suitable polynucleotide encoding the human FVIII protein of the invention in such a way that the polynucleotide is stably integrated into the DNA of the germ line cell of the mature animal and inherited in a normal mendelian fashion. The transgenic animals of the invention will have a phenotype that produces FVIII protein in body fluids and/or tissues. FVIII protein may be removed from these fluids and/or tissues and processed, e.g., for therapeutic use. (see, e.g., Clark et al, "Expression of human anti-viral factor IX in the mil of transgenic sheet" Bio/Technology 7: 487) 492 (1989); Van Cott et al, "Haemophilus factors produced by genetic library: absolute thermal processes world 10(4):70-77 (2004)), the entire contents of which are incorporated herein by reference).

The DNA molecule may be introduced into the embryo by a variety of means including, but not limited to, microinjection, calcium phosphate-mediated precipitation, liposome fusion, or reverse transcription of pluripotent or multipotent stem cellsAnd (5) toxic infection. The transformed cells are then introduced into embryos and incorporated therein to form transgenic animals. E.g. of L.M.HoudebineTransgenic Animal Generation and UseMethods for making transgenic animals are described in Harwood Academic Press, 1997. Transgenic animals may also be generated using methods of nuclear transfer or cloning of embryonic or adult cell lines, as described, for example, in Campbell et al, Nature 380:64-66(1996) and Wilmut et al, Nature 385:810-813 (1997). In addition, as described in U.S. Pat. No. 5,523,222, a technique of injecting DNA using cytoplasm may be used.

FVIII producing transgenic animals can be obtained by introducing chimeric constructs comprising FVIII coding sequences. Methods for obtaining transgenic animals are well known. See, e.g., Hogan et al,MANIPULATING THE MOUSE EMBRYO,(Cold Spring Harbor Press 1986)；Krimpenfort et al.,Bio/Technology 9:88(1991)；Palmiter et al.,Cell 41:343(1985),Kraemer et al.,GENETIC MANIPULATION OF THE EARLY MAMMALIAN EMBRYO(Cold Spring Harbor Laboratory Press 1985); hammer et al, Nature 315:680 (1985); wagner et al, U.S. patent No. 5,175,385; krimpen et al, U.S. Pat. No. 5,175,384, Janne et al, Ann. Med.24:273(1992), Brem et al, Chim. Oggi.11:21(1993), Clark et al, U.S. Pat. No. 5,476,995, the entire contents of which are incorporated herein by reference.

In some embodiments, cis-acting regulatory regions that are "active" in breast tissue may be used, as promoters are more active in breast tissue than in other tissues under the physiological conditions of synthetic milk. These promoters include, but are not limited to, the short and long Whey Acidic Protein (WAP), short and long alpha, beta and kappa caseins, alpha-lactalbumin and beta-lactoglobulin ("BLG") promoters. It is also possible to use according to the invention signal sequences which secrete the expressed protein directly into other body fluids, in particular blood and urine. Examples of such sequences include signal peptides of secreted coagulation factors, including FVIII, protein C, and signal peptides of tissue plasminogen activator.

In addition to the promoters discussed above, useful sequences for regulating transcription are enhancers, splicing signals, transcription termination signals, polyadenylation sites, buffer sequences, RNA processing sequences and other sequences that regulate expression of the transgene.

Preferably, the expression system or construct comprises a 3' untranslated region downstream of the nucleotide sequence encoding the desired recombinant protein. This region can increase the expression of the transgene. In this regard, a useful 3' untranslated region is a sequence that provides a poly A signal.

Suitable heterologous 3' -untranslated sequences can be derived from, for example, SV40 small t antigen, casein 3' untranslated region, or other 3' untranslated sequences well known in the art. The ribosome binding site is also important for increasing the expression efficiency of FVIII. Likewise, sequences that modulate post-translational modifications of FVIII are useful in the invention.

Having described the present invention, the present invention will be explained in more detail in the following examples, which are included in the present invention for illustrative purposes only and are not intended to limit the present invention.

Example 1

Synthetic liver-specific promoters

We designed and completely synthesized a number of artificial promoters containing conserved basic promoter elements and transcription start sites. The basal promoter (basal promoter) is linked at its 5' end to a number of liver-specific transcription factor binding sites for liver-specific expression. The promoter named LXP3.3 (FIG. 1) (SEQ ID NO:1) was chosen because of its small size (200bp) and high activity in the in vitro primary screen using a luciferase reporter and transfection experiments in the human liver cancer cell line Huh 7.

The LXP3.3 promoter was then further tested with LacZ reporter gene packaged in AAV9 vector, which has extensive tissue tropism in liver, heart and muscle etc. (tissue tropism). Parallel in vivo experiments were performed to compare promoter activity and liver specificity to the strong liver-specific promoter thyroxine-binding globulin (TBG), which was previously reported as one of the strongest liver-specific promoters in small and large animal models. AAV9 vector containing LXP3.3-LacZ or TBG-LacZ expression cassettes was injected via tail vein into C56/B6 mice at two different doses. As shown in fig. 2A, X-gal staining of liver and heart showed robust hepatic expression of both promoters and lack of cardiac expression. Quantitative analysis of tissue homogenates showed that although the LXP3.3 promoter was only 200bp in size and the TBG was 681bp in size, both promoters achieved nearly identical LacZ expression levels and tissue specificity to liver (fig. 2B). Specifically, quantitative LacZ enzyme activity analysis showed that gene expression in the liver was greater than 300-fold higher than in the heart for LXP3.3 and TBG promoters. Furthermore, in the liver, the LacZ enzyme activity obtained by the liver-specific promoter was 500 times higher than that obtained by the ubiquitous CMV promoter (fig. 2C). On the other hand, in the heart, the CMV promoter achieved LacZ expression nearly 13 times as high as the liver-specific promoter (fig. 2B). The results show that the synthetic promoter LXP3.3 has high activity and specificity in the liver.

Example 2

Synthetic promoter intron cassettes

The fully synthetic promoter LXP3.3 is linked at its 3' end to a small intron of VH4 with a short non-natural exon junction sequence. This intron was initially tested by in vitro transfection experiments using a weak promoter driving the BDD human FVIII gene. The addition of the VH4 intron provided higher gene expression than the promoter without the intron, and also provided higher expression than the commonly used chimeric intron cin (promega) (fig. 3). Thus, we combined the LXP3.3 promoter and the VH4 intron with an artificial exon junction sequence and named LXP3.3I (SEQ ID NO: 2). To test its activity in driving FVIII expression, we inserted promoter LXP3.3I upstream of the fully synthetic human BDD-deleted FVIII gene (SEQ ID NO:3), which was followed by a small polyadenylation site. Subsequently, the entire gene expression cassette (SEQ ID NO:4) was cloned into the AAV vector plasmid backbone with two AAV inverted terminal repeats for vector DNA replication and vector genome packaging (FIG. 4).

To compare promoter activity in vitro, the ubiquitous CMV promoter linked to the SV40 intron (a powerful combination commonly used) was used in place of LXP3.3I in the FVIII expression cassette. After transfection of the plasmid into the human hepatoma cell line Huh7, FVIII activity was determined using a chromogenic kit. As shown in the transfection experiment in fig. 5, LXP3.3I produced higher FVIII activity in the cell culture medium than the CMV promoter. Furthermore, we compared the fully synthetic human BDD-deleted FVIII gene with the BDD FVIII gene of the native human DNA sequence in transfection experiments. When driven by the same LXP3.3I, in human cells, two genes encoding the same FVIII amino acid sequence but using different codon usage showed no significant difference in FVIII activity (fig. 6). However, when a different liver-specific promoter was used, the BDD FVIII gene with the native codon showed lower expression compared to the LXP3.3I-driven BDD FVIII gene, indicating that higher gene expression was mainly due to the LXP3.3I promoter.

We next tested in vivo gene expression activity in a commonly used FVIII gene knockout hemophilia a mouse model. The LXP3.3I-hF8 gene expression cassette was packaged into the AAV9 capsid and injected into the tail vein of FVIII KO mice. Two different doses of vehicle were used (2x10 per mouse)¹¹And 4x10¹⁰Individual vector genomes) to check for expression in vivo. As shown in fig. 7A, at both doses, high levels and long-term gene expression were achieved for longer than 1 year after vector administration. In addition to the chromogenic assay for FVIII activity, an ELISA assay was used to check the amount of FVIII protein secreted in the plasma (fig. 7B). The results indicate that BDD FVIII protein concentrations are about 50% lower than readings obtained by chromogenic assay of FVIII activity relative to the same full length human FVIII compared to a reference standard for full length wild type human FVIII protein. This difference (lower ELISA readings than FVIII activity readings) is likely due to the lack of long B domain in BDD FVIII, which comprises almost half the length of wild type FVIII. Since the ELISA kit uses polyclonal antibodies against wild-type full-length FVIII including the B domain, BDD FVIII lacking the B domain is expected to haveFewer antibody binding sites and therefore lower ELISA readings. These results indicate that both chromogenic assays and ELISA assays yield consistent measurements of FVIII expression in hemophilia A mice. Partial Thromboplastin Time (PTT) assays were also performed at several time points. The results are essentially identical to those of the other two assays.

Example 3

Factor VIII heavy chain mutations

Previous literature shows that the heavy chain of FVIII is the limiting factor. Heavy chains are much less efficient and/or less stable than light chains, where the precise mechanism remains ambiguous. For example, when expressing heavy and light chain genes separately from two separate vectors, a much higher copy number of the heavy chain gene than the light chain gene is required to obtain a similar protein concentration for coagulation activity. Therefore, we set out to improve the post-translational processing efficiency of the FVIII heavy chain by introducing mutations in its C-terminal region, in particular by introducing new glycosylation sites, since glycosylation is well known for its role in post-translational processing and stability of cell membrane-associated and secreted proteins. We chose to introduce glycosylation at the C-terminus of the heavy chain. The rationale is because the terminal region is amorphous and therefore a flexible region with no defined structure as shown by X-ray crystal structure analysis of BDD FVIII protein. Thus, mutations in this region may not perturb the functional structure of the FVIII protein. Based on this rationale, we used two natural asparagines at positions 734 and 735 and mutated their adjacent amino acids 736 and 737 to threonine (NNAI to NNTT). The mutation minimizes changes in the amino acid sequence and maximizes the degree of glycosylation, resulting in two de novo glycosylation sites (NNT and NTT) in mutant FVIII designated X0 (figure 8).

In addition to the two new glycosylation sites in mutant X0, we added another glycosylation site (with the native glycosylation sequence (YVNRSL) isolated from amino acids 237 to 242) next to the NNTT site by replacing the peptide (EPRSF) at amino acids 738-742 at the N-terminus of the heavy chain. This native glycosylation site (NRS) was chosen because it is one of the most efficient glycosylation sites in the heavy chain (Medzihradszky et al, anal. chem.69:3986(1997)) resulting in mutant FVIII named X1 (FIG. 8).

Alternatively, we have replaced the amino acid EPR at position 738-740 with NNT in mutant X0, resulting in two additional glycosylation sites in mutant X2 (FIG. 8).

Example 4

Mutations at the C-terminal end of the factor VIII heavy chain enhance FVIII activity in vitro

Next, we performed a parallel comparison of FVIII activity of the mutant constructs with their wild type BDD FVIII counterparts. Plasmid transfection was performed in the human hepatoma cell line Huh7, which has no endogenous FVIII expression, but is suitable for gene expression under the control of a liver-specific promoter. As shown in fig. 9A, mutations X1 and X2 increased FVIII activity more than two-fold in vitro compared to the parental gene.

Example 5

Long-term and high-level gene expression of mutant FVIII in vivo in a hemophilia A mouse model

Since FVIII mutants X1 and X2 showed significantly higher FVIII activity in vitro transfection experiments, we next examined their gene expression and FVIII activity in FVIII KO mice in vivo. The X1 and X2 genes are under transcriptional control of the same LXP3.3I promoter and are packaged in AAV8 vector particles. Based on early experiments, a standard vector dose of 5X10 was selected¹⁰v.g./mouse and injected via tail vein into hemophilia a mice aged 2 to 3 months. Plasma samples were collected by the retro-orbital bleeding technique, which is a commonly used method. As shown in fig. 9B, both mutants X1 and X2 achieved higher expression than their wild-type parent BDD FVIII gene (low dose compared to fig. 7A). To monitor long-term gene expression, we retained hemophilia a mice treated with X1 or X2 mutants for 24 weeks of observation. Similar to hemophilia a mice treated with the parent wt BDD FVIII vector (fig. 7A), mice treated with mutant FVIII vector also showed long-term stable gene expression without significant reductionActivity of human FVIII in plasma. Inhibitor testing at different time points showed no inhibitor formation.

It will be understood by those skilled in the art that many and various modifications may be made without departing from the spirit of the invention. Accordingly, it should be clearly understood that the forms of the present invention are illustrative only and are not intended to limit the scope of the present invention.

All publications, patent applications, patents, patent publications, patents, publications, and patents mentioned herein,The sequence of database accession numbers and other references are incorporated by reference in their entirety for their teachings relating to the sentences and/or paragraphs in which the references are presented.

The foregoing is illustrative of the present invention and is not to be construed as limiting thereof.

The sequence is as follows:

1LXP3.3 promoter-200 bp of SEQ ID NO

SEQ ID NO:2 (LXP3.3I-288 bp containing LXP3.3 promoter and VH4 intron

3 Synthesis of human B Domain deleted factor FVIII coding sequence of SEQ ID NO-4374 bp

4 human factor FVIII Gene expression cassette in AAV vector, from left inverted repeat to right inverted terminal repeat-5045 bp

SEQ ID NO 5 human factor FVIII wild type protein sequence without signal peptide-2333 aa.

Sequence listing

<110> University of North Carolina Chapell Hill (The University of North Carolina at Chapel Hill)

Xiao' ao (Xiao )

Li Juan (Li, Juan)

Yuan-Yuan

<120> OPTIMIZED HUMAN coagulation FACTOR VIII GENE EXPRESSION cassette and use thereof (OPTIMIZED HUMAN coagulation FACTOR VIII GENE EXPRESSION cassette

AND THEIR USE)

<130> 5470-734WO

<150> US 62/112,901

<151> 2015-02-06

<160> 34

<170> PatentIn version 3.5

<210> 1

<211> 200

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 1

ctgtttactc tggttaattt ttaaaggagg gtaaacagtg cctgaaagct gacctttgcc 60

cacattcctc cggtagacat taacttatta aattgattct gattacaaat ctgacctttg 120

cccccatctc acccagtaac aatgcaagag ttgatgtcag tctataaaaa gcgaagcgcg 180

cggtgggcgg ggttcgctgc 200

<210> 2

<211> 288

<212> DNA

<213> Artificial

<220>

<223> LXP3.3I synthetic promoter sequence

<400> 2

ctgtttactc tggttaattt ttaaaggagg gtaaacagtg cctgaaagct gacctttgcc 60

cacattcctc cggtagacat taacttatta aattgattct gattacaaat ctgacctttg 120

cccccatctc acccagtaac aatgcaagag ttgatgtcag tctataaaaa gcgaagcgcg 180

cggtgggcgg ggttcgctgc ctgcaggtga gtatctcagg gatccagaca tggggatatg 240

ggaggtgcct ctgatcccag ggctcactgt gggtctctct gttcacag 288

<210> 3

<211> 4374

<212> DNA

<213> Artificial

<220>

<223> Synthesis of human B-Domain deleted factor FVIII coding sequences

<400> 3

atgcagatcg agctgtctac ctgcttcttc ctgtgcctgc tgcggttctg cttcagcgcc 60

accagacggt actatctggg cgccgtggaa ctgagctggg actacatgca gagcgacctg 120

ggcgagctgc ccgtggatgc cagattccct ccaagagtgc ccaagagctt ccccttcaac 180

acctccgtgg tgtacaagaa aaccctgttc gtggaattca ccgaccacct gttcaatatc 240

gccaagccca gacccccctg gatgggcctg ctgggaccta caattcaggc cgaggtgtac 300

gacaccgtcg tgatcaccct gaagaacatg gccagccacc ccgtgtctct gcatgccgtg 360

ggagtgtcct actggaaggc ctctgagggc gccgagtacg acgatcagac cagccagcgc 420

gagaaagagg acgacaaggt gttccctggc ggcagccaca cctacgtgtg gcaggtgctg 480

aaagaaaacg gccccatggc ctccgaccct ctgtgcctga catacagcta cctgagccac 540

gtggacctcg tgaaggacct gaacagcggc ctgatcggag ccctgctcgt gtgtagagag 600

ggcagcctgg ccaaagagaa aacccagacc ctgcacaagt tcatcctgct gttcgccgtg 660

ttcgacgagg gcaagagctg gcacagcgag acaaagaaca gcctgatgca ggaccgggac 720

gccgcctctg ctagagcctg gcccaaaatg cacaccgtga acggctacgt gaacagaagc 780

ctgcccggac tgatcggctg ccaccggaag tctgtgtact ggcacgtgat cggcatgggc 840

accacccctg aggtgcacag catctttctg gaaggacaca cctttctcgt gcggaaccac 900

cggcaggcca gcctggaaat cagccctatc accttcctga ccgcccagac actgctgatg 960

gacctgggcc agtttctgct gttctgccac atcagctccc accagcacga cggcatggaa 1020

gcctacgtga aggtggacag ctgccccgag gaaccccagc tgcggatgaa gaacaacgag 1080

gaagccgagg actacgacga cgacctgacc gacagcgaga tggacgtggt gcgcttcgac 1140

gacgataaca gccccagctt catccagatc agaagcgtgg ccaagaagca ccccaagacc 1200

tgggtgcact atatcgccgc cgaggaagag gactgggatt acgcccctct ggtgctggcc 1260

cccgacgaca gaagctacaa gagccagtac ctgaacaatg gcccccagcg gatcggccgg 1320

aagtataaga aagtgcggtt catggcctac accgacgaga cattcaagac cagagaggcc 1380

atccagcacg agagcggcat cctgggccct ctgctgtatg gcgaagtggg cgacaccctg 1440

ctgatcatct tcaagaacca ggccagcaga ccctacaaca tctaccctca cggcatcacc 1500

gacgtgcggc ccctgtactc tagaaggctg cccaagggcg tgaaacacct gaaggacttc 1560

cccatcctgc ccggcgagat tttcaagtac aagtggaccg tgaccgtgga agatggcccc 1620

accaagagcg accccagatg cctgacacgg tactacagca gcttcgtgaa catggaacgg 1680

gacctggcct ccggcctgat tggcccactg ctgatctgct acaaagaaag cgtggaccag 1740

cggggcaacc agatcatgag cgacaagcgg aacgtgatcc tgtttagcgt gttcgatgag 1800

aaccggtcct ggtatctgac cgagaatatc cagcggttcc tgcccaaccc tgccggcgtg 1860

cagctggaag atcctgagtt ccaggcctcc aacatcatgc actccatcaa tggctatgtg 1920

ttcgacagcc tgcagctgag cgtgtgcctg cacgaggtgg cctactggta catcctgagc 1980

atcggggccc agaccgactt cctgtccgtg ttcttctccg gctacacctt caagcacaag 2040

atggtgtacg aggataccct gaccctgttc ccctttagcg gcgaaaccgt gttcatgagc 2100

atggaaaacc ccggcctgtg gatcctgggc tgccacaaca gcgacttccg gaacagaggc 2160

atgaccgccc tgctgaaggt gtccagctgc gacaagaaca ccggcgacta ctacgaggac 2220

agctatgagg acatcagcgc ctacctgctg agcaagaaca atgccatcga gcccagaagc 2280

ttcagccagc cccctgtgct gaagcggcac cagagagaga tcacccggac caccctgcag 2340

tccgaccagg aagagatcga ttacgacgac accatcagcg tggaaatgaa gaaagaagat 2400

ttcgacatct acgacgagga cgagaaccag agcccccggt cctttcagaa aaagacccgg 2460

cactacttca ttgccgctgt ggaacggctg tgggactacg gcatgagcag cagccctcac 2520

gtgctgagaa acagggccca gagcggcagc gtgccccagt tcaagaaagt ggtgttccag 2580

gaattcacag acggcagctt tacccagcct ctgtaccgcg gcgagctgaa cgaacacctg 2640

ggactgctgg gcccctatat ccgggccgaa gtggaagata acatcatggt caccttccgg 2700

aatcaggcct cccggcccta cagcttctac agctccctga tcagctacga agaggaccag 2760

agacagggcg ctgagccccg gaagaacttc gtgaagccca acgagactaa gacctacttt 2820

tggaaggtgc agcaccacat ggcccctaca aaggacgagt tcgactgcaa ggcctgggcc 2880

tacttctccg atgtggacct ggaaaaggac gtgcactctg ggctgatcgg ccccctgctc 2940

gtgtgccaca ccaacaccct gaatcccgcc cacggcagac aagtgacagt gcaggaattc 3000

gccctgttct tcaccatctt cgacgaaaca aagagctggt acttcaccga aaacatggaa 3060

agaaactgcc gggctccctg caacatccag atggaagatc ccaccttcaa agagaactac 3120

cggttccacg ccatcaacgg ctacatcatg gacacactgc ccggcctcgt gatggctcag 3180

gatcagcgga tccggtggta tctgctgtcc atgggctcca acgagaacat ccacagcatc 3240

cacttcagcg gccacgtgtt caccgtgcgg aaaaaagaag agtacaaaat ggccctgtac 3300

aacctgtacc ctggggtgtt cgagacagtg gaaatgctgc ccagcaaggc cggcatctgg 3360

cgggtggaat gtctgatcgg cgagcatctg cacgctggga tgagcacact gtttctggtg 3420

tacagcaaca agtgccagac acctctgggc atggcctctg gccacatccg ggactttcag 3480

atcacagcca gcggccagta tggccagtgg gccccaaaac tggccagact gcactacagc 3540

ggcagcatca acgcctggtc caccaaagag cccttcagct ggatcaaggt ggacctgctg 3600

gctcccatga tcatccacgg aatcaagacc cagggcgcca gacagaagtt ctccagcctg 3660

tacatctccc agttcatcat catgtactcc ctggacggca agaagtggca gacctaccgg 3720

ggcaatagca ccggcaccct gatggtgttc ttcggcaacg tggactccag cggcattaag 3780

cacaacatct tcaacccccc catcattgcc cggtacatcc ggctgcaccc cacccactac 3840

agcatccggt ccaccctgag aatggaactg atgggctgcg acctgaactc ctgcagcatg 3900

cccctgggga tggaaagcaa ggccatctcc gacgcccaga tcaccgcctc cagctacttc 3960

accaacatgt tcgccacctg gtccccatcc aaggcccggc tgcatctgca gggcagaagc 4020

aatgcttgga ggccccaagt gaacaacccc aaagaatggc tgcaggtgga cttccagaaa 4080

accatgaaag tgaccggcgt gaccacccag ggcgtgaagt ctctgctgac ctctatgtac 4140

gtgaaagagt tcctgatctc cagcagccag gacggccacc agtggaccct gtttttccag 4200

aacggcaaag tgaaagtgtt tcaggggaac caggacagct tcacccccgt cgtgaatagc 4260

ctggaccctc cactgctgac cagatacctg cggatccacc ctcagagttg ggtgcaccag 4320

attgctctgc ggatggaagt gctgggatgc gaggcccagg acctgtactg ataa 4374

<210> 4

<211> 5045

<212> DNA

<213> Artificial

<220>

<223> recombinant AAV vector sequences

<400> 4

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60

cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120

gccaactcca tcactagggg ttcctagatc tacgcgtctg tttactctgg ttaattttta 180

aaggagggta aacagtgcct gaaagctgac ctttgcccac attcctccgg tagacattaa 240

cttattaaat tgattctgat tacaaatctg acctttgccc ccatctcacc cagtaacaat 300

gcaagagttg atgtcagtct ataaaaagcg aagcgcgcgg tgggcggggt tcgctgcctg 360

caggtgagta tctcagggat ccagacatgg ggatatggga ggtgcctctg atcccagggc 420

tcactgtggg tctctctgtt cacagcctgc tagcgccacc atgcagatcg agctgtctac 480

ctgcttcttc ctgtgcctgc tgcggttctg cttcagcgcc accagacggt actatctggg 540

cgccgtggaa ctgagctggg actacatgca gagcgacctg ggcgagctgc ccgtggatgc 600

cagattccct ccaagagtgc ccaagagctt ccccttcaac acctccgtgg tgtacaagaa 660

aaccctgttc gtggaattca ccgaccacct gttcaatatc gccaagccca gacccccctg 720

gatgggcctg ctgggaccta caattcaggc cgaggtgtac gacaccgtcg tgatcaccct 780

gaagaacatg gccagccacc ccgtgtctct gcatgccgtg ggagtgtcct actggaaggc 840

ctctgagggc gccgagtacg acgatcagac cagccagcgc gagaaagagg acgacaaggt 900

gttccctggc ggcagccaca cctacgtgtg gcaggtgctg aaagaaaacg gccccatggc 960

ctccgaccct ctgtgcctga catacagcta cctgagccac gtggacctcg tgaaggacct 1020

gaacagcggc ctgatcggag ccctgctcgt gtgtagagag ggcagcctgg ccaaagagaa 1080

aacccagacc ctgcacaagt tcatcctgct gttcgccgtg ttcgacgagg gcaagagctg 1140

gcacagcgag acaaagaaca gcctgatgca ggaccgggac gccgcctctg ctagagcctg 1200

gcccaaaatg cacaccgtga acggctacgt gaacagaagc ctgcccggac tgatcggctg 1260

ccaccggaag tctgtgtact ggcacgtgat cggcatgggc accacccctg aggtgcacag 1320

catctttctg gaaggacaca cctttctcgt gcggaaccac cggcaggcca gcctggaaat 1380

cagccctatc accttcctga ccgcccagac actgctgatg gacctgggcc agtttctgct 1440

gttctgccac atcagctccc accagcacga cggcatggaa gcctacgtga aggtggacag 1500

ctgccccgag gaaccccagc tgcggatgaa gaacaacgag gaagccgagg actacgacga 1560

cgacctgacc gacagcgaga tggacgtggt gcgcttcgac gacgataaca gccccagctt 1620

catccagatc agaagcgtgg ccaagaagca ccccaagacc tgggtgcact atatcgccgc 1680

cgaggaagag gactgggatt acgcccctct ggtgctggcc cccgacgaca gaagctacaa 1740

gagccagtac ctgaacaatg gcccccagcg gatcggccgg aagtataaga aagtgcggtt 1800

catggcctac accgacgaga cattcaagac cagagaggcc atccagcacg agagcggcat 1860

cctgggccct ctgctgtatg gcgaagtggg cgacaccctg ctgatcatct tcaagaacca 1920

ggccagcaga ccctacaaca tctaccctca cggcatcacc gacgtgcggc ccctgtactc 1980

tagaaggctg cccaagggcg tgaaacacct gaaggacttc cccatcctgc ccggcgagat 2040

tttcaagtac aagtggaccg tgaccgtgga agatggcccc accaagagcg accccagatg 2100

cctgacacgg tactacagca gcttcgtgaa catggaacgg gacctggcct ccggcctgat 2160

tggcccactg ctgatctgct acaaagaaag cgtggaccag cggggcaacc agatcatgag 2220

cgacaagcgg aacgtgatcc tgtttagcgt gttcgatgag aaccggtcct ggtatctgac 2280

cgagaatatc cagcggttcc tgcccaaccc tgccggcgtg cagctggaag atcctgagtt 2340

ccaggcctcc aacatcatgc actccatcaa tggctatgtg ttcgacagcc tgcagctgag 2400

cgtgtgcctg cacgaggtgg cctactggta catcctgagc atcggggccc agaccgactt 2460

cctgtccgtg ttcttctccg gctacacctt caagcacaag atggtgtacg aggataccct 2520

gaccctgttc ccctttagcg gcgaaaccgt gttcatgagc atggaaaacc ccggcctgtg 2580

gatcctgggc tgccacaaca gcgacttccg gaacagaggc atgaccgccc tgctgaaggt 2640

gtccagctgc gacaagaaca ccggcgacta ctacgaggac agctatgagg acatcagcgc 2700

ctacctgctg agcaagaaca atgccatcga gcccagaagc ttcagccagc cccctgtgct 2760

gaagcggcac cagagagaga tcacccggac caccctgcag tccgaccagg aagagatcga 2820

ttacgacgac accatcagcg tggaaatgaa gaaagaagat ttcgacatct acgacgagga 2880

cgagaaccag agcccccggt cctttcagaa aaagacccgg cactacttca ttgccgctgt 2940

ggaacggctg tgggactacg gcatgagcag cagccctcac gtgctgagaa acagggccca 3000

gagcggcagc gtgccccagt tcaagaaagt ggtgttccag gaattcacag acggcagctt 3060

tacccagcct ctgtaccgcg gcgagctgaa cgaacacctg ggactgctgg gcccctatat 3120

ccgggccgaa gtggaagata acatcatggt caccttccgg aatcaggcct cccggcccta 3180

cagcttctac agctccctga tcagctacga agaggaccag agacagggcg ctgagccccg 3240

gaagaacttc gtgaagccca acgagactaa gacctacttt tggaaggtgc agcaccacat 3300

ggcccctaca aaggacgagt tcgactgcaa ggcctgggcc tacttctccg atgtggacct 3360

ggaaaaggac gtgcactctg ggctgatcgg ccccctgctc gtgtgccaca ccaacaccct 3420

gaatcccgcc cacggcagac aagtgacagt gcaggaattc gccctgttct tcaccatctt 3480

cgacgaaaca aagagctggt acttcaccga aaacatggaa agaaactgcc gggctccctg 3540

caacatccag atggaagatc ccaccttcaa agagaactac cggttccacg ccatcaacgg 3600

ctacatcatg gacacactgc ccggcctcgt gatggctcag gatcagcgga tccggtggta 3660

tctgctgtcc atgggctcca acgagaacat ccacagcatc cacttcagcg gccacgtgtt 3720

caccgtgcgg aaaaaagaag agtacaaaat ggccctgtac aacctgtacc ctggggtgtt 3780

cgagacagtg gaaatgctgc ccagcaaggc cggcatctgg cgggtggaat gtctgatcgg 3840

cgagcatctg cacgctggga tgagcacact gtttctggtg tacagcaaca agtgccagac 3900

acctctgggc atggcctctg gccacatccg ggactttcag atcacagcca gcggccagta 3960

tggccagtgg gccccaaaac tggccagact gcactacagc ggcagcatca acgcctggtc 4020

caccaaagag cccttcagct ggatcaaggt ggacctgctg gctcccatga tcatccacgg 4080

aatcaagacc cagggcgcca gacagaagtt ctccagcctg tacatctccc agttcatcat 4140

catgtactcc ctggacggca agaagtggca gacctaccgg ggcaatagca ccggcaccct 4200

gatggtgttc ttcggcaacg tggactccag cggcattaag cacaacatct tcaacccccc 4260

catcattgcc cggtacatcc ggctgcaccc cacccactac agcatccggt ccaccctgag 4320

aatggaactg atgggctgcg acctgaactc ctgcagcatg cccctgggga tggaaagcaa 4380

ggccatctcc gacgcccaga tcaccgcctc cagctacttc accaacatgt tcgccacctg 4440

gtccccatcc aaggcccggc tgcatctgca gggcagaagc aatgcttgga ggccccaagt 4500

gaacaacccc aaagaatggc tgcaggtgga cttccagaaa accatgaaag tgaccggcgt 4560

gaccacccag ggcgtgaagt ctctgctgac ctctatgtac gtgaaagagt tcctgatctc 4620

cagcagccag gacggccacc agtggaccct gtttttccag aacggcaaag tgaaagtgtt 4680

tcaggggaac caggacagct tcacccccgt cgtgaatagc ctggaccctc cactgctgac 4740

cagatacctg cggatccacc ctcagagttg ggtgcaccag attgctctgc ggatggaagt 4800

gctgggatgc gaggcccagg acctgtactg ataagtcgac aggcctaata aagagctcag 4860

atgcatcgat cagagtgtgt tggttttttg tgtgagatct aggaacccct agtgatggag 4920

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg 4980

cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg 5040

gccaa 5045

<210> 5

<211> 2333

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 5

Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr

1 5 10 15

Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro

20 25 30

Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys

35 40 45

Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro

50 55 60

Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val

65 70 75 80

Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val

85 90 95

Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala

100 105 110

Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val

115 120 125

Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn

130 135 140

Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser

145 150 155 160

His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu

165 170 175

Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu

180 185 190

His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp

195 200 205

His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser

210 215 220

Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg

225 230 235 240

Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His

245 250 255

Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu

260 265 270

Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile

275 280 285

Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly

290 295 300

Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met

305 310 315 320

Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg

325 330 335

Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp

340 345 350

Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe

355 360 365

Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His

370 375 380

Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu

385 390 395 400

Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro

405 410 415

Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr

420 425 430

Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile

435 440 445

Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile

450 455 460

Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile

465 470 475 480

Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys

485 490 495

His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys

500 505 510

Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys

515 520 525

Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala

530 535 540

Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp

545 550 555 560

Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe

565 570 575

Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln

580 585 590

Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe

595 600 605

Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser

610 615 620

Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu

625 630 635 640

Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr

645 650 655

Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro

660 665 670

Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp

675 680 685

Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala

690 695 700

Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu

705 710 715 720

Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala

725 730 735

Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg

740 745 750

Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys

755 760 765

Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn

770 775 780

Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro

785 790 795 800

His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe

805 810 815

Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser

820 825 830

Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val

835 840 845

Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly

850 855 860

Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser

865 870 875 880

Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala

885 890 895

Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His

900 905 910

Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro

915 920 925

Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp

930 935 940

Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp

945 950 955 960

Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys

965 970 975

Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys

980 985 990

Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala

995 1000 1005

Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu

1010 1015 1020

Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu

1025 1030 1035

Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp

1040 1045 1050

Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr

1055 1060 1065

Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly

1070 1075 1080

Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe Lys

1085 1090 1095

Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln Arg Thr His

1100 1105 1110

Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro Ser Pro Lys Gln

1115 1120 1125

Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu Gly Gln Asn Phe

1130 1135 1140

Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys Gly Glu Phe Thr

1145 1150 1155

Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn

1160 1165 1170

Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu Asn Asn Thr His

1175 1180 1185

Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr

1190 1195 1200

Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr Val Thr

1205 1210 1215

Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr Arg

1220 1225 1230

Gln Asn Val Glu Gly Ser Tyr Glu Gly Ala Tyr Ala Pro Val Leu

1235 1240 1245

Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys

1250 1255 1260

His Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu

1265 1270 1275

Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys

1280 1285 1290

Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr

1295 1300 1305

Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu

1310 1315 1320

Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser Thr

1325 1330 1335

Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr

1340 1345 1350

Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser

1355 1360 1365

Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala

1370 1375 1380

Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser

1385 1390 1395

Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe Gln Asp Asn Ser

1400 1405 1410

Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val

1415 1420 1425

Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu

1430 1435 1440

Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln Arg Glu

1445 1450 1455

Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr Lys

1460 1465 1470

Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr

1475 1480 1485

Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys

1490 1495 1500

Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu

1505 1510 1515

Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile

1520 1525 1530

Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg

1535 1540 1545

Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp

1550 1555 1560

Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys Glu

1565 1570 1575

Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys

1580 1585 1590

Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys Glu Ser Asn His

1595 1600 1605

Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys Pro Glu Ile Glu

1610 1615 1620

Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser Gln

1625 1630 1635

Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr

1640 1645 1650

Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile

1655 1660 1665

Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp

1670 1675 1680

Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr

1685 1690 1695

Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser

1700 1705 1710

Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro

1715 1720 1725

Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe

1730 1735 1740

Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu

1745 1750 1755

Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val

1760 1765 1770

Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser

1775 1780 1785

Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg

1790 1795 1800

Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys

1805 1810 1815

Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asx Asp Cys

1820 1825 1830

Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val

1835 1840 1845

His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr

1850 1855 1860

Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala

1865 1870 1875

Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr

1880 1885 1890

Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met

1895 1900 1905

Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn

1910 1915 1920

Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp

1925 1930 1935

Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn

1940 1945 1950

Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys

1955 1960 1965

Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val

1970 1975 1980

Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg

1985 1990 1995

Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr

2000 2005 2010

Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met

2015 2020 2025

Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln

2030 2035 2040

Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly

2045 2050 2055

Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys

2060 2065 2070

Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln

2075 2080 2085

Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile

2090 2095 2100

Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly

2105 2110 2115

Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser

2120 2125 2130

Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg

2135 2140 2145

Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu

2150 2155 2160

Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro

2165 2170 2175

Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala

2180 2185 2190

Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys

2195 2200 2205

Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln

2210 2215 2220

Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr

2225 2230 2235

Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu

2240 2245 2250

Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp

2255 2260 2265

Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val

2270 2275 2280

Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu

2285 2290 2295

Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser

2300 2305 2310

Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu

2315 2320 2325

Ala Gln Asp Leu Tyr

2330

<210> 6

<211> 8

<212> PRT

<213> Artificial

<220>

<223> factor VIII consensus insertions

<220>

<221> MISC_FEATURE

<222> (1)..(2)

<223> Xaa can be S or T

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> Xaa can be S or T

<400> 6

Xaa Xaa Tyr Val Asn Arg Xaa Leu

1 5

<210> 7

<211> 8

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 7

Thr Thr Tyr Val Asn Arg Ser Leu

1 5

<210> 8

<211> 8

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 8

Thr Thr Tyr Val Asn Arg Thr Leu

1 5

<210> 9

<211> 8

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 9

Thr Ser Tyr Val Asn Arg Ser Leu

1 5

<210> 10

<211> 8

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 10

Thr Ser Tyr Val Asn Arg Thr Leu

1 5

<210> 11

<211> 8

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 11

Ser Thr Tyr Val Asn Arg Ser Leu

1 5

<210> 12

<211> 8

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 12

Ser Thr Tyr Val Asn Arg Thr Leu

1 5

<210> 13

<211> 8

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 13

Ser Ser Tyr Val Asn Arg Ser Leu

1 5

<210> 14

<211> 8

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 14

Ser Ser Tyr Val Asn Arg Thr Leu

1 5

<210> 15

<211> 5

<212> PRT

<213> Artificial

<220>

<223> factor VIII consensus insertions

<220>

<221> MISC_FEATURE

<222> (1)..(2)

<223> Xaa can be S or T

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Xaa can be S or T

<400> 15

Xaa Xaa Asn Asn Xaa

1 5

<210> 16

<211> 5

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 16

Thr Thr Asn Asn Ser

1 5

<210> 17

<211> 5

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 17

Thr Thr Asn Asn Thr

1 5

<210> 18

<211> 5

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 18

Thr Ser Asn Asn Ser

1 5

<210> 19

<211> 5

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 19

Thr Ser Asn Asn Thr

1 5

<210> 20

<211> 5

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 20

Ser Thr Asn Asn Ser

1 5

<210> 21

<211> 5

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 21

Ser Thr Asn Asn Thr

1 5

<210> 22

<211> 5

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 22

Ser Ser Asn Asn Ser

1 5

<210> 23

<211> 5

<212> PRT

<213> Artificial

<220>

<223> factor VIII insertion sequence

<400> 23

Ser Ser Asn Asn Thr

1 5

<210> 24

<211> 7

<212> PRT

<213> Artificial

<220>

<223> O-linked glycosylation site consensus sequence

<220>

<221> misc_feature

<222> (2)..(3)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> Xaa can be S or T

<400> 24

Cys Xaa Xaa Gly Gly Xaa Cys

1 5

<210> 25

<211> 5

<212> PRT

<213> Artificial

<220>

<223> O-linked glycosylation site sequence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Xaa can be D or E

<400> 25

Asn Ser Thr Xaa Ala

1 5

<210> 26

<211> 5

<212> PRT

<213> Artificial

<220>

<223> O-linked glycosylation site sequence

<400> 26

Asn Ile Thr Gln Ser

1 5

<210> 27

<211> 5

<212> PRT

<213> Artificial

<220>

<223> O-linked glycosylation site sequence

<400> 27

Gln Ser Thr Gln Ser

1 5

<210> 28

<211> 5

<212> PRT

<213> Artificial

<220>

<223> O-linked glycosylation site sequence

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Xaa can be D or E

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Xaa can be R or K

<400> 28

Xaa Phe Thr Xaa Val

1 5

<210> 29

<211> 4

<212> PRT

<213> Artificial

<220>

<223> O-linked glycosylation site sequence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Xaa can be D or E

<400> 29

Cys Xaa Ser Asn

1

<210> 30

<211> 5

<212> PRT

<213> Artificial

<220>

<223> O-linked glycosylation site sequence

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Xaa can be R or K

<400> 30

Gly Gly Ser Cys Xaa

1 5

<210> 31

<211> 30

<212> PRT

<213> Artificial

<220>

<223> B Domain deleted factor VIII sequence

<400> 31

Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro

1 5 10 15

Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr

20 25 30

<210> 32

<211> 30

<212> PRT

<213> Artificial

<220>

<223> mutated B domain deleted factor VIII sequence

<400> 32

Leu Ser Lys Asn Asn Thr Thr Glu Pro Arg Ser Phe Ser Gln Asn Pro

1 5 10 15

Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr

20 25 30

<210> 33

<211> 31

<212> PRT

<213> Artificial

<220>

<223> mutated B domain deleted factor VIII sequence

<400> 33

Leu Ser Lys Asn Asn Thr Thr Tyr Val Asn Arg Ser Leu Ser Gln Asn

1 5 10 15

Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr

20 25 30

<210> 34

<211> 28

<212> PRT

<213> Artificial

<220>

<223> mutated B domain deleted factor VIII sequence

<400> 34

Leu Ser Lys Asn Asn Thr Thr Asn Asn Thr Ser Gln Asn Pro Pro Val

1 5 10 15

Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr

20 25

Claims (22)

1. A modified mammalian factor VIII polypeptide wherein amino acid residues in the heavy chain are modified to create one or more glycosylation sites.

2. The modified factor VIII polypeptide of claim 1, wherein the one or more glycosylation sites are at the C-terminus of the heavy chain.

3. The modified factor VIII polypeptide according to claim 1 or 2, which is a human factor VIII polypeptide.

4. The modified factor VIII polypeptide according to claim 3, wherein amino acid residues 736 and 737 of the wild type human sequence (SEQ ID NO:5) are substituted with amino acid residue XX, wherein X is S or T.

5. The modified factor VIII polypeptide according to claim 3, wherein amino acid residues 736-742 of the wild-type human sequence (SEQ ID NO:5) are substituted by amino acid residues XXYVNRXL, wherein X is S or T.

6. The modified factor VIII polypeptide according to claim 3, wherein amino acid residue 736-740 of the wild-type human sequence (SEQ ID NO:5) is substituted with amino acid residue XXNNX, wherein X is S or T.

7. The modified factor VIII polypeptide of any one of claims 1-6, further comprising a B domain deletion.

8. A polynucleotide encoding the modified factor VIII polypeptide of any one of claims 1-7.

9. The polynucleotide of claim 8, operably linked to a promoter.

10. The polynucleotide of claim 9, wherein the promoter comprises a polynucleotide comprising a synthetic liver-specific promoter, wherein the synthetic liver-specific promoter comprises the nucleotide sequence of SEQ ID No. 1 or a sequence having at least 90% identity thereto.

11. The polynucleotide of claim 10, wherein the synthetic liver-specific promoter is operably linked to an intron.

12. The polynucleotide of claim 11, wherein the intron is from VH 4.

13. The polynucleotide of claim 12, wherein the synthetic liver-specific promoter and the intron together comprise the nucleotide sequence of SEQ ID No.2 or a sequence having at least 90% identity thereto.

14. A vector comprising the polynucleotide of any one of claims 8-13.

15. The vector of claim 14, wherein the vector is a viral vector.

16. The vector of claim 15, wherein the vector is an AAV vector.

17. The vector of claim 16, wherein the AAV vector is an AAV8 vector or an AAV9 vector.

18. A transformed cell comprising the polynucleotide of any one of claims 8-13 and/or the vector of any one of claims 14-17.

19. A transgenic animal comprising the polynucleotide of any one of claims 8-13, the vector of any one of claims 14-17, and/or the transformed cell of claim 18.

20. A method of producing factor VIII in the liver of a subject, comprising delivering to the subject the polynucleotide of any one of claims 8-13, the vector of any one of claims 14-17, and/or the transformed cell of claim 18, thereby producing factor VIII in the liver of the subject.

21. A method of treating hemophilia a or acquired factor VIII deficiency in a subject, comprising delivering to the subject a therapeutically effective amount of the modified human factor VIII polypeptide of any one of claims 1-7, the polynucleotide of any one of claims 8-13, the vector of any one of claims 14-17, and/or the transformed cell of claim 18, thereby treating hemophilia a in the subject.

22. A method of increasing the bioavailability of a factor VIII polypeptide in a subject, comprising delivering to the subject an effective amount of the polynucleotide of any one of claims 8-13, the vector of any one of claims 14-17, and/or the transformed cell of claim 18, thereby increasing the bioavailability of the factor VIII polypeptide in the subject.