WO2025238067A1

WO2025238067A1 - Novel vector system for aav production

Info

Publication number: WO2025238067A1
Application number: PCT/EP2025/063205
Authority: WO
Inventors: Florian Sonntag; Andreas Schulze; Markus HÖRER
Original assignee: Ascend Advanced Therapies GmbH
Current assignee: Ascend Advanced Therapies GmbH
Priority date: 2024-05-15
Filing date: 2025-05-14
Publication date: 2025-11-20
Anticipated expiration: 2026-11-15

Abstract

The present invention relates a novel two or three component linear synthetic DNA vector system for use in methods of adeno-associated virus (AAV) vector production and methods of producing such a vector system. Such a system utilises linear, synthetic DNA molecules generated in an enzymatic in vitro manufacturing process, omitting the use of bacterial fermentation methods and is based on linear, double stranded and preferably covalently closed synthetic DNA.

Description

NOVEL VECTOR SYSTEM FOR AAV PRODUCTION

FIELD OF THE DISCLOSURE

The present invention relates to a novel two or three component linear, synthetic DNA vector system for use in methods of adeno-associated virus (AAV) vector production, suitable for gene therapy, and uses of such a system in adeno-associated virus (AAV) vector production.

BACKGROUND

Adeno-associated virus (AAV) is a member of the Parvoviridae family. The AAV genome is composed of a linear single-stranded DNA molecule which contains approximately 4.7 kilobases (kb) and consists of two major open reading frames encoding the non-structural Rep (replication) and structural Cap (capsid) proteins. Flanking the AAV coding regions are two cis-acting inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, with interrupted palindromic sequences that can fold into hairpin structures that function as primers during initiation of DNA replication. In addition to their role in DNA replication, the ITR sequences have been shown to be necessary for viral integration, rescue from the host genome, and encapsidation of viral nucleic acid into mature virions (Muzyczka; 1992; Curr. Top. Micro. Immunol.; 158:97-129).

Multiple serotypes of AAV exist and offer varied tissue tropism. Known serotypes include, for example, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10 and AAV1 1 .

Recombinant adeno-associated virus (rAAV) vectors are a leading gene delivery platform, have remarkable potential for gene therapy due to their promising safety profile and their ability to transduce many tissues in vivo, and several rAAV-mediated therapies have recently been approved (Ameri; 2018; J. Curr. Ophthalmol. 30, 1-2; Yla-Herttuala; 2012; Mol. Ther. 20:1831-1832). However, despite these advances in the clinic, rAAV vector manufacturing remains a challenge. As an example, a phase 1/2 trial for hemophilia B required over 400 ten-layer cell stacks to generate sufficient material for six patients (Allay et al; 201 1 ; Hum. Gene Ther. 22: 595-604). Although the trial was successful, this study highlights the need for new methods to improve vector generation. Increased production efficiency will reduce manufacturing costs, improve patient access, and make this emerging modality more feasible for large disease indications. Higher gene therapy vector loads are crucial to account for larger patient cohorts systemic diseases, or diseases in less accessible body sites. Thus, beyond the efforts in improving safety, alternative host cells, and alternative viral helpers, an additional area of rAAV research has been in scale-up, moving production from laboratory scale to industrial scale (Clement; 2016; Mol Ther Methods Clin Dev; 3:16002). An important focus of the rAAV vector development field has been to fine-tune the manufacturing process to augment the vector yield, purity, or its potency so that dose of vectors required per patient is low. rAAV transduction requires a reasonable multiplicity of infection of ~10³ to 10⁵ vector genomes (vg) per cell depending on cell type. However, for a clinical trial, an estimated 10¹² to 10¹⁴ viral particles are essential to be efficacious during gene transfer. This high vector dose requirement in the clinical settings has underscored the need for optimizing vector production. Multiple systems for the manufacture of AAV have been developed so far, but currently the cotransfection of HEK293 cells, typically using three or two bacterial plasmids, is the most widely used (Samulski RJ et al;. Annu. Rev. Virol. 2014; 1 : 427-51 ; EP3722434 A1).

Although transient transfection is currently considered the standard approach for clinical and commercial manufacture of AAV, the production and use of plasmid DNA presents a number of challenges in commercial-scale manufacture. First, the cruciform secondary structures of the c/s-acting ITR sequences can be difficult to propagate in Escherichia coli, and plasmid preparations frequently contain deletions in the ITR regions (Yan Z et al;J. Virol. 2005; 79(1): 364-79). Moreover, plasmid DNA production typically requires antibiotic selection, resulting in transfected plasmids containing backbone sequences encoding antibiotic resistance genes. Plasmid-derived sequences are known to be packaged into AAV capsids at a frequency estimated at 1-5% (Chadeuf et al; Mol. Ther. 2005; 12(4): 744-53).

Finally, the majority of production cost of plasmid DNA in E. coli comprises time in GMP suite, capital and labour costs, which represents a challenge for capacity expansion of the existing market in the rapidly advancing gene therapy pipeline.

Taken together there remains a need to develop DNA vector systems for AAV vector production manufacturing, which can efficiently overcome the drawbacks of existing technologies allowing for increased quantity, showing increased expression level, while exhibiting high quality in terms of purity e.g. low levels of empty capsids, host cell protein, and/or contaminating DNA.

SUMMARY OF THE INVENTION

The present invention relates to a novel two or three component linear, synthetic DNA vector system for use in methods of adeno-associated virus (AAV) vector production and methods of producing such a vector system, utilising linear, synthetic DNA molecules generated in an enzymatic in vitro manufacturing process, omitting the use of bacterial fermentation methods.

Applicant has designed a novel two or three DNA vector system to be used in methods where improved and/or increased AAV vector production is needed, e.g. in gene therapy, where traditionally AAV production plasmid systems are used as tools for transferring and expressing desired genetic material. This novel DNA vector system incorporates the split plasmid technology concept disclosed in W02020/208379 A1 , EP3722434 B1 and WO2022/079429 A1 , and described in EP2417591 1.7 (incorporated herein by reference), in the format of linear, double stranded synthetic DNA vectors, suitably covalently closed synthetic DNA vectors.

Based on the disclosure provided herein, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments (E). Specifically, the present disclosure provides the following aspects, advantageous features and specific embodiments (E), respectively alone or in combination:

E1 . A linear synthetic DNA vector system comprising at least a first linear synthetic DNA vector and a second linear synthetic DNA vector, wherein said first vector comprises at least one rep gene encoding at least one functional Rep protein and does not comprise a cap gene encoding a functional set of Cap proteins.

E2. The linear synthetic DNA vector system of E1 , wherein said second vector comprises:

(a) a cap gene encoding at least one functional Cap protein; or

(b) at least one promoter driving a cap gene expression, preferably a cap gene promoter, a cloning site operably linked to said promoter, and an expression cassette flanked on at least one side by an ITR; wherein said second vector does not comprise a rep gene encoding a functional Rep protein and the expression cassette comprises a transgene operably linked to at least one regulatory control element.

E3. The linear synthetic DNA vector system of E1 , wherein said second vector comprises:

(a) a cap gene encoding at least one functional Cap protein; or

(b) at least one promoter driving a cap gene expression, preferably a cap gene promoter, , a cloning site operably linked to said promoter, and does not comprise a rep gene encoding a functional Rep protein; and wherein said system further comprises a third linear synthetic DNA vector comprising an expression cassette flanked on at least one side by an ITR and said expression cassette comprises a transgene operably linked to at least one regulatory control element.

E4. The synthetic DNA vector system of any one of E1 to E3, wherein the at least one rep gene encodes a functional Rep52 and/or Rep 40 protein and at least one gene encoding a functional Rep78 and/or Rep68 protein.

E5. The synthetic DNA vector system of any one of E1 to E4, wherein said first vector comprises at least one helper virus gene, optionally wherein:

(i) the at least one helper virus gene is an adenovirus gene, optionally an Adenovirus 5 or Adenovirus 2 gene; and/or

(ii)the at least one helper virus gene comprises a VA nucleic acid encoding functional VA RNA preferably VA RNA I and II, an E2A gene encoding a functional E2A protein, and an E4 gene encoding a functional E4 protein or a gene encoding functional 22K/33K proteins. E6. The synthetic DNA vector system of any one of E1 to E5 wherein said second vector comprises a cap gene and the cap gene encodes a Cap protein selected from any naturally occurring or genetically engineered AAV serotype.

E7. The synthetic DNA vector system of any one of E1 to E6 wherein:

(i) said second vector does not comprise any dispensable translation initiation codons; and/or

(ii) said second vector does not comprise any dispensable translation initiation codons, wherein the vector plasmid comprises a promoter region comprising one or more promoters, and the promoter region does not comprise ATG or GTG codons, optionally wherein the promoter region comprises p5, p19 and p40 promoters, and wherein ATG or GTG codons at one or more positions corresponding to positions (a) 321-323, (b) 766-768, (c) 955-957, (d) 993-995 and (e) 1014-1016 of SEQ ID NO: 1 are absent or mutated.

E8. Use of the linear synthetic DNA vector system of any one of E1 to E7 for producing a AAV preparation:

(a) having a desired ratio of full to total particles; and/or

(b) at a high or desired yield; and/or

(c) having reduced or completely eliminated undesirable DNA sequences

E9. Use of the linear synthetic DNA vector system of any one of E1 to E7 for:

(a) controlling or maximising the ratio of full to total particles produced during recombinant AAV production; and/or

(b) increasing, optimising or maximising the yield of recombinant AAV produced during recombinant AAV production; and/ or

(c) reducing or completely eliminating undesirable DNA sequences

E10. The use of E8 or E9, wherein the use comprises transfecting a host cell with vector system of any one of E1-E7 and culturing the host cell under conditions suitable for recombinant AAV production.

11 . A method for controlling or maximising the ratio of full to total particles produced during recombinant AAV production comprising:

- obtaining the linear synthetic DNA vector system of any E1-E7;

- transfecting a host cell with said system; and

- culturing the host cell under conditions suitable for recombinant AAV production

- harvesting the recombinant AAV to provide a recombinant AAV preparation comprising a desired ratio of full to total particles.

E12. A method for increasing, optimising or maximising the yield of recombinant AAV produced during recombinant AAV production comprising: - obtaining the linear synthetic DNA vector system of any one of E1-E7;

- transfecting a host cell with said system

E13. A method of reducing or completely eliminating the presence of any undesirable DNA sequences during recombinant AAV production comprising:

- obtaining the linear synthetic DNA vector system of any one of E1-E7;

- transfecting a host cell with said system

BRIEF DESCRIPTON OF THE DRAWINGS

Figure 1 : Schematic illustration of a two-plasmid system for AAV production (“split” plasmid system described in W02020/208379 A1 , EP3722434 B1 , incorporated herein by reference), comprising a helper plasmid and a vector plasmid). Ori = bacterial origin of replication. KanR = kanamycin resistance gene. ITR = inverted terminal repeat. Note the respective plasmid features are not shown to scale.

Figure 2: Schematic illustration of a two-component linear, synthetic DNA vector system for AAV production derived from the two-plasmid system of W02020/208379 A1 , EP3722434 B1. The system comprises a first linear synthetic DNA vector with at least one rep gene encoding at least one functional Rep protein and a helper virus gene; and a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein and an expression cassette flanked on at least one side by an ITR. GOI: gene of interest.

Figure 3: Schematic illustration of a three-component linear, synthetic DNA vector system for AAV production derived from the two-plasmid system of W02020/208379 A1 , EP3722434 B1. The system comprises a first linear synthetic DNA vector with at least one rep gene encoding at least one functional Rep protein and a helper virus gene; a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein; and a third linear, synthetic DNA vector comprising an expression cassette flanked on at least one side by an ITR. GOI: gene of interest.

Figure 4: Schematic illustration of a two-component linear, synthetic DNA vector system for AAV production derived from the two-plasmid system of W02020/208379 A1 , EP3722434 B1. The system comprises a first linear synthetic DNA vector with at least one rep gene encoding at least one functional Rep protein and a helper virus gene, wherein the at least one rep gene transcription orientation is reversed; and a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein and an expression cassette flanked on at least one side by an ITR. GOI: gene of interest. Figure 5. Schematic illustration of a three-component linear, synthetic DNA vector system for AAV production derived from the two-plasmid system of W02020/208379 A1 , EP3722434 B1. The system comprises a first linear synthetic DNA vector with at least one rep gene encoding at least one functional Rep protein and a helper virus gene, wherein the at least one rep gene transcription orientation is reversed; a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein; and a third linear, synthetic DNA vector comprising an expression cassette flanked on at least one side by an ITR. GOI: gene of interest.

Figure 6: Schematic illustration of a two-plasmid system for AAV production (“split” plasmid system described in WO2022/079429 A1 , incorporated herein by reference), comprising a helper plasmid and a vector plasmid). Ori = bacterial origin of replication. KanR = kanamycin resistance gene. ITR = inverted terminal repeat. Note the respective plasmid features are not shown to scale.

Figure 7: Schematic illustration of a two-component linear, synthetic DNA vector system for AAV production derived from a two-plasmid system of WO2022/079429 A1 . The system comprises a first linear synthetic DNA vector with at least one rep gene encoding at least one functional Rep protein and a helper virus gene; and a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein and an expression cassette flanked on at least one side by an ITR. GOI: gene of interest.

Figure 8. Schematic illustration of a three-component linear, synthetic DNA vector system for AAV production derived from a two-plasmid system of WO2022/079429 A1 . The system comprises a first linear synthetic DNA vector with at least one rep gene encoding at least one functional Rep protein and a helper virus gene; a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein; and a third linear, synthetic DNA vector comprising an expression cassette flanked on at least one side by an ITR. GOI: gene of interest.

Figure 9. Schematic illustration of a two-component linear, synthetic DNA vector system for AAV production derived from a two plasmid system of WO2022/079429 A1 .The system comprises a first linear synthetic DNA vector with at least one rep gene encoding at least one functional Rep protein and a helper virus gene, wherein the at least one rep gene transcription orientation is reversed; and a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein and an expression cassette flanked on at least one side by an ITR. GOI: gene of interest.

Figure 10. Schematic illustration of a three-component linear, synthetic DNA system vector for AAV production derived from a two-plasmid system of WO2022/079429 A1 . The system comprises a first linear synthetic DNA vector with at least one rep gene encoding at least one functional Rep protein and a helper virus gene, wherein the at least one rep gene transcription orientation is reversed; a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein; and a third linear, synthetic DNA vector comprising an expression cassette flanked on at least one side by an ITR. GOI: gene of interest.

Figure 11 . Schematic representation of a general process to produce linear synthetic DNA vectors. On the left side is depicted a plasmid comprising seguences to be incorporated or transformed to a linear DNA vector, along with the plasmid backbone seguences. Seguences of interest, included in the initial plasmid, are transferred either to circular (1A) or linear (1 B) template vector. In the next step, said template vector is processed in an in vitro enzymatic amplification (2) in order to obtain a linear DNA vector.

Figure 12 (A-B): Schematic representation of alternative structural configurations of a first linear synthetic DNA vector of the invention. Fig. 12A shows an alternative structural configuration of composing structural elements of a vector described in Example 1A. Fig. 12B shows an alternative structural configuration of composing structural elements of a vector described in Example 1 E.

Figure 13: Schematic illustration of a two-plasmid system for AAV production (“split” plasmid system described in EP24175911 .7, incorporated herein by reference), comprising a helper plasmid and a vector plasmid). Ori = bacterial origin of replication. KanR = kanamycin resistance gene. ITR = inverted terminal repeat. Note the respective plasmid features are not shown to scale.

Figure 14: Schematic illustration of a two-component linear, synthetic DNA vector system for AAV production derived from the two-plasmid system of EP24175911 .7. The system comprises a first linear synthetic DNA vector with at least one rep gene encoding at least one functional Rep protein and a helper virus gene (described in Example 1 H); and a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein and an expression cassette flanked on at least one side by an ITR. GOI: gene of interest (described in Example 1 K or 1 L).

Figure 15: Schematic illustration of a two-component linear, synthetic DNA vector system for AAV production derived from the two-plasmid system of EP24175911 .7. The system comprises a first linear synthetic DNA vector with at least one rep gene encoding at least one functional Rep protein (inverted orientation) and a helper virus gene (described in Example 11); and a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein and an expression cassette flanked on at least one side by an ITR. GOI: gene of interest (described in Example 1 K or 1 L).

Figure 16: Schematic illustration of a two-component linear, synthetic DNA vector system for AAV production derived from the two-plasmid system of EP24175911 .7. The system comprises a first linear synthetic DNA vector with at least one rep gene encoding at least one functional Rep protein (inverted orientation), a spacer seguence and a helper virus gene (described in Example 1 J); and a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein and an expression cassette flanked on at least one side by an ITR. GOI: gene of interest (described in Example 1 K or 1 L).

Figure 17: Schematic illustration of a three-component linear, synthetic DNA vector system for AAV production derived from the two-plasmid system of EP24175911 .7. The system comprises a first linear synthetic DNA vector with at least one rep gene encoding at least one functional Rep protein and a helper virus gene (described in Example 1 H); a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein (described in Example 1 M or 1 N); and a third linear, synthetic DNA vector comprisng an expression cassette flanked on at least one side by an ITR. GOI: gene of interest (described in Example 10).

Figure 18: Schematic illustration of a three-component linear, synthetic DNA vector system for AAV production derived from the two-plasmid system of EP24175911 .7. The system comprises a first linear synthetic DNA vector with at least one rep gene encoding at least one functional Rep protein and a helper virus gene (described in Example 11); a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein (described in Example 1 M or 1 N); and a third linear, synthetic DNA vector comprisng an expression cassette flanked on at least one side by an ITR. GOI: gene of interest (described in Example 10).

Figure 19: Schematic illustration of a three-component linear, synthetic DNA vector system for AAV production derived from the two-plasmid system of EP24175911 .7. The system comprises a first linear synthetic DNA vector with at least one rep gene encoding at least one functional Rep protein and a helper virus gene (described in Example 1J); a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein (described in Example 1 M or 1 N); and a third linear, synthetic DNA vector comprisng an expression cassette flanked on at least one side by an ITR. GOI: gene of interest (described in Example 10).

Figure 20 (A-B): Schematic representation of alternative structural configurations of the first linear synthetic DNA vector of the invention. Fig. 20A shows an alternative structural configuration of composing structural elements of a vector described in Example 1 H. Fig. 12B shows an alternative structural configuration of composing structural elements of a vector described in Example 1J.

Figure 21 (A-D): Functional analysis of rAAV produced using a two-component linear synthetic DNA system. Fig. 21 A: Vector genome yield was measured using a molar ratio for first vector: second vector of 4:3 and a total DNA concentration of 0.52 pg/cell. Vector genome (vg) titre per ml was measured by ddPCR. The vector genome yield was determined 72 h after transfection. Fig. 21 B: Yield of rAAV particles (“capsid yield”) using a molar ratio for first vector: second vector of 4:3 and a total DNA concentration of 0.52 pg/cell. The number of rAAV particles per ml was measured by Gyrolab xPlore assay. The capsid yield was determined 72 h after transfection. Fig. 21C: Levels of KanR impurities were determined for rAAV produced using a molar ratio for first vector: second vector of 4:3 and a total DNA concentration of 0.52 pg/cell 72 h after transfection. Fig. 21 D: Transgene activity following the transduction of HuH-7 cell with rAAV produced using a molar ratio for first vector: second vector of 4:3 and a total DNA concentration of 0.52 pg/cell. *Left column shows values for the control plasmid system and right column values for the tested two component, linear synthetic DNA vector system.

Figure 22(A-D): Functional analysis of rAAV produced using a three-component linear synthetic DNA system. Fig. 22A: Vector genome yield was determined for rAAV produced using a molar ratio first vector: second vector: third vector of 4:3:3 and a total DNA concentration of 0.52 pg/cell. Vector genome (vg) titre per ml was measured by ddPCR. The vector genome yield was determined 72 h after transfection. Fig. 22B: Yield of rAAV particles (“capsid yield”) was determined for rAAV produced using using a molar ratio for first vector: second vector: third vector of 4:3:3 and a total DNA concentration of 0.52 pg/cell. The number of rAAV particles per ml was measured by Gyrolab xPlore assay. The capsid yield was determined 72 h after transfection. Fig. 22C: Levels of KanR impurities were determined for rAAV produced using a molar ratio for first vector: second vector: third vector of 4:3:3 and a total DNA concentration of 0.52 pg/cell 72 h after transfection. Fig. 22D: Transgene activity following the transduction of HuH-7 cell with rAAV produced using a molar ratio for first vector: second vector: third vector of 4:3 and a total DNA concentration of 0.52 pg/cell. *Left column shows values for the control plasmid system and right column values for the tested three component, linear synthetic DNA vector system.

Figure 23(A-E). Functional analysis of rAAV produced using a two-component linear synthetic DNA system Fig. 23A: Vector genome yield was determined for rAAV produced using a molar fratio for irst vector: second vector of 4:1 for the synthetic DNA vectors and 4:3 for the respective plasmids of the control two plasmid DNA system, as well as a total DNA concentration of 0.42 pg/cell for the synthetic DNA and 0.52 pg/cell for the control system. Vector genome (vg) titre per ml was measured by ddPCR. The vector genome yield was determined 72 h after transfection. Fig. 23B: Yield of rAAV particles (“capsid yield”) was determined for rAAV produced using a molar ratio for first vector: second vector of 4:1 for the synthetic DNA vectors and 4:3 for the respective plasmids of the control two plasmid DNA system, as well as a total DNA concentration of 0.42 pg/cell for the synthetic DNA and 0.52 pg/cell for the control system. The number of rAAV particles per ml was measured by Gyrolab xPlore assay. The capsid yield was determined 72 h after transfection. Fig. 23C: The vector genome to total particle ratio. The vector genome to total particle ratio was determined 72 h after transfection. Fig. 23D: Levels of cap impurities were determined for rAAV produced using a molar ratio for first vector: second vector of 4:1 for the synthetic DNA vectors and 4:3 for the respective plasmids of the control two plasmid DNA system, as well as a total DNA concentration of 0.42 pg/cell for the synthetic DNA and 0.52 pg/cell for the control system. Fig. 23E: Levels of kanR impurities were determined for rAAV produced using using a molar ratio for first vector: second vector ratio of 4:1 for the synthetic DNA vectors and 4:3 for the respective plasmids of the control two plasmid DNA system, as well as a total DNA concentration of 0.42 pg/cell for the synthetic DNA and 0.52 pg/cell for the control system. *Left column shows values for the control plasmid system and right column values for the tested two component, linear synthetic DNA vector system.

DETAILED DESCRIPTION

The present invention relates to a novel two or three component DNA vector system for use in methods of adeno-associated virus (AAV) vector production and methods of producing such a vector system. Such a system utilises linear, synthetic DNA molecules generated in an enzymatic in vitro manufacturing process, omitting the use of bacterial fermentation methods and is based on linear, double stranded and preferably covalently closed synthetic DNA. Closed linear DNA molecules typically comprise covalently closed ends also described as hairpin loops, where base-pairing between complementary DNA strands is not present. The hairpin loops join the ends of complementary DNA strands. Structures of this type typically form at the telomeric ends of chromosomes in order to protect against loss or damage of chromosomal DNA by sequestering the terminal nucleotides in a closed structure. By generating closed linear DNA molecules suitable for use as vectors in an in vitro cell-free environment, the use of DNA templates, having extraneous sequences used in traditional plasmid systems, it becomes feasible to minimize the presence of such associated DNA sequences unintentionally packed into AAV vectors. Known “hotspot signals” responsible for mispacking of “plasmid-derived” DNA impurities are the AAV ITR sequences (read-through and reverse packaging) and the AAV p5 promoter (reverse packaging). Additionally, bacteria originating sequences mainly comprise the so-called plasmid backbone sequences like antibiotic resistance genes used as selection markers (e.g. kanR, ampR) and bacterial replication origin sequences (ori) required for the amplification of the plasmids in bacteria like E. coli. Therefore, utilizing the vector system of the current disclosure, the presence of bacterial impurities like endotoxins, host cell protein, genomic DNA or other contaminants like residual antibiotics is avoided, rendering it particularly suitable for therapeutic uses. Linear, double stranded and covalently closed synthetic DNA molecules, on which the AAV vectors production of the invention is based, can be synthesized using various methods available in the state of the art, like the ones described by Karda et al (doi: 10.1038/s41434-018-0056-1), Barreira et al (Gene Therapy ; 30:122-131 (2023)) or in W02023/006978 A1).

The inventors of the instant disclosure have designed a novel two and/or three component linear synthetic DNA, AAV based vector system, separating the AAV rep and cap gene onto two different DNA molecules aiming to the reduction of the frequency of replication-competent AAV (rcAAV) generation. Suitably, the rep gene is driven by the endogenous AAV promoters p5 (large Rep proteins) and p19 (small Rep proteins). In an alternative configuration the rep gene (large Rep proteins) can as well be driven by a heterologous promoter, such as MMTV-LTR promoter.

The components of the novel vector system of the present disclosure are based on and are directly derived from plasmids used in so called “split-plasmid” systems described in W02020/208379 A1 , EP3722434 B1 , WO2022/079429 A1 and EP24216316 (incorporated herein by reference). Both the two and the three component linear synthetic DNA, AAV vector system comprise the adenoviral helper viral elements and the AAV rep gene on one synthetic DNA molecule, while AAV cap gene sequences and AAV vector genome containing the therapeutic cassette are combined on one DNA linear synthetic molecule in the two-component system, or AAV cap gene sequence and AAV vector genome containing the therapeutic cassette are separated in two synthetic DNA molecules in the three component synthetic DNA AAV based vector system. Suitably, the cap gene is driven by the endogenous AAV promoters p5, p19 and p40. Alternatively, cap gene can also be driven by only one or two of the endogenous AAV promoters (including p40).

General Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs In general, the term “comprising” is intended to mean including but not limited to.

In some embodiments of the invention, the word “comprising” is replaced with the phrase “consisting of or the phrase “consisting essentially of. The term “consisting of is intended to be limiting. The terms “protein" and “polypeptide" are used interchangeably herein and are intended to refer to a polymeric chain of amino acids of any length.

For the purpose of this invention, in order to determine the percent identity of two sequences (such as two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in a first sequence for optimal alignment with a second sequence). The nucleotides or amino acids at each position are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the amino acids or nucleotides are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (/.e., % identity = number of identical positions /total number of positions in the reference sequence x 100).

Typically, the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence is 95% identical to SEQ ID NO: 1 , SEQ ID NO: 1 (Genome of AAV2 (AF043303.1) would be the reference sequence. To assess whether a sequence is at least 80% identical to SEQ ID NO: 1 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 1 , and identify how many positions in the test sequence were identical to those of SEQ ID NO: 1 . If at least 80% of the positions are identical, the test sequence is at least 80% identical to SEQ ID NO: 1. If the sequence is shorter than SEQ ID NO: 1 , the gaps or missing positions should be considered to be non-identical positions. The skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In an embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.

A "vector" as used herein refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which can be used to mediate delivery of the polynucleotide to a cell. Illustrative vectors include, for example, plasmids, viral vectors, liposomes, linear synthetic DNA molecules e.g Doggybone™ DNA (or dbDNA abbreviated), hpDNA™, and other gene delivery vehicles. “Doggybone™ DNA or dbDNA” refers to synthetic, double-stranded DNA molecules that are covalently closed and linear, with terminal hairpin loops at both ends, giving them a characteristic "doggybone" or dumbbell-shaped structure. Unlike plasmid DNA, dbDNA does not contain bacterial elements (like antibiotic resistance genes or bacterial replication origins), which can offer improved safety, reduced immunogenicity, and higher expression efficiency in various genetic applications, including gene therapy, vaccine development, and genetic engineering, (for example, Walters et al, (2014) described such DNA molecules). The term “synthetic AAV vector” and “synthetic production of AAV vector” refers to an AAV vector and synthetic production methods thereof in an entirely cell-free environment. The production may involve one or more molecules in a manner that does not involve replication or other multiplication of the molecule by or inside of a cell or using a cellular extract. Synthetic production avoids contamination of the produced molecule with cellular contaminants, e.g., cellular proteins or cellular nucleic acid, viral protein or DNA, insect protein or DNA and further avoids unwanted cellular-specific modification of the molecule during the production process, e.g., methylation or glycosylation or other post-translational modification.

A “two component linear, synthetic DNA vector system” refers to a vector system suitable for AAV production, produced by an enzymatic, cell free process. Such processes are known to those skilled in the art. Such a process is described, for example by Karda et al (doi: 10.1038/s41434-018-0056-1), Barreira et al (Gene Therapy; 30:122-131 (2023)), in W02019101596A1 or in W02023/006978 A1. In the context of the present disclosure such a system contains a first vector comprising a a first linear synthetic DNA vector at least one rep gene encoding at least one functional Rep protein and a helper virus gene; and a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein and an expression cassette flanked on at least one side by an ITR.

A “three component linear, synthetic DNA vector system” refers to a vector system suitable for AAV production, produced by an enzymatic, cell free process. Such processes are known to those skilled in the art. Such a process is described, for example by Karda et al (doi: 10.1038/s41434-018-0056-1), Barreira et al (Gene Therapy; 30:122-131 (2023)), in WG2019101596A1 or in WG2023/006978 A1. In the context of the present disclosure such a system contains a first linear synthetic DNA vector at least one rep gene encoding at least one functional Rep protein and a helper virus gene; a second linear, synthetic DNA vector comprising a cap gene encoding at least one functional Cap protein; and a third linear, synthetic DNA vector comprising an expression cassette flanked on at least one side by an ITR. "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector").

An "rAAV vector" as used herein refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e. , a polynucleotide heterologous to AAV), typically a sequence of interest for the genetic transformation of a cell.

In preferred vector constructs of this invention, the heterologous polynucleotide is flanked by at least one, preferably two AAV inverted terminal repeat sequences (ITRs).

The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

An "AAV virus" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein (preferably by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector".

"Packaging" refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle.

AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. They have been found in all AAV serotypes examined and are in the art. AAV rep and cap are referred to herein as AAV "packaging genes".

A "helper virus" for AAV refers to a virus that allows AAV (e.g. wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV): which are also available from depositories such as ATCC.

Herein, the term “plasmid" is intended to refer to a nucleic acid molecule that can replicate independently of a cell chromosome. The term “plasmid’ is intended to cover circular nucleic acid molecules and linear nucleic acid molecules. Furthermore, the term “plasmid’ is intended to cover bacterial plasmids, but also cosmids, minicircles (Nehlsen, K., Broil S., Bode, J. (2006), Gene Then Mol. Biol., 10: 233-244; Kay, M.A., He, C.-Y, Chen, Z.-H. (2010), Nature Biotechnology, 28: 1287- 1289) and ministrings (Nafissi N, Alqawlaq S, Lee EA, Foldvari M, Spagnuolo PA, Slavcev RA. (2014), Mol Ther Nucleic Acids, 3:e165). Optionally, the plasmid is a circular nucleic acid molecule. Optionally, the plasmid is a nucleic acid molecule that is of bacterial origin.

The term “helper¹’ is not intended to be limiting. Accordingly, a “helper plasmid’ is any plasmid that comprises at least one rep gene encoding at least one functional Rep protein and may or may not comprise a cap gene encoding a functional set of Cap proteins.

The term “three or triple plasmid AAV production system” refers to a rAAV production system which involves the transfection of host cells with three separate plasmids, namely a rAAV vector plasmid which contains a promoter, a gene of interest (GOI) flanked by inverted terminal repeats (ITRs), which are essential for AAV replication and packaging, a rAAV helper plasmid carrying the Rep and Cap genes, which encode the replication and capsid proteins of AAV, respectively and an adenovirus helper plasmid (Ad helper) containing the necessary adenoviral genes, such as, E2a, E4orf6, and VA RNA, which are required to support AAV replication and packaging. Currently, there are available several variations of the above-mentioned system in the art, which are herein incorporated by reference.

The term “two plasmid AAV production system” in the context of the current disclosure refers to a system that comprises only two plasmids and can be used without the need for additional plasmids to produce rAAV. Optionally, the two-plasmid system can be used to produce rAAV without the need for helper virus such as adenovirus. Optionally, the two-plasmid system can be used to produce rAAV without the need for genetic material originating from a host cell, optionally with the exception of a gene encoding E1A/B. However, the system may comprise additional non-plasmid components. Optionally, the two- plasmid system does not comprise a helper virus. Optionally, the two-plasmid system of the invention comprises all the necessary genetic information for the production of rAAV. For example, the two- plasmid system of the invention may comprise at least one rep gene, at least one cap gene and at least one helper gene or helper gene region. Optionally, the two-plasmid system of the invention comprises all the necessary genetic information required for the production of rAAV suitable for use in gene therapy. For example, the two-plasmid system of the invention may comprise at least one rep gene, at least one cap gene, at least one helper gene and an expression cassette comprising a transgene operably linked to at least one regulatory control element. However, in embodiments the two-plasmid system of the invention may lack a functional cap gene (required for the production of rAAV) and/or an expression cassette comprising a transgene operably linked to at least one regulatory control element (required for the production of rAAV suitable for use in gene therapy). Suitably, a two plasmid system of the invention comprises a helper plasmid comprising at least one AAV rep gene encoding at least one functional AAV Rep protein and at least one helper virus gene, and which does not comprise a cap gene encoding a functional set of Cap proteins and a vector plasmid comprising (a) an AAV cap gene encoding at least one functional AAV Cap protein; or (b) at least one AAV cap gene promoter, a cloning site operably linked to the AAV cap gene promoter, and an expression cassette flanked on at least one side by an inverted terminal repeat (ITR); wherein the vector plasmid does not comprise a rep gene encoding a functional Rep protein and the expression cassette comprises a transgene operably linked to at least one regulatory control element. Suitable two plasmid systems are described in WO 2020/208379 A, EP3722434 B1 , WO 2022/079429 A1 and EP24216316 (incorporated herein by reference).

The term “nucleic acid molecule” refers to a polymeric form of nucleotides of any length. The nucleotides may be deoxyribonucleotides, ribonucleotides or analogues thereof. Preferably, the plasmid is made up of deoxyribonucleotides or ribonucleotides. Even more preferably, the plasmid is made up of deoxyribonucleotides, i.e. the plasmid is a DNA molecule. In all instances herein, the term “nucleic acid sequence” may be replaced by the term “polynucleotide”.

The terms “wild type” and “native” are synonymous and refer to genes present in the genome of a strain/serotype of AAV or adenovirus, or to proteins encoded by genes present in the genome of a strain/serotype of AAV or adenovirus.

The helper plasmid may be useful for producing rAAV. Optionally, the helper plasmid is suitable for use in producing rAAV. Optionally, the helper plasmid is for producing rAAV. Optionally, the helper plasmid is suitable for producing rAAV suitable for use in gene therapy. Optionally, the helper plasmid is for producing rAAV for use in gene therapy.

Transcription regulatory elements are nucleotide sequences which effect the level of expression of a gene, and include, for example, promoters, enhancers, introns, untranslated regions, and transcriptional terminators. Some transcription regulatory elements promote greater levels of transcription compared to others (stronger transcription regulatory elements). For example, some promoters are known to promote transcription at a higher level than others. Generally, a promoter will be a stronger promoter if it comprises a sequence that allows for strong binding to the transcription complex. Promoters which are known to be generally strong promoters in human cells include viral promoters. Promoters that are generally believed to be strong promoters in human cells include the EF1A, CMV, CAG and SV40 promoters. Promoters that are generally believed to be weak promoters in human cells include UBC and PGK promoters.

Cap gene

One of the components of the linear, synthetic DNA vector system of the invention, namely a second vector of the invention, may comprise a cap gene. The cap gene encodes a functional Cap protein. The cap gene may encode a functional set of Cap proteins. AAV generally comprises three Cap proteins, VP1 , VP2 and VP3. These three proteins form a capsid into which the AAV genome is inserted and allow the transfer of the AAV genome into a host cell. All VP1 , VP2 and VP3 are encoded in native AAV by a single gene, the cap gene. The amino acid sequence of VP1 comprises the sequence of VP2. The portion of VP1 which does not form part of VP2 is referred to as VP1 unique or VP1 U. The amino acid sequence of VP2 comprises the sequence of VP3. The portion of VP2 which does not form part of VP3 is referred to as VP2unique or VP2U.

A “functional” set of Cap proteins is one which allows for encapsidation of AAV. As discussed above, it is within the abilities of the skilled person to determine whether a given Cap protein is or a set of Cap proteins are functional.

Optionally, VP2 and/or VP3 proteins are “functional” if an AAV or rAAV comprising the VP2 and/or the VP3 proteins is able to transduce Huh7 cells at a level at least 25%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90% or at least 95% of that of an equivalent AAV o rAAV comprising a wild type VP2 and/or VP3 protein.

Optionally, a second vector of a linear, synthetic DNA vector system of the invention comprises a cap gene that encodes a VP1 , a VP2 and/or a VP3 protein. Optionally, the VP1 , VP2 and VP3 proteins are expressed from more than one cap gene. Optionally, the second vector comprises a cap gene that encodes a VP1 , a VP2 and a VP3 protein. Optionally the second vector comprises a cap gene encoding a functional VP1 , i.e. a VP1 protein capable of assembling with other Cap proteins to encapsidate a viral genome.

Different serotypes of AAV have Cap proteins having different amino acid sequences. A cap gene encoding any (set of) Cap protein(s) is suitable for use in connection with the present invention. The Cap protein can be a native Cap protein expressed in AAV of a certain serotype. Alternatively, the Cap protein can be a non-natural, for example an engineered, Cap protein, which is designed to comprise a sequence different to that of a native AAV Cap protein. Genes encoding non-natural Cap proteins are particularly advantageous, as in the context of gene therapy applications it is possible that fewer potential patients have levels of antibodies that prevent transduction by rAAV comprising non-natural Cap proteins, relative to native capsids. Optionally, the cap gene encodes a Cap protein from a serotype selected from the group comprising serotypes 1 , 2, 3A, 3B, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13. Optionally, the cap gene encodes a Cap protein from a serotype selected from the group consisting of serotypes 2, 5, 8, and 9. Optionally, the cap gene encodes a Cap protein selected from the group consisting of LK03, rh74, rh10 and Mut C (WO 2016/181123; WO 2013/029030; WO 2017/096164). Optionally, the cap gene encodes a Cap protein selected from the group of AAV serotypes consisting of serotypes 2, 5, 8 or 9, and Mut C (SEQ ID NO: 3 from WO 2016/181123). Optionally, the cap gene encodes the Cap protein Mut C (SEQ ID NO: 3 from WO 2016/181 123).

Cap gene promoter

Optionally, the second vector comprises a cap gene promoter. The cap gene promoter may be operably linked to a cap gene. Alternatively, the second vector may not comprise a cap gene, but may comprise a cloning site operably linked (i.e. in close juxtaposition: 5’-[cap gene promoter]-[cloning site]-3’) to the cap gene promoter. The user may wish to have the option to add a specific cap gene for a specific application. For example, if the second vector is to be used to produce rAAV for use in gene therapy, the user may wish the vector to lack a cap gene but comprise a cloning site to allow a specific cap gene to be cloned in for a specific application. The vector could be used in connection with any transgene (in an expression cassette), and the user may find that for certain transgenes, encapsidation of such cassettes into capsids having properties such as liver tropism is advantageous, whereas for other transgenes capsids having different tropisms are advantageous. By designing a vector plasmid that comprises a cap gene promoter linked to a cloning site, the user can readily ‘plug in’ an appropriate cap gene for a specific application (such as use of a specific transgene).

Optionally, a second vector of a linear, synthetic DNA vector system of the invention comprises an at least one cap gene promoter, which is a native cap gene promoter.

The native cap gene (i.e. the cap gene of a wild type AAV) is operably linked to a p40 promoter, a p5 promoter and a p19 promoter. Optionally, the at least one cap gene promoter comprises an AAV p40 promoter, a p5 promoter, and/or a p19 promoter. Optionally, the at least one cap gene promoter comprises an AAV p40 promoter, a p5 promoter, and a p19 promoter. However, any suitable promoter that is able to drive cap gene expression can be used.

Optionally, the cap gene is operably linked to a p5 promoter preferably a wild type AAV promoter, more preferably a wild type AAV2 p5 promoter, wherein said p5 promoter is located either at the 5’ end or the 3’ end of a second vector of the present disclosure.

Rep genes

A first linear synthetic DNA vector may comprise at least one rep gene encoding at least one functional Rep protein. AAV comprises a rep gene region which encodes four Rep proteins (Rep 78, Rep 68, Rep 52 and Rep 40). The gene region is under the control of the p5 and p19 promoters. When the p5 promoter is used, a gene that encodes Rep 78 and Rep 68 is transcribed. Rep 78 and Rep 68 are two alternative splice variants (Rep 78 comprises an intron that is excised in Rep 68). Similarly, when the p19 promoter is used, a gene that encodes Rep 52 and Rep 40 is transcribed. Rep 52 and Rep 40 are alternative splice variants (Rep 52 comprises an intron that is excised in Rep 40).

The four Rep proteins are known to be involved in replication and packaging of the viral genome, and are, therefore, useful in rAAV production.

A “functional” Rep protein is one which allows for production of AAV particles. In particular, Rep 78 or Rep 68 (the large Rep proteins) are believed to be involved in replication of the AAV genome, and Rep 52 and Rep 40 (the small Rep proteins) are believed to be involved in packaging of the AAV genome into a capsid. It is within the abilities of the skilled person to determine whether a given Rep protein is functional.

In an embodiment, the at least one rep gene of a first vector of a linear, synthetic DNA vector system of the invention encodes a “functional” Rep protein. If the Rep protein supports rAAV production at a level at least 25%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90% or at least 95% of the level supported by the wild type Rep protein, i.e. if the yield of rAAV vector genomes produced is at least 25%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90% or at least 95% of the yield of rAAV vector genomes produced using the reference two-plasmid system. Preferably, the at least one rep gene of the first vector is functional if it supports rAAV production at a level at least 80% of the level supported by the wild type Rep protein.

In general, a Rep protein will only be able to support rAAV production if it is compatible with the ITR(s) surrounding the genome of the AAV to be packaged. Some Rep proteins may only be able to package genomic material (such as an expression cassette) when it is flanked by ITR(s) of the same serotype as the Rep protein. Other Rep proteins are cross-compatible, meaning that they can package genomic material that is flanked by ITR(s) of a different serotype. For example, in the case of a two component synthetic DNA vector system, it is preferred that the Rep protein is able to support replication and packaging of an expression cassette comprised within the second vector, and such a Rep protein will be compatible with the at least one ITR flanking the expression cassette (i.e. able to replicate and package the expression cassette flanked on at least one side by an ITR).

Optionally, the at least one rep gene is operably linked to a p5 promoter preferably a wild type AAV promoter, more preferably a wild type AAV2 p5 promoter, wherein said p5 promoter is located either at the 5’ end or the 3’ end of a first vector of the present disclosure. Optionally, the at least one rep gene comprises a gene encoding a functional Rep 52 protein, at least one gene encoding a functional Rep 40 protein, and a gene encoding a functional Rep 68 protein.

Optionally, the at least one rep gene comprises a rep 78 cassette comprising a nucleotide sequence encoding a functional Rep 78 protein and wherein the rep 78 cassette is a modified rep 78 cassette which expresses Rep 78 at a reduced level compared to a rep 78 cassette comprising the wild type AAV rep 78 gene, preferably AAV rep 78 gene, under the control of the wild type AAV p5 promoter, preferably AAV2 p5 promoter. The second vector of a linear, synthetic DNA vector system of the invention may comprise two genes encoding a functional Rep 40 protein. In an embodiment, the at least one rep gene comprises two genes encoding a functional Rep 40 protein. For example, the second vector may comprise two rep genes that are separated on the vector. The first of the two separate rep genes could encode Rep 68 (for example using the p5 promoter or a different promoter situated near the normal position of the p5 promoter in the rep gene) and Rep 40 (for example using the p19 promoter or a different promoter situated near the normal position of the p19 promoter). The second of the two separate rep genes could encode Rep 52 and Rep 40. Rep 52 and Rep 40 are alternative splice variants.

If the second vector comprises two genes encoding a Rep 40 protein, one of the two genes that encodes a functional Rep 40 protein may comprise an intron. In one embodiment, both genes that encode a functional Rep 40 protein comprise an intron. However, in a preferred embodiment, only one of the genes that encodes a functional Rep 40 protein comprises an intron. For example, if the user wishes to avoid the at least one rep gene encoding Rep 78, the rep gene may be split through partial duplication into two genes. One gene could comprise nucleotides corresponding to the full-length native rep gene with the sequence corresponding to the intron removed. Such a gene would encode Rep 68 and Rep 40 but would not encode either Rep 78 or Rep 52, as a portion of each of the Rep 78 and Rep 52 proteins is encoded by the sequence which acts as an intron in the context of rep 40. The second gene could comprise nucleotides corresponding to the region of the native rep gene downstream of the p19 promoter, which would encode Rep 52 (intron spliced in) and Rep 40 (intron spliced out).

SEQ ID NO: 1 provides the sequence of the genome of wild type AAV2, and nucleotides 321-2252 of SEQ ID NO: 1 encode the four Rep proteins. The full length rep gene (nucleotides 321-2252) encodes all four Rep proteins (Rep 78 and Rep 68 from the p5 promoter and Rep 52 and Rep 40 from the p19 promoter). A shorter stretch of the rep gene downstream of the p19 promoter (nucleotides 993-2252) encodes Rep 52 and Rep 40 only (/.e. this stretch of the rep gene reaches from the end of the p19 promoter to the end of the gene). Nucleotides 1907-2227 of SEQ ID NO: 1 correspond to an intron. Rep 78 and Rep 52 comprise amino acids encoded by the intron, but Rep 68 and Rep 40 are alternative splice variants that do not comprise amino acids encoded by the intron.

Optionally, the at least one rep gene comprises a gene encoding a functional Rep protein having any of the nucleic acid sequences disclosed in WO 2020/208379 A, EP3722434 B1 , WO 2022/079429 A1 and EP24216316 (incorporated herein by reference).

Start codons

Start codons are present at the beginning of genes and represent the first codon of the messenger RNA transcript. The start codon effectively provides an instruction to the cell machinery to begin transcription. A variety of start codons are available. For example, a variety of start codons are used for transcription initiation in eukaryotic cells. Different start codons may be more or less efficient, i.e. transcription will initiate at different start codons at different levels. Genes that are initiated using efficient start codons will be transcribed more frequently compared to genes that are initiated using less efficient start codons. ATG is the most common start codon used in eukaryotic DNA, and other start codons will generally be less efficient than ATG at starting transcription in human cells (such as host cells that may be used to produce recombinant AAV). For example, alternative start codons include ACG, ATC, AAG, AGG, CTG, GTG, ATT, ATA, and TTG. Optionally, therefore, the start codon that is less efficient that ATG is selected from the group consisting of ACG, ATC, AAG, AGG, CTG, GTG, ATT, ATA, and TTG. Optionally, the start codon that is less efficient than ATG is an ACG start codon.

In one embodiment the rep 78 cassette may comprise an ACG start codon at the beginning of the nucleotide sequence encoding a functional Rep 78 protein

A two or three-component linear synthetic DNA system may be used in method of the invention to produce a AAV vector for use in gene therapy. A “gene therapy” involves administering AAV/viral particles of the invention that are capable of expressing a transgene (such as a Factor IX-encoding nucleotide sequence) in the host to which it is administered. In such cases, the vector plasmid will comprise an expression cassette.

Optionally, the second vector of a two -component linear, synthetic DNA vector system of the invention or the third vector of a three-component linear, synthetic DNA vector system of the invention comprises at least one ITR. Thus, optionally, the second vector or the third vector comprises at least one ITR, but, more typically, two ITRs (generally with one either end of the expression cassette, i.e. one at the 5’ end and one at the 3’ end). There may be intervening sequences between the expression cassette and one or more of the ITRs. The expression cassette may be incorporated into a viral particle located between two regular ITRs or located on either side of an ITR engineered with two D regions. Optionally, the second vector or the third vector comprises ITR sequences which are derived from AAV1 , AAV2, AAV4 and/or AAV6. Preferably the ITR sequences are AAV2 ITR sequences.

Optionally, the second vector or the third vector comprises an expression cassette. As described herein, an expression cassette refers to a sequence of nucleic acids comprising a transgene and a promoter operably linked to the transgene. Optionally, the cassette further comprises additional transcription regulatory elements, such as enhancers, introns, untranslated regions, transcriptional terminators, etc. The expression cassette should be considered to comprise the stretch of the second vector or the third vector between the ITRs that comprises any transgenes and transcription regulatory elements operably linked to the transgene. In embodiments where the second vector or the third vector comprises two ITRs, the expression cassette is considered to comprise the stretch of the vector plasmid between and including the ITRs. Optionally, the expression cassette is less than 5.0 kbp, less than 4.9 kbp, less than 4.8 kbp, less than 4.75 kbp, less than 4.7 kbp, or less than 4.5 kbp. Optionally, the expression cassette is less than 4.7kbp, less than 4.6kbp, less than 4.5kbp, less than 4.4 kbp, less than 4.3 kbp, less than 4.2 kbp, less than 4.1 kbp, less than 4.0 kbp, less than 3.9 kbp, less than 3.8 kbp, less than 3.7 kbp, less than 3.6 kbp, less than 3.5 kbp, less than 3.4 kbp, less than 3.3 kbp, less than 3.2 kbp, less than 3.1 kbp, less than 3.0 kbp, less than 2.9 kbp, less than 2.8 kbp, less than 2.7 kbp, less than 2.6 kbp, less than 2.5 kbp, less than 2.4 kbp, less than 2.3 kbp, less than 2.2 kbp, less than 2.1 kbp, less than 2.0 kbp, 1.9 kbp, less than 1.8 kbp, less than 1 .7 kbp, less than 1.6 kbp, less than 1.5 kbp, less than

1.4 kbp, less than 1 .3 kbp, less than 1.2 kbp, less than 1.1 kbp, less than 1.0 kbp, less than 0.9 kbp, less than 0.8 kbp, less than 0.7 kbp, less than 0.6 kbp, less than 0.5 kbp, less than 0.4 kbp, less than 0.3 kbp, less than 0.2 kbp, or less than 0.1 kbp. Optionally, the expression cassette is between less than 4.9kb and between less than 4.7kb. Optionally, the expression cassette is between 2.0 kbp and

5.5 kbp, between 2.0 kbp and 4.75 kbp, between 2.0 kbp and 4.7 kbp, between 2.1 kbp and 4.9 kbp, between 2.2 kbp and 4.8 kbp or between 2.3 kbp and 4.5 kbp.

Optionally, the expression cassette comprises a transcription regulatory element comprising the promoter element and/or enhancer element from HLP2, HLP1 , LP1 , HCR-hAAT, ApoE-hAAT, and/or LSP. These transcription regulatory elements are described in more detail in the following references: HLP2: WO16/075473; HLP1 : McIntosh J. et al., Blood 2013 Apr 25, 121 (17):3335-44; LP1 : Nathwani et al., Blood. 2006 April 1 , 107(7): 2653-2661 ; HCR-hAAT: Miao et al., Mol Ther. 2000;1 : 522-532; ApoE-hAAT: Okuyama et al., Human Gene Therapy, 7, 637-645 (1996); and LSP: Wang et al., Proc Natl Acad Sci U S A. 1999 March 30, 96(7): 3906-3910. Each of these transcription regulatory elements comprises a promoter, an enhancer, and optionally other nucleotides. If the polynucleotide is intended for expression in the liver, the promoter may be a liver-specific promoter. Optionally, the promoter is a human liver-specific promoter.

The transgene may be any suitable gene. If the vector comprising said transgene is for use in gene therapy, the transgene may be any gene that comprises or encodes a protein or nucleotide sequence that can be used to treat a disease. For example, the transgene may encode an enzyme, a metabolic protein, a signalling protein, an antibody, an antibody fragment, an antibody-like protein, an antigen, or a non-translated RNA such as a miRNA, siRNA, snRNA, or antisense RNA.

Optionally, the second vector comprises a cap gene and further comprises an expression cassette flanked on at least one side by an ITR.

Dispensable translation initiation codons

Optionally, said second does not comprise any dispensable translation initiation codons. Optionally, the second vector may comprise at least two genes that must be capable of being transcribed and translated (the transgene (in an expression cassette) in the two-component system and the cap gene in both two and three component system of the disclosure). Optionally, the transgene encodes a functional RNA which does not encode a translational protein or polypeptide. The transgene, if proteinencoding, and the cap gene will comprise start (ATG or GTG) codons to promote initiation of translation of the gene. However, the second vector may comprise additional instances of ATG or GTG (either inframe or out of frame with the reading frame of these genes), and it is possible that translation may initiate at one of these positions. Since there is no need for translation to be initiated at the site of these additional instances of ATG or GTG, the ATG or GTG codons may be considered to be “dispensable translation initiation codons”. It is preferred that dispensable translation initiation codons are removed or rendered non-functional, in particular where they occur in the promoter region. The promoter region is a region of the second vector that comprises one or more promoters operably linked to the cap gene. In some embodiments, the cap gene is operably linked to one or more of the p5, p19 and p40 promoters, and in such embodiments the promoter region comprises the p5, p19 and p40 promoters.

Optionally, the second vector comprises a promoter region comprising one or more promoters, and the promoter region does not comprise ATG or GTG codons. Optionally, the promoter region comprises p5, p19 and/or p40 promoters, and wherein ATG or GTG codons at one or more positions corresponding to positions (a) 321-323 (Rep78/68 ATG start codon), (b) 766-768 (ATG codon), (c) 955- 957 (ATG codon), (d) 993-995 (Rep52/40 ATG start codon) and (e) 1014-1016 (GTG codon) of SEQ ID NO: 1 are absent or mutated.

Optionally, in the promoter region:

(a) nucleotides corresponding to nucleotides 321-323 of SEQ ID NO: 1 are absent;

(b) nucleotides corresponding to nucleotides 766-768 of SEQ ID NO: 1 are not ATG and are optionally

ATT;

(c) nucleotides corresponding to nucleotides 955-957 of SEQ ID NO: 1 are absent;

(d) nucleotides corresponding to nucleotides 993-995 of SEQ ID NO: 1 are absent; and/or

(e) nucleotides corresponding to nucleotides 1014-1016 of SEQ ID NO: 1 are absent.

Optionally, in the promoter region:

ATT;

(d) nucleotides corresponding to nucleotides 993-995 of SEQ ID NO: 1 are absent; and

Host cell line

The present invention provides a host cell line.

The host cell line is preferably a host cell line that can be used to produce rAAV. The host cell line may therefore be a host cell line suitable for the production of rAAV. In addition, the host cell line may be for the production of rAAV.

In general, a host cell line that is suitable for the production of rAAV is a host cell line that is derived from a eukaryotic cell line, preferably a vertebrate cell line, preferably a mammalian cell line, preferably a human cell line. Optionally, the host cell is a cell selected from the group consisting of a HEK293T cell, a HEK293 cell, a HEK293EBNA cell, a HEK293F cell, aHEK293S cell, a CAP cell, a CAP-T cell, an AGE1 .CR cell, a PerC6 cell, a C139 cell, an EB66 cell, a BHK cell, a COS cell, a Vero cell, a HeLa cell, and an A549 cell. Preferably, the host cell is selected from the group consisting of a HEK293T cell, a HEK293 cell, a HEK293EBNA cell, a CAP cell, a CAP-T cell, an AGE1 .CR cell, a PerC6 cell, a C139 cell, and an EB66 cell. Even more preferably, the host cell is selected from the group consisting of a HEK293T cell, a HEK293 cell, and a HEK293EBNA cell. For example, the host cell may be a HEK293T cell or a HEK293 cell. Optionally, the host cell is a cell that expresses a functional adenoviral E1A/B protein.

It is within the abilities of the skilled person to determine whether a host cell is suitable for the production of rAAV. The host cell will be considered to be suitable for the production of recombinant AAV if it supports AAV production at a level at least 30%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90%, or at least 95% of the level supported by HEK293T cells, i.e. if the yield of recombinant AAV produced is at least 30%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90%, or at least 95% of the yield of recombinant AAV produced in HEK293T cells.

In the context of the present disclosure, the term “host cell” and “host cell line” are used interchangeably.

High (vector genome) yield

The term “yield’ refers to the amount of rAAV particles that are prepared in the methods or uses of the invention. The “yield’ may be expressed as the number of vector genomes (vg) per ml of medium, as measured on bulk product prior to harvesting, purifying and/or concentrating the rAAV preparation.

The yield of rAAV (such as rAAV particles) may be determined by using qPCR to quantify the number of nucleic acid sequences comprising a cassette comprising a promoter sequence (vg).

As used herein the term “maximising” the yield refers to aiming for the highest possible yield within the limitations of the methods of the invention. As used herein the term “optimising” refers to increasing the yield, but aiming for a desired yield which may not be the maximum possible yield if, for example, the user is keen to ensure a high ratio of full to total particles (i.e. minimising the proportion of empty particles).

Increased viral genome titre

The methods of the disclosure may result in a preparation that comprises recombinant AAV at an improved, increased or higher viral genome titre. Viral genome titre is the concentration of viral genome particles present in a preparation. If the preparation comprises rAAV, the viral genome particles will be AAV viral genome particles. Viral genome titre can be used as a measure of the yield of rAAV.

Viral genome titre assay

The skilled person will understand that there are many suitable methods for determining the viral genome titre of a preparation comprising recombinant AAV. Viral genome titre may be measured using digital droplet polymerase chain reaction (ddPCR). Viral genome titre may be measured by the quantitative polymerase chain reaction (qPCR). qPCR or ddPCR may be carried out with primers specific to the viral genome. For example, if the viral genome is the AAV genome, primers specific to the AAV genome will be used. Viral genome titre may be measured using photometric quantification. The AAV viral genome assay may be based on a quantitative polymerase chain reaction (qPCR) specific for the promoter sequence of the rAAV expression cassette. In principle, the qPCR primers can be designed to bind any part of the recombinant AAV genome which is not common to wild type AAV genomes, but it is recommended against using primer template sequences very close to the ITRs as doing so can lead to an exaggerated vector genome titre measurement. Suitably, qPCR is carried out using a pair of primers that are able to amplify at least a region of the promoter of the expression cassette. Optionally, at least one of the primers is specific for (reverse and complementary to or identical to depending on whether the primer is a forward primer or a reverse primer) a region of at least 12, at least 14, at least 16, or at least 18 nucleotides of the promoter of the expression cassette. Optionally, one primer is specific for the start of the promoter (the first at least 12 nucleotides of the promoter) and the other primer is specific for a region of the expression cassette that is 150 base pairs from the binding site of the first primer. The qPCR may be performed using SYBR green or another intercalating dye that can be used for detection of the amplification product. Alternatively, the qPCR product may be detected using TaqmanTM assay or similar.

Cell lysate test samples may be subjected to a nuclease treatment procedure in order to remove nonpacked vector genomes prior to performing qPCR or ddPCR.

“Droplet digital PCR” (ddPCR) refers to a digital PCR assay that measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions that support PCR amplification (i.e., a plurality of such compartments). Typically, a “droplet” refers to water-in-oil droplet (i.e., an oil droplet that may be generated by emulsifying a sample with droplet generator oil); an individual partition of the droplet digital PCR assay. A droplet supports PCR amplification of template molecule(s) using homogenous assay chemistries and workflows similar to those widely used for real-time PCR applications (Hinson et al (2011) Anal. Chem. 83:8604-8610; Pinheiro et al (2012) Anal. Chem. 84:1003-1011). A single ddPCR reaction may typically be comprised of at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 12,000, at least 14,000, at least 16,000, at least 18,000 or at least 20,000 compartments.

Droplet digital PCR may be performed using any platform that performs a digital PCR assay that measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions that support PCR amplification. The strategy for droplet digital PCR may be summarized as follows: a sample is diluted and partitioned into thousands to millions of separate reaction chambers (water-in-oil droplets) so that each contains one or no copies of the nucleic acid molecule of interest. The number of positive droplets detected, which contain the target amplicon (i.e., nucleic acid molecule of interest), versus the number of negative droplets, which do not contain the target amplicon (i.e., nucleic acid molecule of interest), may be used to determine the number of copies of the nucleic acid molecule of interest that were in the original sample. Examples of droplet digital PCR systems include the QX100™ Droplet Digital PCR System by Bio-Rad, which partitions samples containing nucleic acid template into 20,000 nanolitre-sized droplets.

Droplet digital PCR may thus be used to detect a single target in a sample, for example using a single primer pair. However, ddPCR may also be used to detect two different targets in a sample, for example using two primer pairs, each primer pair hybridising to a different target, i.e., duplexing of targets. Duplexing may be extended to look at more targets in a sample, such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or at least 12 different targets in a sample, i.e., multiplexing of targets. Duplexing and multiplexing with ddPCR allows for improved sensitivity and precision, increased low level detection, and also the inference of the size of a nucleic acid. ddPCR may be quantitative.

The skilled person will understand that there are many suitable methods for determining the capsid titre of a preparation comprising recombinant AAV. Capsid titre may be measured by an enzyme-linked immunosorbent assay (ELISA). For example, the capsid-specific ELISA may comprise exposing the rAAV preparation to an antibody that binds to the capsid protein. If, for example, the vector plasmid comprises a cap gene that encodes a capsid from an AAV S3 serotype, the antibody may be an antibody that binds to the AAV S3 capsid. For example, the user may coat a plate with an antibody that is specific for the capsid. The user may then pass the rAAV preparation over the surface of the plate. The capsids will bind to the antibody and be immobilised on the plate. The plate may then be washed to remove contaminants. The amount of capsids present can then be detected by addition of a detection antibody that can bind to the capsid and is conjugated to a detection agent such as streptavidin peroxidase. The amount of capsids present will be proportional to the colour change obtained when the streptavidin peroxidase is exposed to the chromogenic substrate TMB (tetramethylbenzidine). In one aspect of the present invention, the capsid titre is measured by ELISA.

Nucleic acid impurities

The term “nucleic acid impurities” refers to genetic material that has been packaged into rAAV and which was not intended to be packaged. For example, if the second vector or the third vector comprises two ITRs, the genetic material which was intended to be packaged into the rAAV is any genetic material between the two ITRs of said vector, i.e. the “nucleic acid impurities” is any genetic material that is not the genetic material between the two ITRs. The level of nucleic acid impurities may be as measured on bulk product prior to harvesting, purifying and/or concentrating the rAAV preparation, or may be measured after the rAAV have been harvested, purified and/or concentrated.

The level of nucleic acid impurities may be measured using a similar method to the qPCR method for quantifying the number of vector genomes but using qPCR primers specific for the nucleic acid impurities of interest. This qPCR method will provide the copy number of the nucleic acid impurities of interest per ml. The level of nucleic acid impurities may be expressed as percentage of the copy number of vector genomes per ml (as determined using the qPCR method for quantifying the number of vector genomes), i.e. the percentage/level of recombinant AAV comprising nucleic acid impurities may be expressed as the copy number of nucleic acid impurities of interest per ml / the copy number of vector genomes (vg) per ml x 100. When the level of nucleic acid impurities is expressed as a percentage of the copy number of vector genomes, it will be considered to be normalised to vector genome level. In the context of the present disclosure the term “any undesirable DNA sequences” comprises nucleic acid impurities or any other DNA sequence that may contaminate or inadvertently affect the purity of a final product during AAV production.

“Full” particles are rAAV particles comprising both a capsid and the intended vector genome, or at least a partial such genome as determined using the qPCR method described below. “Empty” particles (i.e. particles which are not full) comprise capsids but do not comprise a genome or comprise only a partial genome (thereby not forming a complete rAAV particle) which is not detected using the qPCR method. However, the rAAV preparations may comprise both full particles and empty particles. Generally, a low or minimised proportion of empty particles is desired. For example, if the rAAV are to be used in gene therapy, any empty particles will not comprise the entire expression cassette of interest and so will not be effective in therapy. On the other hand, there are circumstances where the presence of empty particles could be desirable. In some instances, and in some patient groups, it may be the case that the empty particles behave as “decoys” to reduce the immune response in a patient to the administered rAAV particles (WO2013/078400).

The ratio of full to total particles may be expressed herein as the percentage of the total number of particles (capsids) that notionally comprise a vector genome or at least a partial such genome (assuming one (partial) genome per capsid) as determined using the following qPCR assay. qPCR is carried out using a pair of primers that are able to amplify at least a region of the promoter of the expression cassette. Optionally, at least one of the primers is specific for (reverse and complementary to or identical to depending on whether the primer is a forward primer or a reverse primer) a region of at least 12, at least 14, at least 16, or at least 18 nucleotides ofthe promoter of the expression cassette. Optionally, one primer is specific for the start of the promoter (the first at least 12 nucleotides of the promoter) and the other primer is specific for a region of the expression cassette that is 150 base pairs from the binding site of the first primer.

The ratio of full to total particles (as measured as a percentage of the total number of particles that are full particles) may be determined using qPCR to determine the number of vector genomes (as discussed in the previous paragraph) and using a capsid-specific immunoassay to measure the total number of particles. For example, the capsid-specific ELISA may comprise exposing the rAAV preparation to an antibody that binds to the capsid protein. If, for example, the vector plasmid comprises a cap gene that encodes a capsid from an AAV3 serotype, the antibody may be an antibody that binds to the AAV3 capsid. For example, the user may coat a plate with an antibody that is specific for the capsid. The user may then pass the rAAV preparation over the surface ofthe plate. The particles will bind to the antibody and be immobilised on the plate. The plate may then be washed to remove contaminants. The amount of particle present can then be detected by addition of a detection antibody that can bind to the capsid and is conjugated to a detection agent such as streptavidin peroxidase. The amount of particle present will be proportional to the colour change obtained when the streptavidin peroxidase is exposed to the chromogenic substrate TMB (tetramethylbenzidine). Optionally, the desired ratio of full to total particles (expressed as a percentage of the total number of particles that notionally comprise a vector genome) is at least 2.5%, at least 5%, at least 7.5%, at least 8%, between 2.5% and 10%, between 5% and 9%, between 7.5% and 9%, or between 8% and 9% (as measured on bulk product prior to harvesting, purifying and/or concentrating the rAAV preparation). In many cases, increasing the full to total particle ratio is advantageous, and the present inventors have determined that aiming for a full to total particle ratio that is at least 2.5%, at least 5%, at least 7.5%, at least 8%, between 2.5% and 10%, between 5% and 9%, between 7.5% and 9%, or between 8% and 9% achieves a good balance between maintaining a high full to total particle ratio, whilst also achieving a good yield. Optionally, the desired ratio of full to total particles is a ratio of full to total particles that is at least 20% or at least 30% of the ratio of full to total particles achieved using an equivalent method.

Transfecting, culturing and harvesting

The methods of the invention may comprise steps of obtaining the two or three component linear, synthetic DNA vector system of the invention, transfecting a host cell with said system, and culturing the host cell under conditions suitable for recombinant AAV production.

Transfecting a two or three component linear, synthetic DNA vector system, may comprise exposing the host cell to said system in conditions suitable for transfection. For example, the user of the method may add a transfection agent (addition of a transfection agent would be considered to be a condition suitable for transfection). Alternatively, calcium phosphate transfection, electroporation or cationic liposomes could be used.

Culturing the host cell under conditions suitable for rAAV production refers to culturing the host cell under conditions at which it can grow and AAV can replicate. For example, the host cell may be cultured at a temperature between 32°C and 40°C, between 34°C and 38°C, between 35°C and 38°C, or around 37°C. Optionally, the host cell may be cultured in the presence of a complete cell culture medium such as BalanCD HEK293 medium supplemented with 4 mM L-Glutamine. A complete cell culture medium is a medium that provides all the essential nutrients required for growth of the host cell. Optionally, the complete cell culture medium is supplemented with serum, such as fetal bovine serum or bovine serum albumin.

Optionally, the host cell is a host cell such as those defined above under the heading “host celt’. Optionally, the host cell is selected from the group consisting of a HEK293T cell, a HEK293 cell, a HEK293EBNA cell, a CAP cell, a CAP-T cell, an AGE1 .OR cell, a PerC6 cell, a C139 cell, and an EB66 cell. Optionally, the host cell is a cell that expresses a functional E1A/B protein.

The two or three component linear, synthetic DNA vector system may be used to produce rAAV in any suitable cell culture system. Optionally, culturing the host cell under conditions suitable for rAAV production comprises culturing the host cell using a suspension or an adherent system. Optionally, culturing the host cell under conditions suitable for rAAV production comprise culturing the host cell using a suspension system.

For the purposes of the present invention, the term “suspension system” refers to a system suitable for suspension cell culture, i.e. a system which allows cells to grow free-floating in culture medium. Cells in a suspension system may form aggregates or may be suspended in medium as single cells. For the purposes of the present invention, the term “adherent system” refers to a system suitable for adherent cell culture, i.e. for cells to be cultured whilst anchored to a substrate. Optionally, an adherent system refers to a flask or fermenter which forms a container to which cells can bind, and optionally is specifically treated to allow cell adhesion and spreading. Alternatively, an adherent system may be a “carrier system”, in which the container contains an additional carrier such as a bead or a fibre to which the cells can adhere. In such “carrier¹’ adherent systems, the cells tend to adhere less tightly and to have a morphology that is more similar to the morphology of cells grown using suspension systems compared to cells grown in conventional adherent systems.

The method may further comprise a step of purifying the rAAV. In general, a step of purifying the rAAV will involve increasing the concentration of the rAAV compared to other components of the preparation. Optionally, the step of purifying the rAAV results in a concentrated rAAV preparation. Optionally, the step of purifying the rAAV results in an isolated rAAV.

Any suitable purification method may be used. Optionally, the step of purifying the rAAV comprises a technique selected from the group consisting of gradient density centrifugation (such as CsCI or lodixanol gradient density centrifugation), filtration, ion exchange chromatography, size exclusion chromatography, affinity chromatography and hydrophobic interaction chromatography. Optionally, the step of purifying the rAAV is carried out using a technique selected from the group consisting of gradient density centrifugation (such as CsCI or lodixanol gradient density centrifugation), filtration, ion exchange chromatography, size exclusion chromatography, affinity chromatography and hydrophobic interaction chromatography.

Optionally, the method comprises further concentrating the rAAV using ultracentrifugation, tangential flow filtration, or gel filtration.

Optionally, the method comprises formulating the rAAV with a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipients may comprise carriers, diluents and/or other medicinal agents, pharmaceutical agents or adjuvants, etc. Optionally, the pharmaceutically acceptable excipients comprise saline solution. Optionally, the pharmaceutically acceptable excipients comprise human serum albumin. Based on the disclosure provided herein those skilled in the art will recognise or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention. Specifically, the present disclosure provides the following aspects, advantageous features and specific embodiments respectively alone or in combination.

Thus, in one aspect the present disclosure relates to a linear synthetic DNA vector system comprising at least a first linear synthetic DNA vector and a second linear synthetic DNA vector, wherein said first vector comprises at least one rep gene encoding at least one functional Rep protein and does not comprise a cap gene encoding a functional set of Cap proteins.

In one embodiment said first vector comprises a nucleic acid sequence having at least, 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with any one of SEQ ID NOs: 2, 6, 7, 8,10, 1 1 , 12.

In one embodiment said first vector comprises a nucleic acid having the sequence of any one of SEQ ID NO: 2, 6, 7, 8, 10, 11 , 12.

In one embodiment said first vector comprises a nucleic acid sequence having at least 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 2. In one embodiment said first vector comprises a nucleic acid having the sequence of SEQ ID NO: 2

In one embodiment said first vector comprises a nucleic acid sequence having at least 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 6 In one embodiment said first vector comprises a nucleic acid having the sequence of SEQ ID NO: 6

In one embodiment said first vector comprises a nucleic acid sequence having at least 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 7 In one embodiment said first vector comprises a nucleic acid having the sequence of SEQ ID NO: 7

In one embodiment said first vector comprises a nucleic acid sequence having at least 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 8 In one embodiment said first vector comprises a nucleic acid having the sequence of SEQ ID NO: 8 In one embodiment said first vector comprises a nucleic acid sequence having at least 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 10. In one embodiment said first vector comprises or consists of a nucleic acid having the sequence of SEQ ID NO: 10.

In one embodiment said first vector comprises a nucleic acid sequence having at least 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 11. In one embodiment said first vector comprises or consists of a nucleic acid having the sequence of SEQ ID NO: 11.

In one embodiment said first vector comprises a nucleic acid sequence having at least 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 12. In one embodiment said first vector comprises or consists of a nucleic acid having the sequence of SEQ ID NO: 12.

In a further embodiment said second vector comprises:

(a) a cap gene encoding at least one functional Cap protein; or

(b) at least one promoter driving a cap gene expression, preferably a cap gene promoter, a cloning site operably linked to said promoter; wherein said second vector does not comprise a rep gene encoding a functional Rep protein and the expression cassette comprises a transgene operably linked to at least one regulatory control element. In one embodiment said second vector comprises:

(a) a cap gene encoding at least one functional Cap protein; or

(b) at least one promoter driving a cap gene expression, preferably a cap gene promoter, a cloning site operably linked to said promoter, and an expression cassette flanked on at least one side by an ITR; wherein said second vector does not comprise a rep gene encoding a functional Rep protein and the expression cassette comprises a transgene operably linked to at least one regulatory control element. In one embodiment said second vector comprises a nucleic acid sequence having at least 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with any one of SEQ ID NOs: 3, 4, 13, 14, 15, 16. In one embodiment said second vector comprises a nucleic acid sequence having at least 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 3. In one embodiment said second vector comprises a nucleic acid having the sequence of SEQ ID NO:

3.

In one embodiment said second vector comprises a nucleic acid sequence having at least 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 4. In one embodiment said second vector comprises a nucleic acid having the sequence of SEQ ID NO:

4.

In one embodiment said second vector comprises a nucleic acid sequence having at least 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 13. In one embodiment said second vector comprises of a nucleic acid having the sequence of SEQ ID NO:

13.

In one embodiment said second vector comprises a nucleic acid sequence having at least 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 14. In one embodiment said second vector comprises a nucleic acid having the sequence of SEQ ID NO:

14.

In one embodiment said second vector comprises a nucleic acid sequence having at least 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 15. In one embodiment said second vector comprises of a nucleic acid having the sequence of SEQ ID NO:

15.

In one embodiment said second vector comprises a nucleic acid sequence having at least 80% at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, a least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with SEQ ID NO: 16. In one embodiment said second vector comprises of a nucleic acid having the sequence of SEQ ID NO:

16. In one embodiment said linear synthetic DNA vector system comprises a first vector comprising a nucleic acid sequence of any one of SEQ ID NOs: 2, 6, 7, 8, and a second vector comprising a nucleic acid sequence of any one of SEQ ID NOs: 3, 4.

In one embodiment said linear synthetic DNA vector system consists of a first vector comprising a nucleic acid sequence of any one of SEQ ID NOs: 2, 6, 7, 8, and a second vector comprising a nucleic acid sequence of any one of SEQ ID NOs: 3, 4.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising a nucleic acid sequence of SEQ ID NO: 2, and a second vector comprising a nucleic acid sequence of SEQ ID NO: 3.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising a nucleic acid sequence of SEQ ID NO: 6, and a second vector comprising a nucleic acid sequence of SEQ ID NO: 3.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising a nucleic acid sequence of SEQ ID NO: 7, and a second vector comprising a nucleic acid sequence of SEQ ID NO: 3.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising a nucleic acid sequence of SEQ ID NO: 8, and a second vector comprising a nucleic acid sequence of SEQ ID NO: 3.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of any one of SEQ ID NOs: 10, 11 , 12, and a second vector comprising the nucleic acid sequence of any one of SEQ ID NOs: 13, 14.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 10, and a second vector comprising the nucleic acid sequence of SEQ ID NO: 13.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 11 , and a second vector comprising the nucleic acid sequence of SEQ ID NO: 13.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 12, and a second vector comprising the nucleic acid sequence of SEQ ID NO: 13.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 10, and a second vector comprising the nucleic acid sequence of SEQ ID NO: 14.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 11 , and a second vector comprising the nucleic acid sequence of SEQ ID NO: 14.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 12, and a second vector comprising the nucleic acid sequence of SEQ ID NO: 14. In an even further embodiment said second vector comprises:

(a) a cap gene encoding at least one functional Cap protein; or

(b) at least one promoter driving a cap gene expression, preferably a cap gene promoter, a cloning site operably linked to said promoter, and does not comprise a rep gene encoding a functional Rep protein. In one embodiment said second vector comprises:

(a) a cap gene encoding at least one functional Cap protein; or

(b) at least one promoter driving a cap gene expression, preferably a cap gene promoter, a cloning site operably linked to said promoter, and does not comprise a rep gene encoding a functional Rep protein; and wherein said system further comprises a third linear synthetic DNA vector comprising an expression cassette flanked on at least one side by an ITR and said expression cassette comprises a transgene operably linked to at least one regulatory control element.

In one embodiment said linear synthetic DNA vector system comprises a first vector, wherein said first vector comprises at least one rep gene encoding at least one functional Rep protein and does not comprise a cap gene encoding a functional set of Cap proteins, a second vector, wherein said second vector comprises:

(a) a cap gene encoding at least one functional Cap protein; or

(b) at least one promoter driving a cap gene expression, preferably a cap gene promoter, a cloning site operably linked to said promoter, and does not comprise a rep gene encoding a functional Rep protein; and a third vector, wherein said third vector comprises an expression cassette flanked on at least one side by an ITR and said expression cassette comprises a transgene operably linked to at least one regulatory control element.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising a nucleic acid sequence derivable from any one of SEQ ID NOs: 2, 6, 7, 8, a second vector comprising a nucleic acid sequence derivable from SEQ ID NO: 4, and a third vector comprising a nucleic acid sequence derivable from SEQ ID NOs: 5.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising a nucleic acid sequence of any one of SEQ ID NOs: 2, 6, 7, 8, a second vector comprising a nucleic acid sequence of SEQ ID NO: 4, and a third vector comprising a nucleic acid sequence of SEQ ID NOs: 5.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising a nucleic acid sequence of SEQ ID NO: 2, a second vector comprising a nucleic acid sequence of SEQ ID NO: 4, and a third vector comprising a nucleic acid sequence of SEQ ID NOs: 5.

In one embodiment said linear synthetic DNA vector system consists of a first vector comprising a nucleic acid sequence of SEQ ID NO: 2, a second vector comprising a nucleic acid sequence of SEQ ID NO: 4, and a third vector comprising a nucleic acid sequence of SEQ ID NOs: 5. In one embodiment said linear synthetic DNA vector system comprises a first vector comprising a nucleic acid sequence of SEQ ID NO: 6, a second vector comprising a nucleic acid sequence of SEQ ID NO: 4, and a third vector comprising a nucleic acid sequence of SEQ ID NOs: 5.

In one embodiment said linear synthetic DNA vector system consists of a first vector comprising a nucleic acid sequence of SEQ ID NO: 6, a second vector comprising a nucleic acid sequence of SEQ ID NO: 4, and a third vector comprising a nucleic acid sequence of SEQ ID NOs: 5.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising a nucleic acid sequence of SEQ ID NO: 7, a second vector comprising a nucleic acid sequence of SEQ ID NO: 4, and a third vector comprising a nucleic acid sequence of SEQ ID NOs: 5.

In one embodiment said linear synthetic DNA vector system consists of a first vector comprising a nucleic acid sequence of SEQ ID NO: 7, a second vector comprising a nucleic acid sequence of SEQ ID NO: 4, and a third vector comprising a nucleic acid sequence of SEQ ID NOs: 5.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising a nucleic acid sequence of SEQ ID NO: 8, a second vector comprising a nucleic acid sequence of SEQ ID NO: 4, and a third vector comprising a nucleic acid sequence of SEQ ID NOs: 5.

In one embodiment said linear synthetic DNA vector system consists of a first vector comprising a nucleic acid sequence of SEQ ID NO: 8, a second vector comprising a nucleic acid sequence of SEQ ID NO: 4, and a third vector comprising a nucleic acid sequence of SEQ ID NOs: 5.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of any one of SEQ ID NOs: 10, 11 , 12, and at least a second vector comprising a nucleic acid sequence of any one of SEQ ID NOs: 15, 16.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 10, and at least a second vector comprising the nucleic acid sequence of SEQ ID NOs: 15.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 11 , and at least a second vector comprising the nucleic acid sequence of SEQ ID NOs: 15.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 12, and at least a second vector comprising the nucleic acid sequence of SEQ ID NOs: 15.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 10, and at least a second vector comprising the nucleic acid sequence of SEQ ID NOs: 16. In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 11 , and at least a second vector comprising the nucleic acid sequence of SEQ ID NOs: 16.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 12, and at least a second vector comprising the nucleic acid sequence of SEQ ID NOs: 16.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of any one of SEQ ID NOs: 10, 11 , 12, a second vector comprising the nucleic acid sequence of any one of SEQ ID NO: 15,16, and a third vector comprising the nucleic acid sequence of SEQ ID NOs: 17.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 10, a second vector comprising the nucleic acid sequence of any one of SEQ ID NO: 15, and a third vector comprising the nucleic acid sequence of SEQ ID NOs: 17.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 11 , a second vector comprising the nucleic acid sequence of any one of SEQ ID NO: 15, and a third vector comprising the nucleic acid sequence of SEQ ID NOs: 17.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 12, a second vector comprising the nucleic acid sequence of any one of SEQ ID NO: 15, and a third vector comprising the nucleic acid sequence of SEQ ID NOs: 17.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 10, a second vector comprising the nucleic acid sequence of any one of SEQ ID NO: 16, and a third vector comprising the nucleic acid sequence of SEQ ID NOs: 17.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 11 , a second vector comprising the nucleic acid sequence of any one of SEQ ID NO: 16, and a third vector comprising the nucleic acid sequence of SEQ ID NOs: 17.

In one embodiment said linear synthetic DNA vector system comprises a first vector comprising the nucleic acid sequence of SEQ ID NO: 12, a second vector comprising the nucleic acid sequence of any one of SEQ ID NO: 16, and a third vector comprising the nucleic acid sequence of SEQ ID NOs: 17.

In one embodiment said at least one rep gene encodes a functional Rep52 and/or Rep 40 protein and at least one gene encoding a functional Rep78 and/or Rep68 protein. In one embodiment said first vector comprises at least one helper virus gene, optionally wherein:

(ii) the at least one helper virus gene comprises a VA nucleic acid encoding functional VA RNA preferably VA RNA I and II, an E2A gene encoding a functional E2A protein, and an E4 gene encoding a functional E4 protein or a gene encoding functional 22K/33K proteins.

In one embodiment said second vector comprises a cap gene and the cap gene encodes a Cap protein selected from any naturally occurring or genetically engineered AAV serotypes.

In one embodiment of said synthetic DNA vector system

In another aspect the present disclosure relates to a use of said linear synthetic DNA vector system for producing a AAV preparation:

(a) having a desired ratio of full to total particles; and/or

(b) at a high or desired yield.

(c) having reduced or diminished undesirable DNA sequences.

In another aspect the present invention relates to a use of the linear synthetic DNA vector system for:

(b) increasing, optimising or maximising the yield of recombinant AAV produced during recombinant AAV production.

(c) reducing or diminishing any undesirable DNA sequence.

In one embodiment said use comprises transfecting a host cell with a vector system of the invention and culturing the host cell under conditions suitable for recombinant AAV production.

In another aspect the disclosure relates to a method for controlling or maximising the ratio of full to total particles produced during recombinant AAV production comprising:

-obtaining the linear synthetic DNA vector system of the invention -transfecting a host cell with said system

-culturing the host cell under conditions suitable for recombinant AAV production

In another aspect the disclosure relates to a method for increasing, optimising or maximising the yield of recombinant AAV produced during recombinant AAV production comprising: - obtaining the linear synthetic DNA vector system of the invention

-transfecting a host cell with said system

-culturing the host cell under conditions suitable for recombinant AAV production.

In another aspect the disclosure relates to a method of reducing or diminishing the presence of any undesirable DNA sequences during recombinant AAV production comprising:

- obtaining the linear synthetic DNA vector system of the disclosure;

-transfecting a host cell with said system

Numbered embodiments (E) of the disclosure

E1. A two or three component linear, synthetic DNA system for adeno-associated virus (AAV) production comprising a first vector and at least a second vector, wherein the first vector comprises at least one rep gene encoding at least one functional Rep protein and does not comprise a cap gene encoding a functional set of Cap proteins.

E2. The system of E1 comprising a first vector and at least a second vector, wherein the first vector further comprises at least one helper virus gene and does not comprise a cap gene encoding a functional set of Cap proteins, said first vector comprising the sequence of any one of SEQ ID NOs: 2, 6, 7, 8, 10, 11 , 12.

E3. The system of E1 or E2, wherein the first vector comprises at least one rep gene encoding at least one functional Rep protein.

E4. The system of any one of E1-E3, wherein said system comprises a molar excess of second vector compared to first vector plasmid or a molar excess of first vector compared to second vector plasmid.

E5. A first vector for a two or three component linear, synthetic DNA system comprising at least one rep gene encoding at least one functional Rep protein and at least one helper virus gene, and which does not comprise a cap gene encoding a functional set of Cap proteins.

E6. The first vector of E5 for a two or three component linear, synthetic DNA system further comprising at least one helper virus gene and which does not comprise a cap gene encoding a functional set of Cap proteins, wherein said first vector comprises any one of SEQ ID NOs: 2, 6, 7, 8, 10, 11 , 12.

E7. The vector of E6 wherein said vector comprises at least one rep gene encoding at least one functional Rep protein. E8. A second vector for a two-component linear, synthetic DNA system comprising:

(a) a cap gene encoding at least one functional Cap protein; or

(b) at least one promoter driving a cap gene expression, preferably a cap gene promoter, a cloning site operably linked to said promoter, and an expression cassette flanked on at least one side by an ITR; wherein the vector does not comprise a rep gene encoding a functional Rep protein and the expression cassette comprises a transgene operably linked to at least one regulatory control element.

E9. The system of any one of E1-E8, wherein the second vector comprises:

(a) a cap gene encoding at least one functional Cap protein; or

E10. A second vector for a three-component linear, synthetic DNA system comprising:

(a) a cap gene encoding at least one functional Cap protein; or

(b) at least one promoter driving a cap gene expression, preferably a cap gene promoter, a cloning site operably linked to said promoter, wherein the second vector does not comprise a rep gene encoding a functional Rep protein.

E11. The system of any one of E1-E4, wherein the second vector comprises:

(a) a cap gene encoding at least one functional Cap protein; or

(b) at least one promoter driving a cap gene expression, preferably a cap gene promoter, a cloning site operably linked to said promoter, wherein the second vector does not comprise a rep gene encoding a functional Rep protein; and wherein the system comprises a third vector comprising an expression cassette flanked on at least one side by an ITR and the expression cassette comprises a transgene operably linked to at least one regulatory control element.

E12. The system or first vector of any one of E1-E11 , wherein the at least one rep gene comprises a gene encoding:

(a) a functional Rep 52 protein;

(b) a functional Rep 40 protein; and/or

(c) a functional Rep 68 protein.

E13. The system or first vector of any one of E1-E12, wherein the at least one rep gene encodes a functional Rep52 and/or Rep 40 protein and at least one gene encoding a functional Rep78 and/or Rep68 protein. E14. The system or first vector of any one of E1-E13, wherein the first vector comprises two genes encoding a functional Rep 40 protein.

E15. The system or first vector of any one of E1-E14, wherein only one of the two genes encoding a functional Rep 40 protein comprises an intron.

E16. The system or first vector of any one of E1-E15, wherein the gene encoding a functional Rep 52 protein comprises a nucleic acid sequence having at least 95%, at least 98%, at least 99%, or 100% identity to the full length or a fragment of at least 800, at least 900, at least 1000, or at least 1 100 nucleotides in length of nucleotides 993-2186 of SEQ ID NO: 1 , or to a corresponding stretch of nucleotides in a different serotype of AAV.

E17. The system or first vector of any one of E1-E16, wherein the at least one gene encoding a functional Rep 40 protein comprises a nucleic acid sequence having at least 95%, at least 98%, at least 99%, or 100% identity to the full length or to a fragment of at least 600, at least 700, at least 800, or at least 900 nucleotides in length of a stretch of nucleotides corresponding to nucleotides 993-2252 minus nucleotides 1907-2227 of SEQ ID NO: 1 , or to corresponding stretches of nucleotides in a different serotype of AAV.

E18. The system or first vector of any one of E1-E17 wherein the at least one gene encoding a functional Rep 40 protein comprises a nucleic acid sequence having at least 95%, at least 98%, at least 99%, or 100% identity to the full length or to a fragment of at least 900, at least 1000, at least 1100, or at least 1200 nucleotides in length of nucleotides 993-2252 of SEQ ID NO: 1 , or to a corresponding stretch of nucleotides in a different serotype of AAV.

E19. The system or first vector of any one of E1-E18, wherein the gene encoding a functional Rep 68 protein comprises a nucleic acid sequence having at least 95%, at least 98%, at least 99%, or 100% identity to the full length or to a fragment of at least 1000, at least 1400, at least 1500, or at least 1600 nucleotides in length of a stretch of nucleotides corresponding to nucleotides 321-2252 minus nucleotides 1907-2227 of SEQ ID NO: 1 , or to corresponding stretches of nucleotides in a different serotype of AAV.

E20. The system or first vector of any one of E1-E19, wherein the first vector does not comprise a gene encoding a functional Rep 78 protein.

E21. The system or first vector of any one of E1-E20, wherein the first vector does not comprise a contiguous sequence of at least 1700, at least 1800, or 1866 nucleotides corresponding to a contiguous stretch of nucleotides of equivalent length comprised within nucleotides 321-2186 of SEQ ID NO: 1 or within a corresponding stretch of nucleotides in a different serotype of AAV.

E22. The system or first vector of any one of E1-E21 , wherein the at least one rep gene does not comprise a functional internal p40 promoter.

E23. The system or first vector of any one of E1-E22, wherein the at least one rep gene does not comprise a T nucleotide at a position corresponding to position 1823 of SEQ ID NO: 1.

E24. The system or first vector of any one of E1 -E23, wherein the at least one rep gene comprises a C nucleotide at a position corresponding to position 1823 of SEQ ID NO: 1.

E25. The system or first vector of any one of E1-E24, wherein the at least one rep gene does not comprise AAG at positions corresponding to positions 1826-1828 of SEQ ID NO: 1.

E26. The system or first vector of any one of E1-E25, wherein the at least one rep gene comprises CTC at positions corresponding to positions 1826-1828 of SEQ ID NO: 1.

E27. The system or first vector of any one of E1-E26, wherein the first vector does not comprise a contiguous stretch of exclusively cap gene sequence of more than 250 nucleotides, more than 100 nucleotides, or more than 60 nucleotides.

E28. The system or first vector of any one of E1-E27, wherein the first vector does not comprise a contiguous stretch of exclusively cap gene sequence of more than 60 nucleotides.

E29. The system or first vector of any one of E1 -E28, wherein the first vector comprises a portion of cap gene sequence, and the portion of cap gene sequence does not encode a functional set of Cap proteins.

E30. The system or first vector of any one of E1 -E29, wherein the first vector comprises at least one helper virus gene.

E31. The system or first vector of any one of E1 -E30, wherein the at least one helper virus gene is an adenovirus gene.

E32. The system or first vector of any one of E1-E31 , wherein the at least one helper virus gene is an Adenovirus 5 or Adenovirus 2 gene. E33. The system or first vector of any one of E1-E32, wherein the least one helper virus gene comprises:

(a) a VA (viral associated) nucleic acid encoding functional VA RNA I and II;

(b) an E2A gene encoding a functional E2A protein; and/or

(c) an E4 gene encoding a functional E4 protein

E34. The system or first vector of any one of E1-E33, wherein the at least one helper virus gene comprises a VA nucleic acid and an E2A gene.

E35. The system or first vector of any one of E1-E34, wherein the E4 gene is not located between the VA nucleic acid and the E2A gene.

E36. The system or first vector of any one of E1-E35, wherein the first vector does not comprise a gene encoding a functional adenoviral E1A/B protein.

E37. The system or first vector of any one of E1-E36, wherein the first vector does not comprise a contiguous sequence of at least 200, at least 300, at least 350, or 363 nucleotides of a contiguous stretch of nucleotides of equivalent length comprised within nucleotides 4051-4413 of SEQ ID NO: 1 , or a corresponding stretch of nucleotides in a different serotype of AAV.

E38. The system or first vector of any one of E1-E37, wherein the first vector does not comprise a contiguous sequence of at least 400, at least 500, at least 600, or 647 nucleotides of a contiguous stretch of nucleotides of equivalent length comprised within nucleotides 2301-2947 of SEQ ID NO: 1 , or a corresponding stretch of nucleotides in a different serotype of AAV.

E39. The system or first vector of any one of E1 -E38, wherein the at least one helper virus gene is comprised in the first vector, said vector having at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 2, 6, 7, 8, 10, 11 , 12.

E40. The system or first vector or the second vector of any one of E1 -E39, wherein the second vector and/or the second vector does not comprise an artificial Rep binding site.

E41. The system or second vector of any one of E1-E40, wherein said second vector comprises a cap gene operably linked to at least one cap gene promoter.

E42. The system or second vector of any one of E1-E41 , wherein the second vector comprises a cap gene and further comprises an expression cassette flanked on at least one side by an ITR. E43. The system or second vector of any one of E1-E42, wherein the second vector comprises a cap gene and the cap gene encodes a VP1 , a VP2, and/or a VP3 protein.

E44. The system or sector vector of any one of E1-E43, wherein the second vector comprises a cap gene and the cap gene encodes a Cap protein selected from any naturally occurring or genetically engineered AAV serotype.

E45. The system or second vector of any one of E1-E44, wherein the second vector comprises an at least one gene promoter driving a cap gene expression, preferably a cap gene promoter.

E46. The system or second vector of any one of E1-E45, wherein the second vector comprises an at least one cap gene promoter, which is a native cap gene promoter.

E47. The system or second vector of any one of E1-E46, wherein the second vector comprises an at least one cap gene promoter, which comprises an AAV p40 promoter, a p5 promoter, and/or a p19 promoter.

E48. The system or second vector of E47, wherein the at least one cap gene promoter comprises a p40 promoter.

E49. The system or second vector of any one of E45-E48, wherein the at least one cap gene promoter comprises a p40 promoter, a p5 promoter, and a p19 promoter.

E50. The system or second vector of any one of E1-E49, wherein said vector has at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to any one of SEQ ID NOs: 3, 4, 13, 14, 15, 16.

E51 . A third vector for a three-component linear synthetic DNA system, wherein said vector comprises an expression cassette flanked on at least one side by an ITR and the expression cassette comprises a transgene operably linked to at least one regulatory control element.

E52. The system or second vector or third vector of any one of E1-E51 , wherein the transgene encodes an enzyme, a metabolic protein, a signalling protein, an antibody, an antibody fragment, an antibody-like protein, an antigen, or a non-translated RNA such as a miRNA, siRNA, snRNA, or antisense RNA.

E53. The system or second vector or third vector of any one of E1 -E52, wherein the transgene encodes a protein selected from the group consisting of Factor IX, a-Galactosidase A, beta-Glucocerebrosidase and Factor VIII. E54. The system or second vector or third vector of any one of E1-E53, wherein the cloning site is a multi-cloning site (MCS).

E55. The system or second vector of any one of E1-E54, wherein the second vector does not comprise any dispensable translation initiation codons.

E56. The system or second vector of E55, wherein the vector plasmid comprises a promoter region comprising one or more promoters, and the promoter region does not comprise ATG or GTG codons.

E57. The system or second vector of E56, wherein the promoter region comprises p5, p19 and p40 promoters, and wherein ATG or GTG codons at one or more positions corresponding to positions (a) 321-323, (b) 766-768, (c) 955-957, (d) 993-995 and (e) 1014-1016 of SEQ ID NO: 1 are absent or mutated.

E58. The system or second vector of E57, wherein in the promoter region:

ATT;

E59. The system or second vector of E1-E14 or E58, wherein in the promoter region:

ATT;

E60. The system or second vector of any one of E1-E59, wherein the second vector does not comprise any spacers.

E61 . A host cell comprising the two or three component linear synthetic DNA vector system of any one of the preceding embodiments, preferably said cell is a HEK293 cell.

E62. Use of the two or three component linear synthetic DNA vector system of any one of E1-E60 for producing a recombinant AAV preparation:

(a) having a low level of replication competent AAV (rcAAV); (b) having a desired ratio of full to total particles; and/or

(c) at a high or desired yield

(d) having reduced or completely eliminated undesirable DNA sequences.

E63. The use of E62, wherein the low level of rcAAV comprises a low level of rep-rcAAV.

E64. The use of E62 or E63, wherein the low level of rcAAV comprises a lower level of rep-rcAAV compared to the level of rep-rcAAV produced using a two-plasmid system comprising a plasmid comprising both at least one rep gene and at least one cap gene.

E65. The use of any one of E62-E64, wherein the level of rep-rcAAV is the level of rep-rcAAV detected by qPCR using primers binding to rep68 exon 1 .

E66. The use of any one of E62-E65, wherein the level of cap-rcAAV is the level of cap-rcAAV detected by qPCR using primers binding to a sequence encoding VP3.

E67. Use of the two or three component linear synthetic DNA vector system of any one of E1-E60 for:

(a) reducing or minimising the level of replication competent AAV (rcAAV) produced during recombinant AAV production;

(b) reducing or minimising the level of pseudo-wild type replication competent AAV (rcAAV) produced during recombinant AAV production;

(c) controlling or maximising the ratio of full to total particles produced during recombinant AAV production; and/or

(d) increasing, optimising or maximising the yield of recombinant AAV produced during recombinant AAV production.

(e) reducing or completely eliminating any undesirable DNA sequence in the recombinant AAV produced during recombinant AAV production.

E68. The use of any one of E62- E67, wherein the use comprises transfecting a host cell with the two or three component linear synthetic DNA vector system of any one of E1-E60 and culturing the host cell under conditions suitable for recombinant AAV production.

E69. The use of any one of E62-E68, wherein an excess of rep-rcAAV or cap-rcAAV indicates that the level of pseudo wild-type rcAAV is reduced or minimised.

E70. A method for producing a recombinant AAV preparation comprising:

(a) obtaining the two or three component linear synthetic DNA vector system of any one of E1- E60; (b) transfecting a host cell with the two or three component linear synthetic DNA vector system of any one of E1 -E60; and

(c) culturing the host cell under conditions suitable for recombinant AAV production.

E71 . The method of E70, further comprising a step of harvesting the recombinant AAV to provide a recombinant AAV preparation.

E72. A method for reducing or minimising the level of replication competent AAV (rcAAV) produced during recombinant AAV production comprising:

(a) obtaining the two or three component linear synthetic DNA vector system of any one of E1- E60;

(b) transfecting a host cell with the two or three component linear synthetic DNA vector system of any one of E1 -E60; and

E73. A method for reducing or minimising the level of pseudo-wild type replication competent AAV (rcAAV) produced during recombinant AAV production comprising:

E74. The method of E73, wherein an excess of rep-rcAAV or cap-rcAAV indicates that the level of pseudo wild-type rcAAV is reduced or minimised.

E75. A method for controlling or maximising the ratio of full to total particles produced during recombinant AAV production comprising:

E76. The method of E75, further comprising a step of harvesting the recombinant AAV to provide a recombinant AAV preparation comprising a desired ratio of full to total particles. E77. The method of any one of E70-E76, wherein the method is a method for producing a recombinant AAV preparation at a high or desired yield.

E78. A method for increasing, optimising or maximising the yield of recombinant AAV produced during recombinant AAV production comprising:

E79. The method of E78, further comprising a step of harvesting the recombinant AAV to provide a recombinant AAV preparation comprising a high or desired yield of recombinant AAV.

E80. The use or method of any one of E62-E79, wherein the ratio of first vector to second vector or the ratio of first vector to second vector to third vector is adjusted to obtain the desired ratio of full to total particles and/or the high or desired yield of recombinant AAV.

E81. The use or method of E62-E80, comprising a step of selecting a ratio of first vector to second vector, or selecting the ratio of first vector to second vector to third vector.

E82. The use or method of E81 wherein said ratio is selected or adjusted to a ratio that allows the user to obtain the desired ratio of full to total particles or the high or desired yield of recombinant AAV.

E83. The use or method of E81 or E82, wherein said ratio is selected or adjusted to a ratio that achieves a balanced yield versus full to total particle ratio.

E84. The use or method of any one of E81-E83 wherein said ratio is selected or adjusted to a ratio that achieves a maximum yield of recombinant AAV with the minimum yield of empty particles achievable at such maximum yield of recombinant AAV.

E85. The method or use of any one of E62-E84, wherein the host cell is selected from the group consisting of a HEK293T cell, a HEK293 cell, a HEK293EBNA cell, a CAP cell, a CAP-T cell, an AGE1 .CR cell, a PerC6 cell, a C139 cell, and an EB66 cell.

E86. The method or use of any one of E62-E85, wherein the host cell is a cell that expresses a functional E1A/B protein. E87. The method or use of any one of E62-E86, further comprising a step of purifying the recombinant AAV particles.

E88. The method or use of E87, where the step of purifying the recombinant AAV particles is carried out using a technique selected from the group consisting of gradient density centrifugation (such as CsCI or lodixanol gradient density centrifugation), filtration, ion exchange chromatography, size exclusion chromatography, affinity chromatography and hydrophobic interaction chromatography.

E89. The method or use of E87 or E88, comprising further concentrating the recombinant AAV using ultracentrifugation, tangential flow filtration, or gel filtration.

E90. The method or use of any one of E62-E89, comprising formulating the recombinant AAV with a pharmaceutically acceptable excipient.

E91 . A recombinant AAV preparation obtainable by the method of any one of E70-E90.

E92. A recombinant AAV preparation obtained by the method of any one of E70-E91 .

EXAMPLES

General techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, virology, animal cell culture and biochemistry which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook, Fritsch & Maniatis, 1989); "Animal Cell Culture" (R.l. Freshney, ed., 1987); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987); "Current Protocols in Protein Science" (John E Coligan, et al. eds. Wiley and Sons, 1995); and "Protein Purification: Principles and Practice" (Robert K. Scopes, Springer-Verlag, 1994).

All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby incorporated herein by reference.

Example 1 : Synthetic Production of linear DNA Vectors

General Process

A sequence of interest (namely a sequence to be incorporated into or transformed to a linear synthetic DNA molecule), usually in the form of a double stranded DNA molecule and derived from plasmid DNA, PCR DNA or gBIocks DNA is transferred between flanking sequences in a circular or linear template vector. Such template vectors are available in the prior art and they are incorporated herein by reference e.g Karda et al describe such template vectors (Gene Therapy, volume 26, pages 86-92 (2019)). The flanking sequences are serving in later process steps for example as enzyme recognition sites, spacer sequences and/or repetitive, inverted terminal sequence units. In the case of a linear template vector, the flanking sequences are connecting the two DNA strands of the sequence of interest by a hairpin structure.

The template vector is then used in an enzymatic in vitro amplification reaction. The intermediate DNA product is usually generated by utilization of a DNA polymerase and a rolling circle DNA amplification mechanism. To that end, oligo primers are added, or procedures based on primer-free amplification are used.

Further processing, purification and formulation steps result in the final synthetic DNA vector. By these manipulation steps, unwanted DNA like plasmid backbone sequences, if present, are digested and removed. Moreover, the synthetic DNA vectors containing the sequence of interest can be separated into monomers, for example by the cleavage of long DNA concatemers generated during the rolling circle DNA amplification. The linear molecules usually consist of double-stranded DNA, which exhibit covalently closed ends, open ends or combinations thereof. In special cases, DNA oligomers containing hairpin structures are added to single or double stranded DNA monomers. Figure 11 shows a schematic representation of a general process followed in order to produce a linear, synthetic DNA vector.

A. Construction of a vector (“first” vector) encoding at least one functional Rep protein

In order to construct a vector encoding at least one functional Rep protein (“first” vector of a two or three component linear synthetic DNA vector system), based on the helper plasmid disclosed in W02020/208379 A1 and EP3722434 B1 (incorporated herein by reference), nucleotides 200-4497 of wild type AAV2 (Genbank accession number AF043303; SEQ ID NO: 1) containing the rep and cap genes were cloned into the plasmid pUC19 (Yanisch-Perron et al (1985), Gene, 33:103-1 19). Next, AAV2 nucleotides 4461-4497 were deleted to minimize sequence homology with the “second” vector and/or the “third” vector (the construction of which is described below). To prevent Rep 78 expression while maintaining Rep 68 expression, the intron within the cloned rep gene was deleted. In order to provide for expression of Rep 52 following deletion of the intron, AAV2 nucleotides corresponding to rep 52 including the p19 promoter were cloned immediately at 3’ of the intron-less rep 68 gene. The majority of cap gene sequences were then deleted.

The two p40 promoters (one in each of the Rep 68- and Rep 52-encoding rep gene duplications) were rendered non-functional by ablation of the TATA boxes (mutation of the T corresponding to AAV2 position 1823 and AAG corresponding to AAV2 positions 1826-1828 to C and CTC respectively).

The resulting ‘rep cassette’ was then cloned into a plasmid comprising an 8342-nucleotide stretch comprising functional VA RNA I and II, E2A and E4 genes from adenovirus (i.e. helper virus) serotype 5 (SEQ ID NO: 9). The plasmid backbone, containing kanamycin resistance gene and bacterial origin of replication, was about 2.2kb in length, resulting in a helper plasmid of 14021 nucleotides. The 11879-nucleotide stretch comprising the ‘rep cassette’ and the ‘adenoviral sequences’ was then transferred into a template vector free of the bacterial plasmid backbone from which in an enzymatic in vitro reaction an intermediate DNA product was generated by primer-free isothermal amplification. After processing, purification and buffer exchange steps a synthetic, double stranded linear DNA molecule (comprising a plasmid derived sequence of interest having SEQ ID NO: 2) was obtained, covalently closed with single strand hairpins at the 5’ and 3’ ends.

Figure 2 shows a schematic illustration of a first vector of the invention, encoding at least one functional Rep protein, based on a helper plasmid depicted in Figure 1.

B. Construction of a vector (“second” vector) encoding at least one functional Cap protein and comprising an expression cassette

In order to construct a vector encoding at least one functional Cap protein and comprising an expression cassette (“second” vector of a two- component linear synthetic DNA vector system), based on the vector plasmid disclosed in W02020/208379 A1 and EPEP3722434 B1 (incorporated herein by reference), nucleotides 200-4497 of wild type AAV2 (Genbank accession number AF043303; SEQ ID NO: 1) containing the rep and cap genes were cloned into pUC19. Two portions of rep gene sequence, between the p5 and p19 and between the p19 and p40 promoters respectively, were then deleted to prevent Rep protein expression whilst maintaining the downstream cap gene under the regulation of the three native promoters. Similarly to the first vector described above, AAV2 nucleotides 4461-4497 were deleted to minimize sequence homology.

To minimize or prevent translation of undesired products from potential initiation codons within the remaining promoter region, four ATG codons and one GTG codon were removed: ATGs at positions corresponding to AAV2 nucleotides 321-323, 955-957 and 993-995, and a GTG corresponding to AAV2 nucleotides 1014-1016, were deleted, whilst ATG at AAV2 nucleotides 766-768 was mutated to ATT. The AAV2 cap gene encoding the VPs 1 , 2 and 3 immediately 3’ of the above promoter region was replaced with the corresponding sequence of the AAV3B cap gene (Genbank accession number AF028705), which cap gene is 3 nucleotides (equating to one additional encoded amino acid) longer than the AAV2 cap gene. The resulting ‘promoter-cap’ cassette was cloned into a plasmid backbone of approximately 2.1 kb in length containing kanamycin resistance gene and bacterial origin of replication. Into this backbone was inserted a 2985-nucleotide (ITR-to-ITR) AAV expression cassette, containing a secreted alkaline phosphatase (SEAP) transgene sequence linked to enhancer, promoter, intron and polyA regulatory elements, flanked by AAV2 sequence comprising the native AAV2 ITRs (AAV2 nucleotides 1-145 and 4535-4679). Overall, this resulted in an 8581 -nucleotide AAV expression cassette/capsid plasmid.

The 6513-nucleotide stretch comprising the ‘AAV expression cassette’ and the ‘capsid sequence’ was then transferred into a template vector, free of the bacterial plasmid backbone, from which in an enzymatic in vitro reaction an intermediate DNA product was generated by primer-free isothermal amplification. After processing, purification and buffer exchange steps the second, linear synthetic DNA vector was obtained (comprising a plasmid derived sequence of interest having SEQ ID NO: 3), as a double stranded linear DNA molecule, covalently closed with single strand hairpins at the 5’ and 3’ ends.

Figure 2 shows a schematic illustration of a second vector, encoding at least one functional Cap protein and comprising an expression cassette.

C. Construction of a vector (“second” vector) encoding at least one functional Cap protein without comprising an expression cassette

In order to construct a vector encoding at least one functional Cap protein without comprising an expression cassette (“second” vector of a three- component linear synthetic DNA vector system), based on the vector plasmid disclosed in W02020/208379 A1 and EPEP3722434 B1 (incorporated herein by reference), nucleotides 200-4497 of wild type AAV2 (Genbank accession number AF043303; SEQ ID NO: 1) containing the rep and cap genes were cloned into pUC19. Two portions of rep gene sequence, between the p5 and p19 and between the p19 and p40 promoters respectively, were then deleted to prevent Rep protein expression whilst maintaining the downstream cap gene under the regulation of the three native promoters. As for the first vector described above, AAV2 nucleotides 4461-4497 were deleted to minimize sequence homology with the first vector.

To minimise or prevent translation of undesired products from potential initiation codons within the remaining promoter region, four ATG codons and one GTG codon were removed: ATGs at positions corresponding to AAV2 nucleotides 321-323, 955-957 and 993-995, and a GTG corresponding to AAV2 nucleotides 1014-1016, were deleted, whilst ATG at AAV2 nucleotides 766-768 was mutated to ATT.

The AAV2 cap gene encoding the VPs 1 , 2 and 3 immediately 3’ of the above promoter region was replaced with the corresponding sequence of the AAV3B cap gene (Genbank accession number AF028705), which cap gene is 3 nucleotides (equating to one additional encoded amino acid) longer than the AAV2 cap gene. The resulting ‘promoter-cap’ cassette was cloned into a plasmid backbone of approximately 2.1 kb in length containing kanamycin resistance gene and bacterial origin of replication, resulting in a capsid plasmid of 5582 nucleotides.

The 3296-nucleotide stretch comprising the ‘capsid sequence’ was then transferred into a template vector, free of the bacterial plasmid backbone, from which in an enzymatic in vitro reaction an intermediate DNA product was generated by primer-free isothermal amplification. After processing, purification and buffer exchange steps a synthetic linear DNA vector encoding at least one functional Cap protein, without comprising an expression cassette, was obtained, as a double stranded linear DNA molecule (comprising a plasmid derived sequence of interest having SEQ ID NO; 4), covalently closed with single strand hairpins at the 5’ and 3’ ends.

Figure 3 shows a schematic illustration of a second vector encoding at least one functional Cap protein without comprising an expression cassette.

D. Construction of a vector (“third” vector) comprising an expression cassette In order to construct a vector comprising an expression cassette (“third” vector of a three component linear, synthetic DNA vector system), a 2985-nucleotide (ITR-to-ITR) AAV expression cassette, containing a secreted alkaline phosphatase (SEAP) transgene sequence linked to enhancer, promoter, intron and polyA regulatory elements, flanked by AAV2 sequence (Genbank accession number AF043303; SEQ ID NO: 1) comprising the native AAV2 ITRs (AAV2 nucleotides 1-145 and 4535-4679), was cloned into a plasmid backbone of approximately 2.1 kb in length containing kanamycin resistance gene and bacterial origin of replication. This resulted in an AAV expression cassette plasmid of 5163 nucleotides in length.

The 2985-nucleotide stretch comprising the ‘AAV expression cassette’ was then transferred into a template vector free of the bacterial plasmid backbone, from which in an enzymatic in vitro reaction an intermediate DNA product was generated by primer-free isothermal amplification. After processing, purification and buffer exchange steps the synthetic linear DNA vector comprising an expression cassette was obtained (comprising a plasmid derived sequence of interest having SEQ ID NO:5), as a double stranded linear DNA molecule, covalently closed with single strand hairpins at the 5’ and 3’ ends.

Figure 3 shows a schematic illustration of the synthetic linear DNA vector comprising an expression cassette for a gene of interest (GOI) e.g. secreted alkaline phosphatase (SEAP) transgene sequence.

E. Construction of a vector (“first” vector) comprising an entire rep gene under the control of a modified, ACG start codon.

In order to construct a vector comprising an entire rep gene with the large Rep proteins Rep 78 and Rep 68 under the control of a modified, ACG start codon (“first” vector of a two or three component linear synthetic DNA vector system), based on the helper plasmid disclosed in WO2022/079429 A1 (incorporated herein by reference), nucleotides 200-4460 of wild type AAV2 (Genbank accession number AF043303; SEQ ID NO: 1) containing the rep and cap genes were cloned into pUC19 (Yanisch- Perron et al (1985), Gene, 33:103-119). Next, AAV2 nucleotides 2301-4410 were deleted to remove the majority of the cap gene sequences. The ATG start codon in the rep gene driving Rep78/Rep68 expression was replaced with an ACG codon.

The p40 promoter (contained within the rep gene) was rendered non-functional by ablation of the TATA box (mutation of the T corresponding to AAV2 position 1823 and AAG corresponding to AAV2 positions 1826-1828 to C and CTC respectively).

The resulting ‘rep cassette’ was then cloned into a plasmid comprising an 8342-nucleotide stretch comprising functional VA RNA I and II, E2A and E4 genes from adenovirus (i.e. helper virus) serotype 5 (SEQ ID NO: 9). The plasmid backbone, containing kanamycin resistance gene and bacterial origin of replication, was about 2.2kb in length, resulting in a helper plasmid of 12668 nucleotides.

The 10526-nucleotide stretch comprising the ‘rep cassette’ and the ‘adenoviral sequences’ was then transferred into a template vector, free of the bacterial plasmid backbone, from which in an enzymatic in vitro reaction an intermediate DNA product was generated by primer-free isothermal amplification. After processing, purification and buffer exchange steps a synthetic linear DNA vector encoding an entire rep gene under the control of under the control of a modified ACG start codon (comprising a plasmid derived sequence of interest having SEQ ID NO:6) was obtained, as a double stranded linear DNA molecule, covalently closed with single strand hairpins at the 5’ and 3’ ends.

Figure 7 shows a schematic illustration of a first vector comprising an entire rep gene under the control of a modified start codon (ACG), derived from a helper plasmid, as depicted in Figure 6. For the ‘rep cassette’ the portions of AAV2 sequence, by reference to the nucleotide positions of SEQ ID NO: 1 , are indicated.

F. Construction of a vector (“first” vector) comprising an inverted orientation, entire rep gene under the control of a modified, ACG start codon.

In order to construct a vector comprising an entire rep gene with the large Rep proteins Rep 78 and Rep 68 under the control of a modified, ACG start codon (“first” vector of a two or three component linear synthetic DNA vector system), based on the helper plasmid disclosed in WO2022/079429 A1 (incorporated herein by reference), but with an inverted orientation, nucleotides 200-4460 of wild type AAV2 (Genbank accession number AF043303; SEQ ID NO: 1) containing the rep and cap genes were cloned into pUC19 (Yanisch-Perron et al (1985), Gene, 33:103-119). Next, AAV2 nucleotides 2301- 4410 were deleted to remove the majority of the cap gene sequences. The ATG start codon in the rep gene driving Rep78/Rep68 expression was replaced with an ACG codon.

The resulting ‘rep cassette’ was then cloned in an inverted orientation into a plasmid comprising an 8342-nucleotide stretch comprising functional VA RNA I and II, E2A and E4 genes from adenovirus (i.e. helper virus) serotype 5 (SEQ ID NO: 9). The plasmid backbone, containing kanamycin resistance gene and bacterial origin of replication, was about 2.2kb in length, resulting in a helper plasmid of 12670 nucleotides.

The 10528-nucleotide stretch comprising the ‘inverted rep cassette’ and the ‘adenoviral sequences’ was then transferred into a template vector, free of the bacterial plasmid backbone, from which in an enzymatic in vitro reaction an intermediate DNA product was generated by primer-free isothermal amplification. After processing, purification and buffer exchange steps a synthetic linear DNA vector comprising an inverted orientation, entire rep gene under the control of a modified, ACG start codon (comprising a plasmid derived sequence of interest having SEQ ID NOT) was obtained, as a double stranded linear DNA molecule, covalently closed with single strand hairpins at the 5’ and 3’ ends.

Figure 9 is a schematic illustration of a first vector comprising an entire rep gene under the control of a modified start codon (ACG), but with an inverted orientation, derived from a helper plasmid, as depicted in Figure 6. For the ‘inverted rep cassette’ the portions of AAV2 sequence, by reference to the nucleotide positions of SEQ ID NO: 1 , are indicated.

G. Construction of a vector (“first” vector) comprising an inverted orientation rep gene sequence, encoding at least one functional Rep protein

In order to construct a vector comprising an inverted orientation rep gene sequence, encoding at least one functional Rep protein (“first” vector of a two or three component linear synthetic DNA vector system), based on the helper plasmid disclosed in W02020/208379 A1 and EP3722434 B1 (incorporated herein by reference), nucleotides 200-4497 of wild type AAV2 (Genbank accession number AF043303; SEQ ID NO: 1) containing the rep and cap genes were cloned into the plasmid pUC19 (Yanisch-Perron et al (1985), Gene, 33:103-119). Next, AAV2 nucleotides 4461-4497 were deleted to minimise sequence homology with the “second” vector and/or the “third” vector. To prevent Rep 78 expression while maintaining Rep 68 expression, the intron within the cloned rep gene was deleted. In order to provide for expression of Rep 52 following deletion of the intron, AAV2 nucleotides corresponding to rep 52 including the p19 promoter were cloned immediately 3’ of the intron-less rep 68 gene. The majority of cap gene sequences were then deleted.

The resulting ‘rep cassette’ was then cloned in an inverted orientation into a plasmid comprising an 8342-nucleotide stretch comprising functional VA RNA I and II, E2A and E4 genes from adenovirus (i.e. helper virus) serotype 5 (SEQ ID NO: 9). The plasmid backbone, containing kanamycin resistance gene and bacterial origin of replication, was about 2.2kb in length, resulting in a helper plasmid of 14023 nucleotides.

The 11881 -nucleotide stretch comprising the ‘inverted rep cassette’ and the ‘adenoviral sequences’ was then transferred into a template free of the bacterial plasmid backbone from which in an enzymatic in vitro reaction an intermediate DNA product was generated by primer-free isothermal amplification. After processing, purification and buffer exchange steps a synthetic linear DNA vector comprising an inverted orientation rep gene sequence was obtained, as a double stranded linear DNA molecule (comprising a plasmid derived sequence of interest having SEQ ID NO:8), covalently closed with single strand hairpins at the 5’ and 3’ ends.

Figure 4 is a schematic illustration of a first vector of the invention, with an inverted rep gene sequence, based on a helper plasmid depicted in Figure 1.

H. Construction of a vector (“first” vector) comprising an entire rep gene under the control of an ATG start codon. In order to construct a vector comprising an entire rep gene with the large Rep proteins Rep 78 and Rep 68 under the control of an ATG start codon (“first” vector of a two or three component linear synthetic DNA vector system), based on the helper plasmid described in EP24216316 (incorporated herein by reference), nucleotides 200-4460 of wild type AAV2 (Genbank accession number AF043303; SEQ ID NO: 1) containing the rep and cap genes were cloned into pUC19 plasmid (Yanisch-Perron et al (1985), Gene, 33:103-1 19). Next, AAV2 nucleotides 2301-4410 were deleted to remove the majority of the cap gene sequences. Rep78/Rep68 expression is driven by the native ATG start codon in the rep gene.

The 10526-nucleotide stretch comprising the ‘rep cassette’ and the ‘adenoviral sequences’ was then cloned into a circular starting template. Addition of Phi29 DNA polymerase initiates rolling circle amplification and the generation of long, linear, double stranded concatemers containing copies of the starting template. Protelomerase binds to recognition sites flanking the target sequence and performs a cleavage-joining reaction to produce linear, double stranded and covalently closed dbDNA molecules. Restriction enzymes and exonucleases selectively digest and degrade the non-target backbone sequences. After purification and buffer exchange steps known in the art, the final synthetic DNA vector as a double stranded linear DNA molecule was obtained, having the nucleic acid sequence of SEQ ID NO: 10.

Figure 14 is a schematic illustration of a two-component linear synthetic DNA system showing a first vector comprising an entire rep gene under the control of the native start codon ATG along with the main features.

I. Construction of a vector (“first” vector) comprising an inverted orientation, entire rep gene under the control of an ATG start codon.

In order to construct a vector comprising an entire rep gene with the large Rep proteins Rep 78 and Rep 68 under the control of an ATG start codon (“first” vector of a two or three component linear synthetic DNA vector system), based on the helper plasmid described in EP24216316 (incorporated herein by reference), but with an inverted orientation, nucleotides 200-4460 of wild type AAV2 (Genbank accession number AF043303; SEQ ID NO: 1) containing the rep and cap genes were cloned into pUC19 (Yanisch-Perron et al (1985), Gene, 33:103-119). Next, AAV2 nucleotides 2301-4410 were deleted to remove the majority of the cap gene sequences. Rep78/Rep68 expression is driven by the native ATG start codon in the rep gene. The p40 promoter (contained within the rep gene) was rendered non-functional by ablation of the TATA box (mutation of the T corresponding to AAV2 position 1823 and AAG corresponding to AAV2 positions 1826-1828 to C and CTC respectively).

The 10528-nucleotide stretch comprising the ‘inverted rep cassette’ and the ‘adenoviral sequences’ was then cloned into a circular starting template. Addition of Phi29 DNA polymerase initiates rolling circle amplification and the generation of long, linear, double stranded concatemers containing copies of the starting template. Protelomerase binds to recognition sites flanking the target sequence and performs a cleavage-joining reaction to produce linear, double stranded and covalently closed dbDNA molecules. Restriction enzymes and exonucleases selectively digest and degrade the non-target backbone sequences. After purification and buffer exchange steps known in the art, the final synthetic DNA vector as a double stranded linear DNA molecule was obtained, having the nucleic acid sequence of SEQ ID NO: 11.

Figure 15 is a schematic illustration of a two-component linear synthetic DNA system showing a first vector comprising an inverted orientation entire rep gene under the control of the native start codon ATG along with the main features.

J. Construction of a vector (“first” vector) comprising an inverted orientation, entire rep gene and a spacer sequence under the control of an ATG start codon.

In order to construct a vector comprising an inverted, entire rep gene encoding the large Rep proteins Rep 78 and Rep 68 under the control of the native start codon ATG , (“first” vector of a two or three component linear synthetic DNA vector system), based on the helper plasmid described in EP24216316 (incorporated herein by reference) and linked to a spacer sequence at the 3-prime end, nucleotides 200-4460 of wild type AAV2 (Genbank accession number AF043303; SEQ ID NO: 1) containing the rep and cap genes were cloned into pUC19 (Yanisch-Perron et al (1985), Gene, 33:103-119). Next, AAV2 nucleotides 2301-4410 were deleted to remove the majority of the cap gene sequences. Rep78/Rep68 expression is driven by the native ATG start codon in the rep gene.

The resulting ‘rep cassette’ was then cloned in an inverted orientation together with an additional 117 bp spacer sequence at the 3-prime end into a plasmid comprising an 8342-nucleotide stretch comprising functional VA RNA I and II, E2A and E4 genes from adenovirus (i.e. helper virus) serotype 5 (SEQ ID NO: 9). The spacer sequence aims to minimise potential interferences between adjacent coding sequence regions. The plasmid backbone, containing kanamycin resistance gene and bacterial origin of replication, was about 2.2kb in length, resulting in a helper plasmid of 12670 nucleotides.

The 10645-nucleotide stretch comprising the ‘inverted rep cassette’ linked to the spacer sequence and the ‘adenoviral sequences’ was then cloned into a circular starting template. Addition of Phi29 DNA polymerase initiates rolling circle amplification and the generation of long, linear, double stranded concatemers containing copies of the starting template. Protelomerase binds to recognition sites flanking the target sequence and performs a cleavage-joining reaction to produce linear, double stranded and covalently closed dbDNA molecules. Restriction enzymes and exonucleases selectively digest and degrade the non-target backbone sequences. After purification and buffer exchange steps known in the art, the final synthetic DNA vector as a double stranded linear DNA molecule was obtained, having the nucleic acid sequence of SEQ ID NO:12.

Figure 16 is a schematic illustration of a two-component linear synthetic DNA system showing a first vector comprising an inverted orientation entire rep gene under the control of the native start codon ATG and linked to a spacer sequence at the 3-prime end, along with the main features.

K. Construction of a vector (“second” vector) encoding at least one functional Cap protein and comprising an expression cassette

In order to construct a vector encoding at least one functional Cap protein comprising an expression cassette (“second” vector of a two- component linear synthetic DNA vector system), based on the vector plasmid described in EP24216316 (incorporated herein by reference), nucleotides 200-4497 of wild type AAV2 (Genbank accession number AF043303; SEQ ID NO: 1) containing the rep and cap genes were cloned into a pUC19 plasmid. Two portions of rep gene sequence, between the p5 and p19 and between the p19 and p40 promoters respectively, were then deleted to prevent Rep protein expression whilst maintaining the downstream cap gene under the regulation of the three native promoters. AAV2 nucleotides 4461-4497 were deleted to minimise sequence homology with the different helper vectors. To minimise or prevent translation of undesired products from potential initiation codons within the remaining promoter region, four ATG codons and one GTG codon were removed: ATG codons at positions corresponding to AAV2 nucleotides 321 -323, 955-957 and 993-995, and a GTG corresponding to AAV2 nucleotides 1014-1016, were deleted, whilst ATG at AAV2 nucleotides 766-768 was mutated to ATT.

The AAV2 cap gene encoding the VPs 1 , 2 and 3 immediately 3’ of the above promoter region was replaced with the corresponding sequence of the AAV3B cap gene (Genbank accession number AF028705), which cap gene is 3 nucleotides (equating to one additional encoded amino acid) longer than the AAV2 cap gene. The resulting ‘promoter-cap’ cassette was cloned into a plasmid backbone of approximately 2.1 kb in length containing kanamycin resistance gene and bacterial origin of replication. Into this backbone was inserted a 2985-nucleotide (ITR-to-ITR) AAV expression cassette, containing a secreted alkaline phosphatase (SEAP; an exemplified gene of interest or GOI) transgene sequence linked to enhancer, promoter, intron and polyA regulatory elements, flanked by AAV2 sequence comprising the native AAV2 ITRs (AAV2 nucleotides 1-145 and 4535-4679). Overall, this resulted in an 8581 -nucleotide AAV expression cassette/capsid plasmid.

The 6513-nucleotide stretch comprising the ‘AAV expression cassette’ and the ‘capsid sequence’ was then cloned into a circular starting template. Addition of Phi29 DNA polymerase initiates rolling circle amplification and the generation of long, linear, double stranded concatemers containing copies of the starting template. Protelomerase binds to recognition sites flanking the target sequence and performs a cleavage-joining reaction to produce linear, double stranded and covalently closed dbDNA molecules. Restriction enzymes and exonucleases selectively digest and degrade the non-target backbone sequences. After purification and buffer exchange steps known in the art, the final synthetic DNA vector as a double stranded linear DNA molecule was obtained, having the nucleic acid sequence of SEQ ID NO:13.

Figure 14 is a schematic illustration of a two-component linear synthetic DNA system showing a second vector of the invention comprising a capsid encoding sequence and a gene of interest (GOI) sequence. Such a vector is also depicted in Figures 15 and 16.

L. Construction of a vector (“second” vector) encoding at least one functional Cap protein and comprising an expression cassette

In order to construct a vector encoding at least one functional Cap protein comprising an expression cassette (“second” vector of a two- component linear synthetic DNA vector system), based on the vector plasmid described in EP24216316 (incorporated herein by reference), nucleotides 200-4497 of wild type AAV2 (Genbank accession number AF043303; SEQ ID NO: 1) containing the rep and cap genes were cloned into pUC19 plasmid. Two portions of rep gene sequence, between the p5 and p19 and between the p19 and p40 promoters respectively, were then deleted to prevent Rep protein expression whilst maintaining the downstream cap gene under the regulation of the three native promoters. AAV2 nucleotides 4461-4497 were deleted to minimise sequence homology with the different helper vectors. To minimise or prevent translation of undesired products from potential initiation codons within the remaining promoter region, four ATG codons and one GTG codon were removed: ATG codons at positions corresponding to AAV2 nucleotides 321 -323, 955-957 and 993-995, and a GTG corresponding to AAV2 nucleotides 1014-1016, were deleted, whilst ATG at AAV2 nucleotides 766-768 was mutated to ATT.

The AAV2 cap gene encoding the VPs 1 , 2 and 3 immediately 3’ of the above promoter region was replaced with the corresponding sequence of the AAV9 cap gene (Genbank accession number AY530579), which cap gene is 3 nucleotides (equating to one additional encoded amino acid) longer than the AAV2 cap gene. The resulting ‘promoter-cap’ cassette was cloned into a plasmid backbone of approximately 2.1 kb in length containing kanamycin resistance gene and bacterial origin of replication. Into this backbone was inserted a 2985-nucleotide (ITR-to-ITR) AAV expression cassette, containing a secreted alkaline phosphatase (SEAP) transgene sequence linked to enhancer, promoter, intron and polyA regulatory elements, flanked by AAV2 sequence comprising the native AAV2 ITRs (AAV2 nucleotides 1-145 and 4535-4679). Overall, this resulted in an 8581 -nucleotide AAV expression cassette/capsid plasmid.

The 6513-nucleotide stretch comprising the ‘AAV expression cassette’ and the ‘capsid sequence’ was then cloned into a circular starting template. Addition of Phi29 DNA polymerase initiates rolling circle amplification and the generation of long, linear, double stranded concatemers containing copies of the starting template. Protelomerase binds to recognition sites flanking the target sequence and performs a cleavage-joining reaction to produce linear, double stranded and covalently closed dbDNA molecules. Restriction enzymes and exonucleases selectively digest and degrade the non-target backbone sequences. After purification and buffer exchange steps known in the art, the final synthetic DNA vector as a double stranded linear DNA molecule was obtained, having the nucleic acid sequence of SEQ ID NO: 14.

Figure 14 is a schematic illustration of a two-component linear synthetic DNA system showing a second vector of the invention comprising a capsid encoding sequence and a gene of interest (GOI) sequence. Such a vector is also depicted in Figures 15 and 16

M. Construction of a vector (“second” vector) encoding at least one functional Cap protein without comprising an expression cassette

In order to construct a vector encoding at least one functional Cap protein without comprising an expression cassette (“second” vector of a three- component linear synthetic DNA vector system), based on the vector plasmid disclosed EP24216316 (incorporated herein by reference), nucleotides 200-4497 of wild type AAV2 (Genbank accession number AF043303; SEQ ID NO: 1) containing the rep and cap genes were cloned into pUC19. Two portions of rep gene sequence, between the p5 and p19 and between the p19 and p40 promoters respectively, were then deleted to prevent Rep protein expression whilst maintaining the downstream cap gene under the regulation of the three native promoters. AAV2 nucleotides 4461-4497 were deleted to minimise sequence homology with the different helper vectors. To minimise or prevent translation of undesired products from potential initiation codons within the remaining promoter region, four ATG codons and one GTG codon were removed: ATG codons at positions corresponding to AAV2 nucleotides 321 -323, 955-957 and 993-995, and a GTG corresponding to AAV2 nucleotides 1014-1016, were deleted, whilst ATG at AAV2 nucleotides 766-768 was mutated to ATT.

The 3296-nucleotide stretch comprising the ‘capsid sequence’ was then cloned into a circular starting template. Addition of Phi29 DNA polymerase initiates rolling circle amplification and the generation of long, linear, double stranded concatemers containing copies of the starting template. Protelomerase binds to recognition sites flanking the target sequence and performs a cleavage-joining reaction to produce linear, double stranded and covalently closed dbDNA molecules. Restriction enzymes and exonucleases selectively digest and degrade the non-target backbone sequences. After purification and buffer exchange steps known in the art, the final synthetic DNA vector as a double stranded linear DNA molecule was obtained, having the nucleic acid sequence of SEQ ID NO:15.

Figure 17 is a schematic illustration of a three-component linear synthetic DNA system showing a second vector of the invention comprising a capsid encoding sequence. Such a vector is also depicted in Figures 18 and 19.

N. Construction of a vector (“second” vector) encoding at least one functional Cap protein without comprising an expression cassette

The AAV2 cap gene encoding the VPs 1 , 2 and 3 immediately 3’ of the above promoter region was replaced with the corresponding sequence of the AAV9 cap gene (Genbank accession number AY530579; SEQ ID NO: X), which cap gene is 3 nucleotides (equating to one additional encoded amino acid) longer than the AAV2 cap gene. The resulting ‘promoter-cap’ cassette was cloned into a plasmid backbone of approximately 2.1 kb in length containing kanamycin resistance gene and bacterial origin of replication, resulting in a capsid plasmid of 5582 nucleotides.

The 3296-nucleotide stretch comprising the ‘capsid sequence’ was then cloned into a circular starting template. Addition of Phi29 DNA polymerase initiates rolling circle amplification and the generation of long, linear, double stranded concatemers containing copies of the starting template. Protelomerase binds to recognition sites flanking the target sequence and performs a cleavage-joining reaction to produce linear, double stranded and covalently closed dbDNA molecules. Restriction enzymes and exonucleases selectively digest and degrade the non-target backbone sequences. After purification and buffer exchange steps known in the art, the final synthetic DNA vector as a double stranded linear DNA molecule was obtained, having the nucleic acid sequence of SEQ ID NO:16.

O. Construction of a vector (“third” vector) comprising an expression cassette

In order to construct a vector comprising an expression cassette (“third” vector of a three component linear, synthetic DNA vector system), a 2985-nucleotide (ITR-to-ITR) AAV expression cassette, containing a secreted alkaline phosphatase (SEAP) transgene sequence linked to enhancer, promoter, intron and polyA regulatory elements, flanked by AAV2 sequence (Genbank accession number AF043303; SEQ ID NO: 1) comprising the native AAV2 ITRs (AAV2 nucleotides 1-145 and 4535-4679), was cloned into a plasmid backbone of approximately 2.1 kb in length containing kanamycin resistance gene and bacterial origin of replication. This resulted in an AAV expression cassette plasmid of 5163 nucleotides in length.

The 2985-nucleotide stretch comprising the ‘AAV expression cassette’ was then cloned into a circular starting template. Addition of Phi29 DNA polymerase initiates rolling circle amplification and the generation of long, linear, double stranded concatemers containing copies of the starting template. Protelomerase binds to recognition sites flanking the target sequence and performs a cleavage-joining reaction to produce linear, double stranded and covalently closed dbDNA molecules. Restriction enzymes and exonucleases selectively digest and degrade the non-target backbone sequences. After purification and buffer exchange steps known in the art, the final synthetic DNA vector as a double stranded linear DNA molecule was obtained, having the nucleic acid sequence of SEQ ID NO:17.

Figure 17 is a schematic illustration of a three-component linear synthetic DNA system showing a second vector of the invention comprising a gene of interest (GOI) encoding region, flanked by ITR sequences. Such a vector is also depicted in Figures 18 and 19.

Example 2: Two or three component linear synthetic DNA vector system transfection of HEK293 suspension cells in an Ambr®15 bioreactor system.

Part A

HEK293 suspension cells were seeded in BalanCD HEK293 medium, supplemented with 4 mM L- Glutamine from a 1-day culture, with a seeding density of 1.0E+06 - 4.0E+06 viable cells (vc)/mL (NucleoCounter® NC-202™, Chemometec) in a total volume of 12 mL. Transient transfections of HEK293 suspension cells were performed after 2h in the Ambr®15 bioreactor vessels.

In case of the two-component linear synthetic DNA vector system transfection (the system depicted in Figure 2 and comprising the vectors of Example 1A and 1 B), the molar ratio between the respective DNA molecules was 3:1 - 1 :6 (first vector to second vector). A DNA concentration of 0.25 - 1 .0 pg/vc and a PEIpro®:DNA ratio of 1 :1 - 2:1 were applied. DNA and PEIpro® were diluted in fresh, supplemented BalanCD HEK293 medium in a final volume of 400 pL per bioreactor each. The PEIpro®- mix was added to the DNA-mix and incubated for 10 - 30 min at room temperature. 650 pL transfection mix was added per bioreactor vessel.

In case the of the three -component linear synthetic DNA vector system transfection (the system depicted in Figure 3 and comprising the vectors of Example 1A, 1 C and 1 D), in a molar ratio between the respective DNA vectors suitable for AAV production e.g. including the range of 3:1 :1 - 1 :6:6 (first vector: second vector: third vector). A DNA concentration of 0.25 - 1.0 pg/vc and a PEIpro®:DNA ratio of 1 :1 - 2:1 were applied. DNA and PEIpro® were diluted in fresh, supplemented BalanCD HEK293 medium in a final volume of 400 pL per bioreactor each. The PEIpro®-mix was added to the DNA-mix and incubated for 10 - 30 min at room temperature. 650 pL transfection mix was added per bioreactor vessel.

Control transfections were performed in parallel utilising the 2-plasmid split system, described in detail in WC2020/208379 A1 , EP3722434 B1 , and WO 2022/079429 A1 (incorporated herein by reference), which comprises two plasmids encoding adenoviral helper functions and AAV rep on one plasmid and AAV cap and the respective transgene cassette on a second plasmid.

After 48 - 96h incubation to allow for rAAV production, cells were harvested in the medium and a freezethaw lysis was performed by repeated freezing and thawing at -80°C and 37°C, respectively (repeated three times). The samples were centrifuged for 15 min at 3700x g and room temperature before transferring the supernatants to new vials. The clarified freeze-thaw lysates were subjected to affinity chromatography purification or used directly for subsequent analytical procedures.

Part B

Cell Cultivation

HEK293 cells were maintained in suspension culture at 37 °C, 5% CO2, and 120 rpm in BalanCD HEK293 medium supplemented with 4 mM L-glutamine. Cells were passaged every 3-4 days with a seeding density of 1 .0 x 10⁶ or 0.5 x 10⁶ viable cells/mL.

Transfection of HEK293 suspension cells in an AMBR®15 bioreactor system and preparation of cell lysates

HEK293 suspension cells in BalanCD HEK293 medium supplemented with 4 mM L-glutamine from a 1-day culture were seeded with a seeding density of 2.5 x 10⁶ viable cells/mL (NucleoCounter® NC- 202™, Chemometec) in a total volume of 12 mL. Transient transfections of HEK293 suspension cells were performed 2h after inoculation of the Ambr®15 bioreactor vessels.

HEK293 suspension cells were transfected with a molar ratio between the respective two or three component linear synthetic DNA vector systems 4:3, 4:1 (first vector: second vector) or 4:3:3 (first vector: second vector: third vector). A DNA concentration of 0.52 pg/vc or 0.42 pg/vc and a PEIpro®:DNA ratio of 2:1 were applied. DNA and PEIpro® were diluted separately in fresh, supplemented BalanCD HEK293 medium. The PEIpro®-mix was added to the DNA-mix and incubated for 20 ± 2 min at room temperature. The transfection mix was added per bioreactor vessel with a final volume of 5 % of the total volume. Control transfections were performed in parallel utilizing the two- plasmid system from which the two-component linear synthetic DNA vector system of the invention is derived from, (described in EP24175911.7) and the three plasmid system from which the three component linear synthetic DNA vector system of the invention is derived from.

Cells were cultured until 72 h post transfection to allow for rAAV production, harvested in the medium and lysed by three freeze-thaw cycles at -80°C and 37°C, respectively. Cell debris was removed by centrifugation for 10 min at 4700 x g and room temperature before transferring the supernatants to new vials. The clarified freeze-thaw lysates following nuclease treatment were subjected to one-step affinity purification or used directly for subsequent analytical procedures.

Quantification of rAAV vector genomes by ddPCR

The AAV vector genome assay is based on a droplet digital PCR (ddPCR) specific for a transgene sequence of the rAAV expression cassette.

Cell lysate test samples were subjected to a nuclease treatment procedure to remove non-packed vector genomes prior to performing the ddPCR. To that aim, Denarase® (c-LEcta GmbH, Leipzig, Germany) was added to the samples with a final concentration of 20 U/mL. Incubation was performed over night with shaking at room temperature. Afterwards, the samples were centrifuged for 10 min at 4700 x g and room temperature. One-step affinity purified test samples were directly applied to the ddPCR procedure. The samples were either stored at -80°C until performance of the ddPCR or used directly for the analytical procedure. To control for the quality of the ddPCR, a trending control with known rAAV vector genome titre was measured in parallel. To check for contaminations, a no template control (NTC, EB) was also included.

Per reaction, 10 pl ddPCR™ EvaGreen Supermix (Bio-Rad Laboratories Inc., Hercules, USA) were mixed with 1 pL of primer mix (containing 2 pM of each primer) resulting in a reaction master mix volume of 11 pL per reaction. For the test samples, the optimal copy number per reaction is between 10000 and 40000. Thus, samples and trending control were diluted accordingly in elution buffer. The specific working range of this method was determined to be 150 - 120000 copies per reaction. Three dilutions each (in duplicates) were prepared for each sample and trending control using EB buffer (10 mM Tris- Cl, pH 8.5). Per reaction, 9 pL of test sample or trending control dilution were mixed with 11 pl of the reaction master mix to result in the total reaction volume of 20 pL. Samples were partitioned with droplet generator oil (Bio-Rad Laboratories Inc., Hercules, USA) using a droplet generator (Bio-Rad Laboratories Inc., Hercules, USA) according to manufacturer's instructions.

PCR reaction was performed in a C1000 Touch Thermal Cycler 96 Deep Well (Bio-Rad Laboratories Inc., Hercules, USA) with following program steps: 95°C 5 min; 40 cycles (95°C 30 s, 60°C 1 min); 4°C 5 min; 90°C 5 min; 8°C 30 min. A ramp rate of 2°C per second was applied. Droplet readout was performed using a QX200 or QX600 droplet reader (Bio-Rad Laboratories Inc., Hercules, USA). The cycled droplets are analysed using microfluidics to identify droplets which are positive and negative for the respective template/fluorophore (end point assay). From the overall droplet number and the positive and negative droplet numbers the Bio-Rad QX Manager software calculates the initial DNA concentration based on Poisson statistics.

Quantification of rAAV capsids by Gyrolab xPlore rAAV capsids were quantified using the automated immunoassay platform Gyrolab xPlore™ (Gyros Protein Technologies AB, Uppsala, Sweden) in conjunction with the Gyrolab® AAVX Titer Kit for AAV serotype 3B. The sandwich immunometric technique of the generic Gyrolab® AAVX Titer Kit used to capture and detect AAV capsids of serotype 3B is based on the Thermo Scientific™ CaptureSelect™ Biotin Anti-AAVX Conjugate and Thermo Scientific CaptureSelect Alexa Fluor™ 647 Anti-AAVX Conjugate from Thermo Fisher Scientific (Waltham, Massachusetts, USA). A serial dilution of in-house control capsids was applied to interpolate AAV capsid concentrations in the test samples. A negative control was included to check for contamination of reagents. Denarase® treated cell lysates, purified virus test samples and AAV control were diluted in Gyrolab® AAVX Titer Sample Dilution Buffer. All samples and controls were tested in duplicate. Data were analyzed using the Gyrolab® Evaluator Software Version 3.7.2.5976.

Vector genome to total particle ratio

The ratio of vector genomes to total AAV particles is expressed as a percentage. This is based on the vector genome titre (determined by ddPCR, as described above) and the number of total AAV particles (determined by Gyrolab xPlore™, as described above).

Quantification of plasmid-derived impurities by ddPCR (droplet digital PCR)

Prokaryotic DNA sequences, such as antibiotic resistance genes or parts of them originating from the bacterial backbone of the producer plasmids, can be packaged into the rAAV particles, constituting product-related impurities. Plasmid-derived impurity quantification is based on experiments using droplet digital PCR (ddPCR) techniques specific for defined sequences of the kanamycin resistance gene (kanR), present on both helper and vector plasmid, and the AAV cap gene, which is present on the vector plasmid. Plasmid-derived impurity ddPCRs were performed on one-step, spin-protocol based affinity chromatography purified rAAV material. The spin-procedure was performed using POROS™ CaptureSelect™ AAVX Affinity Resin (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Duplex droplet digital PCR (2D ddPCR) enables the simultaneous quantification of plasmid-derived AAV cap gene impurities and kanamycin resistance gene (kanR) impurities as well as the calculation of percentage cap/kanR impurities based on the respective vector genome titres of purified AAV samples.

Per reaction, 10 pL 2x ddPCR Supermix for Probes (no UTP) (Bio-Rad Laboratories Inc., Hercules, USA) were mixed with 1 pL of primer mix A (containing 18 pM of each primer), 1 pL of primer mix B (containing 18 pM of each primer), 1 pL of probe A (containing 5 pM of a FAM fluorophore-labelled probe) and 1 pL of probe B (containing 5 pM of a HEX fluorophore-labelled probe) resulting in a reaction master mix volume of 14 pL per reaction. To control for the quality of the ddPCR, a trending control with known rAAV vector genome titre was measured in addition to the test samples. To check for contaminations, a no template control (NTC, EB) was also included. For the test samples, the optimal copy number per reaction is between 20000 and 40000. Thus, samples and trending control were diluted accordingly in elution buffer. The specific working range of this method was determined to be 189 - 120000 copies per reaction for AAV cap and 83 - 120000 copies per reaction for kanR. Three dilutions each (in duplicates) were prepared for each sample and trending control using EB buffer (10 mM Tris-CI, pH 8.5). Per reaction, 6 pL of test sample or trending control dilution were mixed with 14 pl of the reaction master mix to result in the total reaction volume of 20 pL. Samples were partitioned with droplet generator oil (Bio-Rad Laboratories Inc., Hercules, USA) using a droplet generator (Bio-Rad Laboratories Inc., Hercules, USA) according to manufacturer's instructions. PCR reaction was performed in a C1000 Touch Thermal Cycler 96 Deep Well (Bio-Rad Laboratories Inc., Hercules, USA) with following program steps: 95°C 10 min; 40 cycles (94°C 30 s, 60°C 1 min); 98°C 10 min. A ramp rate of 2°C per second was applied. Droplet readout was performed using the QX200 or QX600 droplet reader (Bio-Rad Laboratories Inc., Hercules, USA). The cycled droplets are analysed using microfluidics to identify droplets which are positive and negative for the respective template/fluorophore (end point assay). From the overall droplet number and the positive and negative droplet numbers the Bio-Rad QX Manager software calculates the initial DNA concentration based on Poisson statistics.

96-well absolute potency assay

Transduction was performed in a 96-well tissue culture plate seeded with HuH-7 cells at 2.5 x 10⁴ vc/well (manual cell count using a “Neubauer” chamber; vc = viable cells). rAAV for transduction were prepared using the method according to “Transfection of HEK293 suspension cells in an AMBR®15 bioreactor system and preparation of cell lysates” using synthetic DNA or the respective plasmid DNA. The products of the transfection step were nuclease treated and then purified with one-step spinprotocol based affinity chromatography. Buffer exchange was then performed in a buffer that was suitable for cell-based assays. Transduction occurred 5 h after the cells were seeded. Transduction was performed at multiplicity of infection (MOI) of 5.56 x 10³ and 2.78 x 10³ with three replicates per sample and a no transduction control (no virus). The MOI were based on vector genome titre, which were determined as set out above under the heading “Quantification of rAAV vector genomes by ddPCR”. After a production phase of 96 h, the supernatant was harvested. The marker activity in the cell supernatant was determined by a chemiluminescence activity assay.

Experiment B (i): Comparison of a two-component linear synthetic DNA system to a control two-plasmid system.

In order to assess the functional properties of a two-component linear synthetic DNA system of the invention, HEK293 cells were cultivated according to the method for “cell cultivation” outlined above. Transfection of HEK293 suspension cells in an AMBRO15 bioreactor system was performed according to the method for “Transfection of HEK293 suspension cells in an AMBR®15 bioreactor system and preparation of cell lysates” using a molar ratio of 4:3 (first vector: second vector) and a DNA concentration of 0.52 pg DNA/cell. The molar ratio of the respective plasmids of the control two-plasmid system was also 4:3. The first vector has the nucleic acid sequence of SEQ ID NO: 10, while the second vector has the nucleic acid sequence of SEQ ID NO: 13 and encodes for an AAV3B capsid and contains a SEAP gene marker sequence (said second vector for generic use with any transgene of interest, without the gene marker sequence, has the nucleic acid sequence of SEQ ID NO:15). The products of the transfection step were assayed as described in “Quantification of rAAV vector genomes by ddPCR”, “Quantification of rAAV capsids by Gyrolab xPlore”, “Quantification of plasmid-derived impurities by ddPCR", “96-well absolute potency assay".

Results:

As it is shown in from Figure 21 A, vector genome yields of the two-component linear synthetic DNA system were in a similar range compared to the reference two plasmid system. As apparent from Figure 21 B, comparable capsid yields were observed for the two-component linear synthetic DNA system and the reference two plasmid system. Figure 21 C shows that plasmid derived impurities originating from the bacterial kanamycin resistance gene, present in the control two-plasmid system, but not present in the synthetic DNA vectors, were drastically reduced for the two-component linear synthetic DNA system in comparison to the reference plasmid setup. As apparent from Figure 21 D, application of the two- component linear synthetic DNA system resulted in similar transgene activity compared to the reference plasmid system. Left column shows values for the control plasmid system and right column values for the tested two component, linear synthetic DNA vector system.

Experiment B (ii): Comparison of a three-component linear synthetic DNA system to a control three- plasmid system.

In order to assess the functional properties of a three-component linear synthetic DNA system of the invention, HEK293 cells were cultivated according to the method for “cell cultivation” outlined above. Transfection of HEK293 suspension cells in an AMBRO15 bioreactor system was performed according to the method for “Transfection of HEK293 suspension cells in an AMBR®15 bioreactor system and preparation of cell lysates” using a molar ratio of 4:3:3 (first vector: second vector: third vector) and a DNA concentration of 0.52 pg DNA/cell. The molar ratio of the respective plasmids of the control three- plasmid system was also 4:3:3. The first vector has the nucleic acid sequence of SEQ ID NO: 10, the second vector has the nucleic acid sequence of SEQ ID NO: 15 and encodes for an AAV3B capsid, while the third vector has the nucleic acid sequence of SEQ ID NO:17 and contains a SEAP gene marker sequence as a transgene.

The products of the transfection step were assayed as described in “Quantification of rAAV vector genomes by ddPCR”, “Quantification of rAAV capsids by Gyrolab xPlore”, “Quantification of plasmid- derived impurities by ddPCR”, “96-well absolute potency assay”.

Results: As it is shown in Figure 22A, vector genome yields of the three-component linear synthetic DNA system were in a similar range compared to the reference three- plasmid system. Figure 22B shows that the capsid yields were comparable between the three-component linear synthetic DNA system and the reference three- plasmid system. As it can be seen in Figure 22C, plasmid derived impurities originating from the bacterial kanamycin resistance gene, present on the vector and helper plasmid of the control plasmid system, but not present in the three-component linear synthetic DNA vector, were drastically reduced for the three-component linear synthetic DNA system compared to the reference three- plasmid system. As apparent, in Figure 22D, application of two-component linear synthetic DNA system resulted in similar transgene activity compared to the reference plasmid system. Left column shows values for the control plasmid system and right column values for the tested three component, linear synthetic DNA vector system.

Experiment B (Hi): Comparison of a two-component linear synthetic DNA system to a control two- plasmid system.

In order to assess the functional properties of a two-component linear synthetic DNA system of the invention, HEK293 cells were cultivated according to the method for “cell cultivation” outlined above. Transfection of HEK293 suspension cells in an AMBRO15 bioreactor system was performed according to the method for “Transfection of HEK293 suspension cells in an AMBR®15 bioreactor system and preparation of cell lysates” using a molar ratio of 4:1 (first vector: second vector) and a DNA concentration of 0.52 pg DNA/cell. The molar ratio of the respective plasmids of the control two-plasmid system was 4:3. The first vector has the nucleic acid sequence of SEQ ID NO: 12, while the second vector has the nucleic acid sequence of SEQ ID NO: 13 and encodes for an AAV3B capsid and contains a SEAP gene marker sequence (said second vector without the gene marker sequence has the nucleic acid sequence of SEQ ID NO:15 for use with any transgene of interest). The products of the transfection step were assayed as described in “Quantification of rAAV vector genomes by ddPCR”, “Quantification of rAAV capsids by Gyrolab xPlore”, “Quantification of plasmid-derived impurities by ddPCR”, “96-well absolute potency assay”.

Results:

As it is shown in Figure 23A, vector genome yields of the two-component linear synthetic DNA system were in a similar range compared to the reference two plasmid system. As apparent from Figure 23B, comparable capsid yields were observed for the two-component linear synthetic DNA system compared to the reference two plasmid system. Figure 23C shows the that the vector genome to total capsid ratio of the two-component linear synthetic DNA system appears to be in a similar range compared to the reference two plasmid system. As apparent from Figure 23D, plasmid derived impurities originating from the AAV capsid gene, located on the second vector and the vector plasmid respectively were in a similar range for the two systems. Figure 23E shows that plasmid derived impurities originating from the bacterial kanamycin resistance gene, present in the plasmids but not present in the synthetic DNA vectors, were drastically reduced for the two-component linear synthetic DNA system. Left column shows values for the control plasmid system and right column values for the tested two component, linear synthetic DNA vector system.

Claims

1. A linear, synthetic DNA vector system comprising at least a first linear synthetic DNA vector and a second linear synthetic DNA vector, wherein said first vector comprises at least one rep gene encoding at least one functional Rep protein and does not comprise a cap gene encoding a functional set of Cap proteins.

2. The linear synthetic DNA vector system of claim 1 , wherein said second vector comprises:

(a) a cap gene encoding at least one functional Cap protein; or

(b) at least one promoter driving cap gene expression, preferably a cap gene promoter, a cloning site operably linked to said promoter

3. The linear synthetic DNA vector system of claim 1 , wherein said second vector comprises:

(a) a cap gene encoding at least one functional Cap protein; or

(b) at least one promoter driving cap gene expression, preferably a cap gene promoter, a cloning site operably linked to said promoter, and an expression cassette flanked on at least one side by an ITR; wherein said second vector does not comprise a rep gene encoding a functional Rep protein and the expression cassette comprises a transgene operably linked to at least one regulatory control element.

4. The linear synthetic DNA vector system of claim 1 or 2, wherein said second vector comprises:

(a) a cap gene encoding at least one functional Cap protein; or

(b) at least one promoter driving cap gene expression, preferably a cap gene promoter, a cloning site operably linked to said promoter, and does not comprise a rep gene encoding a functional Rep protein; and wherein said system further comprises a third linear synthetic DNA vector comprising an expression cassette flanked on at least one side by an ITR and said expression cassette comprises a transgene operably linked to at least one regulatory control element.

5. The linear synthetic DNA vector system of any one of claims 1 to 3, wherein said first vector comprises a nucleic acid sequence with at least 90% sequence identity with any one of SEQ ID NOs: 10, 11 , 12, and wherein said second vector comprises a nucleic acid sequence with at least 90% sequence identity with any one of SEQ ID NOs: 13, 14, 15, 16, preferably a nucleic acid sequence with at least 90% sequence identity with any one of SEQ ID NOs: 15, 16.

6. The linear synthetic DNA vector system of any one of claims 1 to 2, 4, 5, further comprising a third vector wherein said third vector comprises an expression cassette flanked on at least one side by an ITR.

7. The synthetic DNA vector system of any one of claims 1 to 6, wherein the, at least one gene encodes a functional Rep52 and/or Rep 40 protein and at least one gene encoding a functional Rep78 and/or Rep68 protein.

8. The synthetic DNA vector system of any one of claims 1 to 7, wherein said first vector further comprises at least one helper virus gene, optionally wherein:

9. The synthetic DNA vector system of any one of claims 1 to 8, wherein said second vector comprises a cap gene and the cap gene encodes a Cap protein selected from any naturally occurring or genetically engineered AAV serotype.

10. Use of the linear synthetic DNA vector system of any one of claims 1 to 9 for producing a AAV preparation:

(a) having a desired ratio of full to total particles; and/or

(b) at a high or desired yield; and/or

(c) having reduced or completely eliminated undesirable DNA sequences

11. Use of the linear synthetic DNA vector system of any one of claims 1 to 10 for:

(c) reducing or completely eliminating undesirable DNA sequences

12. The use of claim 10 or 11 , wherein the use comprises transfecting a host cell with vector system of any one of claims 1-10 and culturing the host cell under conditions suitable for recombinant AAV production.

13. A method for controlling or maximising the ratio of full to total particles produced during recombinant AAV production comprising:

- obtaining the linear synthetic DNA vector system of any one of claims 1-10; - transfecting a host cell with said system; and

14. A method for increasing, optimising or maximising the yield of recombinant AAV produced during recombinant AAV production comprising:

- obtaining the linear synthetic DNA vector system of any one of claims 1-10;

- transfecting a host cell with said system

15. A method of reducing or completely eliminating the presence of any undesirable DNA sequences during recombinant AAV production comprising:

- obtaining the linear synthetic DNA vector system of any one of claims 1-10;

- transfecting a host cell with said system