Plasmid for the production of adeno-associated virus (AAV)

Abstract

Adeno-associated virus (AAV) is a promising tool for gene therapy, but its high manufacturing costs, driven by the complexity of the traditional triple plasmid transfection process, limit its widespread use. Even though traditional AAV production have relied in the transfection of three plasmids encoding essential components, like the inverted terminal repeats (ITRs), with the Rep and Cap genes, and the helper genes; putting these elements together in a single plasmid can reduce process complexity. Thus, simplifying the AAV production process by using a single plasmid could reduce costs and streamline production, making gene therapies more accessible. The present application discloses a single-plasmid system for the production of a recombinant parvovirus in a host cells. Host cells and kits comprising the plasmid, as well as methods for producing recombinant parvovirus using the single-plasmid system, are also disclosed.

Description

[0001] PLASMID FOR THE PRODUCTION OF ADENO-ASSOCIATED VIRUS (AAV) CROSS REFERENCE TO RELATED APPLICATIONS

[0002] The present application claims the benefit of U.S. provisional patent application serial No. 63 / 760,789, filed on February 20, 2025, and U.S. provisional patent application serial No.

[0003] 63 / 889,656, filed on September 29, 2025, which are incorporated herein by reference in their entirety.

[0004] SEQUENCE LISTING

[0005] A sequence listing is submitted herewith as an XML file named 147851 -00007_Seq Listing.xml, created on February 20, 2026, and having a size of ~ 358509 bytes. The content of the aforementioned file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] The present disclosure generally relates to production of recombinant viruses, and more particularly to the production of recombinant parvoviruses, such as adeno-associated viruses (AAVs).

[0008] BACKGROUND ART

[0009] Adeno-associated virus (AAV) has emerged as one of the most efficient delivery tools for gene therapy applications, offering great potential in the treatment of various genetic diseases. However, a key challenge to the widespread adoption of AAV in gene therapy is the high manufacturing cost, which arises primarily from the complexity of the production process. Traditionally, the recombinant manufacturing of AAV is carried out by triple plasmid transfection in HEK-293 cells. In this process, the essential viral genes required for AAV packaging are distributed across three separate plasmids: one carrying the Rep and Cap genes, another containing the Adenovirus helper genes, and a third plasmid holding the gene of interest (GOI) flanked by the inverted terminal repeats (ITRs).

[0010] The rationale behind using separate plasmids for these components stems from safety concerns, specifically to reduce the likelihood of generating replication-competent AAV (rcAAV), which could pose risks in gene therapy applications. While this strategy has been successful in maintaining safety, it adds complexity and cost to the manufacturing process. Interestingly, recent research has shown that the risk of generating rcAAV is not significantly increased when the ITRs are placed on the same plasmid as the Rep and Cap genes, challenging the necessity of the traditional triple-plasmid approach.

[0011] Given the high manufacturing costs and the need for greater simplicity, there is an urgent need to streamline the AAV production process. A promising approach is to combine and optimizeall the necessary components into a single plasmid. This approach would significantly reduce the complexity of the transfection step, requiring only one plasmid rather than three. By minimizing the number of plasmids, this method not only reduces the time and labor involved in production but also lowers costs, which is crucial for making gene therapies more accessible. Simplifying the AAV production process could thus lead to significant advancements in the efficiency and scalability of gene therapy manufacturing, offering a more cost-effective alternative for clinical applications.

[0012] Single-plasmid approaches are disclosed, e.g., in US 2004 / 0209364 A1, EP 4242316 A1, and US 2024 / 0229067. However, these plasmids do not include a specific set of genes and sequence able to increase significantly AAV viral genomes and functional viral titers.

[0013] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

[0014] SUMMARY OF THE INVENTION

[0015] In various aspects and embodiments, the present disclosure provides the following items 1 to 37:

[0016] 1. An expression plasmid for the production of a recombinant parvovirus, comprising:

[0017] (i) an adenovirus E4 coding sequence that encodes an E4 open reading frame 6 (E4orf6) protein and an E4 open reading frame 6 / 7 (E4orf6 / 7) protein;

[0018] (ii) an adenovirus virus-associated (VA) RNA coding sequence that encodes an adenovirus VAI RNA;

[0019] (iii) an adenovirus early region 2A (E2a) coding sequence that encodes an E2a protein; (iv) an adenovirus L4 coding sequence that encodes L4 proteins;

[0020] (v) a recombinant parvovirus sequence comprising (1) an expression cassette comprising a cloning site for insertion of a nucleotide sequence encoding a gene of interest operably linked to a regulatory element, and (2) at least one inverted terminal repeat (ITR) at one end of the expression cassette;

[0021] (vi) a parvovirus protein coding sequence that encodes parvovirus proteins necessary for the production of the recombinant parvovirus; and

[0022] (vii) one or more regulatory elements that allow expression of the E4orf6 and E4orf6 / 7, the adenovirus VA RNA, the E2a protein, the L4 proteins, the capsid proteins, and the parvovirus proteins necessary for the production of the recombinant parvovirus in a host cell.

[0023] 2. The expression plasmid of item 1, wherein the recombinant parvovirus is a recombinant adeno-associated virus (AAV), and wherein the parvovirus protein coding region comprises an AAV Rep coding region encoding a AAV Rep protein and an AAV Cap coding region encoding an AAV Cap protein.3. The expression plasmid of item 2, wherein the AAV Rep protein is Rep2.

[0024] 4. The expression plasmid of item 3, wherein the AAV Rep coding sequence comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in FIG.

[0025] 17J (SEQ ID NO:20).

[0026] 5. The expression plasmid of item 4, wherein the AAV Rep coding sequence comprises the nucleotide sequence set forth in FIG. 17J (SEQ ID NO:20).

[0027] 6. The expression plasmid of any one of items 2 to 5, wherein the AAV Cap protein is Cap6.

[0028] 7. The expression plasmid of item 6, wherein the AAV Cap coding sequence comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in FIG.

[0029] 17K (SEQ ID NO:21).

[0030] 8. The expression plasmid of item 7, wherein the AAV Cap coding sequence comprises the nucleotide sequence set forth in FIG. 17K (SEQ ID NO:21).

[0031] 9. The expression plasmid of any one of items 1 to 8, wherein the adenovirus VAI RNA coding sequence comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in FIG. 171 (SEQ ID NO:19).

[0032] 10. The expression plasmid of item 9, wherein the adenovirus VAI RNA coding sequence comprises the nucleotide sequence set forth in FIG. 171 (SEQ ID NO: 19).

[0033] 11. The expression plasmid of any one of items 1 to 10, wherein the coding sequence that encodes E4orf6 comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in FIG. 17G (SEQ ID NO:17).

[0034] 12. The expression plasmid of item 11 , wherein the coding sequence that encodes the E4orf6 protein comprises the nucleotide sequence set forth in FIG. 17G (SEQ ID NO:17).

[0035] 13. The expression plasmid of any one of items 1 to 12, wherein the coding sequence that encodes the E4orf6 / 7 protein comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in FIG. 17H (SEQ ID NO: 18).

[0036] 14. The expression plasmid of item 13, wherein the coding sequence that encodes the E4orf6 / 7 protein comprises the nucleotide sequence set forth in FIG. 17H (SEQ ID NO: 18). 15. The expression plasmid of any one of items 1 to 14, wherein the E2A protein is an adenovirus DNA-binding protein (DBP).

[0037] 16. The expression plasmid of item 15, wherein the E2A coding sequence comprises a sequence having at least 90% sequence identity with one of the nucleotide sequences set forth in FIGs. 17A-C (SEQ ID NOs:11-13).

[0038] 17. The expression plasmid of item 16, wherein the E2A coding sequence comprises one of the nucleotide sequences set forth in FIGs. 17A-C (SEQ ID NOs:11-13), preferably SEQ ID NO:12.

[0039] 18. The expression plasmid of any one of items 1 to 17, wherein the L4 proteins comprise an L4-22K protein and an L4-33K protein.19. The expression plasmid of item 18, wherein the L4 coding sequence comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in FIG.

[0040] 17E (SEQ ID NO:15).

[0041] 20. The expression plasmid of item 19, wherein the L4 coding sequence comprises the nucleotide sequence set forth in FIG. 17E (SEQ ID NO: 15).

[0042] 21. The expression plasmid of any one of items 18 to 20, wherein the L4 proteins further comprise an L4-100K protein.

[0043] 22. The expression plasmid of item 21 , wherein the L4 coding sequence further comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in FIG.

[0044] 17D (SEQ ID NO:14).

[0045] 23. The expression plasmid of item 22, wherein the L4 coding sequence further comprises the nucleotide sequence set forth in FIG. 17D (SEQ ID NO: 14).

[0046] 24. The expression plasmid of any one of items 18 to 23, wherein the L4 proteins further comprise a pre-hexon-linking protein III (pVIII).

[0047] 25. The expression plasmid of item 24, wherein the L4 coding sequence further comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in FIG.

[0048] 17F (SEQ ID NO:16).

[0049] 26. The expression plasmid of item 25, wherein the L4 coding sequence further comprises the nucleotide sequence set forth in FIG. 17F (SEQ ID NO: 16).

[0050] 27. The expression plasmid of any one of items 1 to 26, wherein the recombinant parvovirus sequence comprises an ITR at each end of the expression cassette.

[0051] 28. The expression plasmid of any one of items 1 to 27, wherein the expression plasmid has less than 21000 base pairs, less than 20000 base pairs, less than 19000 base pairs or less than 18000 base pairs.

[0052] 29. The expression plasmid of any one of items 1 to 28, wherein the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with one of the sequences set forth in SEQ ID NOs: 31-37.

[0053] 30. The expression plasmid of any one of items 1 to 29, wherein the expression cassette comprises the nucleotide sequence encoding the gene of interest.

[0054] 31. A host cell comprising the expression plasmid of any one of items 1 to 30.

[0055] 32. The host cell of item 31 , which is a mammalian cell.

[0056] 33. The host cell of item 32, which is an HEK293 cell.

[0057] 34. A method for producing recombinant parvovirus comprising:

[0058] (a) introducing the expression plasmid of any one of items 1 to 30 into a host cell;

[0059] (b) incubating the host cell of (a) under suitable conditions for production of recombinant parvovirus particles; and(c) harvesting the recombinant parvovirus particles after the incubation period

[0060] 35. The method of item 34, wherein the host cell is as defined in item 32 or 33.

[0061] 36. A kit comprising the expression plasmid of any one of items 1 to 30; and instructions for use of the expression plasmid for producing a recombinant parvovirus.

[0062] 37. The kit of item 36, further comprising reagents necessary for host cell transformation or transfection.

[0063] 38. A recombinant parvovirus vector produced using the plasmid of any one of items 1 to 30 or the kit of item 36 or 37, or produced by the method of item 34 or 35.

[0064] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0065] BRIEF DESCRIPTION OF DRAWINGS

[0066] In the appended drawings:

[0067] FIG. 1 shows an exemplary plasmid construct named pHelper-RC6-GFP according to an embodiment of the present disclosure that contains all elements for AAV production system.

[0068] FIG.2 shows an exemplary plasmid construct illustrating the pV1 with reduced backbone for single-plasmid AAV production system.

[0069] FIG. 3 shows an exemplary plasmid construct illustrating pV2 with only known essential genes for single-plasmid AAV production system.

[0070] FIG.4 shows an exemplary plasmid construct illustrating pV3 with reduced helper genes for single-plasmid AAV production system.

[0071] FIG.5A shows an exemplary helper plasmid construct with reduced backbone containing only optimized essential Adenovirus helper genes for AAV production.

[0072] FIG. 5B shows an exemplary plasmid construct illustrating pV4 with reduced helper genes and Rep2 / Cap sequences of AAV2 for single-plasmid AAV production system.

[0073] FIG. 5C shows an exemplary plasmid construct illustrating pV4 with reduced helper genes and Rep2 / Cap sequences of AAV6 for single-plasmid AAV production system.

[0074] FIG. 5D shows an exemplary plasmid construct illustrating pV4 with reduced helper genes and Rep2 / Cap sequences of AAV9 for single-plasmid AAV production system.

[0075] FIG. 5E shows an exemplary plasmid construct illustrating pV5.

[0076] FIG. 5F shows an exemplary plasmid construct illustrating pV6-CO1 comprising a first codon-optimized sequence (CO1) of the DBP gene.

[0077] FIG. 5G shows an exemplary plasmid construct illustrating pV6-CO2 comprising a second codon-optimized sequence (CO2) of the DBP gene.

[0078] FIG.6 depicts a graph showing the production of rAAV in HEK293 suspension cells using the triple and the single plasmid system.FIGs. 7A-C depict graphs showing the production of rAAV in HEK293 suspension cells using the triple and the single plasmid system with pV1, pV2, and pV3 single plasmids. FIG. 7A:

[0079] Viral genomes per mL (VG / mL). FIG. 7B: transducing units per mL (TU / mL). FIG. 7C: Viral genomes per transducing units (VG / TU). Plasmid DNA concentration used was of 1 pg / mL forthe single plasmid and 1.5 pg / mL for the triple. T riple and pV2 (n=3), pV1 and pV3 (n=2).

[0080] FIGs. 8A-D depict graphs showing a comparison between the GFP expression levels obtained following the transduction of HEK293SF-3F6 suspension cells with rAAV6-GFP produced with triple and single plasmid transfection. FIG. 8A: Triple transfection: 39.6% of GFP+cells 24 hours post-transduction with AAV6-GFP with MOI of 28, and adenovirus with MOI of 3.

[0081] FIG. 8B: Single plasmid pV1: 43.9% of GFP+cells 24 hours post-transduction (hpt) with AAV6-GFP with MOI of 25, and adenovirus with MOI of 3. FIG. 8C: Single plasmid pV2: 46.6% of GFP+cells 24 hours post-transduction with AAV6-GFP with MOI of 34, and adenovirus with MOI of 3.

[0082] FIG. 8D: Single plasmid pV3: 56.7% of GFP+cells 24 hours post-transduction with AAV6-GFP with MOI of 50, and adenovirus with MOI of 3.

[0083] FIG. 9 depicts a graph showing the growth curve and viability during rAAV production using the triple and the single transfection with pV3 single plasmid with different plasmid DNA concentrations.

[0084] FIGs. 10A-B depict graphs showing the production of rAAV in HEK293 suspension cells in terms of viral genomes using the triple and the single plasmid system with pV3 single plasmid and different plasmid DNA concentrations (FIG. 10A, n=3). Comparison between production at 48 and 70 hpt for triple and single plasmid (0.75 pg / mL) (FIG. 10B, n=3).

[0085] FIGs. 11 A-B depict graphs showing the production of rAAV in HEK293 suspension cells in terms of functional particles using the triple and the single plasmid system with pV3 single plasmid and different plasmid DNA concentrations (FIG. 11 A, n=3). Comparison between production at 48 and 70 hpt for triple and single plasmid (0.75 pg / mL) (FIG. 11B, n=3).

[0086] FIG. 12 depicts a graph showing the production of rAAV in VPC 2.0 suspension cells in terms of viral genomes (VG / mL) and functional titer (transducing units, TU / mL) using the triple and the single plasmid system with pV3 single plasmid and different plasmid DNA concentrations.

[0087] FIG. 13A depicts a graph showing the production of rAAV in VPC 2.0 suspension cells in terms of viral genomes (VG / mL) using the triple and the single plasmid system with different pV4 single plasmids (pV4-GFP-RC2, pV4-GFP-RC6, and pV4-GFP-RC9).

[0088] FIG. 13B shows the results of a mass photometry analysis of the percentage of full and empty viral particles from AAV9 production with single plasmids and triple plasmid transfection.

[0089] FIG. 13C depicts a graph showing the viral genome titers for AAV2-GFP production using the single plasmid version pV5, pV6-CO1, and pV6-CO2.

[0090] FIGs. 14A-14F show the nucleotide sequence of the pV1 plasmid construct depicted in FIG. 2 (SEQ ID NO:1).FIGs. 15A-15F show the nucleotide sequence of the pV2 plasmid construct depicted in FIG. 3 (SEQ ID NO:2).

[0091] FIGs. 16A-16E show the nucleotide sequence of the pV3 plasmid construct depicted in FIG. 4 (SEQ ID NO:3).

[0092] FIGs. 17A-17M show the nucleotide sequences of genes present in the pV3, pV4, pV5 and pV6 plasmid constructs described herein. FIG. 17A: E2A-DBP (SEQ ID NO: 11); FIG. 17B:

[0093] E2A-DBP-CO1 (SEQ ID NO: 12); FIG. 17C: E2A-DBP-CO2 (SEQ ID NO: 13); FIG. 17D: Hexon assembly - L4 100K (SEQ ID NO:14); FIG. 17E: 22 / 33k (SEQ ID NO:15); FIG. 17F: pVIII (SEQ . 17G: E4orf6 (SEQ ID NO:17); FIG. 17H: E4orf6 / 7 (SEQ ID NO:18); FIG. 171: VA NO:19); FIG. 17J: Rep2 (SEQ ID NO:20); FIG. 17K: Cap6 (SEQ ID NO:21); FIG.

[0094] Q ID NO:22); FIG. 17M: Cap9 (SEQ ID NO:23).

[0095]

[0096] 18A-18C show the nucleotide sequence of the helper plasmid construct depicted in FIG. 5A (SEQ ID NO:4).

[0097] FIGs. 19A-19E show the nucleotide sequence of the pV4-GFP-RC2 plasmid construct depicted in FIG. 5B (SEQ ID NO:5).

[0098] FIGs. 20A-20E show the nucleotide sequence of the pV4-GFP-RC6 plasmid construct depicted in FIG. 5C (SEQ ID NO:6).

[0099] FIGs. 21A-21E show the nucleotide sequence of the pV4-GFP-RC9 plasmid construct depicted in FIG. 5D (SEQ ID NO:7).

[0100] FIGs. 22A-22E show the nucleotide sequence of the pV5 plasmid construct depicted in FIG. 5E (SEQ ID NO:8).

[0101] FIGs. 23A-23E show the nucleotide sequence of the pV6-CO1 plasmid construct depicted in FIG. 5F (SEQ ID NO:9).

[0102] FIGs. 24A-24E show the nucleotide sequence of the pV6-CO2 plasmid construct depicted in FIG. 5G (SEQ ID NQ:10).

[0103] FIGs, 25A-25D show an alignment of E2A coding sequence for the DBP gene from Adenovirus serotype 5 (DBP1 , SEQ ID NO:11), and the codon-optimized sequences CO1 (DBP2, SEQ ID NO:12), and CO2 (DBP3, SEQ ID NO:13) (Clustal 0(1.2.4) multiple sequence alignment).

[0104] DISCLOSURE OF INVENTION

[0105] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0106] The terms "comprising", "having", "including", and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0107] The use of any and all examples, or exemplary language (“e.g.”, "such as") provided herein, is intended merely to better illustrate embodiments of the claimed technology and does not pose a limitation on the scope unless otherwise claimed.

[0108] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology.

[0109] Herein, the term "about" has its ordinary meaning. The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).

[0110] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.

[0111] Where features or aspects of the disclosure are described in terms of Markush groups or list of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member, or subgroup of members, of the Markush group or list of alternatives.

[0112] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0113] Unless otherwise indicated, the recombinant protein, cell culture, molecular biology, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).The current application is based on the identification of non-essential adenovirus regions in traditional plasmids to obtain a plasmid with reduced size, leading to improved production of viral genomes and functional viral particles.

[0114] Accordingly, in a first aspect, the present disclosure provides an expression plasmid for the production of a recombinant parvovirus, comprising:

[0115] (i) an adenovirus E4 coding sequence that encodes an E4 open reading frame 6 (E4orf6) protein and an E4 open reading frame 6 / 7 (E4orf6 / 7) protein;

[0116] (ii) an adenovirus virus associated (VA) RNA coding sequence that encodes an adenovirus VAI RNA;

[0117] (iii) an adenovirus early region 2A (E2A) coding sequence that encodes an E2A protein;

[0118] (iv) an adenovirus L4 coding region that encodes L4 proteins;

[0119] (v) a recombinant parvovirus sequence comprising (1) an expression cassette comprising a cloning site for insertion of a nucleotide sequence encoding a gene of interest operably linked to a regulatory element, and (2) at least one inverted terminal repeat (ITR) at one end of the expression cassette;

[0120] (vi) a parvovirus protein coding sequence that encodes parvovirus proteins necessary for the production of the recombinant parvovirus; and

[0121] (vii) one or more regulatory elements that allow expression of the E4orf6 and E4orf6 / 7, the adenovirus VA RNA, the E2a protein, the L4 proteins, the capsid proteins, and the parvovirus proteins necessary for the production of the recombinant parvovirus in a host cell.

[0122] The expression plasmid described herein allows for production of a recombinant parvovirus by transfecting a host cell with a single plasmid, i.e. the plasmid is expression configured such that, upon introduction of the plasmid into a host cell, the host cell produces recombinant parvovirus (e.g., rAAV) particles encapsidating a vector genome without cotransfection of a separate rep / cap plasmid or a separate helper plasmid.

[0123] The term “coding sequence” as used herein refers to a nucleic acid sequence that encodes a particular protein or functional nucleic acid, such as an RNA (e.g., a non-coding RNA such as a viral-associated (VA) RNA). The nucleic acid sequence is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' terminal end and a translation stop codon at the 3' terminal end. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, as well as synthetic DNA sequences. A transcription termination sequence is usually located 3' to the coding sequence.In an embodiment, the expression plasmid has a size of 24000 base pairs or less, excluding the nucleotide sequence encoding the gene product of interest. In an embodiment, the expression plasmid has a size of 23500 base pairs or less, excluding the nucleotide sequence encoding the gene product of interest. In an embodiment, the expression plasmid has a size of 23000 base pairs or less, excluding the nucleotide sequence encoding the gene product of interest. In an embodiment, the expression plasmid has a size of 22500 base pairs or less, excluding the nucleotide sequence encoding the gene product of interest. In an embodiment, the expression plasmid has a size of 22000 base pairs or less, excluding the nucleotide sequence encoding the gene product of interest. In an embodiment, the expression plasmid has a size of 21500 base pairs or less, excluding the nucleotide sequence encoding the gene product of interest. In an embodiment, the expression plasmid has a size of 21000 base pairs or less, excluding the nucleotide sequence encoding the gene product of interest. In an embodiment, the expression plasmid has a size of 20500 base pairs or less, excluding the nucleotide sequence encoding the gene product of interest. In an embodiment, the expression plasmid has a size of 20000 base pairs or less, excluding the nucleotide sequence encoding the gene product of interest. In an embodiment, the expression plasmid has a size of 19500 base pairs or less, excluding the nucleotide sequence encoding the gene product of interest. In an embodiment, the expression plasmid has a size of 19000 base pairs or less, excluding the nucleotide sequence encoding the gene product of interest. In an embodiment, the expression plasmid has a size of 18000 base pairs or less, excluding the nucleotide sequence encoding the gene product of interest. In an embodiment, the expression plasmid has a size of 17000 base pairs or less, excluding the nucleotide sequence encoding the gene product of interest. In an embodiment, the expression plasmid has a size of 16000 base pairs or less, excluding the nucleotide sequence encoding the gene product of interest. In some embodiments the expression plasmid has a size in the range of 10000, 11000, 12000, 13000, 14000, or 15000 base pairs to 16000, 17000, 18000, 19000, 20000, 21000, 22000, 23000, 24000 or 25000 base pairs.

[0124] The expression plasmid according to the present disclosure does not include coding sequences that encode a functional adenovirus Hexon protein, a functional adenovirus plVa2 protein, a functional adenovirus pV protein, a functional adenovirus pVI protein, a functional adenovirus pVII protein, a functional adenovirus pIX protein, a functional adenovirus pX protein, a functional adenovirus penton base protein, a functional adenovirus pllla protein, a functional adenovirus VA RNA II, a functional adenovirus 52K protein, a functional adenovirus DNA polymerase protein, a functional adenovirus Fiber protein, a functional adenovirus DNA terminal protein, a functional adenovirus protein U (U exon), a functional adenovirus E4orf1 protein, a functional adenovirus E4orf2 protein, a functional adenovirus E4orf3 protein, and a functional E4orf4 protein. The expression plasmid may comprise sequences that encode only non-functional fragments of the above-noted proteins, or the sequences encoding the above-noted proteins maybe completely absent from the expression plasmid. In an embodiment, the sequences encoding one or more of the above-noted proteins are completely absent from the expression plasmid. In an embodiment, the sequences encoding all the above-noted proteins are completely absent from the expression plasmid.

[0125] The term “E4 open reading frame 6 (E4orf6) protein” refers to native E4orf6 proteins from adenoviruses as well as functional homologs and functional equivalents of native E4orf6 proteins. The terms “functional homologs” and “functional equivalents” as used herein refer to proteins that perform similar biological functions across different species or within the same organism, despite potentially having different structures (i.e., different amino acid sequences) or evolutionary origins. These proteins may or may not derived from a common ancestral gene, but they fulfill analogous roles in biological processes. Functional equivalents may be synthetic proteins obtained by introducing mutations (e.g., deletions, substitutions, etc.) in a native E4orf6 protein. Functional homologs and functional equivalents of native E4orf6 proteins maintain the ability to facilitate viral replication. Examples of E4orf6 proteins include the proteins described in UniProtKB Accession Nos. P03239, A0A0U4P939, A0A0U4XAG5, A0A0U5BLK0, A0A0U5BYK0, A0A127WK17, A0A1U9ALN6, A0A2H4PJA2, A0A3Q9HJC8, A0A3S9SPU8, A0A3S9SPY1, A0A3S9SQ43, A0A3S9SSA7, A0A481ZK45, A0A4P2SIG0, A0A513TZT9, A0A516UYM0, A0A5K6WAR8, A0A7D6XV13, A0A7L4WIJ4, A0A7L4WIR4, A0A7L4WJ23, A0A7M3SYU3, A0A7M3SYY1 , A0A9E7TWR6, A0AAF0I8L3, A0AAF0YXC7, A0AAF1BU25, A0AAF1C0D6, E1ARS8, E1U5Q7, J7IDM5, J9Z3F5, J9Z4R0, J9Z4V0, J9Z5D8, Q6VGT3, and T1UHK2. The E4orf6 protein may be derived from any of serotypes 1 to 52 of an adenovirus. In an embodiment, the E4orf6 protein is derived from adenovirus serotype 5 (Ad5) or Ad2. In an embodiment, the sequence that encodes the E4orf6 protein comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence set forth in FIG. 17G (SEQ ID NO: 17, Ad5) or SEQ ID NO:28 (Ad2). In an embodiment, the sequence that encodes the E4orf6 protein comprises the nucleotide sequence set forth in FIG. 17G (SEQ ID NO:17) or SEQ ID NO:28.

[0126] The term “E4 open reading frame 6 / 7 (E4orf6 / 7) protein” refers to native E4orf6 / 7 proteins from adenoviruses as well as functional homologs and functional equivalents of native E4orf6 / 7 proteins. The functional equivalents may be synthetic proteins obtained by introducing mutations (e.g., deletions, substitutions, etc.) in a native E4orf6 / 7 protein. Functional homologs and functional equivalents of native E4orf6 / 7 proteins maintain the ability to facilitate viral replication. Examples of E4orf6 / 7 proteins include the proteins described in UniProtKB Accession Nos. P03238, A0A0G2R237, A0A0P0LY41, A0A0U4XAB1, A0A0U5BPW3, A0A0U5CNF1, A0A127WK23, A0A1U9ALN2, A0A3G8WH84, A0A3Q9HJN1, A0A3Q9HKG4, A0A3Q9HKK6, A0A3Q9HLP3, A0A3S9SQW9, A0A3S9SR06, A0A4P2SHE1, A0A513TZT8, A0A516UYL3, A0A5K6WAT2, A0A6M4W5H0, A0A6M6ACK0, A0A7D6XT86, A0A7L4WGX0, A0A7L4WHV5,A0A7L4WID4, A0A7L4WII8, A0A7L4WIV8, A0A7L4WIZ3, A0A7L4WJF7, A0A7L4WKB2, A0A7M3SYU2, A0A8F9RAB4, A0A9E7TVZ2, A0AAF0I7R6, A0AAF0I8U3, A0AAF0Z020, A0AAF1BTF5, A0AAF1BU00, E1ARS7, E1U5Q6, J7I6W8, J9Z4Y3, J9Z5N2, J9Z5S3, and Q6VGT4. The E4orf6 / 7 protein may be derived from any of serotypes 1 to 52 of an adenovirus, for example Ad2 or Ad5. In an embodiment, the E4orf6 / 7 protein is derived from Ad5. In an embodiment, the sequence that encodes the E4orf6 / 7 protein comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence set forth in FIG. 17H (SEQ ID NO: 18, Ad5) orSEQ ID NO:29 (Ad2). In an embodiment, the sequence that encodes the E4orf6 protein comprises the nucleotide sequence set forth in FIG. 17H (SEQ ID NO:18) or SEQ ID NO:29.

[0127] The term “adenovirus E2A protein” as used herein refers to the protein encoded by the E2A region of an adenovirus genome. The adenovirus E2A protein is a DNA-binding protein (DBP) that plays a role in the elongation phase of viral strand displacement replication by unwinding the template in an ATP-independent fashion. As used herein, the term “adenovirus E2A protein” encompasses native adenovirus E2A proteins as well as functional homologs and functional equivalents of adenovirus E2A proteins. The functional equivalents may be synthetic proteins obtained by introducing mutations (e.g., deletions, substitutions, etc.) in native adenovirus E2A proteins. Functional homologs and functional equivalents of E2A proteins maintain the ability to bind DNA and to stimulate viral DNA replication and gene transcription. Examples of E2A proteins include the proteins described in UniProtKB Accession Nos. B9A5C2, B9A5N0, B9A5R6, C4P220, D4N3J2, E5RW13, G1FBX9, G3CK86, G9JUX0, H0PPG2, H5T755, H6WWL6, M0QTT9, M0QU16, M0QUC9, M0QUK7, M0QUY3, M0QV22, M0QV62, M0QVA2, M0QVR0, Q5TJ02, T1UGU8, T1UH02, T1UJ28, T1UJG7, T1UK28, T1UKX8, and W8CZB6. The E2A protein may be derived from any of serotypes 1 to 52 of an adenovirus. In an embodiment, the sequence that encodes the E2A protein comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with one of the nucleotide sequences set forth in FIGs. 17A-C (SEQ ID NOs:11-13, Ad5) orSEQ ID NO:24 (Ad2). In an embodiment, the sequence that encodes the E2A protein comprises one of the nucleotide sequences set forth in FIGs. 17A-C (SEQ ID NOs:11-13) or SEQ ID NO:24. In further embodiments, the sequence that encodes the E2A protein comprises one of the nucleotide sequences set forth in FIGs. 17B-C (SEQ ID NO:12 or 13), preferably SEQ ID NO:12.

[0128] The term “adenovirus VAI RNA” refers a type of non-coding RNA found in adenovirus that plays a role in regulating translation of mRNAs late after infection. As used herein, the term “adenovirus VAI RNA” encompasses native VAI RNA as well as functional homologs and functional equivalents of adenovirus VAI RNA. The functional equivalents may be synthetic RNAs obtained by introducing mutations (e.g., deletions, substitutions, etc.) in native VAI RNAs. Functional homologs and functional equivalents of native VAI RNAs maintain the ability toregulate translation of mRNAs late after infection. Examples of VAI RNA includes those described in RNAcentral Accession Nos. URS00028F278C_45659, URS00001CC771_1643649, URS0000202376_28280, URS00005DCFAB_52275, URS00005A5C13_3043599, URS00005A5C13_3088344, URS000290A029_10515, URS00028E7756_28282, URS00022C6907_10541 , and URS0000E5A95E_28285. The VAI RNA may be derived from any of serotypes 1 to 52 of an adenovirus, for example Ad2 or Ad5. In an embodiment, the sequence that encodes the VAI RNA comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence set forth in FIG. 171 (SEQ ID NO:19, Ad5) or SEQ ID NO:30 (Ad2). In an embodiment, the sequence that encodes the VAI RNA comprises the nucleotide sequence set forth in FIG. 171 (SEQ ID NO:19) or SEQ ID NQ:30.

[0129] The term “L4 proteins” as used herein refers to proteins encoded by the adenoviral late genes 4 and includes an L4-22K protein, L4-33K protein, an L4-100K protein and pre-hexon-linking protein VIII (pVIII).

[0130] In an embodiment, the L4 proteins comprise an L4-22K protein. The term “L4-22K protein” refers to native L4-22K proteins from adenoviruses as well as functional homologs and functional equivalents of native L4-22K proteins. The functional equivalents may be synthetic proteins obtained by introducing mutations (e.g., deletions, substitutions, etc.) in a native L4-22K protein. Functional homologs and functional equivalents of native L4-22K proteins maintain the ability to regulate expression of early and late adenoviral genes and / or promote the packaging of the viral genome. Examples of L4-22K proteins include the proteins described in UniProtKB Accession Nos. A0A1W2KFL8, A0A2L1F3C2, A0A2R3WN40, A0A2R3WNE0, A0A3G1S5X8, A0A3G9CMP5, A0A3G9JZX3, A0A3G9K3W8, A0A3G9K9B3, G9G859, Q2KSE3, Q2KSQ0, Q5GFA3, and Q6H1B9. The L4-22K protein may be derived from any of serotypes 1 to 52 of an adenovirus, for example Ad2 or Ad5.

[0131] In an embodiment, the L4 proteins comprise an L4-33K protein. The term “L4-33K protein” refers to native L4-33K proteins from adenoviruses as well as functional homologs and functional equivalents of native L4-33K proteins. The functional equivalents may be synthetic proteins obtained by introducing mutations (e.g., deletions, substitutions, etc.) in a native L4-33K protein. Functional homologs and functional equivalents of native L4-33K proteins maintain the ability to induce alternative splicing of late transcripts and / or promote the packaging of the viral genome. Examples of L4-33K proteins include the proteins described in UniProtKB Accession Nos. P24940, I6LEV2, I6LEV9, J7I5U1 , J7IA99, K7NFE9, Q2KRW6, Q2KS34, Q2KSA8, Q2KSI2, Q2KST3, Q2Y0H0, Q3ZKU0, Q4JEM9, Q5EY58, R4HL98, R4HLD8, R4HM45, R4HML1, T1UGQ6, T1UIT1, and T1UIX3. The L4-33K protein may be derived from any of serotypes 1 to 52 of an adenovirus, for example Ad2 or Ad5.In an embodiment, the sequence that encodes the L4-22K / 33K proteins comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence set forth in FIG. 17E (SEQ ID NO: 15, Ad5) or SEQ ID NO:26 (Ad2). In an embodiment, the sequence that encodes the L4-22K protein comprises the nucleotide sequence set forth in FIG. 17E (SEQ ID NO: 15) or SEQ ID NO:26.

[0132] In an embodiment, the L4 proteins comprise an L4-100K protein. The term “L4-100K protein” refers to native L4-100K proteins from adenoviruses as well as functional homologs and functional equivalents of native L4-100K proteins. The functional equivalents may be synthetic proteins obtained by introducing mutations (e.g., deletions, substitutions, etc.) in a native L4-100K protein. Functional homologs and functional equivalents of native L4-100K proteins maintain the ability to help hexon proteins assembly into trimers. Examples of L4-100K proteins include the proteins described in UniProtKB Accession Nos. A4ZKK4, A4ZKL9, B2VQF9, B6C6Z0, B6DUA6, B8XLH5, B9A5C3, B9A5F9, B9A5J5, B9A5N1, B9A5R7, B9A5Y9, B9A6D3, C4P221, D4N3J3, E1AI26, E1CIK6, E1CIP2, E1CIS7, E5RW14, E5RW85, E5RWC0, E9P5A1, F1DT73, F8UFR1, G1FBY0, G1FC17, G3CK87, G9JUX1, H0PPG3, H5T756, H6WWL7, K7ZJW7, M0QTU0, M0QU17, M0QU56, M0QU91, M0QUD0, M0QUG9, M0QUK8, M0QUP7, M0QUU4, M0QUY4, M0QV23, M0QV63, M0QVA3, M0QVE2, M0QVI2, M0QVM1 , M0QVR1, M0QW0, Q09TY1, Q4KSJ5, Q5TJ01 , T1 UDL3, T1 UFZ7, T1 UGU5, T1 UGY8, T1 UGZ1 , T1 UH47, T1 UH92, T1 UHC1 , T1UHI5, T1UI12, T1UIE4, T1UIZ6, T1UJH7, T1UJJ3, T1UJU8, T1UK37, T1UK66, T1UK82, T1UKC5, T1UKD8, T1UKP0, T1UL93, T1ULC3, T1ULS5, T1UM31, T1UMA6, W8CZ85, W8VZ34, X4Y9F3, and X4YFL5. The L4-100K protein may be derived from any of serotypes 1 to 52 of an adenovirus, for example from Ad2 or Ad5. In an embodiment, the sequence that encodes the L4-100K protein comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence set forth in FIG. 17D (SEQ ID NO:14, Ad5) or SEQ ID NO:25 (Ad2). In an embodiment, the sequence that encodes the L4-100K protein comprises the nucleotide sequence set forth in FIG.

[0133] 17D (SEQ ID NO:14) or SEQ ID NO:25.

[0134] In an embodiment, the L4 proteins comprise a pre-hexon-linking protein VIII (pVIII) protein. The term “pVIII protein” refers to native pVIII proteins from adenoviruses as well as functional homologs and functional equivalents of native pVIII proteins. The functional equivalents may be synthetic proteins obtained by introducing mutations (e.g., deletions, substitutions, etc.) in a native pVIII protein. Functional homologs and functional equivalents of native pVIII proteins maintain the ability to connect the core protein with the capsid. Examples of pVIII proteins include the proteins described in UniProtKB Accession Nos. P24936, A0A0U5BNW0, A0A0U5BX25, A0A0U5BZF5, A0A1 U9ALM1 , A0A3Q9HLK9, A0A3S9SS30, A0A513TZS8, A0A516UYK9, A0A6M6AAD0, A0A7D6XQT4, A0A7L4WGW1, A0A7L4WJF0, A0A9E7Q7E4, A0AAF0CWU6, A0AAF0YUB7, A0AAF0Z333, E1ARR7, E1U5P6, J7IDL8, J9Z5R3, P03280, Q6VGU2, Q71BW3,T1UG92, T1UM71, Q779D1, Q779D9, Q779E3, Q779E4, Q779F8, and Q779G6. The pVIII protein may be derived from any of serotypes 1 to 52 of an adenovirus, for example from Ad2 or Ad5. In an embodiment, the sequence that encodes the pVIII protein comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence set forth in FIG. 17F (SEQ ID NO: 16, Ad5) or SEQ ID NO:27 (Ad2). In an embodiment, the sequence that encodes the pVIII protein comprises the nucleotide sequence set forth in FIG. 17F (SEQ ID NO: 16) or SEQ ID NO:27.

[0135] In an embodiment, the parvovirus proteins necessary for the production of the recombinant parvovirus comprise a parvovirus Rep protein and a parvovirus Cap protein. The term “Rep protein” refers to native Rep proteins from parvoviruses as well as functional homologs and functional equivalents of native Rep proteins. The functional equivalents may be synthetic proteins obtained by introducing mutations (e.g., deletions, substitutions, etc.) in a Rep protein. Functional homologs and functional equivalents of native Rep proteins maintain the ability to facilitate viral replication and insertion of a viral genome into a host genome during latent infection. Examples of Rep proteins include the proteins described in UniProtKB Accession Nos. P03132, Q89268, Q89269, Q89270, A9RAH9, B5SUY6, C7FFR9, G1JYZ2, 041854, 056136, 056138, Q1I031, Q1I033, Q2LD61, Q5Y9B3, Q5Y9B5, Q65310, Q670Q9, Q670R1, Q670R3, Q670R5, Q670R7, Q6GWF3, Q6JL80, Q7TG44, Q8JQF9, Q8JQG1, Q9WBP7, Q9YJC1, and W8GKL5. The Rep proteins may be from any AAV, including but not limited to natural serotypes 1 , 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11 , or 13 and any other AAV serotypes or variants. In an embodiment, the Rep protein is from AAV2. In an embodiment, the sequence that encodes the Rep protein comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleotide sequence set forth in FIG. 17J (SEQ ID NO:20). In an embodiment, the sequence that encodes the Rep protein comprises the nucleotide sequence set forth in FIG. 17J (SEQ ID NQ:20).

[0136] The term “Cap protein” refers to native Cap proteins from parvoviruses as well as functional homologs and functional equivalents of native Cap proteins. The functional equivalents may be synthetic proteins obtained by introducing mutations (e.g., deletions, substitutions, etc.) in a Cap protein. Functional homologs and functional equivalents of native Cap proteins maintain the ability to package the viral genome. Examples of Cap proteins include the proteins described in UniProtKB Accession Nos. B4Y884, B4Y886, B4Y887, B4Y889, C7FFS0, Q5Y9B4, Q6GWF2, Q6JBZ2, Q6JBZ3, Q6JBZ8, Q6JC14, Q6JC15, Q6JC16, Q6JC18, Q6JC19, Q6JC37, Q6JC45, Q6JC47, Q6JC49, Q6JC50, Q6JC53, Q6JC54, Q6JC55, Q6JC56, Q6JC57, Q6JC60, Q6JC61, Q6JC62, Q808V7, Q808V8, Q808W5, Q808X1 , Q808X3, Q8JQF8, W8GGS2, B4Y888, Q6JC13, Q6JC46, Q6JC51, Q6JC52, Q6JC58, Q6JC63, Q808W0, Q8JQG0, B4Y874, B4Y875, B4Y876, B4Y881, B4Y882, B4Y883, B4Y890, 056137, 056139, Q1I030, Q1I032, Q65311, Q6JC08, Q6JC10, Q6JC12, Q6JC22, Q6JC23, Q6JC24, Q6JC40, Q6JC59, Q6JL79, Q808Y3, Q9WBP8,B4Y877, B4Y880, P03135, Q670Q6, Q670Q7, Q670Q8, Q670R0, Q670R4, Q670R6, Q670R8, Q670S0, Q6JBZ0, Q6JBZ1, Q6JBZ4, Q6JBZ5, Q6JBZ6, Q6JBZ7, Q6JBZ9, Q6JC01, Q6JC05, Q6JC06, Q6JC07, Q6JC09, Q6JC11, Q6JC17, Q6JC20, Q6JC21, Q6JC25, Q6JC26, Q6JC27, Q6JC28, Q6JC29, Q6JC30, Q6JC31, Q6JC33, Q6JC34, Q6JC35, Q6JC36, Q6JC38, Q6JC39, Q6JC41, Q6JC42, Q6JC43, Q6JC44, Q6JC48, Q808V9, Q808X2, 041855, Q2VJ48, Q6JC00, Q6JC02, Q6JC03, Q6JC04, A0A513ZUK1, B4Y891, B5SUY7, Q5Y9B2, Q808W6, Q808W8, Q808X6, Q808X7, Q808X8, Q808X9, A5A0V7, A7UMZ4, A7UMZ7, A7UN00, B5LAD6, B5LAD8, G0ZSH7, G8EME6, G8EME8, G8EMF0, G8EMF2, H9M8D1, M1U1K6, Q65444, Q67666, Q6R5J0, Q6R5P9, Q6R967, Q6TY07, Q6TY08, Q6TY09, Q6TY10, Q6TY11 , Q6TY12, Q6TY13, Q6TY14, Q6TY15, Q6TY16, Q6TY17, Q83289, Q83290, Q8V395, S4V558, T1TCQ8, T1TD63, Q6JC64, Q6JC65, Q6JC66, Q808W9, Q808X0, B4Y885, Q808Y0, Q808Y1 , Q808Y2, B4Y878, B4Y879, Q808W1, Q808W2, Q808W3, Q808W4, Q808W7, Q808X4, M4IQQ4, M4IRL1, M4ISG9, Q5XXZ6, Q6V7U2, E0XJJ5, Q9YIJ1, G1JYZ3, G1JYZ5, C0LA97, M4IS86, and Q2VJ52. The Cap proteins may be from any AAV, including but not limited to natural serotypes 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11 , or 13 and any other AAV serotypes or variants. In an embodiment, the Cap protein is from AAV2, AAV6 or AAV9, preferably AAV9. In an embodiment, the sequence that encodes the Cap protein comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the one of the nucleotide sequences set forth in FIGs. 17K-M (SEQ ID NOs:21-23) , preferably SEQ ID NO:23. In an embodiment, the sequence that encodes the Cap protein comprises one the nucleotide sequences set forth in FIGs. 17K-M (SEQ ID NOs:21-23), preferably SEQ ID NO:23.

[0137] In an embodiment, at least one coding sequence is codon optimized for expression in a host cell, e.g., a mammalian host cell. Codon optimization refers to the process of redesigning a gene’s DNA sequence so it uses codons that are preferred by a chosen host organism, while keeping the amino acid sequence of the encoded product (e.g., protein) the same. Typically, codon optimization aims to improve expression by matching the host’s codon usage bias (use of more frequently translated codons, adjusting GC content and avoiding problematic sequence patterns, removing / avoiding mRNA secondary structures, cryptic splice sites, premature poly(A) signals, repeats, or unwanted restriction sites and / or reducing risks of ribosome stalling or poor translation due to rare codons. Representative codon-optimized versions of the E2A-DBP gene are described herein (SEQ ID NO :12 and 13), but the skilled person would understand that codon-optimized versions of any of the genes / sequences described herein could be designed by the skilled person and are encompassed by the present disclosure.

[0138] The term “parvovirus” refers to a family of animal viruses that constitute the family Parvoviridae. They have linear, single-stranded DNA (ssDNA) genomes that typically contain two genes encoding for a replication initiator protein, called NS1, and the protein the viral capsid is made of. The coding portion of the genome is flanked by an inverted terminal repeat (ITR) at eachend that form into hairpin loops that are important during replication. Parvovirus virions are small compared to most viruses, at 23-28 nanometers in diameter, and contain the genome enclosed in an icosahedral capsid that has a rugged surface. The parvovirus family contains three subfamilies and 126 species, including adeno-associated virus (AAV), bocavirus and protoparvovirus. In an embodiment, the recombinant parvovirus is a recombinant AAV.

[0139] In this context, the term "adenovirus" is used interchangeably with "adenovirus vector" and denotes a member of the Adenoviridae family. The Adenoviridae family encompasses all animal adenoviruses within the Mastadenovirus genus. Specifically, human adenoviruses are categorized into subgenera A-F, which include various serotypes. Subgenera A-F comprise human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Ad11A and Ad11P), 12, 13, 14, 15, 16, 17, 18, 19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, and 51.

[0140] The term “adeno-associated virus” (AAV) as used herein includes without limitation AAV type 1 , AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, caprine AAV, porcine AAV, ovine AAV and any other AAV now known or later discovered serotypes and variants.

[0141] The terms “regulatory element” or “regulatory sequence” as used herein refer to promoters, enhancers, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), and the like, which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell. It is not necessary for all of these regulatory sequences to be present, as long as the chosen coding sequence can be replicated, transcribed, and translated within a suitable host cell.

[0142] The term “promoter” as used herein refers to a DNA regulatory sequence to which RNA polymerase binds, initiating transcription of a downstream (3' direction) coding sequence. Moreover, these terms should be interpreted broadly to include additional regulatory elements, such as enhancer regions, intron splice donors and acceptors, other 5' untranslated regions, and similar components. A promoter can be either homologous or heterologous to the target gene. The field offers a wide array of known promoters for use in this context, encompassing a variety of viral and mammalian promoters. Promoters that are selective for certain cell types or specific tissues can be employed to target or enhance gene sequence expression in particular cell populations compared to others. Promoters can be constitutively active, conditionally active, or inducible, depending on the cell type. Viral promoters include, for example, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); adenovirus E4 promoter, adenovirus E2a promoter, herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter region (CMV-IE),SFFV promoter, and Rous Sarcoma Virus (RSV) promoter. Mammalian promoters include, for example, those for expressing EF1a, ubiquitin (e.g., ubiquitin B or C), globin, actin, phosphoglycerate kinase (PGK), NSE (neuronal specific enolase), synapsin or NeuN, as well as composite promoters, such as the CAG promoter (combination of the CMV early enhancer element and chicken beta-actin promoter). In an embodiment, the plasmid comprises a p5 promoter operably linked to the Rep coding sequence.

[0143] The term “enhancer” as used herein refers to a polynucleotide sequence which acts on the activity of a promoter and thus stimulates the transcription of a gene or coding sequence functionally connected to this promoter. Unlike promoters, enhancers exert their effect regardless of position and orientation, allowing them to be placed before or after a transcription unit, within an intron, or even within the coding region. Enhancers can be located in close proximity to the transcription unit or at a significant distance from the promoter. There can also be a physical and functional overlap with the promoter. Numerous enhancers from various sources are known in the art, which may be found in databases such as GenBank (e.g., SV40 enhancers, CMV enhancers, polyoma enhancers, adenovirus enhancers, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE)). These enhancers are available as standalone elements or within cloned polynucleotide sequences (e.g., available from ATCC or other commercial and individual sources). Additionally, some promoters, such as the commonly used CMV promoter, contain enhancers. In an embodiment, the plasmid comprises a CMV enhancer operably linked to the gene of interest. In an embodiment, the plasmid comprises a WPRE operably linked to the gene of interest, e.g., in the 3' UTR.

[0144] The term "operably linked" as used herein refers to the functional relationship between regulatory elements such as promoters and enhancers and a gene of interest such that the regulatory elements can regulate the transcription of the gene. Specifically, it means that the regulatory element(s) is / are positioned relative to the gene in a way that allows it / them to initiate and control the transcription process effectively. This relationship does not necessarily require the regulatory elements to be immediately adjacent to the gene, as long as they can still influence gene expression. The concept ensures that the genetic elements are configured to perform their intended biological functions within a cell.

[0145] The term "inverted terminal repeats" or "ITRs" refers to short, palindromic nucleotide sequences found at both ends of a DNA molecule, such as a viral genome. These sequences are characterized by their ability to form secondary structures due to their palindromic nature, meaning that they read the same backward as forward. In the context of adeno-associated viruses (AAV), ITRs play a role in the replication and packaging of the viral genome. They serve as origins of DNA replication and are involved in the integration and excision of the viral DNA within host genomes. The nucleotide sequences of AAV ITR regions are well-established in the field. As used herein, an ITR may correspond to native ITRs from viruses (e.g., AAV), or to variants thereofcomprising nucleotide insertion(s), deletion(s), and / or substitution(s) and that maintain the ability to mediate the replication and packaging of the viral genome. Moreover, the ITR may originate from various AAV serotypes, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, etc. Additionally, the 5' and 3' ITRs flanking a selected nucleotide sequence in an AAV vector do not necessarily need to be identical or derived from the same AAV serotype or isolate, provided they perform their intended functions, namely, allowing for the excision and rescue of the sequence of interest from a host cell genome or vector, and enabling the integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell. In some embodiments, the ITRs comprise sequence modifications that reduce recombination during bacterial propagation while retaining function in replication and packaging.

[0146] "Identity" refers to sequence identity between two polypeptides or nucleic acids. Percent (%) sequence identity with respect to a reference polypeptide or nucleotide sequence is the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference polypeptide or nucleotides sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid or nucleotide sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLASTp, BLAST-2, CLUSTALW, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0147] In certain embodiments, the expression construct described herein comprises one or more nucleic acids that encode one or more codon-optimized proteins for expression in cells such as human, mammalian, or primate cells, including HuH7, HEK293T, or CHO cells. The nucleic acids encoding the proteins described herein may undergo codon optimization to enhance their activity, stability, or expression in host cells, while maintaining the original amino acid sequence. Codon optimization involves substituting codons in a nucleotide sequence with preferred codons that encode the same amino acid, such as those favored or optimal for mammalian expression, thereby preserving the amino acid sequence. This process can be carried out using gene optimization software. Codon optimization techniques are well-established in the art.

[0148] In some embodiments, the expression construct further comprises one or more additional elements, such as plasmid replication origin sequences and selectable markers. Examples of selectable markers include, but are not limited to, ampicillin resistant genes, chloramphenicol resistant genes, tetracycline resistant genes, neomycin resistant genes,blasticidin resistant genes, hygromycin resistant genes, puromycin resistant genes, zeocin resistant genes, and kanamycin resistant genes.

[0149] The term “gene of interest” as used herein refers to a nucleotide sequence encoding a gene product of interest. The term “gene product” as used herein refers to the biochemical material, either RNA or protein, that is the result of gene expression. The gene product may be a functional RNA molecule, such as a guide RNA, a ribosomal RNA (rRNA), transfer RNA (tRNA), or microRNA (miRNA) which is not translated into protein but exhibits biological activity, or a peptide, polypeptide or protein such as a therapeutic protein, an antibody or antibody fragment, an enzyme, a receptor, a secreted factor, or a nuclease (e.g., Cas). The term "gene product" encompasses all these end products of gene expression.

[0150] In an embodiment, the expression plasmid has the configuration or architecture according to any one of FIGs. 2-4 and 5B-5G. The term “configuration” or “architecture” as used herein refers to the physical form and topology (shape / arrangement) of the plasmid molecule, layout (relative arrangement and orientation) of the functional DNA elements on the plasmid, i.e., how parts are positioned with respect to one another on the circular map, including the order of elements around the plasmid, the orientation / strand direction of each feature, the positioning relative to key landmarks (e.g., origin of replication (ori), selectable marker(s), restriction sites, and / or regulatory elements).

[0151] In an embodiment, the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with one of the sequences set forth in SEQ ID NOs: 5-10 and 31-37. In an embodiment, the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the sequence set forth in SEQ ID NO: 5. In an embodiment, the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the sequence set forth in SEQ ID NO: 5. In an embodiment, the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the sequence set forth in SEQ ID NO:6. In an embodiment, the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the sequence set forth in SEQ ID NO:7. In an embodiment, the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the sequence set forth in SEQ ID NO:8. In an embodiment, the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the sequence set forth in SEQ ID NO:9. In an embodiment, the expression plasmid comprises a sequence having at least 95%,96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the sequence set forth in SEQ ID NO: 10. In an embodiment, the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the sequence set forth in SEQ ID NO:31. In an embodiment, the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the sequence set forth in SEQ ID NO:32. In an embodiment, the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the sequence set forth in SEQ ID NO:33. In an embodiment, the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the sequence set forth in SEQ ID NO:34. In an embodiment, the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the sequence set forth in SEQ ID NO:35. In an embodiment, the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the sequence set forth in SEQ ID NO:36. In an embodiment, the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the sequence set forth in SEQ ID NO:37.

[0152] In another aspect, the present disclosure provides a host cell, such as a cell line, comprising the expression plasmid described herein. In an embodiment, the host cell is stably transformed with the expression plasmid described herein.

[0153] In another embodiment, the present disclosure provides a kit for producing recombinant AAV particles, the kit comprising the expression plasmid described herein. In some embodiments, the kit further comprises instructions for use, e.g., instructions for use in a method described herein. In some embodiments, the kit further comprises one or more tubes or other types of containers. In some embodiments, the kit further comprises various reagents, such as solutions, buffers, enzymes, etc., for example reagent for transfection of the expression plasmid into a host cell.

[0154] The present application further relates to methods for producing a recombinant parvovirus particle using the expression plasmid described herein. The method comprises the step of introducing the expression plasmid described herein into a host cell, incubating the host cell harboring the expression cassette for a desired period of time and under suitable conditions to produce recombinant parvovirus particles, and harvesting recombinant parvovirus particles after the incubation period. In some embodiments, the parvovirus is AAV. The method may furthercomprise harvesting cells and / or supernatant, purifying the parvovirus (e.g., rAAV) using an appropriate method or combination thereof (e.g., affinity chromatography, ion exchange, ultracentrifugation, filtration);

[0155] A host cell for producing parvovirus particles can contain the expression plasmid described herein in the form of e.g., episomal plasmids. The expression plasmid described herein may also be stably integrated into the host cell genome. Examples of host cells encompass, but are not limited to, microorganisms, yeast cells, insect cells, and animal cells. In certain embodiments, the host cell is a mammalian host cell, such as human HuH7 and HEK293 cells, Chinese hamster ovary cells ("CHO"), and baby hamster kidney ("BHK") cells. Suitable mammalian cells for implementing the present application include, among others: COS cells (e.g., ATCC No. CRL 1650 or 1651), BHK cells (e.g., ATCC No. CRL 6281), CHO cells (ATCC No. CCL 61), HeLa cells (e.g., ATCC No. CCL 2), 293 cells (ATCC No. 1573), CHOP, HuH7, A549, HeLa, HEK293, NIH3T3, HT1080, Vero, and NS-1 cells. In an embodiment, the host cell expresses the adenoviral E1A and E1B-55k proteins. In an embodiment, the host cell is an HEK293 cell, such as Gibco™ Viral Production Cells 2.0 (VPCs 2.0). The term "host cell" encompasses the progeny of the original cell that has been transfected. It is acknowledged that the progeny of a single parental cell may not be completely identical in morphology or in genomic or total DNA content to the original parent, due to natural, accidental, or intentional mutations. A "mammalian host cell" refers to a cell originally derived from a mammal or its progeny.

[0156] The expression plasmid described herein may be transfected into the host cell using any methods known in the art. As used herein, the terms "transformation", "transduction", and "transfection" are interchangeably used with each other, unless specifically noted otherwise, and refer to introduction of a nucleic acid into a host cell (via a virus or a virus vector as required). Any transformation method can be used as long as it is a method for introducing a nucleic acid into a host cell. Examples thereof include various well-known techniques such as use of a cell that has been made competent, electroporation method, method using a particle gun (gene gun), CaPO4-mediated transfection, or using lipids or polymeric molecules such as Polyethylenimine (PEI).

[0157] The AAV vectors produced using the plasmids and methods described herein may be used as gene therapy vectors in vivo or used to modify cells ex vivo for cell-based therapies. Gene transfer involves the delivery of new genetic information into cells. Genetic information may be transiently or stably inserted into cells. The AAV vectors produced using the plasmids and methods described herein are used for gene transfer. Thus, the AAV vectors produced using the plasmids and methods described herein may be capable of non-site-specific or site-specific integration into a mammalian chromosome without substantial cytotoxicity, and which direct host cell-specific expression of a therapeutic polynucleotide. Preferably the AAV vectors produced using the plasmids and methods described herein are used in gene therapy in mammals, more preferably humans.In an embodiment, the expression plasmids and methods described herein permits to obtain yields of at least 1x109viral genome copy per mL (VG / mL), for example at least 2x109, 3x109, 4x109, 5x109VG / mL, 6x109VG / mL, 7x109VG / mL, 8x109VG / mL, 9x109VG / mL, 1x1010VG / mL, 2x1010VG / mL, 3x1010VG / mL, 4x1010VG / mL, 5x1010VG / mL, 6x1010VG / mL, 7x1010VG / mL, 8x1010VG / mL, 9x1010VG / mL, or 1x1011VG / mL.

[0158] Table 1 : List of sequences according to the present disclosure

[0159]

[0160] MODE(S) FOR CARRYING OUT THE INVENTION

[0161] The present invention is illustrated in further details by the following non-limiting examples.Example 1: Materials and Methods

[0162] Generation of sinqle plasmid pHelper-RC6-GFP

[0163] Recombinant AAV (rAAV) production in HEK293 cells is usually performed using three different plasmids carrying genes for: AAV Rep and Cap; the gene of interest flanked by ITR sequences; and a set of helper genes, mainly coming from the adenovirus genome. The steps below were followed to generate a single plasmid containing all necessary genes to produce recombinant AAV:

[0164] 1) An insert containing Rep2Cap6 sequence was amplified by PCR from commercially available plasmid pDGM6 and later digested with restriction enzymes Not\ / Xba\. A Not\ / Xba\ digested kanamycin-resistant plasmid backbone (pShuttle) and the Rep2Cap6 insert were ligated with T4 DNA ligase generating the plasmid pRC6.

[0165] 2) An insert containing ITR-CAG-GFP-ITR sequence was digested from commercially available plasmid pAAV-CAG-GFP with restriction enzyme PvuW. The backbone, already containing Rep2Cap6 sequence, was digested with EcoRV. Digested pRC6 and the ITR- CAG-GFP-ITR insert were ligated with T4 DNA ligase, generating the plasmid pRC6- GFP.

[0166] 3) Finally, the pRC6-GFP plasmid was cloned by homologous recombination in a plasmid containing all genes for adenovirus 5 production in HEK293 cells using the pAdEasy system (Agilent), generating the pHelper-RC6-GFP (FIG. 1).

[0167] The plasmid pHelper-RC6-GFP contains the following adenovirus genes: Hexon, pV, pVI, pVII, pVIII, pIX, pX, penton base protein, pllla, plVa2, 52K, protease C5, DNApol, DNA terminal protein, sequences of E3, U exon, Fiber, E4orfs, DBP, Hexon assembly, 22 / 33k, VA RNA region, including VA RNA I and VA RNA II.

[0168] Generation of sinqle plasmids pV1, pV2, and pV3

[0169] To generate a single plasmid for production of recombinant AAV only, without possible contaminants from adenovirus proteins and reduce plasmid size, deletions of non-essential adenovirus helper genes were made.

[0170] Generation of pV1

[0171] Construction of Version 1 (pV1) (SEQ ID NO: 1, FIGs. 14A-14F): pHelper-RC6-GFP was digested with As / SI and Seal for the removal of non-essential genes from adenovirus, generating pV1 by Gibson assembly technique, and removing 14,220 bp (FIG. 2).

[0172] For pV1 , the following adenovirus genes were removed: complete sequences of Hexon, pV, pVI, pVII, pX, penton base protein, pllla, 52K; partial sequences of DNApol, and DNA terminalprotein. The deleted genes are non-essential genes for AAV production and can generate contaminants in the final product.

[0173] Generation of pV2

[0174] Construction of Version 2 (pV2) (SEQ ID NO: 2, FIGs. 15A-15F): pV1 was digested with Spel and Avril for the removal of genes around the E4 genes, generating pV2 by Gibson assembly, and removing 3,769 bp (FIG. 3).

[0175] For pV2, the following adenovirus genes were removed: complete sequences of E3, U exon, fiber, E4orf3, E4orf2, E4orf1; partial sequence of E4orf4. The E4 region was optimized by deleting adenovirus structural proteins, such as Fiber, that could generate contaminants in the final product. E4orf4 was removed since it autoregulates its transcription acting as an inhibitor of E1a activation of E4 genes. The known commercially available adenovirus helper plasmids, such as pXX6-80 and pAdDeltaF6, still contain adenovirus Fiber genes and E4orf1, 2, 3, and 4.

[0176] A construct similar to pV2 but without all the E4 genes (E4orf1-4, 6, and 6 / 7) was also generated. However, the construct demonstrated a significant decrease in viral production, 17-times lower than triple transient transfection control. Therefore, E4orf6 and E4orf6 / 7 were maintained as part of the single plasmid.

[0177] Generation of pV3

[0178] Construction of Version 3 (pV3) (SEQ ID NO: 3, FIGs. 16A-16E): pV2 was digested with As / SI and Hpa\ for the removal of non-essential sequences around the VA RNA region, generating pV3 by Gibson assembly, and removing 3,384 bp (FIG. 4).

[0179] For pV3, the VA RNA region was reduced to only VA RNA I (i.e., VA RNA II was deleted). The following adenovirus genes around the region were removed entirely: DNApol, plVa2, and pIX.

[0180] In summary, the plasmid pV3 contains unmodified adenovirus serotype 5 helper sequences of: E2a genes comprehending DBP, Hexon assembly, 22 / 33k, and pVIII; VA RNA I sequence; and E4 genes comprehending E4orf6 and E4orf6 / 7; comprising 8.4 kb. The AAV Rep and Cap portion of the pV3 plasmid contains unmodified Rep2 and Cap6 sequences of the commercially available pDGM6; comprising 4.5 kb. Finally, the cassette for the production of recombinant AAV present in the pV3 plasmid is the unmodified CAG-GFP sequence from the commercially available pAAV-CAG-GFP plasmid; comprising 2.9 kb. The final construct of the pV3 single plasmid is 20,853 bp in size, including sequences for origin of replication and antibiotic resistance genes for propagation in bacteria.Generation of Helper plasmid with reduced backbone and pV4

[0181] Helper plasmid containing only optimized essential Adenovirus helper genes for AAV production with reduced backbone was generated by digesting the pV3 with Nru\ for the removal of Rep / Cap, GFP cassette, and unnecessary backbone sequences, and reassembling. In summary, the Helper plasmid contains unmodified adenovirus serotype 5 helper sequences of: E2a genes comprehending DBP, Hexon assembly (100K), 22 / 33k, and pVIII; VA RNA I sequence; and E4 genes comprehending E4orf6 and E4orf6 / 7; and includes sequences for origin of replication and antibiotic resistance genes for propagation in bacteria. Restriction sites were included to facilitate cloning of Rep / Cap and GOI. The Helper plasmid construct is 11,397 bp in size (FIG. 5A). Single plasmids pV4 (version 4) were generated by cloning the GFP cassette into the pHelper with reduced backbone digested with Seal. Then, the addition of the Rep2 / Cap sequences of AAV2, AAV6, and AAV9 into pV4-GFP digested with Nru\ generated the following single plasmids: pV4-GFP-RC2, pV4-GFP-RC6, pV4-GFP-RC9, with a size of 18,812 bp, 18,916 bp, and 18,742 bp, respectively (FIGs. 5B-D, SEQ ID NOs:5-7).

[0182] Generation of pV5 sinqle plasmid

[0183] The Helper plasmid construct (11 ,397 bp in size) was digested with As / SI and Xmn\ restriction enzymes to remove unnecessary plasmid backbone and assembled again to introduce a short sequence containing restriction sites As / SI, Pac\, and Xmn\ between the regions of the VAI RNA and the origin of replication. Then, this construct (11 ,225 bp in size) was digested with Seal and had a GFP cassette, of 2,324 bp in size, cloned into it, resulting in 13,549 bp in size. Lastly, the construct was digested with Nru\ and Avril restriction enzymes to remove unnecessary backbone sequence and allow the assembly of the Rep2Cap2 cassette, resulting in the single plasmid construct version 5 (pV5) with 17,470 bp in size (FIGs. 5E and 22A-22E), smaller than pV4.

[0184] Generation of the pV6 sinqle plasmid

[0185] The pV5 single plasmid was digested with Fsel and Asci restriction enzymes to remove the DBP portion of the E2a gene originally from the adenovirus serotype 5 genome and assembled again to introduce the codon-optimized sequences (CO1 and CO2) of the DBP gene (FIGs. 17B, C). Alignment of the two codon-optimized sequences of DBP was done by comparing to the original sequence (FIGs.25A-D). The single plasmid constructs version 6 (pV6) with codon-optimized sequences pV6-CO1 (FIGs. 5F and 23A-23E) and pV6-CO2 (FIGs. 5G and 24A-24E) are 17,187 bp in size.AAV production by transient transfection

[0186] HEK293SF-3F6 (NRC) suspension cells were cultured in serum-free conditions, in HyCell™ TransFx-H medium (Cytiva) in 20 mL working volume in 125 mL shake flasks. PEI-25K (Polysciences) was used as the transfection reagent at a ratio of 2:1 PEI:DNA. Around 1 x 106cells / mL were transfected with a concentration of plasmid DNA of 1 g / mL, unless otherwise specified. Triple transient transfection was performed using pAdDeltaF6 helper plasmid, pRepCap6, and pAAV-CAG-GFP (Addgene) at equal molar plasmid ratios. Alternatively, VPC 2.0 suspension cells (ThermoFisher) were cultured in serum-free conditions, in VPM medium (Gibco) in 30 mL working volume in 125 mL shake flasks. AAVMax™ transfection kit (Gibco), including transfection reagent and booster, was used following the manufacturer’s recommended protocol. Around 3E6 cells / mL were transfected with a concentration of plasmid DNA of 1.5 pg / mL, unless otherwise specified. Alternatively, HEK293SF-3F6 (NRC) cells were cultured in serum-free conditions, in HyCell™ TransFx-H medium (Cytiva) in 5 mL working volume in T-25 flasks in static mode. PEI-25K (Polysciences) was used as the transfection reagent at a ratio of 3:1 PEI:DNA. Around 1.25 x 106cells / mL were transfected with a concentration of plasmid DNA of 1.8 pg / mL, unless otherwise specified.

[0187] Cells were collected 24-, 48-, 72-, or 96-hours post-transfections, cells were lysed using chemical lysis buffer and treated with endonuclease; crude lysate was centrifuged to pellet cell debris.

[0188]

[0189] Clarified crude lysate was used to determine viral genome content (VG / mL) by droplet digital PCR (ddPCR). Capsid productivity (VP / mL) was determined using a commercially available ELISA kit for AAV6 (Progen). The functional viral particles quantification (TU / mL) was determined using a cell-based assay where HEK293SF-3F6 cells are co-infected with dilutions of rAAV and Adenovirus at low MOI; 24 hours post-transduction, GFP positive cells are assessed via flow cytometry and the transducing units are determined.

[0190] Example 2: Results

[0191] Production of rAAV using the pHelper-RC6-GFP single plasmid was comparable to triple transient transfection using the three plasmids: pAdDeltaF6, pAAV-CAG-GFP, and pRepCap6 (Addgene). rAAV viral genomes (n=3), functional particles (n=2), and total particles (n=1) produced using the triple and the single transfection were evaluated (FIG. 6).

[0192] Productions of rAAV using the pV1, and pV2 after the reduction of the plasmid size and non-essential genes were similar to the triple transient transfection in terms of viral genome copy (VG / mL) measured by ddPCR and transducing unit (TU / mL). rAAV yields using pV3 were higher relative to the triple transient transfection (FIG. 7).Production of rAAV using a similar pV2 construct without E4orf6 and E4orf6 / 7 demonstrated significant reduction in titer (2.6 x 108VG / mL) relative to pV2 with E4orf6 and E4orf6 / 7 (1.3 x 109VG / mL) and triple transfection (4.6 x 109VG / mL).

[0193] Transduction of HEK293SF-3F6 suspension cells with AAV6-GFP produced from single plasmid transfection (pV1 , pV2, pV3) and from triple transfection was assessed (FIG.8). A higher percentage of transduced cells expressing GFP was obtained 24 hours after transfection with tested recombinant AAV produced with pV3 single plasmid, demonstrating the maintenance of the transduction capacity of the recombinant AAV.

[0194] Production of recombinant AAV was carried out using the single plasmid pV3 at various DNA concentrations and compared to triple transient transfection. Plasmid DNA concentrations of 1 pg / mL, 0.75 pg / mL, and 0.5 pg / mL were tested with the single plasmid and the control triple transient transfection was performed using 1 pg / mL of plasmid DNA. Higher viability at time of harvest was observed when using the single plasmid (FIG. 9). A higher viral genome titer was obtained using the single plasmid compared to the control (FIG. 10). A comparison of the triple transfection and the single transfection using 0.75 pg / mL demonstrated a 2-fold increase in viral genome titer for production harvested 48 hours post-transfection (hpt) and a 5-fold increase for production harvested 70 hours post-transfection (FIG. 10). Functional titer (transducing units) was determined by co-infecting HEK293SF-3F6 suspension cells with adenovirus with low MOI and rAAV produced with various concentrations of plasmids and harvested 48- and 70-hours posttransfection (FIG. 11). Production of functional rAAV particles was 2.3 times higher (48hpt) and 4.6 times higher (70hpt) than the control using 0.75 pg / mL of the pV3 single plasmid (FIG. 11).

[0195] Production of recombinant AAV6-GFP in VPC 2.0 cells using the AAVMax transfection kit was carried out with the single plasmid pV3 at various DNA concentrations and compared to triple transient transfection. Plasmid DNA concentrations of 1.5 pg / mL, 2 pg / mL, and 3 pg / mL were tested with the single plasmid, and the control triple transient transfection was performed using 1.5 pg / mL of plasmid DNA. A higher viral genome titer (VG / mL) was obtained using the single plasmid compared to the control. rAAV production using 3 pg / mL of the pV3 demonstrated an increase of up to 5 times in viral genome titer relative to control (FIG. 12). Functional titer (transducing units, TU / mL) was determined by co-infecting suspension cells with adenovirus with low MOI and rAAV produced with single and triple plasmids. Production of functional rAAV particles increased up to 1.6-fold using the single plasmid-produced rAAV relative to the control (FIG. 12).

[0196] Productions of rAAV using the single plasmids pV4-GFP-RC2, pV4-GFP-RC6, and pV4-GFP-RC9 in VPC 2.0 suspension cells using the AAVMax transfection kit were quantified. Improvement in viral yield is seen more significantly for AAV9 production with a 2-fold increase in viral genomes per mL (FIG. 13A).AAV9 productions from VPC 2.0 cells transfected with the single-plasmid construct pV4-GFP-RC9 and standard triple-plasmid transfection system were harvested, purified by affinity chromatography, and analyzed by mass photometry (Refeyn TwoMP) to determine the ratio of full to empty capsids. The single-plasmid transfection condition produced an approximately two-fold higher proportion of full capsids relative to the triple-plasmid transfection condition (FIG.13B).

[0197] Productions of rAAV using the single plasmids pV5, pV6-CO1, and pV6-CO2 in HEK293SF cells using the PEI 25K transfection reagent in static mode were harvested 96 hours post-transfection, and the viral genome titers were quantified. Improvement in viral yield is seen for production with pV6-CO1 relative to the pV5 (FIG. 13C).

[0198] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise.

Claims

WHAT IS CLAIMED IS:

1. An expression plasmid for the production of a recombinant parvovirus, comprising:(i) an adenovirus E4 coding sequence that encodes an E4 open reading frame 6 (E4orf6) protein and an E4 open reading frame 6 / 7 (E4orf6 / 7) protein;(ii) an adenovirus virus-associated (VA) RNA coding sequence that encodes an adenovirus VAI RNA;(iii) an adenovirus early region 2A (E2a) coding sequence that encodes an E2a protein; (iv) an adenovirus L4 coding sequence that encodes L4 proteins;(v) a recombinant parvovirus sequence comprising (1) an expression cassette comprising a cloning site for insertion of a nucleotide sequence encoding a gene of interest operably linked to a regulatory element, and (2) at least one inverted terminal repeat (ITR) at one end of the expression cassette;(vi) a parvovirus protein coding sequence that encodes parvovirus proteins necessary for the production of the recombinant parvovirus; and(vii) one or more regulatory elements that allow expression of the E4orf6 and E4orf6 / 7, the adenovirus VA RNA, the E2a protein, the L4 proteins, the capsid proteins, and the parvovirus proteins necessary for the production of the recombinant parvovirus in a host cell.

2. The expression plasmid of claim 1 , wherein the recombinant parvovirus is a recombinant adeno-associated virus (AAV), and wherein the parvovirus protein coding region comprises an AAV Rep coding region encoding a AAV Rep protein and an AAV Cap coding region encoding an AAV Cap protein.

3. The expression plasmid of claim 2, wherein the AAV Rep protein is Rep2.

4. The expression plasmid of claim 3, wherein the AAV Rep coding sequence comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in SEQ ID NO:20.

5. The expression plasmid of claim 4, wherein the AAV Rep coding sequence comprises the nucleotide sequence set forth in SEQ ID NO:20.

6. The expression plasmid of any one of claims 2 to 5, wherein the AAV Cap protein is Cap2, Cap6 or Cap9.

7. The expression plasmid of claim 6, wherein the AAV Cap coding sequence comprises a sequence having at least 90% sequence identity with one of the nucleotide sequences set forth in SEQ ID NOs:21-23.

8. The expression plasmid of claim 7, wherein the AAV Cap coding sequence comprises one of the nucleotide sequences set forth in SEQ ID NOs:21-23.

9. The expression plasmid of any one of claims 1 to 8, wherein the adenovirus VAI RNA coding sequence comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 19 or 30.

10. The expression plasmid of claim 9, wherein the adenovirus VAI RNA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 19 or 30.

11. The expression plasmid of any one of claims 1 to 10, wherein the coding sequence that encodes E4orf6 comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 17 or 28.

12. The expression plasmid of claim 11, wherein the coding sequence that encodes the E4orf6 protein comprises the nucleotide sequence set forth in SEQ ID NO: 17 or 28.

13. The expression plasmid of any one of claims 1 to 12, wherein the coding sequence that encodes the E4orf6 / 7 protein comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 18 or 29.

14. The expression plasmid of claim 13, wherein the coding sequence that encodes the E4orf6 / 7 protein comprises the nucleotide sequence set forth in SEQ ID NO: 18 or 29.

15. The expression plasmid of any one of claims 1 to 14, wherein the E2A protein is an adenovirus DNA-binding protein (DBP).

16. The expression plasmid of claim 15, wherein the E2A coding sequence comprises a sequence having at least 90% sequence identity with one of the nucleotide sequences set forth in SEQ ID NOs:11-13 and 24.

17. The expression plasmid of claim 16, wherein the E2A coding sequence comprises one of the nucleotide sequences set forth in SEQ ID NOs:11-13 and 24.

18. The expression plasmid of any one of claims 1 to 17, wherein the L4 proteins comprise an L4-22K protein and an L4-33K protein.

19. The expression plasmid of claim 18, wherein the L4 coding sequence comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in SEQ ID NO:15 or 26.

20. The expression plasmid of claim 19, wherein the L4 coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 15 or 26.

21. The expression plasmid of any one of claims 18 to 20, wherein the L4 proteins further comprise an L4-100K protein.

22. The expression plasmid of claim 21 , wherein the L4 coding sequence further comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in SEQ ID NO:14 or25.

23. The expression plasmid of claim 22, wherein the L4 coding sequence further comprises the nucleotide sequence set forth in SEQ ID NO: 14 or 25.

24. The expression plasmid of any one of claims 18 to 23, wherein the L4 proteins further comprise a pre-hexon-linking protein VIII (pVIII).

25. The expression plasmid of claim 24, wherein the L4 coding sequence further comprises a sequence having at least 90% sequence identity with the nucleotide sequence set forth in SEQ ID NO:16 or 27.

26. The expression plasmid of claim 25, wherein the L4 coding sequence further comprises the nucleotide sequence set forth in SEQ ID NO: 16 or 27.

27. The expression plasmid of any one of claims 1 to 26, wherein the recombinant parvovirus sequence comprises an ITR at each end of the expression cassette.

28. The expression plasmid of any one of claims 1 to 27, wherein the expression plasmid has less than 21000 base pairs, less than 20000 base pairs, less than 19000 base pairs, less than 18000 base pairs, less than 17000 base pairs, or less than 16000 base pairs.

29. The expression plasmid of any one of claims 1 to 28, wherein the expression plasmid comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with one of the sequences set forth in SEQ ID NOs: 31-37.

30. The expression plasmid of any one of claims 1 to 29, wherein the expression cassette comprises the nucleotide sequence encoding the gene of interest.

31. A host cell comprising the expression plasmid of any one of claims 1 to 30.

32. The host cell of claim 31 , which is a mammalian cell.

33. The host cell of claim 32, which is an HEK293.

34. A method for producing recombinant parvovirus comprising:(a) introducing the expression plasmid of any one of claims 1 to 30 into a host cell;(b) incubating the host cell of (a) under suitable conditions for production of recombinant parvovirus particles; and(c) harvesting the recombinant parvovirus particles after the incubation period35. The method of claim 31 , wherein the host cell is as defined in claim 33 or 34.

36. A kit comprising the expression plasmid of any one of claims 1 to 30; and instructions for use of the expression plasmid for producing a recombinant parvovirus.

37. The kit of claim 36, further comprising reagents necessary for host cell transformation or transfection.

38. A recombinant parvovirus vector produced using the plasmid of any one of claims 1 to 30 or the kit of claim 36 or 37, or produced by the method of claim 34 or 35.

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