Chimeric helper nucleic acid molecules containing helper elements from three different helper viruses

Abstract

The present invention relates to chimeric helper nucleic acid molecules containing helper elements from three different helper viruses, and to helper-free virus methods and systems for producing infectious recombinant adeno-associated viruses (rAAVs). The present invention is directed to chimeric helper nucleic acid molecules suitable for use in methods for producing infectious rAAVs, comprising one or more polynucleotides encoding adenovirus helper virus functions, including an E2A gene from adenovirus (Ad), an E4 gene from Ad, and a virus-associated ribonucleic acid (VA-RNA) polynucleotide from Ad; and one or more polynucleotides comprising an open reading frame (ORF) from a gene of herpes simplex virus (HSV), particularly a polynucleotide encoding at least the UL12 protein from HSV; and an ORF from a gene of human bocavirus (HBoV), particularly a polynucleotide encoding at least the NS2 protein from HBoV, wherein the polynucleotide encodes additional helper virus functions, and the polynucleotide further comprises non-endogenous sequences for controlling expression of the ORFs. The present invention also relates to packaging systems, rAAV producer cells, helper-free virus methods for producing rAAV producer cell lines, methods for producing replication-deficient infectious rAAV vector particles, and in vitro uses of rAAV producer cells in producing replication-deficient infectious rAAV vector particles.

Description

[Technical Field]

[0001] The present invention relates to chimeric helper nucleic acid molecules containing helper elements from three different helper viruses, and to helper-free virus methods and systems for producing infectious recombinant adeno-associated viruses (rAAVs). The present invention is directed to chimeric helper nucleic acid molecules suitable for use in methods for producing infectious rAAVs, comprising one or more polynucleotides encoding adenovirus helper virus functions, including an E2A gene from adenovirus (Ad), an E4 gene from Ad, and a virus-associated ribonucleic acid (VA-RNA) polynucleotide from Ad; and one or more polynucleotides comprising an open reading frame (ORF) from a gene of herpes simplex virus (HSV), particularly a polynucleotide encoding at least the UL12 protein from HSV; and an ORF from a gene of human bocavirus (HBoV), particularly a polynucleotide encoding at least the NS2 protein from HBoV, wherein the polynucleotide encodes additional helper virus functions, and the polynucleotide further comprises non-endogenous sequences for controlling expression of the ORFs. The present invention also relates to packaging systems, rAAV producer cells, helper-free virus methods for producing rAAV producer cell lines, methods for producing replication-deficient infectious rAAV vector particles, and in vitro uses of rAAV producer cells in producing replication-deficient infectious rAAV vector particles. [Background technology]

[0002] Genetic diseases are caused by missing or defective genes. Gene therapy aims to treat this condition by delivering functional copies of the affected gene to the patient's cells. Gene delivery can be carried out by recombinant viruses such as adeno-associated viruses (AAV). AAV is preferred due to its safety, low pathogenicity, and ability to infect multiple selective tissues.

[0003] Genetic defects are usually present in every cell in a patient's body. Therefore, to achieve therapeutic efficacy, AAVs for gene therapy must be delivered to the majority of cells in an organ. As a result, AAV-based treatments require the administration of very large amounts of virus. Producing sufficient AAVs at an acceptable cost without compromising safety is a major challenge in biological manufacturing. Increasing or improving AAV production will enable the treatment of a wide variety of genetic diseases. Optimization strategies are focusing on the genetic components required for AAV construction, producer cell lines, and omics-based approaches (Cell Culture Engineering and Technology, pp. 335–364, Chapter 1, Bio-Production of Adeno-Associated Virus for Gene Therapy).

[0004] In cultured cells, AAV replication requires coinfection with a helper virus. In the absence of helper virus coinfection, AAV can specifically integrate its genome into the AAVS1 region of chromosome 19. Subsequent helper virus infection releases the AAV genome from chromosome 19 in a process called rescue, allowing productive replication to occur. AAV genomes cloned into plasmid vectors can also serve to initiate productive AAV replication. When such constructs are transfected into cells and the cells are simultaneously or subsequently infected with a helper virus, the AAV genome is released from the plasmid (J. Virol., 2003, 77 (21), pp. 11480-11490).

[0005] AAV, a member of the Parvoviridae family, contains a single-stranded genome of approximately 4,700 bases. The AAV genome contains two genes: one encoding a nonstructural protein (Rep gene) and the other encoding a structural protein (Cap gene). The Rep gene encodes four Rep proteins (i.e., Rep78, Rep68, Rep50, and Rep42) that are primarily involved in viral DNA replication, capsid assembly, and transcription. The Cap gene encodes three proteins (VP1, VP2, and VP3) that are components of the mature capsid, a protein (assembly activator protein) involved in capsid assembly from the VP proteins, and finally, a recently discovered protein (membrane-bound accessory protein) involved in virus release.

[0006] The AAV life cycle depends on the presence or absence of a helper virus. Helper viruses induce active replication of the viral genome and the formation of new infectious AAVs, and are therefore necessary for the production of large quantities of rAAV. Initially, helper elements were provided by infectious adenoviruses used to transduce cells transfected with plasmids encoding the Rep and Cap genes. However, infectious adenoviruses were found in purified final rAAV vector stocks. To overcome this problem, minimal adenovirus helper elements were identified and cloned into plasmids, leading to helper virus-free production methods such as the so-called triple transfection method. Engineering and optimization of helper plasmids, which also depend on the AAV serotype, can dramatically affect rAAV production efficiency because some helper viruses are preferentially found in certain serotypes based on seroepidemiological data.

[0007] U.S. Patent No. 11,142,775 describes a helper virus-free method for producing rAAV or a helper virus-free method for preparing a helper virus-free chimeric RAAV / human bocavirus 1 (HBoV1).

[0008] Combining the NP1 and NS2 genes of HBoV1 with Ad helper genes to create a dual helper plasmid for rAAV vector production in a conventional three-plasmid transfection system has been reported ( Mol. Ther. 2018, 11, pp. 40-51 ).

[0009] U.S. Patent Application Publication No. US20220073947 discloses a circular nucleic acid comprising at least one AAV promoter that can be activated by at least one helper polypeptide or polynucleotide originating from or derived from a virus selected from the Adenoviridae and Herpesviridae families.

[0010] International patent application WO 2022 / 173944 discloses a method for producing AAV in E1 complementary producer cells, the method comprising transfecting the producer cells with one or more vectors comprising the E1A Ad helper gene; Ad helper genes selected from E2A, E4, or both; virus-associated non-coding RNA (VA RNA); and AAV genes selected from Rep, Cap, or both.

[0011] Examples of commercially available helper plasmids (pHelpers) are pALD-X80 from Aldevron, pALD-HELP from Aldevron, and VPK-402 from Cellbiolabs.

[0012] rAAV is currently the most widely used viral vector for in vivo gene therapy. rAAV production is based on cell culture and a defined set of genetic elements, including the transgene and the structural and nonstructural elements used in rAAV production.

[0013] Despite years of bioprocess optimization to improve both the quantity and quality of rAAV production processes, meeting the requirements of rAAV-based therapeutics at sufficient scale and cost remains a challenge. Therefore, it is an object of the present invention to provide a helper virus-free method for rAAV production, which enables the production of high-titer, high-quality rAAV. Another object of the present invention is to develop a versatile helper nucleic acid molecule (e.g., a helper plasmid (pHelper)) suitable for the production of various AAV serotypes and various producer cell lines. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] U.S. Patent No. 11,142,775 [Patent Document 2] U.S. Patent Application Publication No. US20220073947 [Patent Document 3] International Publication No. 2022 / 173944 [Patent Document 4] European Patent No. 3256583B1 [Non-patent literature]

[0015] [Non-Patent Document 1] Cell Culture Engineering and Technology, pp. 335-364, Bio-Production of Adeno-Associated Virus for Gene Therapy chapter [Non-patent document 2] J. Virol., 2003, 77 (21), pp. 11480-11490 [Non-patent document 3] Mol. Ther. 2018, 11, pp. 40-51 [Non-patent document 4] Wang et al., Mol. Ther. 2018, 11, pp. 40-51 [Non-patent document 5] Marie-Claude Geoffroy et al., Current Gene Therapy, 2005, 5 (3), pp. 265-271 Summary of the Invention

[0016] In this invention, the inventors describe the design and use of novel helper nucleic acid molecules and such helper plasmids for use in producing rAAV using a triple transfection method in mammalian cells. The novel helper nucleic acid molecules (e.g., helper plasmids) contain genes originating from three different helper viruses (herein, adenovirus, bocavirus, and herpesvirus). The novel helper nucleic acid molecules (e.g., helper plasmids) led to enhanced production of infectious rAAV compared to plasmids containing only adenovirus genes, or adenovirus and bocavirus genes, or adenovirus and herpesvirus genes on the same helper nucleic acid molecule (e.g., helper plasmid). Furthermore, this novel nucleic acid molecule (e.g., helper plasmid) has been proven compatible with various AAV serotypes (2, 5, 8, and 9), packaging cell lines (HEK-293T, VPC 2.0, and Expi293F), and transfection reagents (FectoVIR-AAV, PEIpro, and TransIT-VirusGEN), highlighting the high versatility of this designed helper nucleic acid molecule in various production systems.

[0017] Specifically, the present invention describes a triple transfection method for mammalian cells, in which three different plasmids are used: an AAV transfer plasmid (pTransfer) containing the gene of interest flanked by AAV2 inverted terminal repeats (ITRs), a Rep / Cap plasmid (pRC) containing the Rep gene from AAV2 and the Cap gene from the desired AAV serotype to be produced, and a helper plasmid (pHelper) containing helper elements from helper viruses (i.e., adenovirus, bocavirus, and herpesvirus) that support AAV production.

[0018] In one embodiment, we describe a method using a new type of pHelper to replace the current pHelper used in rAAV production. This new pHelper was transfected under the same conditions as the classical triple transfection method, but produced better infectious AAV particle titer yields than other pHelpers.

[0019] The combination of helper elements from three different viruses, specifically pHelper (i.e., adenovirus, bocavirus, and herpesvirus), on the same helper nucleic acid molecule enabled the production of high infectious virus yields in all conditions tested (varying AAV serotypes, transfection reagents, and packaging cell lines).

[0020] The present invention provides chimeric helper nucleic acid molecules suitable for use in methods for producing infectious recombinant adeno-associated viruses (rAAV), comprising: - one or more polynucleotides encoding adenovirus helper virus (auxiliary) functions for activating the expression of Rep and Cap proteins from an adeno-associated virus, the polynucleotide comprising the E2A gene from an adenovirus (Ad), preferably from an adenovirus of serotype 5 (Ad5), the E4 gene from Ad, preferably from Ad5, in particular the E4orf6 sequence; and a virus-associated ribonucleic acid (VA-RNA) polynucleotide from Ad, preferably from Ad5; and / or - one or more polynucleotides encoding helper virus functions for AAV production, comprising an open reading frame (ORF) derived from a gene of herpes simplex virus (HSV), preferably from a gene of herpes simplex virus serotype 1 (HSV1), and an ORF derived from a gene of human bocavirus (HBoV), preferably from a gene of human bocavirus serotype 1 (HBoV1), and further comprising endogenous or non-endogenous sequences for controlling the expression of these ORFs; The present invention relates to a chimeric helper nucleic acid molecule comprising:

[0021] In certain embodiments, the present invention provides a chimeric helper nucleic acid molecule suitable for use in a method for producing an infectious recombinant adeno-associated virus (rAAV), comprising: (i) an ORF derived from a gene of HSV, which comprises a polynucleotide encoding at least one protein of HSV, in particular a polynucleotide encoding at least the UL12 protein derived from HSV or the ICP8 protein derived from HSV, preferably a polynucleotide encoding the UL12 protein derived from HSV, preferably HSV1, or the ICP8 protein derived from HSV, preferably HSV1; (ii) an ORF derived from the gene of HBoV, consisting of a polynucleotide encoding the NS2 protein derived from HBoV, preferably derived from HBoV1, or the NP1 protein derived from HBoV, preferably derived from HBoV1; (iii) a VA-RNA polynucleotide derived from Ad, preferably Ad5; (iv) the E2A gene from Ad, preferably from Ad5; (v) the E4 gene from Ad, preferably from Ad5, in particular the E4orf6 sequence Including, Each of the polynucleotides (i), (ii), (iii), (iv), and (v) is inserted as a separate expression cassette within the chimeric helper nucleic acid molecule, and the polynucleotides (i) to (v) are arranged in any order and / or orientation relative to each other; However, this chimeric helper nucleic acid molecule (ii) does not contain an ORF derived from an HBoV gene and is composed of a polynucleotide encoding the NS2 protein derived from HBoV and the NP1 protein derived from HBoV; Concerning chimeric helper nucleic acid molecules.

[0022] In certain embodiments of the invention, chimeric helper nucleic acid molecules suitable for use in methods for producing infectious recombinant adeno-associated virus (rAAV) comprise: (i) an ORF derived from a gene of HSV, which consists of a polynucleotide encoding at least one protein of HSV, preferably a polynucleotide encoding one or two proteins of HSV, in particular a polynucleotide encoding at least the UL12 protein derived from HSV or the ICP8 protein derived from HSV, preferably a polynucleotide encoding the UL12 protein derived from HSV, preferably HSV1, and / or the ICP8 protein derived from HSV, preferably HSV1; (ii) an ORF derived from the gene of HBoV, consisting of a polynucleotide encoding the NS2 protein derived from HBoV, preferably derived from HBoV1, or the NP1 protein derived from HBoV, preferably derived from HBoV1; (iii) a VA-RNA polynucleotide derived from Ad, preferably Ad5; (iv) the E2A gene from Ad, preferably from Ad5; (v) the E4 gene from Ad, preferably from Ad5, in particular the E4orf6 sequence Including, Each of the polynucleotides (i), (ii), (iii), (iv), and (v) is inserted as a separate expression cassette within the chimeric helper nucleic acid molecule, and the polynucleotides (i) to (v) are arranged in any order and / or orientation relative to each other; However, this chimeric helper nucleic acid molecule (ii) does not contain an ORF derived from an HBoV gene, and is composed of a polynucleotide encoding the NS2 protein derived from HBoV and the NP1 protein derived from HBoV.

[0023] As defined herein, the term "chimeric nucleic acid molecule" refers to a nucleic acid molecule that contains sequences from different organisms.

[0024] As defined herein, the term "nucleic acid molecule" or "nucleic acid" refers to a polymeric compound of covalently linked nucleotides that form a chain structure. The nucleic acid may be single-stranded, double-stranded, or a combination of single-stranded and double-stranded in different regions of the nucleic acid chain; it may be selected from the group consisting of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), Dicer substrate small interfering RNA (dsiRNA), small hairpin RNA (shRNA), RNA transcripts, microRNA (miRNA), messenger RNA (mRNA), circular RNA (circRNA), guide RNA (gRNA), small activating RNA (saRNA), small regulatory RNA (srRNA), long non-coding RNA (lncRNA), and antisense oligonucleotide. The term "polynucleotide" may be used interchangeably with the term "nucleic acid molecule." Certain nucleic acid molecules are open reading frames (ORFs), which encode the amino acid sequence of a polypeptide and are capable of expressing such a polypeptide when placed under the control of transcriptional and translational regulatory elements, such as a promoter.

[0025] As defined herein, the terms "helper," "helper function," or "helper functions," when referring to a helper nucleic acid molecule suitable for AAV production, mean that the molecule provides a function, i.e., a "helper virus function," required for AAV replication, viral gene expression for transduction, and viral particle assembly (i.e., viral particle assembly for AAV genomes packaged in cells, particularly mammalian cells) (Marie-Claude Geoffroy et al., Current Gene Therapy, 2005, 5(3), pp. 265-271). In certain embodiments, such helper functions may be provided by polypeptides encoded by nucleic acid molecules. In certain embodiments, such helper functions, when provided by nucleic acid molecules originating from HSV or HBoV, may be similar in nature to the helper functions provided by Ad nucleic acids (particularly E2A, E4, and / or VA-RNA) used in accordance with the present invention. In certain embodiments, helper functions, when provided by nucleic acid molecules originating from HSV or HBoV, may enhance or improve the helper functions provided by the Ad nucleic acids used in accordance with the present invention, i.e., may allow for an increase in the rAAV viral titer in producer cells and / or the transduction titer (TU / ml or VG / ml) of the produced rAAV, determined after infection of permissive cells. In certain embodiments, helper functions, when provided by nucleic acid molecules originating from HSV or HBoV, may provide new activities in producer cells and / or infected cells relative to those provided by the Ad nucleic acids used in accordance with the present invention. Such new activities may also lead to an increase in the rAAV titer in producer cells and / or the transduction titer (TU / ml or VG / ml) of the produced rAAV, determined after infection of permissive cells (e.g., HT-1080 or CHO-K1). The increase in rAAV titer can be assessed or measured by any suitable known method by comparing it to the titer of the produced rAAV obtained when nucleic acid molecules originating from HSV and HBoV are not used in accordance with the present invention.Examples of measuring rAAV titer are disclosed in the Examples herein. In certain embodiments, helper functions provided by HSV or HBoV helper genes or ORFs improve the versatility of the produced rAAV with respect to producer cells, transfection agents, and / or rAAV serotypes (particularly considering serotypes 2, 5, 8, and / or 9). rAAV titer can be determined as disclosed in the Examples using capsid titer (VP / ml), e.g., using a titration ELISA kit, genomic titer (VG / ml) by qPCR performed on a reporter gene product, etc.

[0026] As used herein, the term "expression cassette" refers to a functional expression unit capable of driving the expression of one or more integrated polynucleotides.

[0027] As defined herein, the term "VA-RNA from Ad" refers to the VA-RNA element from Ad, which is a small non-coding RNA transcribed by Pol III. In a preferred embodiment, the term refers to the VA-RNA from Ad5 (positions 10,545-11,029 of the NCBI reference sequence AC_000008.1).

[0028] As defined herein, the term "E2A gene from Ad" refers to the E2A gene encoding the E2A protein from AD that is transcribed by pol III. In a preferred embodiment, the term refers to the E2A gene from Ad5 (positions 22,388-27,121 of the NCBI reference sequence AC_000008.1).

[0029] As defined herein, the term "E4orf6 sequence from Ad" refers to E4orf6, which is one of the proteins encoded by the E4 gene from Ad. In a preferred embodiment, the term refers to the E4orf6 coding sequence from Ad5 (positions 33,188-34,072 of the NCBI reference sequence AC_000008.1).

[0030] The present inventors have demonstrated that the use of the triple-chimeric helper element of the present invention advantageously yields greater amounts of infectious recombinant viruses of various AAV serotypes (particularly serotypes 2, 5, 8, and / or 9) in a variety of producer cells, thereby demonstrating versatility. The present inventors have also demonstrated that the infectious titer (measured as TU / VP / VG) of the produced rAAV can be significantly increased using the triple-chimeric helper element of the present invention. Thus, helper elements provided by nucleic acid molecules originating from HSV and HBoV are considered to provide additional helper virus functions to those provided by Ad nucleic acid molecules.

[0031] The phrases "originating" or "originates," in the plural or singular, as used herein to disclose nucleic acid molecules, particularly those originating from the HSV or HBoV viruses, refer to the fact that the expressed polypeptides encoded by nucleic acids originating from the HSV or HBoV viruses have the properties of the native viral proteins expressed by these respective viruses to the extent that they confer a similar or identical effect as helper elements when used in the present invention. The nucleotide sequences of such nucleic acid molecules may be identical to the wild-type sequences found in the genome of the viruses or may be modified with respect to such wild-type sequences that have been specifically modified for optimization of expression in a particular production cell. Nucleic acid molecules originating from the HSV and HBoV viruses may be purified or synthetic molecules.

[0032] As defined herein, the term "recombinant adeno-associated virus (rAAV)" refers to an AAV virus that contains one or more heterologous genes or heterologous polynucleotides (i.e., not of AAV origin) in its genome. Thus, rAAV can be used as a vector that allows for the expression of this heterologous gene or heterologous polynucleotide in a target cell.

[0033] As defined herein, the term "gene" refers to a nucleic acid molecule or open reading frame (ORF) that is transcribed (in the case of DNA) and translated (with the production of mRNA) into a polypeptide in vitro or in vivo under the control of its endogenous or native regulatory sequences (e.g., a promoter).

[0034] In certain embodiments of the invention, the chimeric helper nucleic acid molecule is a recombinant helper nucleic acid molecule.

[0035] As defined herein, the term "recombinant nucleic acid molecule" means that a nucleic acid molecule has been genetically modified, e.g., by the addition or insertion of a heterologous nucleic acid construct into the molecule.

[0036] In another particular embodiment of the invention, the chimeric helper nucleic acid molecule is a chimeric helper expression vector, in particular a chimeric helper expression plasmid.

[0037] As defined herein, the term "vector" relates to a biological or chemical entity suitable for delivering a polynucleotide of interest (particularly a polynucleotide encoding a polypeptide of interest) into a cell (particularly a producer cell or target cell as defined herein). Accordingly, the term "vector" refers to a nucleic acid molecule that contains genetic information and is capable of transferring the nucleic acid molecule carrying said genetic information, or that contains this nucleic acid molecule in its structure. In some embodiments, a vector allows for the expression of the nucleic acid molecule carrying the genetic information, and in particular allows for the expression of a polypeptide from said molecule. In one embodiment, the vector is an expression vector that contains regulatory elements for the expression of a nucleic acid molecule contained in this expression vector, in particular for expressing a heterologous polypeptide. Thus, in the context of the present invention, the term "vector" relates to a nucleic acid molecule carrying a viral helper function as disclosed herein, or to a nucleic acid molecule carrying a transgene of interest, e.g., in a plasmid transfer as disclosed herein, or to a nucleic acid molecule carrying a molecule encoding a Rep protein and / or a Cap protein, such as pRep / Cap, and also to the recombinant AAV particles produced. A vector may, for example, be or contain a replicon. Vectors can be episomal or non-episomal. In one embodiment, vectors can be plasmids that have been genetically modified and are intended for the introduction of nucleic acid molecules into cells. In one embodiment, vectors can be recombinant viral particles, the genome of which contains nucleic acids carrying the genetic information to be introduced into target cells and, if necessary, expressed in such cells. In one embodiment, vectors of the present invention contain at least a bacterial functional unit (ori) and may also contain one or more expression units and / or one or more integration units.

[0038] As defined herein, the term "plasmid" refers to a DNA molecule distinct from chromosomal DNA and capable of autonomous replication. Plasmids are generally circular double-stranded DNA.

[0039] In certain embodiments of the present invention, the rAAV is selected from the group consisting of serotype 1 rAAV (rAAV1), serotype 2 rAAV (rAAV2), serotype 3 rAAV (rAAV3), serotype 4 rAAV (rAAV4), serotype 5 rAAV (rAAV5), serotype 6 rAAV (rAAV6), serotype 7 rAAV (rAAV7), serotype 8 rAAV (rAAV8), serotype 9 rAAV (rAAV9), serotype 10 rAAV (rAAV10), serotype 11 rAAV (rAAV11), serotype 12 rAAV (rAAV12), and serotype 13 rAAV (rAAV13). In certain embodiments, the rAAV produced is derived from a serotype selected from among rAAV2, rAAV5, rAAV8, and rAAV9, preferably from a serotype selected from among rAAV2, rAAV5, and rAAV9.

[0040] In certain embodiments of the invention, the Ad providing the polynucleotide encoding a polypeptide possessing AAV helper function is a serotype 2 Ad (Ad2) or a serotype 5 Ad (Ad5), preferably Ad5. In certain embodiments, the Ad demonstrates the helper function of serotype 2 AAV (AAV2), serotype 5 AAV (AAV5), or serotype 9 AAV (AAV9), particularly a polynucleotide encoding the Cap protein of AAV2.

[0041] Herpes simplex virus (HSV) is a human herpesvirus, an encapsidated double-stranded DNA virus classified into two known serotypes: HSV-1 (also known as human alphaherpesvirus 1, HHV1; its complete genome sequence, along with annotated amino acid sequences of the encoded proteins, is available at GenBank under accession number MN136524), and HSV-2 (human alphaherpesvirus 2; its complete genome sequence, along with annotated amino acid sequences of the encoded proteins, is available at EMBL under accession number Z86099). The DNA genome consists of two covalently linked components, termed the L (long) unique region and the S (short) unique region. HSV virions are particles containing a capsid and an envelope. Among its nonstructural proteins, HSV encodes UL12, an alkaline exonuclease (DNAse) that is involved in viral nucleic acid metabolism by processing linear or branched viral DNA intermediates to promote the generation of mature progeny viral DNA molecules. This protein possesses endonuclease and exonuclease activity toward both double- and single-stranded DNA substrates.

[0042] In a specific embodiment of the present invention, the ORF derived from an HSV gene, which comprises a polynucleotide encoding at least one HSV protein, is a polynucleotide encoding an HSV structural protein and / or a nonstructural protein, particularly a polynucleotide encoding a protein selected from the group consisting of HSV UL5, UL8, UL12, UL29, UL52, and ICP8. Preferably, the polynucleotide encodes the HSV UL12 protein alone or in combination with other HSV proteins such as the above-mentioned HSV proteins; or the polynucleotide encodes the HSV ICP8 protein alone or in combination with other HSV proteins such as the above-mentioned HSV proteins. In particular, the polynucleotide encodes the HSV UL12 protein and the HSV ICP8 protein.

[0043] In specific embodiments of the invention, the HSV is serotype 1 HSV (HSV1) or serotype 2 HSV (HSV2), preferably HSV1. In one embodiment, the polynucleotide encodes the UL12 protein from HSV1. In another embodiment, the polynucleotide encodes the ICP8 protein from HSV1. In another embodiment, the polynucleotide encodes the UL12 protein and the ICP8 protein from HSV1.

[0044] In a preferred embodiment of the present invention, the ORF derived from an HSV gene consists of a polynucleotide encoding the UL12 protein derived from HSV, preferably from HSV1.

[0045] Human bocavirus (HBoV) is a single-stranded DNA virus of the Parvoviridae family, including serotypes HBoV1, HBoV2, HBoV3, and HBoV4, some of which can infect humans. Several sequences of viral genomes from strains isolated from patients, along with the amino acid sequences of the encoded proteins, are available in GenBank under accession numbers MG953829, MG953830, MG953831, MG953832, MG953833, and MG953834. The HBoV genome encodes three ORFs (ORF1, ORF2, and ORF3). These encode four nonstructural proteins (NS1, NS2, NS3, and NS4), including the small nonstructural protein NP1, and the capsid proteins VP1, VP2, and VP3. The NP1 gene is an alternative reading frame of VP1 and overlaps with the beginning of VP1. NP1 is characterized by its ability to induce apoptosis in cells.

[0046] In one embodiment, the helper functions provided by the nucleic acid molecules originating from HSV or HBoV are achieved by expression of HSV or HBoV polypeptides, respectively.

[0047] The terms "polypeptide" or "protein," used interchangeably, are defined herein as a wild-type or naturally occurring polypeptide of a virus, such as an HSV virus or an HBoV virus, or a fragment of such a wild-type polypeptide, or a mutant polypeptide, or a synthetic polypeptide, wherein such a fragment, mutant, or synthetic polypeptide maintains the helper effect of the wild-type polypeptide (or the wild-type polypeptide used in the population) on rAAV production (particularly the titer of the rAAV produced). In one embodiment, the fragment or mutant polypeptide may have 80% or more, or 90% or more (particularly greater than 95%) identity to the wild-type sequence of the reference polypeptide.

[0048] In a specific embodiment of the present invention, the polynucleotide encoding the additional helper virus functions provided by the polynucleotide originating from HBoV is a polynucleotide encoding a structural and / or nonstructural protein of HBoV, in particular a polynucleotide encoding a protein selected from the group consisting of the NS1, NS2, NS3, NS4, NP1, VP1, VP2, and VP3 proteins of HBoV. Preferably, the polynucleotide encodes the HBoV NS2 protein alone or in combination with other HBoV proteins, or the HBoV NP1 protein alone or in combination with other HBoV proteins.

[0049] In a preferred embodiment of the present invention, the ORF derived from an HBoV gene consists of a polynucleotide encoding the NS2 protein or the NP1 protein of HBoV. Preferably, the ORF derived from an HBoV gene consists of a polynucleotide encoding the NS2 protein of HBoV.

[0050] In a specific embodiment of the present invention, the HBoV is serotype 1 HBoV (HBoV1), serotype 2 HBoV (HBoV2), serotype 3 HBoV (HBoV3), or serotype 4 HBoV (HBoV4), preferably HBoV1. In one embodiment, the polynucleotide encodes an HBoV1 helper protein, in particular the NS2 protein, and in particular the NS2 protein alone or in combination with another protein, such as HBoV1 NP1. Preferably, the ORF derived from an HBoV gene consists of a polynucleotide encoding the NS2 protein of HBoV1.

[0051] In one embodiment, the polynucleotide encoding the additional helper functions encodes the UL12 protein and the NS2 protein of an HSV serotype, particularly the NS2 protein alone or in combination with other proteins such as HBoV NP1. In a specific embodiment, the polynucleotide encodes the UL12 protein and the NS2 protein of an HSV1 serotype, particularly the NS2 protein alone or in combination with other proteins such as HBoV NP1.

[0052] In a specific embodiment of the present invention, the ORF derived from an HSV gene consists of a polynucleotide encoding a UL12 protein derived from HSV, preferably HSV1, and the ORF derived from an HBoV gene consists of a polynucleotide encoding an NP1 protein derived from HBoV, preferably HBoV1.

[0053] In a preferred embodiment of the present invention, the ORF derived from an HSV gene consists of a polynucleotide encoding the UL12 protein derived from HSV, preferably from HSV1, and the ORF derived from an HBoV gene consists of a polynucleotide encoding the NS2 protein derived from HBoV, preferably from HBoV1.

[0054] In a specific embodiment of the present invention, the ORF derived from an HSV gene consists of a polynucleotide encoding an ICP8 protein derived from HSV, preferably from HSV1, and the ORF derived from an HBoV gene consists of a polynucleotide encoding an NP1 protein derived from HBoV, preferably from HBoV1.

[0055] In a preferred embodiment of the present invention, the ORF derived from an HSV gene consists of a polynucleotide encoding an ICP8 protein derived from HSV, preferably from HSV1, and the ORF derived from an HBoV gene consists of a polynucleotide encoding an NS2 protein derived from HBoV, preferably from HBoV1.

[0056] In a specific embodiment of the present invention, the ORF derived from an HSV gene comprises a polynucleotide encoding the UL12 protein derived from HSV in combination with other HSV proteins such as the above-mentioned proteins, and the ORF derived from an HBoV gene comprises a polynucleotide encoding the NP1 protein derived from HBoV, preferably HBoV1. More specifically, the ORF derived from an HSV gene comprises polynucleotides encoding the UL12 protein derived from HSV and the ICP8 protein derived from HSV, preferably HSV1, and the ORF derived from an HBoV gene comprises a polynucleotide encoding the NP1 protein derived from HBoV, preferably HBoV1.

[0057] In a preferred embodiment of the present invention, the ORF derived from an HSV gene consists of a polynucleotide encoding the UL12 protein derived from HSV in combination with other HSV proteins such as the above-mentioned proteins, and the ORF derived from an HBoV gene consists of a polynucleotide encoding the NS2 protein derived from HBoV, preferably HBoV1. In particular, the ORF derived from an HSV gene consists of polynucleotides encoding the UL12 protein derived from HSV and the ICP8 protein derived from HSV, preferably HSV1, and the ORF derived from an HBoV gene consists of a polynucleotide encoding the NS2 protein derived from HBoV, preferably HBoV1.

[0058] In a particular embodiment of the present invention, nucleic acids (i) and (ii) are under the control of non-endogenous promoters, in particular under the control of different promoters. In a preferred embodiment of the present invention, the NS2 protein from HBoV, preferably from HBoV1, is under the control of the pCMV promoter. In another preferred embodiment of the present invention, the NP1 protein from HBoV, preferably from HBoV1, is under the control of the pCMV promoter.

[0059] In another preferred embodiment of the invention, at least the UL12 protein from HSV, preferably from HSV1, is under the control of the EFS promoter (ie the core promoter of the eukaryotic translation elongation factor 1 alpha).

[0060] In another preferred embodiment of the invention, at least the ICP8 protein from HSV, preferably from HSV1, is under the control of the EFS promoter (ie the core promoter of the eukaryotic translation elongation factor 1 alpha).

[0061] In another preferred embodiment of the present invention, at least the ICP8 protein from HSV, particularly from HSV1, and at least the UL12 protein from HSV, particularly from HSV1, preferably the ICP8 protein and the UL12 protein from HSV, particularly from HSV1, are under the control of the EFS promoter (i.e., the core promoter of eukaryotic translation elongation factor 1α).

[0062] As defined herein, the term "promoter" refers to a nucleic acid sequence that drives gene expression. Examples of promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, CMV enhancer, EFS promoter, SV40 viral promoter, CBA promoter, CAG promoter, E2A promoter, and E4 promoter.

[0063] The term "non-endogenous promoter" refers to a promoter derived from a source other than the native promoter of a coding sequence.

[0064] In a preferred embodiment of the invention, the polynucleotides in the chimeric nucleic acid molecule comprise, in 5' to 3' order, (i), (ii), (iii), (iv), and (v), wherein nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, particularly under the control of different promoters.

[0065] In certain embodiments of the invention, the chimeric helper nucleic acid molecule further comprises additional adenoviral genes, for example, the E1 gene from Ad, preferably from Ad5.

[0066] In certain embodiments of the invention, the chimeric helper nucleic acid molecule comprises: - origin of replication (ori); and / or - herpes simplex virus thymidine kinase (HSV TK polyA) signal downstream of the HSV UL12 ORF; and / or - a bovine growth hormone polyadenylation (bGH polyA) signal downstream of the HBoV NS2 ORF; and / or - transcriptional regulatory elements for controlling the transcription of the HSV and HBoV ORFs, such as the cytomegalovirus (CMV) promoter, the CMV enhancer, the EFS promoter, the SV40 viral promoter, the CBA promoter, the CAG promoter, the E2A promoter, and / or the E4 promoter; and / or - optionally a nucleic acid encoding a marker gene, for example encoding a kanamycin resistance gene Further includes:

[0067] As defined herein, the term "ori" refers to an isolated origin of replication based on high-copy pmB1 and flanked by two bacterial transcription terminators: a T7 terminator at the 5' position and a LuxIA terminator at the 3' position. Such transcriptional isolation is designed to minimize transcriptional interference from adjacent content, thereby ensuring higher amplification rates in bacteria.

[0068] As defined herein, the term "nucleic acid encoding a kanamycin resistance gene" refers to an antibiotic resistance cassette consisting of the nptII ORF, which confers resistance to kanamycin treatment, under the control of the promoter and terminator of pAmpR followed by a hairpin terminator.

[0069] In a preferred embodiment of the invention, in the chimeric helper nucleic acid molecule: - the polynucleotide encoding the UL12 protein from HSV1 is that of SEQ ID NO: 1; - the polynucleotide encoding the NS2 protein from HBoV1 is that of SEQ ID NO: 2; - the VA-RNA polynucleotide from Ad5 is that of SEQ ID NO: 3; - the E2A gene from Ad5 is that of SEQ ID NO: 4; - the E4 gene from Ad5 is that of SEQ ID NO: 5, in particular the E4orf6 sequence from Ad5 is that of SEQ ID NO: 6; and / or - the polynucleotide encoding the ICP8 protein from HSV1 is that of SEQ ID NO: 32.

[0070] In a preferred embodiment of the invention, in the chimeric helper nucleic acid molecule: - ori is the nucleotide sequence of SEQ ID NO: 7; - the HSV TK polyA signal is the polynucleotide of sequence SEQ ID NO: 8; - the bGH polyA signal is the polynucleotide of sequence SEQ ID NO: 9; - the CMV promoter is the polynucleotide of sequence SEQ ID NO: 10; - the CMV enhancer is the polynucleotide of sequence SEQ ID NO: 11; - the EFS promoter is the polynucleotide of sequence SEQ ID NO: 12; - the E2A promoter is the polynucleotide of sequence SEQ ID NO: 13; - the E4 promoter is a polynucleotide of sequence SEQ ID NO: 14; and / or the kanamycin resistance gene is of the sequence SEQ ID NO: 15.

[0071] In a preferred embodiment of the present invention, the chimeric helper nucleic acid molecule comprises (i) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding the UL12 protein from HSV1, and an HSV TK polyA signal, the sequence of which is SEQ ID NO: 16, and (ii) a polynucleotide insert comprising a CMV promoter, a polynucleotide encoding the NS2 protein from HBoV1, and a bGH polyA signal, the sequence of which is SEQ ID NO: 17.

[0072] In a specific embodiment of the present invention, the chimeric helper nucleic acid molecule comprises (i) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding the UL12 protein from HSV1, and an HSV TK polyA signal, the sequence of which is SEQ ID NO: 16, and (ii) a polynucleotide insert comprising a CMV promoter, a polynucleotide encoding the NP1 protein from HBoV1, and a bGH polyA signal, the sequence of which is SEQ ID NO: 17.

[0073] In another specific embodiment of the invention, the chimeric helper nucleic acid molecule comprises (i) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding the ICP8 protein from HSV1, and an HSV TK polyA signal, the sequence of which is SEQ ID NO: 16, and (ii) a polynucleotide insert comprising a CMV promoter, a polynucleotide encoding the NS2 protein from HBoV1, and a bGH polyA signal, the sequence of which is SEQ ID NO: 17.

[0074] In another specific embodiment of the invention, the chimeric helper nucleic acid molecule comprises (i) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding the ICP8 protein from HSV1, and an HSV TK polyA signal, the sequence of which is SEQ ID NO: 16, and (ii) a polynucleotide insert comprising a CMV promoter, a polynucleotide encoding the NP1 protein from HBoV1, and a bGH polyA signal, the sequence of which is SEQ ID NO: 17.

[0075] In another specific embodiment of the invention, the chimeric helper nucleic acid molecule comprises: (i) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding the UL12 protein from HSV1, and an HSV TK polyA signal, the sequence of which is SEQ ID NO: 16; (ii) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding the ICP8 protein from HSV1, and an HSV TK polyA signal, the sequence of which is SEQ ID NO: 16; and (iii) a polynucleotide insert comprising a CMV promoter, a polynucleotide encoding the NS2 protein from HBoV1, and a bGH polyA signal, the sequence of which is SEQ ID NO: 17.

[0076] In another specific embodiment of the invention, the chimeric helper nucleic acid molecule comprises: (i) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding the UL12 protein from HSV1, and an HSV TK polyA signal, the sequence of which is SEQ ID NO: 16; (ii) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding the ICP8 protein from HSV1, and an HSV TK polyA signal, the sequence of which is SEQ ID NO: 16; and (iii) a polynucleotide insert comprising a CMV promoter, a polynucleotide encoding the NP1 protein from HBoV1, and a bGH polyA signal, the sequence of which is SEQ ID NO: 17.

[0077] In a preferred embodiment of the invention, the chimeric helper nucleic acid molecule is a plasmid, in particular the plasmid Ad-HBoV-HSV of SEQ ID NO: 18 or the plasmid Ad-HBoV-HSV5 of SEQ ID NO: 35, preferably the plasmid Ad-HBoV-HSV of SEQ ID NO: 18.

[0078] In a preferred embodiment of the invention, in the chimeric helper nucleic acid molecule: - the amino acid sequence of the UL12 protein from HSV1 is that of SEQ ID NO: 19; - the amino acid sequence of the NS2 protein from HBoV1 is that of SEQ ID NO: 20; - the amino acid sequence of the E2A protein from Ad5 is that of SEQ ID NO: 21; - the amino acid sequence of the E4orf1 sequence from Ad5 is that of SEQ ID NO: 22; - the amino acid sequence of the E4orf6 sequence from Ad5 is that of SEQ ID NO: 23; - the nucleotide sequence encoding the NP1 protein from HBoV1 is that of SEQ ID NO: 24; - the amino acid sequence of the NP1 protein from HBoV1 is that of SEQ ID NO: 25; - the E1 gene from Ad5 is the polynucleotide of SEQ ID NO: 26; - the amino acid sequence of the E1A protein from Ad5 is that of SEQ ID NO: 27; - the amino acid sequence of the E1B55k protein from Ad5 is that of SEQ ID NO: 28; - the amino acid sequence of the E4orf2 sequence from Ad5 is that of SEQ ID NO: 29; - the amino acid sequence of the E4orf3 sequence from Ad5 is that of SEQ ID NO: 30; - the amino acid sequence of the E4orf4 sequence from Ad5 is that of SEQ ID NO: 31; - the amino acid sequence of the E4 protein from Ad5 is that of SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31; - the amino acid sequence of the E1 protein from Ad5 is that of SEQ ID NO: 27 or SEQ ID NO: 28; and / or - the amino acid sequence of the ICP8 protein from HSV1 is that of SEQ ID NO: 33.

[0079] The E4 protein may consist of the E4orf1, E4orf2, E4orf3, E4orf4, and E4orf6 sequences defined above.

[0080] The E1 protein may consist of the E1A protein and the E1B55k protein as defined above.

[0081] In a preferred embodiment of the invention, the chimeric helper nucleic acid molecule comprises: (i) an ORF derived from a gene of HSV, consisting of a polynucleotide encoding a UL12 protein derived from HSV, preferably derived from HSV1; (ii) an ORF derived from the gene of HBoV, consisting of a polynucleotide encoding the NS2 protein derived from HBoV, preferably from HBoV1; (iii) a VA-RNA polynucleotide derived from Ad, preferably Ad5; (iv) the E2A gene from Ad, preferably from Ad5; (v) the E4 gene from Ad, preferably from Ad5, in particular the E4orf6 sequence Including, Each of the polynucleotides (i), (ii), (iii), (iv), and (v) is inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and the polynucleotides (i) through (v) are arranged in any order and / or orientation relative to each other; preferably, each of the polynucleotides (i), (ii), (iii), (iv), and (v) is inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and the polynucleotides in the chimeric nucleic acid molecule have, in 5' to 3' direction, the following order: (i), (ii), (iii), (iv), and (v), and nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, particularly under the control of a different promoter; However, this chimeric helper nucleic acid molecule (ii) does not contain an ORF derived from an HBoV gene, and is composed of a polynucleotide encoding the NS2 protein derived from HBoV and the NP1 protein derived from HBoV; and However, this chimeric helper nucleic acid molecule does not include an additional polynucleotide encoding a protein from HSV.

[0082] In another preferred embodiment of the invention, the chimeric helper nucleic acid molecule comprises: (i) an ORF derived from a gene of HSV, consisting of a polynucleotide encoding a UL12 protein derived from HSV, preferably derived from HSV1; (ii) an ORF derived from the gene of HBoV, consisting of a polynucleotide encoding the NP1 protein derived from HBoV, preferably from HBoV1; (iii) a VA-RNA polynucleotide derived from Ad, preferably Ad5; (iv) the E2A gene from Ad, preferably from Ad5; (v) the E4 gene from Ad, preferably from Ad5, in particular the E4orf6 sequence Including, Each of the polynucleotides (i), (ii), (iii), (iv), and (v) is inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and the polynucleotides (i) through (v) are arranged in any order and / or orientation relative to each other; preferably, each of the polynucleotides (i), (ii), (iii), (iv), and (v) is inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and the polynucleotides in the chimeric nucleic acid molecule have, in 5' to 3' direction, the following order: (i), (ii), (iii), (iv), and (v), and nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, particularly under the control of a different promoter; However, this chimeric helper nucleic acid molecule (ii) does not contain an ORF derived from an HBoV gene, and is composed of a polynucleotide encoding the NS2 protein derived from HBoV and the NP1 protein derived from HBoV; and However, this chimeric helper nucleic acid molecule does not include an additional polynucleotide encoding a protein from HSV.

[0083] In another preferred embodiment of the invention, the chimeric helper nucleic acid molecule comprises: (i) an ORF derived from a gene of HSV, consisting of a polynucleotide encoding an ICP8 protein derived from HSV, preferably from HSV1; (ii) an ORF derived from the gene of HBoV, consisting of a polynucleotide encoding the NS2 protein derived from HBoV, preferably from HBoV1; (iii) a VA-RNA polynucleotide derived from Ad, preferably Ad5; (iv) the E2A gene from Ad, preferably from Ad5; (v) the E4 gene from Ad, preferably from Ad5, in particular the E4orf6 sequence Including, Each of the polynucleotides (i), (ii), (iii), (iv), and (v) is inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and the polynucleotides (i) through (v) are arranged in any order and / or orientation relative to each other; preferably, each of the polynucleotides (i), (ii), (iii), (iv), and (v) is inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and the polynucleotides in the chimeric nucleic acid molecule have, in 5' to 3' direction, the following order: (i), (ii), (iii), (iv), and (v), and nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, particularly under the control of a different promoter; However, this chimeric helper nucleic acid molecule (ii) does not contain an ORF derived from an HBoV gene, and is composed of a polynucleotide encoding the NS2 protein derived from HBoV and the NP1 protein derived from HBoV; and However, this chimeric helper nucleic acid molecule does not include an additional polynucleotide encoding a protein from HSV.

[0084] In another preferred embodiment of the invention, the chimeric helper nucleic acid molecule comprises: (i) an ORF derived from a gene of HSV, consisting of a polynucleotide encoding an ICP8 protein derived from HSV, preferably from HSV1; (ii) an ORF derived from the gene of HBoV, consisting of a polynucleotide encoding the NP1 protein derived from HBoV, preferably from HBoV1; (iii) a VA-RNA polynucleotide derived from Ad, preferably Ad5; (iv) the E2A gene from Ad, preferably from Ad5; (v) the E4 gene from Ad, preferably from Ad5, in particular the E4orf6 sequence Including, Each of the polynucleotides (i), (ii), (iii), (iv), and (v) is inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and the polynucleotides (i) through (v) are arranged in any order and / or orientation relative to each other; preferably, each of the polynucleotides (i), (ii), (iii), (iv), and (v) is inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and the polynucleotides in the chimeric nucleic acid molecule have, in 5' to 3' direction, the following order: (i), (ii), (iii), (iv), and (v), and nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, particularly under the control of a different promoter; However, this chimeric helper nucleic acid molecule (ii) does not contain an ORF derived from an HBoV gene, and is composed of a polynucleotide encoding the NS2 protein derived from HBoV and the NP1 protein derived from HBoV; and However, this chimeric helper nucleic acid molecule does not include an additional polynucleotide encoding a protein from HSV.

[0085] In another preferred embodiment of the invention, the chimeric helper nucleic acid molecule comprises: (i) an ORF derived from a gene of HSV, consisting of a polynucleotide encoding a UL12 protein derived from HSV, preferably derived from HSV1, and an ICP8 protein derived from HSV, preferably derived from HSV1; (ii) an ORF derived from the gene of HBoV, consisting of a polynucleotide encoding the NS2 protein derived from HBoV, preferably from HBoV1; (iii) a VA-RNA polynucleotide derived from Ad, preferably Ad5; (iv) the E2A gene from Ad, preferably from Ad5; (v) the E4 gene from Ad, preferably from Ad5, in particular the E4orf6 sequence Including, Each of the polynucleotides (i), (ii), (iii), (iv), and (v) is inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and the polynucleotides (i) through (v) are arranged in any order and / or orientation relative to each other; preferably, each of the polynucleotides (i), (ii), (iii), (iv), and (v) is inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and the polynucleotides in the chimeric nucleic acid molecule have, in 5' to 3' direction, the following order: (i), (ii), (iii), (iv), and (v), and nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, particularly under the control of a different promoter; However, this chimeric helper nucleic acid molecule (ii) does not contain an ORF derived from an HBoV gene, and is composed of a polynucleotide encoding the NS2 protein derived from HBoV and the NP1 protein derived from HBoV; and However, this chimeric helper nucleic acid molecule does not include an additional polynucleotide encoding a protein from HSV.

[0086] In another preferred embodiment of the invention, the chimeric helper nucleic acid molecule comprises: (i) an ORF derived from a gene of HSV, consisting of a polynucleotide encoding a UL12 protein derived from HSV, preferably derived from HSV1, and an ICP8 protein derived from HSV, preferably derived from HSV1; (ii) an ORF derived from the gene of HBoV, consisting of a polynucleotide encoding the NP1 protein derived from HBoV, preferably from HBoV1; (iii) a VA-RNA polynucleotide derived from Ad, preferably Ad5; (iv) the E2A gene from Ad, preferably from Ad5; (v) the E4 gene from Ad, preferably from Ad5, in particular the E4orf6 sequence Including, Each of the polynucleotides (i), (ii), (iii), (iv), and (v) is inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and the polynucleotides (i) through (v) are arranged in any order and / or orientation relative to each other; preferably, each of the polynucleotides (i), (ii), (iii), (iv), and (v) is inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and the polynucleotides in the chimeric nucleic acid molecule have, in 5' to 3' direction, the following order: (i), (ii), (iii), (iv), and (v), and nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, particularly under the control of a different promoter; However, this chimeric helper nucleic acid molecule (ii) does not contain an ORF derived from an HBoV gene, and is composed of a polynucleotide encoding the NS2 protein derived from HBoV and the NP1 protein derived from HBoV; and However, this chimeric helper nucleic acid molecule does not include an additional polynucleotide encoding a protein from HSV.

[0087] The location, order and / or orientation of functional sequences in a chimeric helper nucleic acid molecule is shown, for example, in FIG. 10 (plasmids Ad-HBoV-HSV and Ad-HBoV-HSV5 according to the invention).

[0088] The present invention also provides (a) an AAV transfer nucleic acid molecule, in particular a plasmid (pTransfer) comprising a transgene of interest comprising an open reading frame (ORF) under the control of transcriptional and translational regulatory elements, wherein the transgene of interest is flanked by inverted terminal repeats (ITRs) from AAV, in particular from serotype 2 AAV (AAV2), serotype 5 AAV (AAV5), serotype 8 AAV (AAV8), or serotype 9 AAV (AAV9), preferably from AAV2, AAV5, or AAV9, more preferably from AAV2; (b) a Rep / Cap nucleic acid molecule, in particular a plasmid (pPackaging, or pRC, or pAAVRep / Cap) that provides AAV viral functions and contains the replication (Rep) gene from AAV2 and the capsid (Cap) gene from an AAV, preferably from AAV serotype 2, 5, 8, or 9; and (c) a chimeric helper nucleic acid molecule according to the present invention A packaging system comprising: The nucleic acid molecules (a), (b), and (c) may be provided as one molecule, two different molecules, or three different molecules, preferably three different molecules consisting of or comprising (a), (b), and (c), respectively. It also relates to packaging systems.

[0089] As defined herein, the term "packaging system" refers to a system, such as a plasmid, that contains structural genes and packaging genes.

[0090] As defined herein, the term "AAV-derived inverted terminal repeats (ITRs)" refers to regions found at both ends of the AAV genome that function together in cis as origins of DNA replication and as packaging signals for the viral genome.

[0091] As defined herein, the term "transgene" refers to a gene to be delivered by an AAV. Examples of transgenes of interest include, but are not limited to, therapeutic genes or ORFs.

[0092] In certain embodiments of the present invention, the AAV is an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, preferably an AAV of serotype 2, 5, 8, or 9, more preferably an AAV of serotype 2, 5, or 9, and even more preferably an AAV of serotype 2.

[0093] The present invention also relates to recombinant adeno-associated virus (rAAV) producer cells transformed, in particular transfected, with a packaging system according to the invention, in particular rAAV producer cells that are recombinant E1-complementing producer cells.

[0094] In certain embodiments of the invention, the rAAV producer cells are mammalian cells, preferably human cells, more preferably HEK-293 cells, even more preferably HEK-293T cells, and in particular recombinant E1-complementing producer cells.

[0095] The present invention also provides a helper-free virus method for producing infectious recombinant adeno-associated virus (rAAV) in a producer cell line, comprising: - transforming, in particular transfecting, a cell or cell line, preferably a mammalian cell or an insect cell, with a packaging system according to the invention, in particular stably transfecting said cell with a packaging system according to the invention, and allowing the transfected cell to produce rAAV virions; - harvesting the transformed, particularly transfected, cells and lysing them to recover the rAAV virions; and - collecting the rAAV virions in the cell lysate or supernatant and, optionally, purifying the rAAV virions. The present invention also relates to a method comprising:

[0096] In certain embodiments of the invention, the helper-free virus method includes purifying rAAV virions.

[0097] As defined herein, the term "AAV virion" refers to a complete virus particle, e.g., a wild-type AAV virus particle that contains single-stranded genomic DNA packaged in AAV capsid proteins.

[0098] As defined herein, the term "rAAV virion" refers to a recombinant AAV virion (i.e., an infectious, replication-defective virus composed of an AAV protein shell) that encapsidates a DNA molecule of interest flanked on both sides by AAV ITRs.

[0099] In certain embodiments of the invention, the producer cell line is selected from the group consisting of a HEK-293 cell line, e.g., a HEK-293T cell line, a VPC 2.0 cell line, an Expi293F cell line, and an adherent cell line, and the cells are grown in suspension.

[0100] As defined herein, the term "adherent cell line" refers to a cell line that requires a solid support for growth and is therefore anchorage-dependent. Examples of adherent cells include, but are not limited to, MRC-5 cells, HeLa cells, Vero cells, NIH-3T3 cells, L293 cells, CHO cells, BHK-21 cells, MCF-7 cells, A549 cells, COS cells, HEK 293 cells, Hep G2 cells, SNN-BE(2) cells, BAE-1 cells, and SH-SY5Y cells.

[0101] In certain embodiments of the invention, transfection is carried out by chemical transfection, electroporation, or sonoporation.

[0102] The present invention also provides a method for producing a replication-deficient infectious recombinant adeno-associated virus (rAAV) vector particle, comprising: - transforming, in particular transfecting, cells, preferably mammalian or insect cells, with a packaging system according to the invention, in particular stably transfecting said cells with a packaging system according to the invention, and allowing the transformed, in particular transfected, cells to produce rAAV vector particles; - harvesting the transformed, particularly transfected, cells and lysing them to recover the rAAV vector particles; and - collecting the rAAV vector particles in the cell lysate or supernatant and optionally purifying the rAAV vector particles. The present invention also relates to a method comprising:

[0103] In certain embodiments of the invention, the method of producing replication-defective infectious rAAV vector particles comprises purifying the rAAV vector particles.

[0104] The present invention also relates to the in vitro use of rAAV producer cells according to the invention in the production of replication-deficient infectious rAAV vector particles.

[0105] Other features and advantages of the present invention will become apparent from the following examples and will be illustrated in the drawings. [Brief explanation of the drawings]

[0106] [Figure 1] Overview of the various helper plasmids used in this study. The overall structure of the plasmids is shown as a diagram, including genes derived from the AAV helper virus (light grey = adenovirus, dark grey = bocavirus, and white = herpesvirus). The name and size of each plasmid is indicated on the left. The use of heterologous promoters is indicated by arrows (pCMV = cytomegalovirus promoter, and pEFS = eukaryotic translation elongation factor 1 alpha core promoter). pALD-X80 and pHelper ALD were purchased from Aldevron, and pHelper CB was purchased from Cellbiolabs. All other plasmids were constructed for the purposes of this study using E-zyvec's proprietary technology, as described in EP 3256583 B1. [Figure 2]Determining the optimal plasmid ratio for rAAV2 production in HEK-293T suspension. rAAV2 vectors expressing the eGFP reporter gene were produced in HEK-293T cells grown in suspension. Cells were transfected with three plasmids (pRC2 vector expressing Rep and Cap, pAAV-GFP control vector expressing GFP under the control of a CMV promoter flanked by AAV2 ITRs, and a helper vector (pALD-X80) expressing at least Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors) and FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent. rAAV titers (transducing units, TU / mL) were determined 72 hours posttransfection by transduction of H1080 cells and quantification of eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transducing units / mL (TU / mL). Various mass ratios of this plasmid combination were used to determine the optimal ratio between these plasmids to obtain the highest infectious titer, and the resulting copy number of each plasmid was then used in further experiments to keep the copy number of the plasmids comparable in each condition. [Figure 3] Comparison of various helper plasmids for the efficiency of rAAV2 production. rAAV2 vectors expressing the eGFP reporter gene were produced in HEK-293T cells grown in suspension. Cells were transfected with three plasmids (pRC2 vector expressing Rep and Cap, pAAV-GFP control vector expressing GFP under the control of a CMV promoter flanked by AAV2 ITRs, and a helper vector expressing at least Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors) and FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent. AAV titers (transducing units, TU / mL) were determined 72 hours posttransfection by transducing H1080 cells and quantifying eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transducing units / mL (TU / mL). [Figure 4A]Addition of genes from other helper viruses to the helper plasmid and its effect on rAAV2 production. rAAV2 vectors expressing the eGFP reporter gene were produced in HEK-293T cells grown in suspension. Cells were transfected with three plasmids (pRC2 vector expressing Rep and Cap, pAAV-GFP control vector expressing GFP under the control of a CMV promoter flanked by AAV2 ITRs, and a helper vector expressing at least Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors) and FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent. AAV titers (transducing units, TU / mL) were determined 72 hours posttransfection by transducing H1080 cells and quantifying eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transducing units / mL (TU / mL) normalized to the infectivity of rAAV produced by pALD-X80 (containing only adenovirus genes) and expressed as fold increase in panels A, B, and F. A) The infectivity of rAAV produced by plasmids carrying different helper genes derived from either bocavirus (Ad-HBoV1 and Ad-HBoV2 in gray) or herpesvirus (Ad-HSV1 and Ad-HSV2 in white) was added to the adenovirus genes to generate double chimeric helper plasmids. [Figure 4B]Addition of genes from other helper viruses to the helper plasmid and its effect on rAAV2 production. rAAV2 vectors expressing the eGFP reporter gene were produced in HEK-293T cells grown in suspension. Cells were transfected with three plasmids (pRC2 vector expressing Rep and Cap, pAAV-GFP control vector expressing GFP under the control of a CMV promoter flanked by AAV2 ITRs, and a helper vector expressing at least Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors) and FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent. AAV titers (transducing units, TU / mL) were determined 72 hours posttransfection by transducing H1080 cells and quantifying eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transducing units / mL (TU / mL) normalized to the infectivity of rAAV produced by pALD-X80 (containing genes from adenovirus only) and expressed as fold increase in panels A, B, and F. B) Infectivity of rAAV produced by a plasmid carrying genes from adenovirus, bocavirus, and herpesvirus (triple chimeric plasmid). [Figure 4C]Addition of genes from other helper viruses to the helper plasmid and its effect on rAAV2 production. rAAV2 vectors expressing the eGFP reporter gene were produced in HEK-293T cells grown in suspension. Cells were transfected with three plasmids (pRC2 vector expressing Rep and Cap, pAAV-GFP control vector expressing GFP under the control of a CMV promoter flanked by AAV2 ITRs, and a helper vector expressing at least Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors) and FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent. AAV titers (transducing units, TU / mL) were determined 72 hours posttransfection by transducing H1080 cells and quantifying eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transducing units / mL (TU / mL) normalized to the infectivity of rAAV produced by pALD-X80 (containing only adenovirus genes) and expressed as fold increase in panels A, B, and F. C) Quantification of genome copies per mL (GC / mL). [Figure 4D]Addition of genes from other helper viruses to the helper plasmid and its effect on rAAV2 production. rAAV2 vectors expressing the eGFP reporter gene were produced in HEK-293T cells grown in suspension. Cells were transfected with three plasmids (pRC2 vector expressing Rep and Cap, pAAV-GFP control vector expressing GFP under the control of a CMV promoter flanked by AAV2 ITRs, and a helper vector expressing at least Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors) and FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent. AAV titers (transducing units, TU / mL) were determined 72 hours posttransfection by transducing H1080 cells and quantifying eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transducing units / mL (TU / mL) normalized to the infectivity of rAAV produced by pALD-X80 (containing only adenovirus genes) and expressed as fold increase in panels A, B, and F. D) Viral particles per mL (VP / mL). [Figure 4E]Addition of genes from other helper viruses to the helper plasmid and its effect on rAAV2 production. rAAV2 vectors expressing the eGFP reporter gene were produced in HEK-293T cells grown in suspension. Cells were transfected with three plasmids (pRC2 vector expressing Rep and Cap, pAAV-GFP control vector expressing GFP under the control of a CMV promoter flanked by AAV2 ITRs, and a helper vector expressing at least Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors) and FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent. AAV titers (transducing units, TU / mL) were determined 72 hours posttransfection by transducing H1080 cells and quantifying eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transducing units / mL (TU / mL) normalized to the infectivity of rAAV produced by pALD-X80 (containing only adenovirus genes) and expressed as fold increase in panels A, B, and F. E) Full / empty ratio (GC / VP). [Figure 4F]Addition of genes from other helper viruses to the helper plasmid and its effect on rAAV2 production. rAAV2 vectors expressing the eGFP reporter gene were produced in HEK-293T cells grown in suspension. Cells were transfected with three plasmids (pRC2 vector expressing Rep and Cap, pAAV-GFP control vector expressing GFP under the control of a CMV promoter flanked by AAV2 ITRs, and a helper vector expressing at least Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors) and FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent. AAV titers (transducing units, TU / mL) were determined 72 hours posttransfection by transducing H1080 cells and quantifying eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transducing units / mL (TU / mL) normalized to the infectivity of rAAV produced by pALD-X80 (containing only adenovirus genes) and expressed as fold increase in panels A, B, and F. F) TU / VP of rAAV2 produced by either pALD-X80 or the triple chimeric plasmid. [Figure 5]Versatility and efficiency of triple chimeric helper plasmids for rAAV2 production in various HEK-293-derived packaging cell lines were evaluated. rAAV2 vectors expressing the eGFP reporter gene were produced in A) VPC 2.0 cells grown in suspension in virus production medium, B) 293-F cells grown in suspension in Freestyle™ 293 medium, or C) Expi293F™ cells grown in suspension in Expi293™ Expression medium. Cells were transfected with three plasmids (pRC2 vector expressing Rep and Cap, pAAV-GFP control vector expressing GFP under the control of a CMV promoter flanked by AAV2 ITRs, and helper vectors expressing at least Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors) and FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent. AAV titers (transducing units, TU / mL) were determined 72 hours post-transfection by transduction of H1080 cells and quantification of eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transducing units / mL (TU / mL) normalized to the infectivity of rAAV produced by pALD-X80 and expressed as fold increase. [Figure 6]Versatility and efficiency of triple chimeric helper plasmids for rAAV2 production using various transfection reagents were evaluated. rAAV2 vectors expressing the eGFP reporter gene were produced in HEK-293T cells grown in suspension. Cells were transfected with three plasmids (pRC2 vector expressing Rep and Cap, pAAV-GFP control vector expressing GFP under the control of a CMV promoter flanked by AAV2 ITRs, and a helper vector expressing at least Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors) at a ratio of 1:1 μg DNA / μL reagent, along with either FectoVIR-AAV, PEIpro, or TransIT-VirusGEN. AAV titers (transducing units, TU / mL) were determined 72 hours posttransfection by transducing H1080 cells and quantifying eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transducing units / mL (TU / mL) normalized to the infectivity of rAAV produced by pALD-X80 and expressed as fold increase. [Figure 7]Versatility and efficiency of triple chimeric helper plasmids for producing rAAV of various serotypes were evaluated. A) rAAV5, B) rAAV8, or C) rAAV9 vectors expressing the eGFP reporter gene were produced in HEK-293T cells grown in suspension. Cells were transfected with three plasmids (pRC5, pRC8, or pRC9 vectors expressing Rep and serotype-specific Cap, pAAV-GFP control vector expressing GFP under the control of a CMV promoter flanked by AAV2 ITRs, and helper vectors expressing at least Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors) and FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent. AAV titers (transducing units, TU / mL) were determined 72 hours posttransfection by transducing H1080 cells and quantifying eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transducing units / mL (TU / mL) normalized to the infectivity of rAAV produced by pALD-X80 and expressed as fold increase. [Figure 8] A) Aldevron's pALD-X80 (18,876 bp), B) Aldevron's pALD-HELP (11,584 bp), and C) Cellbiolabs' VPK-402 (11,635 bp). [Figure 9] The plasmid Ad-HBoV-HSV (13324 bp) according to the present invention. [Figure 10]Helper plasmids used for rAAV production: the plasmid Ad-HBoV-HSV (13,324 bp) of the present invention and SEQ ID NO: 18; the plasmid Ad-HBoV-HSV5 (15,034 bp) of the present invention and SEQ ID NO: 35; and the comparative plasmid Ad-HBoV5 (11,651 bp) of SEQ ID NO: 36 disclosed in Wang et al., Mol. Ther. 2018, 11, pp. 40-51. All three plasmids exhibit identical structures with respect to adenoviral elements (VA-RNA, E2A, and E4orf6). Plasmid Ad-HBoV-HSV further contains a polynucleotide encoding the UL12 protein derived from HSV1 and a polynucleotide encoding the NS2 protein derived from HBoV1. Plasmid Ad-HBoV-HSV5 further contains a polynucleotide encoding the ICP8 protein derived from HSV1 and a polynucleotide encoding the NS2 protein derived from HBoV1. The comparative plasmid Ad-HBoV5 further contains a polynucleotide encoding the NS2 and NP1 proteins from HBoV1 and a polynucleotide encoding the self-cleaving peptide P2A of SEQ ID NO: 34. The HSV and HBoV proteins are driven by either the pCMV promoter or the pEFS promoter. [Figure 11] rAAV production efficiency (rAAV VG / mL titer produced in HEK293T). 11A) rAAV2, or 11B) rAAV5, or 11C) rAAV8, or 11D) rAAV9 production efficiency using the following pHelper: plasmid Ad-HBoV-HSV and plasmid Ad-HBoV-HSV5 of the present invention, and comparative plasmid Ad-HBoV5 disclosed in Wang et al., Mol. Ther. 2018, 11, pp. 40-51. For easier representation, the HBoV- and HSV-derived proteins present in pHelper are shown above the graph. [Example]

[0107] Materials and Methods cell culture HEK-293T (ATCC® CRL-3216™): Human embryonic kidney cells were grown in suspension in Freestyle F17 medium supplemented with 8 mM glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.1% Pluronic. Cells were incubated at 37°C in an 8% CO2 in air atmosphere under agitation (130 rpm, 50 mm orbit).

[0108] Freestyle™ 293-F cells (Gibco™ R79007): Human embryonic kidney cells are derived from the HEK-293 parent cell line. Cells were cultured in suspension in Freestyle™ 293 medium supplemented with 8 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.1% Pluronic. Cells were incubated at 37°C in an 8% CO2 in air atmosphere under agitation (130 rpm - 50 mm orbit).

[0109] Viral Production Cells 2.0 (Gibco™ A49784): Human embryonic kidney cells are derived from the HEK-293F parent cell line (derived from the HEK-293 cell line). Cells were grown in suspension in viral production medium supplemented with 8 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were incubated at 37°C in an 8% CO2 in air atmosphere under agitation (130 rpm, 50 mm orbit).

[0110] Expi293F™ (Gibco™ A14527): Human embryonic kidney cells are derived from the HEK-293 parent cell line. Cells were cultured in suspension in Expi293™ Expression medium supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin. Cells were incubated at 37°C in an 8% CO2 in air atmosphere under agitation (130 rpm - 50 mm orbit).

[0111] HT-1080 (ATCC® CCL-121™): Human fibrosarcoma cells were grown in DMEM 4.5 g / L 10% FBS glucose supplemented with 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin at 37°C in 5% CO2 in air atmosphere.

[0112] CHO-K1 (ATCC® CCL-61™): A cell line derived as a subclone from a parent CHO cell line established from an ovarian biopsy of an adult female Chinese hamster. Cells were grown in RPMI containing 10% FBS supplemented with 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin at 37°C in a 5% CO2 atmosphere in air.

[0113] Recombinant virus production HEK-293T (ATCC® CRL-3216™): Human embryonic kidney cells are a highly transfectable derivative of human embryonic kidney 293 cells and contain the SV40 T antigen. HEK-293T cells are widely used for recombinant virus production, gene expression, and protein production.

[0114] HEK-293T cells were cultured at 1 × 10 in 28.5 mL of Freestyle F17 supplemented with 8 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.1% Pluronic in a 125 mL Erlenmeyer flask. 6 Cells were incubated at 37°C in 8% CO2 in air atmosphere under agitation (130 rpm - 50 mm orbit).

[0115] Freestyle™ 293-F cells (Gibco™ R79007): Human embryonic kidney cells are a highly transfectable derivative of human embryonic kidney 293 cells. Freestyle™ 293F cells are part of the Freestyle™ MAX 293 Expression System and are used for gene expression and recombinant protein production.

[0116] Freestyle™ 293-F cells were cultured at 1 x 10 in 28.5 mL of Freestyle™ 293 supplemented with 8 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.1% Pluronic in a 125 mL Erlenmeyer flask. 6 Cells were seeded at 100 cells / mL. Cells were grown under agitation (130 rpm, 50 mm orbit) in an 8% CO2 atmosphere in air. 2v The mixture was incubated at 37°C in a PBS.

[0117] Viral Production Cells 2.0 (Gibco™ A49784): Human embryonic kidney cells are a clonal cell line derived from the HEK293F parent cell line and are the core component of the AAV-MAX Helper-Free AAV Production System. Viral Production Cells 2.0 are highly transfectable, do not contain the SV40 T antigen, and have been used for adeno-associated virus (AAV) production.

[0118] Viral Production Cells 2.0 cells were cultured at 1 × 10 in 28.5 mL of Viral Production Medium supplemented with 8 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 125 mL Erlenmeyer flask. 6 Cells were incubated at 37°C in 8% CO2 in air atmosphere under agitation (130 rpm - 50 mm orbit).

[0119] Expi293F™ (Gibco™ A14527): Human embryonic kidney cells are derived from the HEK-293 parent cell line. Expi293F™ are highly transfectable and are primarily used for recombinant protein production and recombinant virus production.

[0120] Expi293F™ cells were cultured at 1 x 10 in 28.5 mL of Expi293™ Expression medium supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin in a 125 mL Erlenmeyer flask. 6 Cells were incubated at 37°C in 8% CO2 in air atmosphere under agitation (130 rpm - 50 mm orbit).

[0121] Recombinant adeno-associated virus (rAAV) was produced in HEK-293T, VPC 2.0, or Expi293F™ cells and expressed at 1 × 10 6 Cells were seeded at 7.23 × 10 cells / mL and cultured at 37°C, 8% CO for 24 hours. Then, they were co-transfected with three plasmids: the pRC vector expressing Rep and Cap, the pHelper vector expressing at least the Adenovirus E2A, E4, and VA helper factors, and the pAAV-GFP (Cat. No. AAV-400, Cell BioLabs) control vector, which expresses GFP under the control of the CMV promoter. Plasmids were diluted in 1.5 mL of unsupplemented culture medium (standard: pAAV-GFP 7.23 × 10 cells / mL). 4 copies / cell - pRC 5.32 x 10 4 pHelper 1 x 10 copies / cell 4 The diluted DNA was then added to FectoVIR-AAV (1 μL per μg of total DNA), vortexed, and incubated at room temperature for 30 minutes. The transfection complex was added to the cells, and the Erlenmeyer flask was incubated at 37°C in an 8% CO2 atmosphere in air under agitation (130 rpm, 50 mm orbit) for 72 hours.

[0122] Three days after transfection, the transfected cells were collected and centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the pellet was resuspended in 2 mL of PBS. The cells were then lysed using three consecutive freeze / thaw cycles at -80°C and 37°C to release the rAAV. 2 mL of the lysate was then collected and centrifuged at 14000 rpm for 30 minutes to separate the rAAV from the cell debris. The rAAV-containing supernatant was then analyzed.

[0123] Transducing unit titers (TU / mL) were determined using recombinant adeno-associated viruses expressing a GFP reporter gene after infection of permissive cells (HT-1080 or CHO-K1) depending on the rAAV serotype produced in 96-well plates. Briefly, permissive cells were cultured at 7 × 10 per well. 3 Cells were seeded with 1000 cells and incubated for 4 hours at 37°C in a 5% CO2 atmosphere in air. The collected rAAV was then serially diluted in supplemented culture medium and added to the permissive cells in place of the previous culture medium used. 72 hours after transduction, GFP expression was analyzed by flow cytometry to determine transducing units.

[0124] Capsid titers (VP / mL) were determined using AAV Titration ELISA kits (catalog numbers PRAAV2 / PRAAV5 / PRAAV8 / PRAAV9, PROGEN) according to the manufacturer's recommendations and protocols and according to the serotype of the recombinant adeno-associated virus produced and analyzed.

[0125] Genomic titers (VG / mL) were determined by qPCR (QuantStudio™ 3 - ThermoFisher). Plasmid DNA encoding GFP was added at 2 × 10 8 copies / µL ~ 2 x 10 2A standard curve was generated using primers (Qiagen) targeting the GFP reporter gene expressed by the produced rAAV, used at a concentration of 10 copies / μL, and the SensiFAST probe Lo-ROX kit (Ozyme).

[0126] Figure 1 provides an overview of all helper plasmid maps used in this study. All helper plasmids were constructed with at least the minimal Ad helper elements (VA-RNA, E2A, and E4). Additional elements from other helper viruses (HBoV and HSV) were also added to compare their ability to produce rAAV compared to commercially available helper plasmids.

[0127] Figure 2 shows the viral titers (transducing units per mL) of rAAV2 produced after transfection of HEK 293t cells with various ratios of pHelper (pALD-X80). The mass ratio of 2:2:1 (pTransgene:pRC:pHelper) showed the best viral titer compared to the other ratios tested and was therefore selected for further experiments to compare the various pHelper variants presented in this study.

[0128] Figure 3 discloses the viral titers (transducing units per mL) of rAAV2 produced after transfection of HEK 293t cells using various pHelpers. Three commercially available helper plasmids (pALD-X80 and pALD-HELP from Aldevron and VPK-402 from Cellbiolabs) were used as benchmarks and compared with the minimal adenovirus-only element pHelper (mpH3sV2). The minimal pHelper (mpH3sV2) exhibited similar viral titers to the other commercially available helper plasmids, suggesting that none of the sequences removed in the mpH3sV2 construct were necessary to support efficient rAAV production. Therefore, the mpH3sV2 (approximately 8 kb) pHelper was used as a frame for further optimization of pHelper.

[0129] Figure 4 discloses the following: A) Viral titers (transducing units per mL) of rAAV2 produced after transfection of HEK 293t cells with various pHelpers containing either Ad-only elements (pALD-X80, pALD-HELP, and mpH3sV2, black), Ad and HBoV elements (Ad-HBoV1 and Ad-HBoV2, white), or Ad and HSV elements (Ad-HSV2 and Ad-HSV6, gray) (referred to as double-chimeric vectors). The addition of specific helper elements from HBoV and HSV could enhance the viral titers of viruses produced with these pHelpers compared to Ad-only pHelpers, suggesting that the elements introduced into the double-chimeric vectors were beneficial for efficient rAAV2 production (see Ad-HBoV1 and Ad-HSV6 compared to the optional black plots).

[0130] B) rAAV2 viral titers (transducing units per mL) produced after transfection of HEK 293t cells with various pHelpers containing either Ad-only elements (pALD-X80, pALD-HELP, and mpH3sV2) or Ad, HBoV, and HSV elements (Ad-HBoV-HSV). As shown in Figure 4A, the addition of either HBoV or HSV elements is beneficial for efficient rAAV2 production. Therefore, we evaluated the efficacy of pHelpers containing all of the Ad, HBoV, and HSV elements known to be involved in rAAV production to determine whether they could further improve rAAV2 production. Figure 4B shows that combinations of elements from all three helper viruses (referred to as triple-chimeric vectors or triple-chimeric pHelpers) performed better than double-chimeric vectors (approximately 1.6-fold increase compared to a 1.2-fold increase, respectively, when using pALD-X80 as the reference). These results suggest that the combination of helper elements from three different viruses is the best combination we have been able to obtain so far for efficiently producing infectious rAAV2.

[0131] C) GC titers (genome copies per mL) of rAAV2 produced with either pALD-X80 or Ad-HBoV-HSV, analyzed by qPCR. Because virus produced with triple-chimera pHelper was more infectious than virus produced with commercially available helper plasmids, the purpose of this experiment was to determine whether this enhanced viral titer was dependent on the amount of encapsidated DNA. The results showed no significant difference between rAAV2 produced with either pALD-X80 or our triple-chimera pHelper.

[0132] D) VP titers (viral particles per mL) of rAAV2 produced with either pALD-X80 or Ad-HBoV-HSV analyzed by ELISA. rAAV2 produced with both pALD-X80 and triple-chimera pHelper showed similar VP titers, suggesting that the complete empty ratio (filled capsids vs. empty capsids) of rAAV2 produced with triple-chimera pHelper was unchanged compared to the commercially available helper plasmid.

[0133] E) Percentage of capsids containing viral DNA (GC / VP) for rAAV2 produced with either pALD-X80 or Ad-HBoV-HSV. As shown in Figures 4C and 4D, the GC and VP titers were similar between rAAV2 produced with either pALD-X80 or the triple chimera pHelper, and therefore the GC / VP ratio (corresponding to the total number of capsids containing viral DNA copies) was also similar.

[0134] F) Overall quality (TU / VP) of rAAV2 produced by either pALD-X80 or Ad-HBoV-HSV pHelper. Because rAAV2 produced by triple-chimera pHelper had higher infectivity compared to rAAV2 produced by pALD-X80 (Figure 4B) but did not exhibit enhanced GC or VP titers (Figures 4C and 4D), the quality of rAAV2 production was analyzed in terms of the ratio of infectious to non-infectious particles (TU / VP). As shown in Figure 4F, this ratio increased when rAAV2 was produced by triple-chimera pHelper compared to pALD-X80. This increase in ratio suggested that triple-chimera pHelper enabled the production of better quality virus compared to its pALD-X80 counterpart.

[0135] Figure 5 shows the viral titers (transducing units / mL) of rAAV2 produced after transfection of various producer cell lines: VPC 2.0 (Figure 5A), HEK 293F (Figure 5B), and Expi293F (Figure 5C) using pHelper containing Ad-only elements (pALD-X80, pALD-HELP) or Ad+HBoV+HSV elements (Ad-HBoV-HSV). Results were normalized to the infectivity of rAAV produced with pALD-X80 and expressed as fold increases. The results suggest that the enhancing effect of combining Ad, HBoV, and HSV elements in a single plasmid, previously observed in HEK293T, is not cell line specific and can be observed across the three producer cell lines tested, with fold increases ranging from approximately 1.8 to approximately 4.5. Figures 5A–5C demonstrate the versatility of the triple-chimeric pHelper across various cell lines.

[0136] Figure 6 discloses the viral titers (transducing units / mL) of rAAV2 produced after transfection of HEK 293T cells with pHelper containing Ad-only elements or Ad+HboV+HSV elements (Ad-HBoV-HSV). Cells were transfected using three different transfection reagents: FectoVIR-AAV (Polyplus), PEIpro (Polyplus), and TransIT-VirusGEN (Mirus Bio). Results were normalized to the infectivity of rAAV produced with pALD-X80 and expressed as fold increase.

[0137] Because all previous results were obtained using FectoVIR-AAV for transfection of HEK293T cells, the aim was to determine whether the increased viral titers obtained with the triple chimera pHelper (Ad-HBoV-HSV) were related to the specific transfection reagent. Therefore, HEK293T cells were transfected using two other transfection reagents: PEIpro and TransIT-VirusGEN.

[0138] Comparison of titers obtained with pALD-X80 and with triple chimeric pHelper (Ad-HBoV-HSV) using various transfection reagents suggested that the increase observed with triple chimeric pHelper was independent of the transfection reagent used.

[0139] Figure 7 shows the viral titers (transducing units / mL) of various serotypes of rAAV: rAAV5 (Figure 7A), rAAV8 (Figure 7B), and rAAV9 (Figure 7C) using pHelper containing Ad-only elements (pALD-X80, pALD-HELP) or Ad, HBoV, and HSV elements (Ad-HBoV-HSV). Results were normalized to the infectivity of rAAV produced with pALD-X80 and expressed as fold increases. The results suggest that the enhancing effect of combining Ad, HBoV, and HSV elements in a single plasmid, previously observed in the production of rAAV2, is not specific to that serotype and can also be observed in the production of other rAAV serotypes, with fold increases ranging from approximately 1.4 to approximately 1.8. Figures 7A–7C demonstrate the versatility of the triple-chimeric pHelper for various AAV serotypes.

[0140] Absence / limitation of unnecessary sequences: Some commercially available pHelpers contain unnecessary sequences derived from the adenovirus genome. For gene therapy purposes, these sequences should be avoided to ensure better safety and less toxicity. To this end, we have defined and removed all unnecessary sequences from the adenovirus genome, retaining only the elements involved in rAAV production.

[0141] Unnecessary sequences refer to sequences not involved in rAAV production, i.e., sequences that typically flank required genes present in commercially available plasmids due to technical cloning limitations. Therefore, any nucleotides / sequences other than those found in the gene of interest (from the promoter to the polyadenylation signal) were excluded from the plasmids of the present invention.

[0142] Increased titer: One of the major concerns in rAAV manufacturing has been increasing viral titer to meet the requirement for large amounts of infectious virus necessary for rAAV-based gene therapy. The plasmids of the present invention resulted in an increased percentage of infectious virus compared to other commercially available helper plasmids (up to approximately 2-fold increase depending on the AAV serotype).

[0143] Virus quality: The plasmids of the present invention resulted in increased numbers of infectious viruses compared to the benchmark commercial pHelper. Furthermore, the inventors demonstrated that virus production performed with the plasmids of the present invention resulted in improved infectious titers per total capsid (TU / VP), suggesting that the rAAV produced with the plasmids of the present invention was of better quality (more infectious). Given that current virus production methods result in very low quality virus batches (most of the viruses produced are noninfectious), finding ways to improve the infectivity of rAAV could improve the overall efficacy of rAAV-based gene therapy while reducing immune responses targeting these noninfectious viruses.

[0144] Versatile pHelper for Various AAV Serotypes: The helper plasmids of the present invention were tested with various AAV serotypes to evaluate their efficiency relative to currently used methods for producing the serotypes of interest (i.e., serotypes 2, 5, 8, and 9). The plasmids of the present invention demonstrated increased infectious titers for all of the above serotypes compared to commercially available helper plasmids. The extent of this increase was serotype-dependent, ranging from 2-fold for AAV2 to 3-fold for AAV5, AAV8, and AAV9, with a range of 1.5-fold to 2-fold for AAV2, AAV5, AAV8, or AAV9.

[0145] Versatile pHelper for various cell lines: The helper plasmids of the present invention were tested in several mammalian cell lines derived from HEK-293 (i.e., HEK-293T cells, HEK-293F cells, VPC 2.0 cells, and Expi293F cells). In four cell lines tested, the helper plasmids of the present invention showed increased infectious titers compared to commercially available helper plasmids, suggesting that the plasmids of the present invention function under different cell culture conditions as needed.

[0146] The present inventors compared the rAAV production efficiency between the triple-chimeric pHelper of the present invention (plasmid Ad-HBoV-HSV and plasmid Ad-HBoV-HSV5) and the comparative plasmid Ad-HBoV5 disclosed in Wang et al., Mol. Ther. 2018, 11, pp. 40-51. The triple-chimeric pHelper of the present invention resulted in highly efficient production of multiple rAAV serotypes (2, 5, 8, and 9) (Figures 10 and 11).

[0147] While the present invention has been described in terms of various preferred embodiments, those skilled in the art will understand that various modifications, substitutions, omissions, and alterations may be made therein without departing from the scope of the present invention. Accordingly, it is intended that the scope of the present invention be limited by the following claims (including equivalents thereof).

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