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

Abstract

The present invention relates to a chimeric helper nucleic acid molecule comprising helper elements from three different helper viruses, as well as methods and systems for producing an infectious recombinant adeno-associated virus (rAAV) without helper viruses. The present invention claims a chimeric helper nucleic acid molecule suitable for use in a method for producing an infectious rAAV, the chimeric helper nucleic acid molecule comprising one or more polynucleotides encoding an adenovirus helper viral function, wherein the polynucleotides comprise an E2A gene from an adenovirus (Ad), an E4 gene from the Ad, and a virus-related ribonucleic acid (VA-RNA) polynucleotide from the Ad; and one or more polynucleotides comprising an open reading frame (ORF) from a herpes simplex virus (HSV) gene, in particular a polynucleotide encoding at least a UL12 protein from HSV; and an ORF from the human bocavirus (HBoV) gene, in particular a polynucleotide encoding at least the NS2 protein from HBoV, where the polynucleotide further encodes a helper viral function, and where the polynucleotide further comprises a non-endogenous sequence for controlling the expression of ORF. The invention also relates to a packaging system, an rAAV-producing cell, a helper-virus-free method for producing an rAAV-producing cell line, a method for producing a replication-deficient infectious rAAV vector particle, and an in vitro use of an rAAV-producing cell for producing a replication-deficient infectious rAAV vector particle.

Description

Technical Field

[0001] The present invention relates to a chimeric auxiliary nucleic acid molecule comprising auxiliary elements from three different auxiliary viruses, and a method and system for producing an infectious recombinant adeno-associated virus (rAAV) without an auxiliary virus. The present invention claims a chimeric auxiliary nucleic acid molecule suitable for a method for producing an infectious rAAV, wherein the chimeric auxiliary nucleic acid molecule comprises one or more polynucleotides encoding adenovirus auxiliary virus functions, wherein the polynucleotides comprise E2A genes from adenovirus (Ad), E4 genes from Ad, and virus-associated ribonucleic acid (VA-RNA) polynucleotides from Ad; and one or more polynucleotides comprising an open reading frame (ORF) from a herpes simplex virus (HSV) gene, in particular a polynucleotide encoding at least UL12 protein from HSV; and an ORF from a human bocavirus (HBoV) gene, in particular a polynucleotide encoding at least NS2 protein from HBoV, wherein the polynucleotide further encodes auxiliary virus functions, and wherein the polynucleotide further comprises a non-endogenous sequence for controlling the expression of the ORF. The present invention also relates to packaging systems, rAAV producer cells, helper virus-free methods for producing rAAV producer cell lines, methods for producing replication-defective infectious rAAV vector particles, and in vitro use of rAAV producer cells in producing replication-defective infectious rAAV vector particles. Background Art

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

[0003] Genetic defects are usually present in every cell of the patient's body. Therefore, gene therapy AAV must be delivered to most cells in the organ to achieve a therapeutic effect. Therefore, AAV-based treatment requires the administration of a very large amount of virus. Producing enough AAV at an acceptable cost without compromising safety is a major challenge in bioproduction. Increasing or improving AAV production will help treat various genetic diseases. Optimization strategies focus on the genetic components required for AAV assembly, production cell lines, and omics-based methods (Cell Culture Engineering and Technology, 335-364, Chapter Bio-Production of Adeno-Associated Virus for Gene Therapy).

[0004] In cultured cells, the replication of AAV requires co-infection with a helper virus. In the absence of a helper virus co-infection, AAV can integrate its genome site-specifically into the AAVS1 region of chromosome 19. When subsequently infected with a helper virus, the AAV genome is released from chromosome 19 through a process called rescue and begins productive replication. The AAV genome cloned into a plasmid vector can also be used to initiate productive AAV replication. When this construct is transfected into cells and these cells are simultaneously or subsequently infected with a helper virus, the AAV genome is released from the plasmid (J. Virol., 2003, 77 (21), 11480-11490).

[0005] AAV is a member of the Parvoviridae family and contains a single-stranded genome of approximately 4,700 bases. The genome of AAV 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, namely Rep78, Rep68, Rep50 and Rep42, which are mainly involved in viral DNA replication, packaging, integration and transcription. The Cap gene encodes three proteins that are the building blocks of the mature capsid (VP1, VP2 and VP3), proteins involved in assembling the capsid from the VP protein (assembly activation protein), and recently discovered proteins involved in viral budding (membrane-associated accessory protein).

[0006] The life cycle of AAV depends on the presence of a helper virus. Helper viruses induce active replication of the viral genome and the formation of new infectious AAV and are therefore necessary for the production of large quantities of rAAV. Initially, helper elements were provided from infectious adenoviruses that were used to transduce cells transfected with plasmids encoding the Rep and Cap genes. However, infectious adenoviruses were found in purified rAAV final vector stocks. To address this issue, minimal helper elements of adenovirus were identified and cloned into plasmids, giving rise to helper-free production methods, such as the so-called triple transfection method. Engineering and optimization of helper plasmids have a significant impact on rAAV production efficiency, which also depends on the AAV serotype, as some helper viruses prefer to bind to specific serotypes based on seroepidemiological data.

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

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

[0009] U.S. patent application US20220073947 discloses a circular nucleic acid comprising at least one AAV promoter, which can be activated by at least one auxiliary polypeptide or auxiliary polynucleotide, which is derived from or derived from a virus selected from adenovirus and herpes virus.

[0010] International patent application WO2022 / 173944 discloses a method for producing AAV in E1 complementing production cells, which includes the step of transfecting the production cells with one or more vectors comprising: E1A Ad helper gene; Ad helper gene selected from E2A, E4 or both; virus-associated non-coding RNA (VA RNA); and AAV gene selected from Rep, Cap or both.

[0011] Examples of commercially available helper plasmids (pHelper) include 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. The production of rAAV is based on cell culture and a defined set of genetic elements, including the transgene and structural / nonstructural elements for rAAV production.

[0013] Although bioprocess optimization has been carried out for many years to improve the quantity and quality of rAAV production methods, it is still difficult to meet the requirements of sufficient scale and cost for rAAV-based therapies. Therefore, one object of the present invention is to provide a method for producing rAAV without helper virus, wherein the method is capable of producing high titer and high quality rAAV. Another object of the present invention is to develop a universal helper nucleic acid molecule, such as a helper plasmid (pHelper) suitable for the production of different AAV serotypes and different production cell lines. Summary of the invention

[0014] In the present invention, the inventors describe the design and use of a novel auxiliary nucleic acid molecule, and illustrate that this auxiliary plasmid is used to produce rAAV using a triple transfection method in mammalian cells. This novel auxiliary nucleic acid molecule (such as an auxiliary plasmid) includes genes derived from three different auxiliary viruses, which are adenovirus, bocavirus and herpes virus herein. Compared with plasmids (such as auxiliary plasmids) that only include adenovirus genes, even include adenovirus and bocavirus, or include adenovirus and herpes virus genes on the same auxiliary nucleic acid molecule, this novel auxiliary nucleic acid molecule (such as an auxiliary plasmid) causes the output of infectious rAAV to increase. In addition, this novel nucleic acid molecule (such as an auxiliary plasmid) is shown to be compatible with multiple 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 the designed auxiliary nucleic acid molecules in various production systems.

[0015] Specifically, the present invention is illustrated by a triple transfection method of mammalian cells, in which three different plasmids are used: an AAV transfer plasmid (pTransfer), which contains the gene of interest flanked by AAV2 inverted terminal repeats (ITRs); a Rep / Cap plasmid (pRC), which contains the Rep gene from AAV2 and the Cap gene from the desired AAV serotype to be produced; and a helper plasmid (pHelper), which contains helper elements from helper viruses (i.e., adenovirus, bocavirus, and herpes virus), which support the production of AAV.

[0016] In one embodiment, the inventors describe a method using a novel pHelper that replaces the current pHelper for rAAV production. This novel pHelper performs transfection under the same conditions as the classical triple transfection method, but achieves higher titers of infectious AAV particles than other pHelpers.

[0017] Combining helper elements from three different viruses on the same helper nucleic acid molecule, specifically pHelper (i.e., adenovirus, bocavirus, and herpesvirus), allowed the production of highly infectious virus yields under all conditions tested (varying AAV serotypes, transfection reagents, and packaging cell lines).

[0018] The present invention relates to a chimeric auxiliary nucleic acid molecule, which is suitable for a method for producing an infectious recombinant adeno-associated virus (rAAV), wherein the chimeric auxiliary nucleic acid molecule comprises:

[0019] - one or more polynucleotides encoding adenoviral helper virus (helper) functions for activating the expression of Rep and Cap proteins from an adeno-associated virus, wherein the polynucleotides comprise 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

[0020] - One or more polynucleotides encoding helper virus functions for AAV production, wherein the polynucleotides comprise an open reading frame (ORF) from a herpes simplex virus (HSV), preferably a herpes simplex virus serotype 1 (HSV1) gene; and an ORF from a human bocavirus (HBoV), preferably a human bocavirus serotype 1 (HBoV1) gene; and wherein the polynucleotides further comprise an endogenous or non-endogenous sequence for controlling the expression of the ORF.

[0021] In a specific embodiment, the present invention relates to a chimeric helper nucleic acid molecule suitable for use in a method for producing an infectious recombinant adeno-associated virus (rAAV), the chimeric helper nucleic acid molecule comprising:

[0022] (i) an ORF from an HSV gene, which consists of a polynucleotide encoding at least one HSV protein, in particular a polynucleotide encoding at least a UL12 protein from HSV or at least an ICP8 protein from HSV, preferably a polynucleotide encoding a UL12 protein from HSV, preferably from HSV1, or a polynucleotide encoding an ICP8 protein from HSV, preferably from HSV1;

[0023] (ii) an ORF from a HBoV gene, which consists of a polynucleotide encoding an NS2 protein from HBoV, preferably from HBoV1, or an NP1 protein from HBoV, preferably from HBoV1;

[0024] (iii) a VA-RNA polynucleotide from Ad, preferably from Ad5;

[0025] (iv) an E2A gene from Ad, preferably from Ad5;

[0026] (v) an E4 gene from Ad, preferably from Ad5, in particular an E4orf6 sequence;

[0027] wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and wherein the polynucleotides of (i) to (v) are arranged in any order and / or orientation relative to each other;

[0028] Provided that the chimeric auxiliary nucleic acid molecule does not comprise (ii) an ORF from an HBoV gene, which consists of a polynucleotide encoding an NS2 protein from HBoV and an NP1 protein from HBoV.

[0029] In a specific embodiment of the invention, the chimeric helper nucleic acid molecule suitable for use in the method for producing an infectious recombinant adeno-associated virus (rAAV) comprises:

[0030] (i) an ORF from an HSV gene, which consists of a polynucleotide encoding at least one HSV protein, preferably one or two proteins of HSV, in particular a polynucleotide encoding at least a UL12 protein from HSV or at least an ICP8 protein from HSV, preferably a polynucleotide encoding a UL12 protein from HSV, preferably from HSV1, and / or a polynucleotide encoding an ICP8 protein from HSV, preferably from HSV1;

[0031] (ii) an ORF from a HBoV gene, which consists of a polynucleotide encoding an NS2 protein from HBoV, preferably from HBoV1, or an NP1 protein from HBoV, preferably from HBoV1;

[0032] (iii) a VA-RNA polynucleotide from Ad, preferably from Ad5;

[0033] (iv) an E2A gene from Ad, preferably from Ad5;

[0034] (v) an E4 gene from Ad, preferably from Ad5, in particular an E4orf6 sequence;

[0035] wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and wherein the polynucleotides of (i) to (v) are arranged in any order and / or orientation relative to each other;

[0036] Provided that the chimeric auxiliary nucleic acid molecule does not comprise (ii) an ORF from an HBoV gene, which consists of a polynucleotide encoding an NS2 protein from HBoV and an NP1 protein from HBoV.

[0037] As defined herein, the term "chimeric nucleic acid molecule" refers to a nucleic acid molecule comprising sequences from different organisms.

[0038] As defined herein, the term "nucleic acid molecule" or "nucleic acid" refers to a polymer composed of covalently linked nucleotides to form a chain-based structure. Nucleic acids are single-stranded, or double-stranded, or a combination of single and double strands on different regions of the nucleic acid chain; and are selected from deoxyribonucleic acid (DNA), ribonucleic acid (RNA), small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), dicer substrate short 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 oligonucleotides. The term "polynucleotide" can be used interchangeably with the term "nucleic acid molecule". A specific nucleic acid molecule is an open reading frame (ORF) encoding an amino acid sequence of a polypeptide, and these polypeptides can be expressed when placed under the control of transcriptional and translational regulatory elements (including promoters).

[0039] As defined herein, when the term "helper" or "helper function" refers to an auxiliary nucleic acid molecule suitable for AAV production, it means that the molecule provides functions, i.e., "helper virus functions" necessary for AAV to be replicated and for viral gene expression for transduction and assembly of viral particles, i.e., necessary for packaging the AAV genome in cells (particularly mammalian cells) (Marie-Claude Geoffroy, et al. Current Gene Therapy, 2005, 5(3), 265-271). In one embodiment, these helper functions can be provided by a polypeptide encoded by a nucleic acid molecule. In one embodiment, when these helper functions are provided by a nucleic acid molecule derived from HSV or HBoV, they are the same as the helper functions provided by the Ad nucleic acid (particularly E2A, E4 and / or VA-RNA) used according to the present invention. In one embodiment, when the auxiliary function is provided by a nucleic acid molecule derived from HSV or HBoV, it can enhance or improve the auxiliary function provided by the Ad nucleic acid used according to the present invention, that is, it can increase the rAAV virus titer in the production cell and / or the transduction titer (TU / ml or VG / ml) of the produced rAAV determined after infection of the permissive cells. In one embodiment, when the auxiliary function is provided by a nucleic acid molecule derived from HSV or HBoV, it can be a function that provides new activities (relative to those provided by the Ad nucleic acid used according to the present invention) in the production cell and / or infected cell. These new activities can also increase the rAAV titer in the production cell and / or the transduction titer (TU / ml or VG / ml) of the produced rAAV determined after infection of the permissive cells. The increase in rAAV titer can be evaluated or measured by any appropriate known method by comparing the rAAV titer produced when the nucleic acid molecules derived from HSV and HBoV are not used according to the present invention. Examples of measurement of rAAV titers are disclosed in the embodiments of this article. In one embodiment, the helper functions provided by the HSV or HBoV helper genes or ORFs increase the versatility of the rAAV produced in terms of production cells, transfection reagents and / or rAAV serotypes, particularly types 2, 5, 8 and / or 9. rAAV titers can be determined using capsid titers (VP / ml) as disclosed in the Examples, for example using a titration ELISA kit, by performing qPCR on a reporter gene product to determine genome titers (VG / ml).

[0040] As defined herein, the term "expression cassette" refers to a functional expression unit capable of driving the expression of one or more incorporated polynucleotides.

[0041] 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 polIII. In a preferred embodiment, it refers to the VA-RNA from Ad5 (positions 10,545 to 11,029 of the NCBI reference sequence AC_000008.1).

[0042] As defined herein, the term "E2A gene from Ad" refers to an E2A gene encoding an E2A protein from Ad transcribed by pol III. In a preferred embodiment, it refers to the E2A gene from Ad5 (positions 22,388 to 27,121 of the NCBI reference sequence AC_000008.1).

[0043] As defined herein, "E4orf6 sequence from Ad" refers to E4orf6, which is one of the proteins encoded by the E4 gene from Ad. In a preferred embodiment, it refers to the E4orf6 coding sequence from Ad5 (positions 33,188 to 34,072 of NCBI reference sequence AC_000008.1).

[0044] The inventors have demonstrated that the use of the triple chimeric auxiliary element according to the present invention advantageously provides higher quantities of infectious recombinant viruses of various AAV serotypes (particularly types 2, 5, 8 and / or 9) and shows versatility in different production cells. The inventors have also demonstrated that the use of the triple chimeric auxiliary element according to the present invention can significantly increase the infectious titer (measured as TU / VP / VG) of the produced rAAV. Therefore, the auxiliary element provided by the nucleic acid molecule derived from HSV and HBoV is considered to provide further auxiliary virus function compared to the auxiliary virus function provided by the Ad nucleic acid molecule.

[0045] The expressions "derived from" or "derived from" (singular or plural) as used herein, in particular nucleic acid molecules derived from HSV or HBoV viruses are disclosed, refer to the fact that the expressed polypeptides encoded by nucleic acids derived from HSV or HBoV viruses have the characteristics of natural viral proteins expressed by these corresponding viruses, so that they confer similar or identical effects to the auxiliary elements used in the present invention. The nucleotide sequence of such nucleic acid molecules may be identical to the wild-type sequence found in the viral genome, or may be modified relative to the wild-type sequence, in particular in order to optimize expression in defined production cells. Nucleic acid molecules derived from HSV and HBoV viruses may be purified or synthetic molecules.

[0046] As defined herein, the term "recombinant adeno-associated virus (rAAV)" refers to an AAV virus that contains one or more heterologous genes or polynucleotides in its genome, i.e., these heterologous genes or polynucleotides are not derived from AAV. Therefore, rAAV can be used as a vector to express heterologous genes or polynucleotides in target cells.

[0047] 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 (involving the production of mRNA) into a polypeptide in vitro or in vivo under the control of endogenous or native regulatory sequences (such as a promoter).

[0048] In a specific embodiment of the invention, the chimeric helper nucleic acid molecule is a recombinant helper nucleic acid molecule.

[0049] As defined herein, the term "recombinant nucleic acid molecule" refers to a nucleic acid molecule that has been modified by genetic engineering, for example, by the addition or insertion of a heterologous nucleic acid construct into the molecule.

[0050] In another specific embodiment of the present invention, the chimeric helper nucleic acid molecule is a chimeric helper expression vector, in particular a chimeric helper expression plasmid.

[0051] As defined herein, the term "vector" refers to a biological or chemical entity suitable for delivering a polynucleotide of interest, in particular a polynucleotide encoding a polypeptide of interest, to a cell, in particular a production cell or a target cell as described herein. Therefore, the term "vector" refers to or contains genetic information in its structure and is capable of delivering a nucleic acid molecule carrying the genetic information. In some embodiments, the vector is capable of expressing the nucleic acid molecule carrying the genetic information, in particular enabling the expression of polypeptides from these molecules. In one embodiment, the vector is an expression vector, which comprises regulatory elements for expressing the nucleic acid molecule it contains, in particular expressing heterologous polypeptides. In the context of the present invention, the term "vector" is therefore related to a nucleic acid molecule carrying a viral auxiliary function disclosed herein, or to a nucleic acid molecule carrying a transgene of interest (e.g., a plasmid transfer disclosed herein), or to a nucleic acid molecule carrying a molecule encoding a Rep and / or Cap protein (such as pRep / Cap), and it is also related to the recombinant AAV particles produced. A vector may be, for example, a replicon, or may contain a replicon. A vector may be free or non-free. In one embodiment, a vector may be a plasmid modified by genetic engineering, and is intended to transfer a nucleic acid molecule to a cell. In one embodiment, the vector may be a recombinant viral particle, the genome of which comprises the nucleic acid to be transferred into the target cell and, if desired, expressed in such a cell. In one embodiment, the vector according to the invention comprises at least one bacterial functional unit (ori), and may also comprise one or more expression units and / or one or more integration units.

[0052] As defined herein, the term "plasmid" refers to a DNA molecule that is different from chromosomal DNA and is capable of autonomous replication. Plasmids are usually circular double-stranded DNA.

[0053] In a specific embodiment of the present invention, rAAV is selected from the following serotypes: rAAV of serotype 1 (rAAV1), rAAV of serotype 2 (rAAV2), rAAV of serotype 3 (rAAV3), rAAV of serotype 4 (rAAV4), rAAV of serotype 5 (rAAV5), rAAV of serotype 6 (rAAV6), rAAV of serotype 7 (rAAV7), rAAV of serotype 8 (rAAV8), rAAV of serotype 9 (rAAV9), rAAV of serotype 10 (rAAV10), rAAV of serotype 11 (rAAV11), rAAV of serotype 12 (rAAV12) and rAAV of serotype 13 (rAAV13). In a specific embodiment, the produced rAAV is selected from rAAV2, rAAV5, rAAV8 and rAAV9, preferably rAAV2, rAAV5 and rAAV9.

[0054] In a specific embodiment of the invention, the Ad providing a polynucleotide encoding a polypeptide having AAV helper function is Ad of serotype 2 (Ad2) or Ad of serotype 5 (Ad5), preferably Ad5. In a specific embodiment, Ad demonstrates helper function of a polynucleotide encoding the Cap protein of AAV serotype 2 (AAV2), AAV serotype 5 (AAV5) or AAV serotype 9 (AAV9), particularly AAV2.

[0055] Herpes simplex virus (HSV) is a human herpesvirus, which is an enveloped double-stranded DNA virus divided into two known serotypes: HSV-1 (human alphaherpesvirus 1, also known as HHV1, whose complete genome sequence can be found in GenBank with accession number MN136524, and annotated amino acid sequences of encoded proteins) and HSV-2 (human alphaherpesvirus 2, whose complete genome sequence can be found in EMBL with accession number Z86099, and annotated amino acid sequences of encoded proteins). The DNA genome consists of two covalently linked components, respectively referred to as L (long) and S (short) unique regions. The HSV virion is a particle comprising a capsid and an envelope. Among the nonstructural proteins, HSV encodes UL12, which is an alkaline exonuclease (DNA enzyme) that participates in viral nucleic acid metabolism by processing linear or branched viral DNA intermediates to promote the production of mature progeny viral DNA molecules. The protein has endonuclease and exonuclease activity on both double-stranded and single-stranded DNA substrates.

[0056] In a specific embodiment of the present invention, the ORF from the HSV gene composed of a polynucleotide encoding at least one protein of HSV is a polynucleotide encoding a structural and / or non-structural protein of HSV, in particular a protein selected from UL5, UL8, UL12, UL29, UL52 and ICP8 of HSV. Preferably, the polynucleotide encodes a single UL12 protein from HSV, or a combination of a UL12 protein from HSV with other HSV proteins (proteins from HSV as described above); or the polynucleotide encodes a single ICP8 protein from HSV, or a combination of an ICP8 protein from HSV with other HSV proteins (proteins from HSV as described above). In particular, the polynucleotide encodes a UL12 protein from HSV and an ICP8 protein from HSV.

[0057] In a specific embodiment of the present invention, the HSV is HSV of serotype 1 (HSV1) or HSV of serotype 2 (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 and ICP8 proteins from HSV1.

[0058] In a preferred embodiment of the present invention, the ORF from the HSV gene consists of a polynucleotide encoding the UL12 protein from HSV, preferably from HSV1.

[0059] Human bocavirus (HBoV) is a single-stranded DNA virus of the Parvoviridae family, covering the HBoV1, HBoV2, HBoV3 and HBoV4 serotypes, some of which are capable of infecting humans. The genome sequences of the virus strains isolated from patients can be found in GenBank under accession numbers MG953829, MG953830, MG953831, MG953832, MG953833 and MG953834, as well as the amino acid sequences of the encoded proteins. The genome of HBoV encodes 3 ORFs (ORF1, ORF2, ORF3). One of them encodes 4 nonstructural proteins (NS1, NS2, NS3 and NS4), one of which encodes the small nonstructural protein NP1 protein, and the last one in the genome encodes the capsid proteins (VP1, VP2 and VP3). The NP1 gene is an alternative reading frame of VP1 and overlaps with the start of VP1. The ability of NP1 to induce apoptosis in cells has been characterized.

[0060] In one embodiment, the helper functions provided by the nucleic acid molecule derived from HSV or HBoV are achieved by expressing a polypeptide of HSV or HBoV, respectively.

[0061] The terms "polypeptide" or "protein" are used interchangeably and are defined herein as a wild-type or natural polypeptide in an HSV or 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 retains the auxiliary effect of the wild-type polypeptide (or a commonly used wild-type polypeptide) on rAAV production, especially on the titer of the produced rAAV. In one embodiment, the fragment or mutant polypeptide may have more than 80% or more than 90%, especially more than 95% identity with the wild-type sequence of the reference polypeptide.

[0062] In a specific embodiment of the invention, the polynucleotide encoding the additional helper virus functions provided by the polynucleotide derived from HBoV is a polynucleotide encoding a structural and / or non-structural protein of HBoV, in particular a protein selected from the group consisting of NS1, NS2, NS3, NS4, NP1, VP1, VP2 and VP3 proteins of HBoV. Preferably, the polynucleotide encodes a single HBoV NS2 protein, or a combination of HBoV NS2 protein with other HBoV proteins, or a single HBoV NP1 protein, or a combination of HBoV NP1 protein with other HBoV proteins.

[0063] In a preferred embodiment of the present invention, the ORF from the HBoV gene consists of a polynucleotide encoding the NS2 protein from HBoV or the NP1 protein from HBoV. Preferably, the ORF from the HBoV gene consists of a polynucleotide encoding the NS2 protein from HBoV.

[0064] In a specific embodiment of the present invention, the HBoV is HBoV of serotype 1 (HBoV1), HBoV of serotype 2 (HBoV2), HBoV of serotype 3 (HBoV3) or HBoV of serotype 4 (HBoV4), preferably HBoV1. In one embodiment, the polynucleotide encodes an HBoV1 accessory protein, in particular the NS2 protein, in particular a single NS2 protein, or a combination of NS2 and other proteins (such as NP1 of HBoV1). Preferably, the ORF from the HBoV gene consists of a polynucleotide encoding the NS2 protein of HBoV1.

[0065] In one embodiment, the polynucleotide encoding additional auxiliary functions encodes the UL12 protein and NS2 protein of the HSV serotype, in particular a single NS2 protein, or a combination of NS2 with other proteins (such as NP1 of HBoV). In a specific embodiment, the polynucleotide encodes the UL12 protein and NS2 protein of the HSV1 serotype, in particular a single NS2 protein, or a combination of NS2 with other proteins (such as NP1 of HBoV1).

[0066] In a specific embodiment of the present invention, the ORF from the HSV gene consists of a polynucleotide encoding the UL12 protein from HSV, preferably from HSV1; and the ORF from the HBoV gene consists of a polynucleotide encoding the NP1 protein from HBoV, preferably from HBoV1.

[0067] In a preferred embodiment of the present invention, the ORF from the HSV gene consists of a polynucleotide encoding the UL12 protein from HSV, preferably from HSV1; and the ORF from the HBoV gene consists of a polynucleotide encoding the NS2 protein from HBoV, preferably from HBoV1.

[0068] In a specific embodiment of the present invention, the ORF from the HSV gene consists of a polynucleotide encoding the ICP8 protein from HSV, preferably from HSV1; and the ORF from the HBoV gene consists of a polynucleotide encoding the NP1 protein from HBoV, preferably from HBoV1.

[0069] In a preferred embodiment of the present invention, the ORF from the HSV gene consists of a polynucleotide encoding the ICP8 protein from HSV, preferably from HSV1; and the ORF from the HBoV gene consists of a polynucleotide encoding the NS2 protein from HBoV, preferably from HBoV1.

[0070] In a specific embodiment of the present invention, the ORF from the HSV gene consists of a polynucleotide encoding a combination of a UL12 protein from HSV and other HSV proteins (e.g., the above-mentioned proteins from HSV); and the ORF from the HBoV gene consists of a polynucleotide encoding an NP1 protein from HBoV, preferably from HBoV1. In particular, the ORF from the HSV gene consists of a polynucleotide encoding a UL12 protein from HSV, preferably from HSV1, and an ICP8 protein from HSV, preferably from HSV1; and the ORF from the HBoV gene consists of a polynucleotide encoding an NP1 protein from HBoV, preferably from HBoV1.

[0071] In a preferred embodiment of the present invention, the ORF from the HSV gene consists of a polynucleotide encoding a combination of a UL12 protein from HSV and other HSV proteins (e.g., the above-mentioned HSV proteins); and the ORF from the HBoV gene consists of a polynucleotide encoding an NS2 protein from HBoV, preferably from HBoV1. In particular, the ORF from the HSV gene consists of a polynucleotide encoding a UL12 protein from HSV, preferably from HSV1, and an ICP8 protein from HSV, preferably from HSV1; and the ORF from the HBoV gene consists of a polynucleotide encoding an NS2 protein from HBoV, preferably from HBoV1.

[0072] In a specific embodiment of the invention, nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, in particular different promoters. In a preferred embodiment of the invention, the NS2 protein from HBoV, preferably from HBoV1, is under the control of the pCMV promoter. In another preferred embodiment of the invention, the NP1 protein from HBoV, preferably from HBoV1, is under the control of the pCMV promoter.

[0073] In another preferred embodiment of the present 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.

[0074] In another preferred embodiment of the present 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.

[0075] In another preferred embodiment of the present invention, at least ICP8 protein from HSV, in particular from HSV1 and at least UL12 protein from HSV, in particular from HSV1, preferably ICP8 and UL12 proteins from HSV, in particular from HSV1, are under the control of EFS promoter (i.e., the core promoter of eukaryotic translation elongation factor 1α).

[0076] 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, cytomegalovirus (CMV) promoter, CMV enhancer, EFS promoter, SV40 virus promoter, CBA promoter, CAG promoter, E2A promoter, and E4 promoter.

[0077] The term "non-endogenous promoter" refers to a promoter that is derived from a different source than the native promoter of the coding sequence.

[0078] In a preferred embodiment of the present invention, the polynucleotides in the chimeric nucleic acid molecule have the following order from 5' to 3' direction: (i), (ii), (iii), (iv) and (v), wherein nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, in particular different promoters.

[0079] In a specific embodiment of the present invention, the chimeric helper nucleic acid molecule further comprises an additional adenoviral gene, such as the E1 gene from Ad, preferably from Ad5.

[0080] In a specific embodiment of the present invention, the chimeric helper nucleic acid molecule further comprises:

[0081] - origin of replication (ori); and / or

[0082] - Herpes simplex virus thymidine kinase (HSV TK polyA) signal from downstream of the HSV UL12 ORF; and / or

[0083] - bovine growth hormone polyadenylation (bGH polyA) signal from downstream of HBoV NS2 ORF; and / or

[0084] - transcriptional regulatory elements, such as cytomegalovirus (CMV) promoter, CMV enhancer, EFS promoter, SV40 virus promoter, CBA promoter, CAG promoter, E2A promoter and / or E4 promoter, to control the transcription of HSV ORF and HBoV ORF; and / or

[0085] -optionally a nucleic acid encoding a marker protein such as a kanamycin resistance gene.

[0086] As defined herein, the term "ori" refers to an insulating replication origin based on high-copy pmB1 and flanked by two bacterial transcription terminators, namely, a T7 terminator at the 5' end and a LuxIA terminator at the 3' end. This transcriptional insulation is designed to minimize transcriptional interference from neighboring content, thereby ensuring higher amplification rates in bacteria.

[0087] As defined herein, the term "nucleic acid encoding a kanamycin resistance gene" refers to an antibiotic resistance cassette, located in nptIIORF, conferring resistance to kanamycin treatment under the control of the pAmpR promoter and terminated by a hairpin terminator.

[0088] In a preferred embodiment of the present invention, in the chimeric helper nucleic acid molecule:

[0089] - the polynucleotide encoding the UL12 protein from HSV1 is SEQ ID NO: 1;

[0090] - the polynucleotide encoding the NS2 protein from HBoV1 is SEQ ID NO: 2;

[0091] - the VA-RNA polynucleotide from Ad5 is SEQ ID NO: 3;

[0092] - the E2A gene from Ad5 is SEQ ID NO: 4;

[0093] - the E4 gene from Ad5 is SEQ ID NO: 5, in particular the E4orf6 sequence from Ad5 is SEQ ID NO: 6; and / or

[0094] - The polynucleotide encoding the ICP8 protein from HSV1 is SEQ ID NO:32.

[0095] In a preferred embodiment of the present invention, in the chimeric helper nucleic acid molecule:

[0096] -ori is the nucleotide sequence of SEQ ID NO:7;

[0097] -HSV TK polyA signal is a polynucleotide of sequence SEQ ID NO: 8;

[0098] - bGH polyA signal is a polynucleotide of sequence SEQ ID NO: 9;

[0099] -CMV promoter is a polynucleotide of sequence SEQ ID NO: 10;

[0100] -CMV enhancer is a polynucleotide of sequence SEQ ID NO:11;

[0101] -EFS promoter is a polynucleotide of sequence SEQ ID NO:12;

[0102] - E2A promoter is a polynucleotide of sequence SEQ ID NO: 13;

[0103] - E4 promoter is a polynucleotide of sequence SEQ ID NO: 14; and / or

[0104] - kanamycin resistance gene having the sequence SEQ ID NO: 15;

[0105] In a preferred embodiment of the present invention, the chimeric auxiliary nucleic acid molecule comprises (i) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding a UL12 protein from HSV1 and an HSV TK polyA signal, the sequence of the HSV TK polyA signal being the sequence of SEQ ID NO:16, and (ii) a polynucleotide insert comprising a CMV promoter, a polynucleotide encoding a NS2 protein from HBoV1 and a bGH polyA signal, the sequence of the bGH polyA signal being the sequence of SEQ ID NO:17.

[0106] In a specific embodiment of the present invention, the chimeric auxiliary nucleic acid molecule comprises (i) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding a UL12 protein from HSV1 and an HSV TK polyA signal, the sequence of the HSV TK polyA signal being the sequence of SEQ ID NO:16, and (ii) a polynucleotide insert comprising a CMV promoter, a polynucleotide encoding an NP1 protein from HBoV1 and a bGH polyA signal, the sequence of the bGH polyA signal being the sequence of SEQ ID NO:17.

[0107] In another specific embodiment of the present invention, the chimeric auxiliary nucleic acid molecule comprises (i) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding an ICP8 protein from HSV1 and an HSV TK polyA signal, the sequence of which is the sequence of SEQ ID NO:16, and (ii) a polynucleotide insert comprising a CMV promoter, a polynucleotide encoding an NS2 protein from HBoV1 and a fragment of a bGH polyA signal, the sequence of which is the sequence of SEQ ID NO:17.

[0108] In another specific embodiment of the present invention, the chimeric auxiliary nucleic acid molecule comprises (i) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding an ICP8 protein from HSV1 and an HSV TK polyA signal, the sequence of the HSV TKpolyA signal being the sequence of SEQ ID NO:16, and (ii) a polynucleotide insert comprising a CMV promoter, a polynucleotide encoding an NP1 protein from HBoV1 and a bGH polyA signal, the sequence of the bGH polyA signal being the sequence of SEQ IDNO:17.

[0109] In another specific embodiment of the present invention, the chimeric auxiliary nucleic acid molecule comprises (i) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding a UL12 protein from HSV1 and an HSV TK polyA signal, the sequence of which is the sequence of SEQ ID NO:16, (ii) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding an ICP8 protein from HSV1 and an HSV TK polyA signal, the sequence of the HSV TK polyA signal being the sequence of SEQ ID NO:16, and (iii) a polynucleotide insert comprising a CMV promoter, a polynucleotide encoding an NS2 protein from HBoV1 and a bGH polyA signal, the sequence of the bGH polyA signal being the sequence of SEQ ID NO:17.

[0110] In another specific embodiment of the present invention, the chimeric auxiliary nucleic acid molecule comprises (i) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding a UL12 protein from HSV1, and an HSV TK polyA signal, the sequence of the HSV TK polyA signal being the sequence of SEQ ID NO:16, (ii) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding an ICP8 protein from HSV1, and an HSV TK polyA signal, the sequence of the HSV TK polyA signal being the sequence of SEQ ID NO:16, and (iii) a polynucleotide insert comprising a CMV promoter, a polynucleotide encoding an NP1 protein from HBoV1, and a bGH polyA signal, the sequence of the bGH polyA signal being the sequence of SEQ ID NO:17.

[0111] In a preferred embodiment of the present invention, the chimeric auxiliary nucleic acid molecule is a plasmid, in particular the Ad-HBoV-HSV plasmid of SEQ ID NO:18, or the Ad-HBoV-HSV5 plasmid of SEQ ID NO:35, preferably the Ad-HBoV-HSV plasmid of SEQ ID NO:18.

[0112] In a preferred embodiment of the present invention, in the chimeric helper nucleic acid molecule:

[0113] - the amino acid sequence of the UL12 protein from HSV1 is SEQ ID NO: 19;

[0114] - the amino acid sequence of the NS2 protein from HBoV1 is SEQ ID NO: 20;

[0115] - the amino acid sequence of the E2A protein from Ad5 is SEQ ID NO: 21;

[0116] - the amino acid sequence of the E4orf1 sequence from Ad5 is SEQ ID NO: 22;

[0117] - the amino acid sequence of the E4orf6 sequence from Ad5 is SEQ ID NO: 23;

[0118] - the nucleotide sequence encoding the NP1 protein from HBoV1 is SEQ ID NO: 24;

[0119] - the amino acid sequence of the NP1 protein from HBoV1 is SEQ ID NO: 25;

[0120] - the E1 gene from Ad5 is a polynucleotide of SEQ ID NO: 26;

[0121] - the amino acid sequence of the E1A protein from Ad5 is SEQ ID NO: 27;

[0122] - the amino acid sequence of the E1B55k protein from Ad5 is SEQ ID NO: 28;

[0123] - the amino acid sequence of the E4orf2 sequence from Ad5 is SEQ ID NO: 29;

[0124] - the amino acid sequence of the E4orf3 sequence from Ad5 is SEQ ID NO: 30;

[0125] - the amino acid sequence of the E4orf4 sequence from Ad5 is SEQ ID NO: 31;

[0126] - the amino acid sequence of the E4 protein from Ad5 is SEQ ID NO:22, SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:31;

[0127] - the amino acid sequence of the E1 protein from Ad5 is SEQ ID NO: 27 or SEQ ID NO: 28; and / or

[0128] -The amino acid sequence of the ICP8 protein from HSV1 is SEQ ID NO:33.

[0129] The E4 protein may be composed of the E4orf1 sequence, E4orf2 sequence, E4orf3 sequence, E4orf4 sequence and E4orf6 sequence defined above.

[0130] The E1 protein may consist of the E1A protein and the E1B55k protein defined above.

[0131] In a preferred embodiment of the present invention, the chimeric auxiliary nucleic acid molecule comprises:

[0132] (i) an ORF from an HSV gene, which consists of a polynucleotide encoding a UL12 protein from HSV, preferably from HSV1;

[0133] (ii) an ORF from an HBoV gene, which consists of a polynucleotide encoding an NS2 protein from HBoV, preferably from HBoV1;

[0134] (iii) a VA-RNA polynucleotide from Ad, preferably from Ad5;

[0135] (iv) an E2A gene from Ad, preferably from Ad5;

[0136] (v) an E4 gene from Ad, preferably from Ad5, in particular an E4orf6 sequence;

[0137] wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric auxiliary nucleic acid molecule as a separate expression cassette, and wherein the polynucleotides of (i) to (v) are arranged in any order and / or orientation relative to each other; preferably, wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric auxiliary nucleic acid molecule as a separate expression cassette, wherein the polynucleotides in the chimeric nucleic acid molecule have the following order from 5' to 3' direction: (i), (ii), (iii), (iv) and (v), and nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, in particular different promoters;

[0138] Provided that the chimeric helper nucleic acid molecule does not comprise (ii) an ORF from a HBoV gene consisting of a polynucleotide encoding an NS2 protein from HBoV and an NP1 protein from HBoV; and

[0139] The proviso is that the chimeric auxiliary nucleic acid molecule does not contain a further polynucleotide encoding a protein from HSV.

[0140] In another preferred embodiment of the present invention, the chimeric auxiliary nucleic acid molecule comprises:

[0141] (i) an ORF from an HSV gene, which consists of a polynucleotide encoding a UL12 protein from HSV, preferably from HSV1;

[0142] (ii) an ORF from an HBoV gene, which consists of a polynucleotide encoding the NP1 protein from HBoV, preferably from HBoV1;

[0143] (iii) a VA-RNA polynucleotide from Ad, preferably from Ad5;

[0144] (iv) an E2A gene from Ad, preferably from Ad5;

[0145] (v) an E4 gene from Ad, preferably from Ad5, in particular an E4orf6 sequence;

[0146] wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric auxiliary nucleic acid molecule as a separate expression cassette, and wherein the polynucleotides of (i) to (v) are arranged in any order and / or orientation relative to each other; preferably, wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric auxiliary nucleic acid molecule as a separate expression cassette, wherein the polynucleotides in the chimeric nucleic acid molecule have the following order from 5' to 3' direction: (i), (ii), (iii), (iv) and (v), and nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, in particular different promoters;

[0147] Provided that the chimeric helper nucleic acid molecule does not comprise (ii) an ORF from a HBoV gene consisting of a polynucleotide encoding an NS2 protein from HBoV and an NP1 protein from HBoV; and

[0148] The proviso is that the chimeric auxiliary nucleic acid molecule does not contain a further polynucleotide encoding a protein from HSV.

[0149] In another preferred embodiment of the present invention, the chimeric auxiliary nucleic acid molecule comprises:

[0150] (i) an ORF from an HSV gene, which consists of a polynucleotide encoding an ICP8 protein from HSV, preferably from HSV1;

[0151] (ii) an ORF from an HBoV gene, which consists of a polynucleotide encoding an NS2 protein from HBoV, preferably from HBoV1;

[0152] (iii) a VA-RNA polynucleotide from Ad, preferably from Ad5;

[0153] (iv) an E2A gene from Ad, preferably from Ad5;

[0154] (v) an E4 gene from Ad, preferably from Ad5, in particular an E4orf6 sequence;

[0155] wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric auxiliary nucleic acid molecule as a separate expression cassette, and wherein the polynucleotides of (i) to (v) are arranged in any order and / or orientation relative to each other; preferably, wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric auxiliary nucleic acid molecule as a separate expression cassette, wherein the polynucleotides in the chimeric nucleic acid molecule have the following order from 5' to 3' direction: (i), (ii), (iii), (iv) and (v), and nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, in particular different promoters;

[0156] Provided that the chimeric helper nucleic acid molecule does not comprise (ii) an ORF from a HBoV gene consisting of a polynucleotide encoding an NS2 protein from HBoV and an NP1 protein from HBoV; and

[0157] The proviso is that the chimeric auxiliary nucleic acid molecule does not contain a further polynucleotide encoding a protein from HSV.

[0158] In another preferred embodiment of the present invention, the chimeric auxiliary nucleic acid molecule comprises:

[0159] (i) an ORF from an HSV gene, which consists of a polynucleotide encoding an ICP8 protein from HSV, preferably from HSV1;

[0160] (ii) an ORF from an HBoV gene, which consists of a polynucleotide encoding the NP1 protein from HBoV, preferably from HBoV1;

[0161] (iii) a VA-RNA polynucleotide from Ad, preferably from Ad5;

[0162] (iv) an E2A gene from Ad, preferably from Ad5;

[0163] (v) an E4 gene from Ad, preferably from Ad5, in particular an E4orf6 sequence;

[0164] wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric auxiliary nucleic acid molecule as a separate expression cassette, and wherein the polynucleotides of (i) to (v) are arranged in any order and / or orientation relative to each other; preferably, wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric auxiliary nucleic acid molecule as a separate expression cassette, wherein the polynucleotides in the chimeric nucleic acid molecule have the following order from 5' to 3' direction: (i), (ii), (iii), (iv) and (v), and nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, in particular different promoters;

[0165] Provided that the chimeric helper nucleic acid molecule does not comprise (ii) an ORF from a HBoV gene consisting of a polynucleotide encoding an NS2 protein from HBoV and an NP1 protein from HBoV; and

[0166] The proviso is that the chimeric auxiliary nucleic acid molecule does not contain a further polynucleotide encoding a protein from HSV.

[0167] In another preferred embodiment of the present invention, the chimeric auxiliary nucleic acid molecule comprises:

[0168] (i) an ORF from an HSV gene, which consists of a polynucleotide encoding a UL12 protein from HSV, preferably from HSV1, and an ICP8 protein from HSV, preferably from HSV1;

[0169] (ii) an ORF from an HBoV gene, which consists of a polynucleotide encoding an NS2 protein from HBoV, preferably from HBoV1;

[0170] (iii) a VA-RNA polynucleotide from Ad, preferably from Ad5;

[0171] (iv) an E2A gene from Ad, preferably from Ad5;

[0172] (v) an E4 gene from Ad, preferably from Ad5, in particular an E4orf6 sequence;

[0173] wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric auxiliary nucleic acid molecule as a separate expression cassette, and wherein the polynucleotides of (i) to (v) are arranged in any order and / or orientation relative to each other; preferably, wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric auxiliary nucleic acid molecule as a separate expression cassette, wherein the polynucleotides in the chimeric nucleic acid molecule have the following order from 5' to 3' direction: (i), (ii), (iii), (iv) and (v), and nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, in particular different promoters;

[0174] Provided that the chimeric helper nucleic acid molecule does not comprise (ii) an ORF from a HBoV gene consisting of a polynucleotide encoding an NS2 protein from HBoV and an NP1 protein from HBoV; and

[0175] The proviso is that the chimeric auxiliary nucleic acid molecule does not contain a further polynucleotide encoding a protein from HSV.

[0176] In another preferred embodiment of the present invention, the chimeric auxiliary nucleic acid molecule comprises:

[0177] (i) an ORF from an HSV gene, which consists of a polynucleotide encoding a UL12 protein from HSV, preferably from HSV1, and an ICP8 protein from HSV, preferably from HSV1;

[0178] (ii) an ORF from an HBoV gene, which consists of a polynucleotide encoding the NP1 protein from HBoV, preferably from HBoV1;

[0179] (iii) a VA-RNA polynucleotide from Ad, preferably from Ad5;

[0180] (iv) an E2A gene from Ad, preferably from Ad5;

[0181] (v) an E4 gene from Ad, preferably from Ad5, in particular an E4orf6 sequence;

[0182] wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric auxiliary nucleic acid molecule as a separate expression cassette, and wherein the polynucleotides of (i) to (v) are arranged in any order and / or orientation relative to each other; preferably, wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric auxiliary nucleic acid molecule as a separate expression cassette, wherein the polynucleotides in the chimeric nucleic acid molecule have the following order from 5' to 3' direction: (i), (ii), (iii), (iv) and (v), and nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, in particular different promoters;

[0183] Provided that the chimeric helper nucleic acid molecule does not comprise (ii) an ORF from a HBoV gene consisting of a polynucleotide encoding an NS2 protein from HBoV and an NP1 protein from HBoV; and

[0184] The proviso is that the chimeric auxiliary nucleic acid molecule does not contain a further polynucleotide encoding a protein from HSV.

[0185] The position, order and / or orientation of the functional sequences in the chimeric auxiliary nucleic acid molecule are, for example, Fig.10 (plasmids Ad-HBoV-HSV and Ad-HBoV-HSV5 according to the present invention).

[0186] The present invention also relates to a packaging system, comprising:

[0187] (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 AAV serotype 2 (AAV2), AAV serotype 5 (AAV5), AAV serotype 8 (AAV8) or AAV serotype 9 (AAV9), preferably from AAV2, AAV5 or AAV9, more preferably from AAV2;

[0188] (b) a Rep / Cap nucleic acid molecule, in particular a plasmid (pPackaging or pRC or pAAVRep / Cap) that provides AAV viral functions and comprises the replication (Rep) gene from AAV2 and the capsid (Cap) gene from AAV, preferably from AAV serotype 2, 5, 8 or 9; and

[0189] (c) a chimeric helper nucleic acid molecule according to the present invention;

[0190] The nucleic acid molecules (a), (b) and (c) are provided as one molecule, two different molecules or three different molecules, preferably three different molecules consisting of (a), (b) and (c) or comprising (a), (b) and (c) respectively.

[0191] As defined herein, the term "packaging system" refers to a system, such as a plasmid, that comprises structural and packaging genes.

[0192] As defined herein, the term "inverted terminal repeats (ITRs) from AAV" refers to regions at both ends of the AAV genome that together serve as origins of DNA replication in the cis direction and packaging signals for the viral genome.

[0193] As defined herein, the term "transgene" refers to a gene to be delivered by AAV. Transgenes of interest include, but are not limited to, genes or ORFs of therapeutic interest.

[0194] In a specific embodiment 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, even more preferably an AAV of serotype 2.

[0195] The present invention also relates to a recombinant adeno-associated virus (rAAV) producer cell, in particular a recombinant E1 complementation producer cell, which is transformed, in particular transfected, with the packaging system according to the present invention.

[0196] In a specific embodiment of the present invention, the rAAV producer cell is a mammalian cell, preferably a human cell, more preferably a HEK-293 cell, even more preferably a HEK-293T cell, in particular a recombinant E1 complementation producer cell.

[0197] The present invention also relates to a helper virus-free method for producing infectious recombinant adeno-associated virus (rAAV) in a production cell line, comprising the following steps:

[0198] - transforming, in particular transfecting, cells or cell lines, preferably mammalian cells 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 production of rAAV virions by the transfected cells;

[0199] - harvesting transformed, in particular transfected, cells and lysing them to recover rAAV virions; and

[0200] - Collecting rAAV virions in cell lysates or supernatants and optionally purifying said rAAV virions.

[0201] In a specific embodiment of the invention, the helper virus-free method comprises a step of purifying the rAAV virions.

[0202] As defined herein, the term "AAV virion" refers to an intact viral particle, such as a wild-type AAV viral particle comprising single-stranded genomic DNA packaged in AAV capsid proteins.

[0203] As defined herein, the term "rAAV virion" refers to a recombinant AAV virion, ie, an infectious, replication-defective virus consisting of an AAV protein coat encapsulating a DNA molecule of interest flanked on both sides by AAV ITRs.

[0204] In a specific embodiment of the invention, the production cell line is selected from the group consisting of a HEK-293 cell line (eg, a HEK-293T cell line), a VPC 2.0 cell line, an Expi293F cell line, and an adherent cell line, wherein the cells are grown in suspension.

[0205] As defined herein, the term "adherent cell line" refers to a cell line that requires 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.

[0206] In a specific embodiment of the invention, transfection is performed by chemical transfection, electroporation or sonoporation.

[0207] The present invention also relates to a method for producing replication-defective infectious recombinant adeno-associated virus (rAAV) vector particles, comprising the following steps:

[0208] - transforming, in particular transfecting, cells, preferably mammalian cells 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;

[0209] - harvesting transformed, in particular transfected, cells and lysing them to recover rAAV vector particles; and

[0210] - collecting rAAV vector particles in cell lysates or supernatants and optionally purifying said rAAV vector particles.

[0211] In a specific embodiment of the invention, the method for producing replication-defective infectious rAAV vector particles comprises the step of purifying the rAAV vector particles.

[0212] The present invention also relates to the in vitro use of a rAAV producer cell according to the present invention for producing replication-defective infectious rAAV vector particles.

[0213] Other features and advantages of the present invention will be apparent from the following examples and will be further illustrated in the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0214] Figure 1 .Overview of the different helper plasmids used in this study. The overall architecture of the plasmid is shown in a schematic diagram, which contains genes from the AAV helper virus (light gray = adenovirus, dark gray = parvovirus, white = herpes virus). The name and size of each plasmid are shown on the left. The use of heterologous promoters is indicated by arrows (pCMV = cytomegalovirus promoter, pEFS = core promoter of eukaryotic translation elongation factor 1α). pALD-X80 and pHelper ALD were purchased from Aldevron, and pHelper CB was purchased from Cellbiolabs. All other plasmids were constructed by E-zyvec proprietary technology for the purpose of this study, as described in patent EP3256583B1.

[0215] Figure 2 .Determination of the optimal plasmid ratio for rAAV2 production in suspension HEK-293T. rAAV2 vectors expressing the eGFP reporter gene were produced in HEK-293T cells grown in suspension. Cells were transfected with FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent using 3 plasmids (pRC2 vector expressing Rep and Cap, pAAV-GFP control vector expressing GFP under the control of the CMV promoter and flanked by AAV2 ITRs, and a helper vector (pALD-X80) expressing at least adenoviral E2A, adenoviral E4, and adenoviral VA helper factors). 72 hours after transfection, rAAV titers (transduction units, TU / mL) were determined by transducing H1080 cells and quantifying eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transduction units / mL (TU / mL). Different mass ratios were used for this plasmid to determine the optimal ratio between these plasmids to produce the highest infection titer. The resulting copy number of each plasmid was then used for further experiments to keep the copy number of the plasmid the same in each condition.

[0216] Figure 3.Comparison of the efficiency of rAAV2 production with different helper plasmids. rAAV2 vectors expressing the eGFP reporter gene were produced in HEK-293T cells grown in suspension. Cells were transfected with FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent using 3 plasmids (pRC2 vector expressing Rep and Cap, pAAV-GFP control vector expressing GFP under the control of the CMV promoter and flanked by AAV2 ITRs, and a helper vector expressing at least adenoviral E2A, adenoviral E4, and adenoviral VA helpers). AAV titers (transduction units, TU / mL) were determined 72 hours after transfection by transducing H1080 cells and quantifying eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transduction units / mL (TU / mL).

[0217] Figure 4. Addition of genes from other helper viruses to the helper plasmid and the effect on rAAV2 production. rAAV2 vectors expressing the eGFP reporter gene were produced in HEK-293T cells grown in suspension. Cells were transfected with FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent using three plasmids (pRC2 vector expressing Rep and Cap, pAAV-GFP control vector expressing GFP under the control of the CMV promoter and flanked by AAV2 ITRs, and helper vectors expressing at least adenoviral E2A, adenoviral E4, and adenoviral VA helper factors). 72 hours after transfection, AAV titers (transduction units, TU / mL) were determined by transducing H1080 cells and quantifying eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transduction units / mL (TU / mL), which are normalized to the infectivity of rAAV produced with pALD-X80 (containing only adenoviral genes), and are expressed as fold increase in Figures A, B, and F. A) Infectivity of rAAV produced with plasmids carrying different helper genes from parvovirus (Ad-HBoV1 and Ad-HBoV2, grey) or herpesvirus (Ad-HSV1 and Ad-HSV2, white) added to adenovirus genes to generate dual chimeric helper plasmids. B) Infectivity of rAAV produced with plasmids carrying genes from adenovirus, parvovirus, and herpesvirus (triplet chimeric plasmid). C) Quantification of genome copies per mL (GC / mL). D) Viral particles per mL (VP / mL). E) Complete / empty capsid ratio (GC / VP). F) TU / VP of rAAV2 produced with pALD-X80 or the triple chimeric plasmid.

[0218] Figure 5. Evaluation of the versatility and efficiency of triple chimeric helper plasmids for rAAV2 production in different packaging cell lines derived from HEK-293. VPC 2.0 cells grown in suspension in A) virus production medium or B) FreestyleTM 293-F cells grown in suspension in 293 medium or C) Expi293 TM Expi293F grown in suspension in expression medium TM rAAV2 vectors expressing the eGFP reporter gene were produced in cells. Cells were transfected with FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent using 3 plasmids (pRC2 vector expressing Rep and Cap, pAAV-GFP control vector expressing GFP under the control of the CMV promoter and flanked by AAV2 ITRs, and a helper vector expressing at least adenoviral E2A, adenoviral E4, and adenoviral VA helper factors). 72 hours after transfection, AAV titers (transduction units, TU / mL) were determined by transducing H1080 cells and quantifying eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transduction units / mL (TU / mL), which are normalized to the infectivity of rAAV produced with pALD-X80, and are expressed as fold increase in the figures.

[0219] Figure 6 .Evaluation of the versatility and efficiency of triple chimeric helper plasmids for rAAV2 production under different transfection reagents. 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 the CMV promoter and 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 with FectoVIR-AAV, PEIpro, or TransIT-VirusGEN. 72 hours after transfection, AAV titers (transduction units, TU / mL) were determined by transducing H1080 cells and quantifying eGFP+ cells by flow cytometry. Results are expressed as relative rAAV2 transduction units / mL (TU / mL), which are normalized to the infectivity of rAAV produced with pALD-X80 and are expressed as fold increase in the figures.

[0220] Figure 7. Evaluation of the versatility and efficiency of triple chimeric helper plasmids for producing rAAV of different serotypes. 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 FectoVIR-AAV at a ratio of 1:1 μg DNA / μL reagent using 3 plasmids (pRC5, pRC8, or pRC9 vectors expressing Rep and serotype-specific Cap, pAAV-GFP control vector expressing GFP under the control of the CMV promoter and flanked by AAV2 ITRs, and a helper vector expressing at least adenoviral E2A, adenoviral E4, and adenoviral VA helper factors). 72 hours after transfection, AAV titers (transduction units, TU / mL) were determined 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 with pALD-X80, and are presented in the graphs as fold increase.

[0221] Figure 8. A) pALD-X80 from Aldevron (18876 bp); B) pALD-HELP from Aldevron (11584 bp); C) VPK-402 from Cellbiolabs (11635 bp).

[0222] Fig. 9 .Plasmid Ad-HBoV-HSV (13324 bp) according to the present invention.

[0223] Fig.10.Helper plasmids for rAAV production: plasmid Ad-HBoV-HSV according to the present invention (13324 bp, SEQ ID NO: 18), plasmid Ad-HBoV-HSV5 according to the present invention (15034 bp, SEQ ID NO: 35) and comparative plasmid Ad-HBoV5 (11651 bp, SEQ ID NO: 36) disclosed in Wang et al., Mol. Ther. 2018, 11, 40-51. All three plasmids have the same architecture in terms of adenoviral elements (VA-RNA, E2A and E4orf6). Plasmid Ad-HBoV-HSV further comprises a polynucleotide encoding the UL12 protein from HSV1 and a polynucleotide encoding the NS2 protein from HBoV1. Plasmid Ad-HBoV-HSV5 further comprises a polynucleotide encoding the ICP8 protein from HSV1 and a polynucleotide encoding the NS2 protein from HBoV1. Comparative plasmid Ad-HBoV5 further comprises polynucleotides encoding NS2 and NP1 proteins from HBoV1 and a polynucleotide encoding the self-cleaving peptide P2A (SEQ ID NO: 34). The HSV and HBoV proteins are driven by pCMV or pEFS promoters.

[0224] Figure 11. rAAV production efficiency (rAAV VG / mL titer produced in HEK293T). Production efficiency of 11A) rAAV2, or 11B) rAAV5, or 11C) rAAV8, or 11D) rAAV9 using the following pHelpers: plasmid Ad-HBoV-HSV and plasmid Ad-HBoV-HSV5 according to the present invention, and comparative plasmid Ad-HBoV5 disclosed by Wang et al., Mol. Ther. 2018, 11, 40-51. For ease of representation, proteins from HBoV and HSV present in pHelpers are shown at the top of the chart. DETAILED DESCRIPTION

[0225] Example

[0226] Materials and Methods

[0227] Cell culture

[0228] HEK-293T( CRL-3216 TM ): 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. The cells were incubated at 37°C and 8% CO 2Incubate with stirring in air (130 rpm - 50 mm orbit).

[0229] Freestyle TM 293-F cells (Gibco TM R79007): Human embryonic kidney cells were derived from the HEK-293 parental cell line. The cells were cultured in Freestyle 400 cells supplemented with 8 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.1% Pluronic. TM The cells were cultured in suspension in 293 medium at 37°C and 8% CO 2 Incubate with stirring in air (130 rpm - 50 mm orbit).

[0230] Virus Growth Cell Pluronic 2.0 (Gibco TM A49784): Human embryonic kidney cells were derived from the HEK-293F parental cell line, which in turn was derived from the HEK-293 cell line. The cells were grown in suspension in virus production medium supplemented with 8 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were incubated at 37°C, 8% CO 2 Incubate with stirring in air (130 rpm - 50 mm orbit).

[0231] Expi293F TM (Gibco TM A14527): Human embryonic kidney cells were derived from the HEK-293 parental cell line. The cells were cultured in Expi293 medium supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin. TM The cells were cultured in suspension in expression medium. 2 Incubate with stirring in air (130 rpm - 50 mm orbit).

[0232] HT-1080( CCL-121 TM ): Human fibrosarcoma cells were cultured in DMEM medium supplemented with 4.5 g / L glucose, 10% fetal bovine serum, 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO 2 Growth in suspension in an air environment.

[0233] CHO-K1( CCL-61 TM): The cell line is derived from a subclone of a parental CHO cell line originally obtained from an ovarian biopsy sample of an adult female Chinese hamster. The cells were grown in RPMI medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin at 37°C, 5% CO 2 grow in an air environment.

[0234] Recombinant virus production

[0235] HEK-293T( CRL-3216 TM ): Human embryonic kidney cells are highly transfective derivatives of human embryonic kidney 293 cells and contain SV40 T antigen. HEK-293T cells are widely used in recombinant virus production, gene expression and protein production.

[0236] HEK-293T cells were cultured at 1x10 6 The cells were inoculated at a density of 10 cells / mL in 28.5 mL 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. The cells were incubated at 37°C, 8% CO 2 Incubate with stirring in air (130 rpm - 50 mm orbit).

[0237] Freestyle TM 293-F cells (Gibco TM R79007): Human embryonic kidney cells are highly transfectable derivatives of human embryonic kidney 293 cells. Freestyle TM 293F cells are Freestyle TM Part of the MAX 293 Expression System and is used for gene expression and recombinant protein production.

[0238] Freestyle TM 293-F cells were cultured at 1x10 6 The cells were seeded at a density of 10 cells / mL in 28.5 mL of Freestyle 500 μL medium supplemented with 8 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.1% Pluronic in a 125 mL Erlenmeyer flask. TM 293. The cells were cultured at 37°C and 8% CO 2 Incubate with stirring in air (130 rpm - 50 mm orbit).

[0239] Virus Growth Cell 2.0 (GibcoTM A49784): Human embryonic kidney cells are a clonal cell line derived from the HEK293F parental cell line and are the core component of the AAV-MAX Helper-Free AAV production system. Virus Growth Cells 2.0 are highly transfective, do not contain SV40 T antigen, and are used for adeno-associated virus (AAV) production.

[0240] The virus was grown in 2.0 cells at 1x10 6 The cells were inoculated at a density of 10 cells / mL in 28.5 mL of ViralProduction medium supplemented with 8 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 125 mL Erlenmeyer flask. The cells were incubated at 37°C, 8% CO 2 Incubate with stirring in air (130 rpm - 50 mm orbit).

[0241] Expi293F TM (Gibco TM A14527): Human embryonic kidney cells derived from the HEK-293 parental cell line. Expi293F TM The cells are highly transfectable and are primarily used for recombinant protein production and recombinant virus production.

[0242] Expi293F TM The cells were 1x10 6 The cells were inoculated at a density of 10 cells / mL into 28.5 mL of Expi293 medium supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin in a 125 mL Erlenmeyer flask. TM The cells were cultured in 37°C, 8% CO 2 Incubate with stirring in air (130 rpm - 50 mm orbit).

[0243] In HEK-293T, VPC 2.0, or Expi293F TM Recombinant adeno-associated virus (rAAV) was produced in cells at 1x10 6 The cells were seeded at a density of 10 cells / mL and incubated at 37°C and 8% CO 2The cells were cultured in an environment of 4% paraformaldehyde for 24 hours and then co-transfected with three plasmids (pRC vector expressing Rep and Cap, pHelper vector expressing at least Adeno E2A, Adeno E4 and Adeno VA auxiliary factors, and pAAV-GFP (Catalog No. AAV-400, Cell BIOLABS, INC.) control vector expressing GFP under the control of the CMV promoter). The plasmids were diluted in 1.5 mL of unsupplemented medium (standard: pAAV-GFP 7.23x10 4 copies / cell – pRC 5.32x10 4 Copies / cell – pHelper 1x10 4 The transfection complex was added to the cells and the Erlenmeyer flask was incubated at 37°C, 8% CO 2 Incubate for 72 hours in an air environment with agitation (130 rpm - 50 mm orbit).

[0244] The transfected cells were harvested 3 days after transfection, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the pellet was resuspended in 2 mL PBS. The cells were then lysed using 3 consecutive freeze-thaw cycles (-80°C to 37°C) to release rAAV. 2 mL of lysate was then collected and centrifuged at 14000 rpm for 30 minutes to separate rAAV from cell debris. The supernatant containing rAAV was then analyzed.

[0245] Transducing unit titers (TU / mL) were determined by infecting permissive cells (HT-1080 or CHO-K1, depending on the rAAV serotype produced) in 96-well plates with a recombinant adeno-associated virus expressing the GFP reporter gene. Briefly, permissive cells were plated at 7×10 3 The cells were seeded at a density of 10 cells / mL and incubated at 37°C with 5% CO 2 The cells were incubated in an air environment for 4 h. The harvested rAAV was then serially diluted in supplemented medium and added to the permissive cells as a replacement for the medium used previously. GFP expression was analyzed by flow cytometry 72 h after transduction to determine transducing units.

[0246] The capsid titer (VP / mL) was determined by using an AAV titration ELISA kit (Catalog No. PRAAV2 / PRAAV5 / PRAAV8 / PRAAV9, PROGENINC) following the manufacturer's recommendations and protocol and depending on the serotype of the recombinant adeno-associated virus produced and analyzed.

[0247] By qPCR (QuantStudio TM 3–ThermoFisher) to measure the genome titer (VG / mL). The concentration used was 2x10 8 copies / μL to 2x10 2 A standard curve was generated using primers targeting the GFP reporter gene expressed by the produced rAAV (Qiagen) and the SensiFAST probe Lo-ROX kit (Ozyme).

[0248] Figure 1 An overview of all the helper plasmid maps used in this study is disclosed. All helper plasmids were constructed with at least minimal Ad helper elements (VA-RNA, E2A, and E4). Additional elements from other helper viruses (HBoV and HSV) were also added to compare their efficacy with commercial helper plasmids in producing rAAV.

[0249] Figure 2 The viral titers (transducing units per mL) of rAAV2 produced after transfection of HEK 293t cells using different ratios of pHelper (pALD-X80) are disclosed. The mass ratio of 2:2:1 (pTransgene:pRC:pHelper) gave the best viral titers compared to the other tested ratios and was therefore selected for further experiments to compare the different pHelpers shown in this study.

[0250] Figure 3 The viral titers (transduction units per mL) of rAAV2 produced after transfection of HEK 293t cells using different pHelpers are disclosed. Three commercial helper plasmids were used as references (pALD-X80 and pALD-HELP from Aldevron and VPK-402 from Cellbiolabs) for comparison with the minimal pHelper (mpH3sV2) containing only adenoviral elements. The minimal pHelper (mpH3sV2) showed similar viral titers to other commercial helper plasmids, indicating that all sequences removed in the mpH3sV2 construct are not necessary to support efficient rAAV production. Therefore, the mpH3sV2 (approximately 8kb) pHelper was used as a framework for further optimization of pHelper.

[0251] Figure 4 is public:

[0252] A) Viral titers (transducing units per mL) of rAAV2 produced after transfection of HEK 293t cells with various pHelpers containing only Ad elements (pALD-X80, pALD-HELP, and mpH3sV2, black), or containing Ad and HBoV elements (Ad-HBoV1 and Ad-HBoV2, white), or containing Ad and HSV elements (Ad-HSV2 and Ad-HSV6, gray) (referred to as dual chimeric vectors). Addition of specific auxiliary elements from HBoV and HSV enhanced the viral titers of the viruses produced by these pHelpers compared to the Ad-only pHelper, indicating that the elements introduced into the dual chimeric vectors are beneficial for the efficient production of rAAV2 (see Ad-HBoV1 and Ad-HSV6 compared to any black graph).

[0253] B) Virus titers (transducing units per mL) of rAAV2 produced after transfection of HEK 293t cells with various pHelpers containing only Ad elements (pALD-X80, pALD-HELP, and mpH3sV2) or containing Ad, HBoV, and HSV elements (Ad-HBoV-HSV). Figure 4A As shown, the addition of HBoV or HSV elements is beneficial to the efficient production of rAAV2, so the inventors evaluated the effectiveness of pHelper containing all Ad, HBoV and HSV elements involved in rAAV production to examine whether it can further improve the production of rAAV2. Figure 4B The results showed that the combination of elements from all three helper viruses (called triple chimeric vector or triple chimeric pHelper) outperformed the dual chimeric vector (by about 1.6-fold and 1.2-fold, respectively, compared to when pALD-X80 was used as a reference). These results indicate that the combination of helper elements from three different viruses is the best combination currently available to the inventors for the efficient production of infectious rAAV2.

[0254] C) GC titers (genomic copies per mL) of rAAV2 produced using pALD-X80 or Ad-HBoV-HSV were analyzed by qPCR. Since the virus produced using the triple chimeric pHelper was more infectious than the virus produced using the commercial helper plasmid, the purpose of this experiment was to examine whether this enhanced viral titer was dependent on the amount of encapsulated DNA. The results showed no significant differences between rAAV2 produced using pALD-X80 or our triple chimeric pHelper.

[0255] D) VP titer (viral particles per mL) of rAAV2 produced using pALD-X80 or Ad-HBoV-HSV was analyzed by ELISA. rAAV2 produced using pALD-X80 and triple chimeric pHelper both showed similar VP titers, indicating that the full / empty capsid ratio (full capsids to empty capsids) of rAAV2 produced using triple chimeric pHelper was unchanged compared to the commercial helper plasmid.

[0256] E) The percentage of viral DNA contained in the capsid of rAAV2 produced using pALD-X80 or Ad-HBoV-HSV (GC / VP). Figure 4C and 4D As shown, the GC and VP titers of rAAV2 produced using pALD-X80 or the triple chimeric pHelper were similar, and thus the GC / VP ratio (corresponding to the total number of capsids containing viral DNA copies) was also similar.

[0257] F) Overall quality (TU / VP) of rAAV2 produced using pALD-X80 or Ad-HBoV-HSV pHelper. Since rAAV2 produced using triple chimeric pHelper is more infectious than that produced using pALD-X80 ( Figure 4B ), but did not show enhanced GC or VP titers ( Figure 4C and 4D ), the quality of rAAV2 production was therefore analyzed in terms of the ratio of infectious to non-infectious particles (TU / VP). Figure 4F As shown, this ratio increased when rAAV2 was produced using the triple chimeric pHelper compared to when pALD-X80 was used. This increased ratio indicates that the triple chimeric pHelper is able to produce higher quality viruses compared to its counterpart pALD-X80.

[0258] FIG5 discloses the viral titers (transducing units per mL) of rAAV2 produced after transfection of different production cell lines with pHelper containing only Ad elements (pALD-X80, pALD-HELP) or containing Ad+HBoV+HSV elements (Ad-HBoV-HSV): VPC 2.0 ( Figure 5A )、HEK 293F( Figure 5B ) and Expi293F( Figure 5CThe results were normalized to the infectivity of rAAV produced by pALD-X80 and expressed as fold increase. The results indicate that the enhancing effect of the combination of Ad, HBoV and HSV elements in a single plasmid previously observed in HEK293T is not cell line specific and can also be observed in the three production cell lines tested, with fold increase ranging from about 1.8 to about 4.5. Figures 5A to 5C This indicates the versatility of the triple chimeric pHelper for different cell lines.

[0259] Figure 6 The viral titers (transducing units per mL) of rAAV2 produced after transfection of HEK 293T using pHelper containing only Ad elements or containing Ad + HBoV + HSV elements (Ad-HBoV-HSV) are disclosed. Cells were transfected using three different transfection reagents: FectoVIR-AAV (Polyplus), PEIpro (Polyplus), and TransIT-VirusGEN (Mirus Bio). The results were normalized to the infectivity of rAAV produced by pALD-X80 and expressed as fold increase.

[0260] Since all previous results were obtained using FectoVIR-AAV transfection of HEK293T cells, the aim was to examine whether the increased viral titers obtained using the triple chimeric pHelper (Ad-HBoV-HSV) were related to specific transfection reagents. Therefore, two additional transfection reagents were used to transfect HEK293T cells: PEIpro and TransIT-VirusGEN.

[0261] The titers obtained using different transfection reagents and with pALD-X80 as well as the titers obtained with the triple chimeric pHelper (Ad-HBoV-HSV) indicate that the increase observed with the triple chimeric pHelper is independent of the transfection reagent used.

[0262] FIG. 7 discloses the viral titers (transducing units per mL) of rAAV of different serotypes produced after transfection using pHelper containing only Ad elements (pALD-X80, pALD-HELP) or Ad and HBoV and HSV elements (Ad-HBoV-HSV): rAAV5 ( Fig. 7A )、rAAV8( Figure 7B ) and rAAV9( Figure 7C). The results were normalized to the infectivity of rAAV produced by pALD-X80 and expressed as fold increase. The results suggest that the enhancing effect of the combination of Ad, HBoV, and HSV elements in a single plasmid, previously observed in the production of rAAV2, is not serotype specific and can also be observed in the production of other rAAV serotypes, with fold increases ranging from about 1.4 to about 1.8. Figures 7A to 7C This indicates that the triple chimeric pHelper is universal for different AAV serotypes.

[0263] Deletion / limitation of unnecessary sequences: Some commercial pHelpers contain unnecessary sequences from the adenoviral genome. For gene therapy purposes, these sequences should be avoided to improve safety and reduce toxicity. To this end, the inventors delineated all unnecessary sequences in the adenoviral genome and removed them to retain only the elements involved in rAAV production.

[0264] Unnecessary sequences refer to sequences that are not involved in rAAV production, i.e., sequences that are usually present on the sides of the desired genes in commercial plasmids due to technical cloning limitations. Therefore, any nucleotides / sequences in the plasmid other than the target gene (from the promoter to the polyadenylation signal) are excluded from the plasmid of the present invention.

[0265] Titer increase: One of the major problems in rAAV production is to increase the viral titer to meet the requirements of large amounts of infectious virus required for rAAV-based gene therapy. The plasmids of the present invention result in an increased proportion of infectious virus compared to other commercial helper plasmids (up to about 2-fold depending on the AAV serotype).

[0266] Virus quality: The plasmids of the present invention resulted in an increase in the amount of infectious virus compared to the reference commercial pHelper. In addition, the inventors showed that virus production using the plasmids of the present invention had a higher infectious titer per total capsid (TU / VP), indicating that the rAAV produced using the plasmids of the present invention is of better quality (more infectious). Considering that current virus production methods result in very low quality of virus batches (most of the produced viruses are non-infectious), finding ways to increase the infectivity of rAAV could improve the overall efficiency of rAAV-based gene therapy while reducing the immune response against these non-infectious viruses.

[0267] Universal pHelper for different AAV serotypes: The helper plasmids of the invention have been tested on various AAV serotypes to evaluate their efficiency compared to current methods for producing the serotypes of interest (i.e., serotypes 2, 5, 8, and 9). The plasmids of the invention showed increased infectivity titers on all of the above serotypes compared to the commercial helper plasmids. The extent of the increase was serotype dependent, ranging from 2-fold for AAV2, up to 3-fold for AAV5, AAV8, and AAV9, and particularly 1.5 to 2-fold for AAV2, AAV5, AAV8, or AAV9.

[0268] Universal pHelper for different cell lines: The helper plasmid of the present invention has been tested in several mammalian cell lines derived from HEK-293, namely HEK-293T, HEK-293F, VPC 2.0 and Expi293F cells. In the four cell lines tested, the plasmid of the present invention showed increased infectivity titers compared to the commercial helper plasmid, indicating that the plasmid of the present invention works in different cell culture conditions as required.

[0269] The inventors compared the rAAV production efficiency between the triple chimeric pHelper according to 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, 40-51. The triple chimeric pHelper according to the present invention resulted in efficient production of multiple rAAV serotypes (2, 5, 8 and 9) ( Fig.10 and 11).

[0270] Although the present invention has been described with various preferred embodiments, it will be appreciated by those skilled in the art that various modifications, substitutions, omissions and changes may be made without departing from the scope of the present invention. Therefore, the scope of the present invention is intended to be limited by the scope of the following claims, including their equivalents.

Claims

1. A chimeric auxiliary nucleic acid molecule suitable for use in a method for producing an infectious recombinant adeno-associated virus (rAAV), the chimeric auxiliary nucleic acid molecule comprising: (i) an ORF from an HSV gene, which consists of a polynucleotide encoding at least one HSV protein, in particular a polynucleotide encoding a UL12 protein from HSV, preferably from HSV1, or an ICP8 protein from HSV, preferably from HSV1; (ii) an ORF from a HBoV gene, which consists of a polynucleotide encoding an NS2 protein from HBoV, preferably from HBoV1, or an NP1 protein from HBoV, preferably from HBoV1; (iii) a VA-RNA polynucleotide from Ad, preferably from Ad5; (iv) an E2A gene from Ad, preferably from Ad5; (v) an E4 gene from Ad, preferably from Ad5, in particular an E4orf6 sequence; wherein the polynucleotides of (i), (ii), (iii), (iv) and (v) are each inserted into the chimeric helper nucleic acid molecule as a separate expression cassette, and wherein the polynucleotides of (i) to (v) are arranged in any order and / or orientation relative to each other; Provided that the chimeric auxiliary nucleic acid molecule does not comprise (ii) an ORF from an HBoV gene, which consists of a polynucleotide encoding an NS2 protein from HBoV and an NP1 protein from HBoV.

2. The chimeric auxiliary nucleic acid molecule according to claim 1, wherein the ORF from the HSV gene consists of a polynucleotide encoding the UL12 protein from HSV, preferably from HSV1.

3. The chimeric helper nucleic acid molecule according to claim 1 or 2, wherein the ORF from the HBoV gene consists of a polynucleotide encoding the NS2 protein from HBoV, preferably from HBoV1.

4. A chimeric auxiliary nucleic acid molecule according to any one of claims 1 to 3, wherein the polynucleotides in the chimeric nucleic acid molecule have the following order from 5' to 3' direction: (i), (ii), (iii), (iv) and (v), and wherein nucleic acids (i) and (ii) are under the control of a non-endogenous promoter, particularly different promoters.

5. The chimeric helper nucleic acid molecule according to any one of claims 1 to 4, further comprising an additional adenoviral gene, such as the E1 gene from Ad, preferably from Ad5.

6. The chimeric auxiliary nucleic acid molecule according to any one of claims 1 to 5, further comprising: - origin of replication (ori); and / or - Herpes simplex virus thymidine kinase (HSV TK polyA) signal downstream of the HSV UL12 ORF; and / or - bovine growth hormone polyadenylation (bGH polyA) signal from downstream of HBoV NS2 ORF; and / or - transcriptional regulatory elements, such as cytomegalovirus (CMV) promoter, CMV enhancer, EFS promoter, SV40 virus promoter, CBA promoter, CAG promoter, E2A promoter and / or E4 promoter, to control the transcription of HSV ORF and HBoV ORF; and / or -optionally a nucleic acid encoding a marker protein such as a kanamycin resistance gene.

7. The chimeric auxiliary nucleic acid molecule according to any one of claims 1 to 6, wherein: - the polynucleotide encoding the UL12 protein from HSV1 is SEQ ID NO: 1; - the polynucleotide encoding the NS2 protein from HBoV1 is SEQ ID NO: 2; - the VA-RNA polynucleotide from Ad5 is SEQ ID NO: 3; - the E2A gene from Ad5 is SEQ ID NO: 4; - the E4 gene from Ad5 is SEQ ID NO: 5, in particular the E4orf6 sequence from Ad5 is SEQ ID NO: 6; and / or - The polynucleotide encoding the ICP8 protein from HSV1 is SEQ ID NO:

32.

8. The chimeric auxiliary nucleic acid molecule according to any one of claims 1 to 7, wherein: -ori is the nucleotide sequence of SEQ ID NO:7; -HSV TK polyA signal is a polynucleotide of sequence SEQ ID NO: 8; - bGH polyA signal is a polynucleotide of sequence SEQ ID NO: 9; -CMV promoter is a polynucleotide of sequence SEQ ID NO: 10; -CMV enhancer is a polynucleotide of sequence SEQ ID NO:11; -EFS promoter is a polynucleotide of sequence SEQ ID NO:12; - E2A promoter is a polynucleotide of sequence SEQ ID NO: 13; -E4 promoter is a polynucleotide of sequence SEQ ID NO:14; - Kanamycin resistance gene is the sequence SEQ ID NO: 15; - the amino acid sequence of the UL12 protein from HSV1 is SEQ ID NO: 19; - the amino acid sequence of the NS2 protein from HBoV1 is SEQ ID NO: 20; - the amino acid sequence of the E2A protein from Ad5 is SEQ ID NO: 21; - the amino acid sequence of the E4 protein from Ad5 is SEQ ID NO:22, SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:31; - the amino acid sequence of the E4orf6 sequence from Ad5 is SEQ ID NO: 23; - the nucleotide sequence encoding the NP1 protein from HBoV1 is SEQ ID NO: 24; - the amino acid sequence of the NP1 protein from HBoV1 is SEQ ID NO: 25; - the E1 gene from Ad5 is a polynucleotide of SEQ ID NO: 26; - the amino acid sequence of the E1 protein from Ad5 is SEQ ID NO: 27 or SEQ ID NO: 28; and / or -The amino acid sequence of the ICP8 protein from HSV1 is SEQ ID NO:

33.

9. The chimeric auxiliary nucleic acid molecule of any one of claims 1 to 8, comprising (i) a polynucleotide insert comprising an EFS promoter, a polynucleotide encoding a UL12 protein from HSV1, and an HSV TK polyA signal, the sequence of which is the sequence of SEQ ID NO: 16, and (ii) a polynucleotide insert comprising a CMV promoter, a polynucleotide encoding a NS2 protein from HBoV1, and a bGH polyA signal, the sequence of which is the sequence of SEQ ID NO:

17.

10. The chimeric auxiliary nucleic acid molecule according to any one of claims 1 to 9, which 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.

11. A packaging system comprising: (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 AAV serotype 2 (AAV2), AAV serotype 5 (AAV5), AAV serotype 8 (AAV8) or AAV serotype 9 (AAV9), preferably from AAV serotype 2 (AAV2); (b) a Rep / Cap nucleic acid molecule, in particular a plasmid (pPackaging or pRC or pAAVRep / Cap) that provides AAV viral functions and comprises the replication (Rep) gene from AAV2 and the capsid (Cap) gene from AAV, preferably from AAV serotype 2, 5, 8 or 9; and (c) a chimeric auxiliary nucleic acid molecule according to any one of claims 1 to 10; The nucleic acid molecules (a), (b) and (c) are provided as one molecule, two different molecules or three different molecules, preferably three different molecules consisting of (a), (b) and (c) or comprising (a), (b) and (c) respectively.

12. A recombinant adeno-associated virus (rAAV) producer cell, in particular a recombinant E1 complementation producer cell, which is transformed, in particular transfected, with the packaging system according to claim 11.

13. The rAAV producer cell according to claim 12, which is a mammalian cell, preferably a human cell, more preferably a HEK-293 cell, in particular a recombinant E1 complementation producer cell.

14. A helper virus-free method for producing infectious recombinant adeno-associated virus (rAAV) in a producer cell line, comprising the following steps: - transforming, in particular transfecting, a cell or a cell line, preferably a mammalian cell or an insect cell, with a packaging system according to claim 11, in particular stably transfecting said cell with a packaging system according to claim 11, and allowing the transfected cell to produce rAAV virions; - harvesting transformed, in particular transfected, cells and lysing them to recover rAAV virions; and - Collecting rAAV virions in cell lysates or supernatants and optionally purifying said rAAV virions.

15. The method of claim 14, wherein the production cell line is selected from the group consisting of a HEK-293 cell line (eg, a HEK-293T cell line), a VPC 2.0 cell line, an Expi293F cell line, and an adherent cell line, and wherein the cells are grown in suspension.

16. The method of claim 14 or 15, wherein transfection is performed by chemical transfection, electroporation or sonoporation.

17. A method for producing replication-defective infectious recombinant adeno-associated virus (rAAV) vector particles, comprising the following steps: - transforming, in particular transfecting, a cell, preferably a mammalian cell or an insect cell, with a packaging system according to claim 11, in particular stably transfecting the cell with a packaging system according to claim 11, and allowing the transformed, in particular transfected cell to produce rAAV vector particles; - harvesting transformed, in particular transfected, cells and lysing them to recover rAAV vector particles; and - collecting rAAV vector particles in cell lysates or supernatants and optionally purifying said rAAV vector particles.

18. Use of the rAAV producer cell according to claim 12 or 13 in vitro for producing replication-defective infectious rAAV vector particles.

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