Methods and packaging / producing cells for producing recombinant adeno-associated viral vectors

Abstract

The present disclosure provides a method of producing a recombinant adeno-associated viral vector in which no adenovirus is used as a helper virus, and which comprises using a nucleic acid sequence encoding an adenovirus L4-22K protein and / or a nucleic acid sequence encoding an adenovirus L4-33K protein as a helper gene sequence for the production of a recombinant adeno-associated viral vector. The present disclosure also provides a recombinant adeno-associated viral vector packaging / producing cell for producing a recombinant adeno-associated viral vector without using an adenovirus as a helper virus, the packaging / producing cell comprising an adenovirus L4-22K protein or a nucleic acid encoding the same and / or an adenovirus L4-33K protein or a nucleic acid encoding the same.

Description

Technical Field

[0001] The present disclosure relates to a method for producing a recombinant adeno-associated viral vector and a packaging / production cell for producing a recombinant adeno-associated viral vector. Specifically, the present disclosure relates to improving the yield of a recombinant adeno-associated viral vector by using a specific gene sequence as an auxiliary gene in a method for producing a recombinant adeno-associated viral vector and in the construction of a packaging / production cell for a recombinant adeno-associated viral vector. Background Art

[0002] Adeno-associated virus (AAV) belongs to the genus Dependoparvovirus of the Parvoviridae family, and it requires the presence of a helper virus (such as adenovirus) to complete viral replication. AAV is a small non-enveloped virus whose genome consists of single-stranded DNA, is about 4.7kb in length, and contains two gene expression frames, Rep and Cap. The Rep gene encodes the protein required for viral replication, and the Cap gene encodes the capsid protein. On both sides of the Rep and Cap gene expression frames are inverted terminal repeats (ITRs), which are the initiation signals for viral DNA replication. Different capsid proteins determine the serotype of AAV, such as AAV2, AAV5, AAV8, AAV9, etc.

[0003] Rep and Cap gene expression frames in the AAV genome can be replaced with exogenous target nucleic acid fragments through genetic engineering methods to produce recombinant adeno-associated virus (rAAV) vectors containing exogenous target genes. rAAV is widely used in gene therapy because it can effectively deliver genes to target cells and has the characteristics of low toxicity and low immunogenicity.

[0004] Since AAV has replication defects, in the production process of rAAV, a helper virus (for example, adenovirus, herpes virus, papillomavirus) that can provide necessary auxiliary factors to help rAAV replication is required, or when a helper virus is not used, a helper gene (helper gene) derived from other viruses to promote AAV replication and assembly needs to be provided to the host cells used for rAAV production. The helper gene is usually from adenovirus (for example, from adenovirus E1A, E1B, E2A, E4 or VA RNA gene), and may also be from herpes virus, papillomavirus, etc. In practical applications, a transgenic plasmid carrying an exogenous GOI, a plasmid carrying a Rep gene and a Cap gene (RC plasmid), and a helper plasmid carrying a helper gene can be transferred into host cells to produce rAAV. HEK293 and its derivative cell lines are currently the most commonly used rAAV production cell lines. Because the adenovirus E1A and E1B genes have been stably inserted into their genome, during virus production, only the auxiliary gene plasmid pHelper containing the adenovirus E4 gene, E2A gene and VA RNA gene needs to be additionally transfected.

[0005] The L4 gene is part of the adenovirus genome and belongs to the late transcription unit. It mainly encodes proteins that constitute the viral capsid or proteins involved in capsid assembly. The L4 gene is about 3.7kbp long. It accounts for about 10% of the Ad5 genome. The L4 gene encodes 4 important proteins: L4-100K, L4-33K, L4-22K and pVIII. The L4-33K protein plays a role in the conversion of early to late gene expression in the viral life cycle. It is a splicing factor that affects the processing of viral RNA transcripts, allowing the virus to produce proteins required for assembling new viral particles (Wu K et al., The adenovirus L4-33K protein regulates both late gene expression patterns and viral DNA packaging. J Virol. 2013; 87 (12): 6739-6747). In the L4 region of Ad5, the L4-22K coding region overlaps with the L4-33K coding region. Like L4-33K protein, L4-22K protein is also involved in the regulation of viral gene expression and participates in the transition from early to late stages of the viral life cycle by affecting the splicing of viral RNA transcripts (Wu K et al., supra).

[0006] The rapid increase in rAAV gene therapy products has created a huge demand for large-scale production capacity. However, the existing industrial production capacity is still insufficient to meet the needs of future commercialization and widespread patient use. The current virus-free assisted production system can only produce rAAV vectors at low titers even under optimized cell culture conditions. Therefore, there is an urgent need for a method to increase the yield of rAAV vectors. Summary of the invention

[0007] The inventors have discovered for the first time that adding specific genes in the adenovirus genome into the rAAV production system as auxiliary genes can significantly increase the yield of rAAV vectors, thereby hopefully solving the yield bottleneck problem faced by rAAV vectors in commercial production.

[0008] In one aspect, the present disclosure provides a method for producing rAAV vectors, in which adenovirus is not used as a helper virus, and the method includes using a nucleic acid sequence encoding adenovirus L4-22K protein and / or a nucleic acid sequence encoding adenovirus L4-33K protein as an auxiliary gene sequence for rAAV vector production. The adenovirus L4-22K protein can be a wild-type L4-22K protein derived from the Adenoviridae family, or it can be a variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% sequence identity with the amino acid sequence of the wild-type L4-22K protein derived from the Adenoviridae family and capable of producing the same or similar effect of increasing the yield of rAAV vectors. The adenovirus L4-33K protein can be a wild-type L4-33K protein derived from the Adenoviridae family, or a variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% sequence identity with the amino acid sequence of the wild-type L4-33K protein derived from the Adenoviridae family and capable of producing the same or similar effect of increasing the yield of rAAV vectors. In one example, the adenovirus is a human adenovirus. In one example, the adenovirus is adenovirus Ad5.

[0009] In one example, when both the nucleic acid sequence encoding the adenovirus L4-22K protein and the nucleic acid sequence encoding the adenovirus L4-33K protein are used, the nucleic acid sequence encoding the adenovirus L4-22K protein and the nucleic acid sequence encoding the adenovirus L4-33K protein form one nucleic acid sequence. In one example, when both the nucleic acid sequence encoding the adenovirus L4-22K protein and the nucleic acid sequence encoding the adenovirus L4-33K protein are used, the nucleic acid sequence encoding the adenovirus L4-22K protein overlaps with the nucleic acid sequence encoding the adenovirus L4-33K protein.

[0010] In one embodiment, the method for producing a rAAV vector further comprises using a plurality of additional helper gene sequences.

[0011] In one embodiment, the plurality of additional helper gene sequences comprises one or more nucleic acid sequences derived from adenovirus, herpes simplex virus, hepatitis C virus, influenza virus, human herpes virus, bocavirus, or human papillomavirus.

[0012] In one embodiment, the plurality of additional auxiliary gene sequences include nucleic acid sequences derived from adenovirus E1A, E1B, E2A, E4 or VA RNA genes or combinations thereof. In one embodiment, the additional auxiliary gene sequence may be a nucleic acid sequence derived from adenovirus E1A gene, a nucleic acid sequence derived from adenovirus E1B gene, a nucleic acid sequence derived from adenovirus E2A gene, a nucleic acid sequence derived from adenovirus E4 gene, or a nucleic acid sequence derived from adenovirus VA RNA gene, or a combination of these nucleic acid sequences. In one embodiment, the nucleic acid sequence derived from adenovirus E4 gene may be a nucleic acid sequence derived from adenovirus E4orf1, E4orf2, E4orf3, E4orf3 / 4, E4orf4, E4orf6 or E4orf6 / 7 gene, or a combination of these nucleic acid sequences.

[0013] In one embodiment, the plurality of additional auxiliary gene sequences include nucleic acid sequences derived from herpes simplex virus (HSV) UL5, UL8, UL12, UL29, UL30, UL42, UL52, ICP0, ICP4, ICP22, ICP34.5 or US11 genes or a combination thereof. In one example, the herpes simplex virus is herpes simplex virus type 1 (HSV-1).

[0014] In one embodiment, the nucleic acid sequence derived from hepatitis C virus (HCV) may be a nucleic acid sequence derived from the NS5A gene of hepatitis C virus.

[0015] In one embodiment, the nucleic acid sequence derived from influenza virus may be a nucleic acid sequence derived from the NS1 gene of influenza virus.

[0016] In one embodiment, the nucleic acid sequence derived from human herpesvirus (HHV) can be a nucleic acid sequence derived from a human herpesvirus Rep gene. In one example, the human herpesvirus is human herpesvirus type 6 (HHV-6).

[0017] In one embodiment, the nucleic acid sequence derived from Bocavirus (HBoV) can be a nucleic acid sequence derived from Bocavirus NS2, NS4, NP1 or BocaSR gene or a combination thereof. In one example, the Bocavirus is Bocavirus type I (HBoV-1).

[0018] In one embodiment, the nucleic acid sequence derived from human papillomavirus (HPV) can be a nucleic acid sequence derived from human papillomavirus E1, E2 or E6 gene or a combination thereof. In one example, the human papillomavirus is human papillomavirus type 16 (HPV-16).

[0019] In one embodiment, the method for producing rAAV vector comprises transferring the nucleic acid sequence encoding adenovirus L4-22K protein and / or the nucleic acid sequence encoding adenovirus L4-33K protein into a host cell.

[0020] In one example, the method for producing rAAV vectors comprises transferring a vector (e.g., plasmid) carrying a nucleic acid sequence encoding adenovirus L4-22K protein and / or a vector (e.g., plasmid) carrying a nucleic acid sequence encoding adenovirus L4-33K protein into a host cell. In one example, the method for producing rAAV vectors comprises transferring a vector (e.g., plasmid) carrying both a nucleic acid sequence encoding adenovirus L4-22K protein and a nucleic acid sequence encoding adenovirus L4-33K protein into a host cell.

[0021] In one embodiment, the host cell is a mammalian cell. In one embodiment, the mammalian cell can be selected from HEK293 cells, A549 cells, Hela cells, HepG2 cells, BHK cells, COS cells, Vero cells, MDCK cells, Per.C6 cells, CAP cells, and derivatives thereof.

[0022] In another aspect, the present disclosure provides a recombinant adeno-associated virus vector packaging / production cell for producing a recombinant adeno-associated virus vector without using adenovirus as a helper virus, wherein the packaging / production cell comprises adenovirus L4-22K protein or its encoding nucleic acid and / or adenovirus L4-33K protein or its encoding nucleic acid. The adenovirus L4-22K protein may be a wild-type L4-22K protein derived from the Adenoviridae family, or may be a variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% sequence identity with the amino acid sequence of the wild-type L4-22K protein derived from the Adenoviridae family and capable of producing the same or similar effect of increasing the yield of rAAV vectors. The adenovirus L4-33K protein can be a wild-type L4-33K protein derived from the Adenoviridae family, or a variant having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% sequence identity with the amino acid sequence of the wild-type L4-33K protein derived from the Adenoviridae family and capable of producing the same or similar effect of increasing the yield of rAAV vectors. In one example, the adenovirus is a human adenovirus. In one example, the adenovirus is adenovirus Ad5.

[0023] In one embodiment, the packaging / production cell further comprises a plurality of additional helper gene sequences. In one embodiment, the plurality of additional helper gene sequences comprise one or more nucleic acid sequences derived from adenovirus, herpes simplex virus, hepatitis C virus, influenza virus, human herpes virus, bocavirus, or human papillomavirus.

[0024] In one embodiment, the packaging / production cell further comprises a nucleic acid sequence derived from an adenovirus E1A, E1B, E2A, E4 or VA RNA gene or a combination thereof. In one embodiment, the packaging / production cell comprises a nucleic acid sequence derived from an adenovirus E1A gene, a nucleic acid sequence derived from an adenovirus E1B gene, a nucleic acid sequence derived from an adenovirus E2A gene, a nucleic acid sequence derived from an adenovirus E4 gene, or a nucleic acid sequence derived from an adenovirus VA RNA gene, or a combination of these nucleic acid sequences. In one embodiment, the nucleic acid sequence derived from an adenovirus E4 gene may be a nucleic acid sequence derived from an adenovirus E4orf1, E4orf2, E4orf3, E4orf3 / 4, E4orf4, E4orf6 or E4orf6 / 7 gene, or a combination of these nucleic acid sequences.

[0025] In one embodiment, the packaging / production cell further comprises a nucleic acid sequence derived from a herpes simplex virus UL5, UL8, UL12, UL29, UL30, UL42, UL52, ICP0, ICP4, ICP22, ICP34.5 or US11 gene or a combination thereof. In an example, the herpes simplex virus is herpes simplex virus type 1.

[0026] In one embodiment, the packaging / production cell further comprises a nucleic acid sequence derived from the NS5A gene of the Hepatitis C virus.

[0027] In one embodiment, the packaging / production cell further comprises a nucleic acid sequence derived from the influenza virus NS1 gene.

[0028] In one embodiment, the packaging / production cell further comprises a nucleic acid sequence derived from a human herpesvirus Rep gene. In one example, the human herpesvirus is human herpesvirus type 6.

[0029] In one embodiment, the packaging / production cell further comprises a nucleic acid sequence derived from a Bocavirus NS2, NS4, NP1 or BocaSR gene or a combination thereof. In one example, the Bocavirus is Bocavirus type I.

[0030] In one embodiment, the packaging / production cell further comprises a nucleic acid sequence derived from the human papillomavirus E1, E2 or E6 gene or a combination thereof. In one example, the human papillomavirus is human papillomavirus type 16.

[0031] In another aspect, the present disclosure provides the use of adenovirus L4-22K protein or its encoding nucleic acid and / or adenovirus L4-33K protein or its encoding nucleic acid for producing a recombinant adeno-associated virus vector without using adenovirus as a helper virus.

[0032] The present invention significantly improves the yield of the rAAV vector production method by using a nucleic acid sequence encoding adenovirus L4-22K protein and / or a nucleic acid sequence encoding adenovirus L4-33K protein as an auxiliary gene sequence for rAAV vector production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The effect of adding L4-22K and / or L4-33K genes as helper genes on rAAV vector production according to one embodiment of the present disclosure is shown.

[0034] Figure 2 The effect of adding L4-22K and / or L4-33K genes as helper genes on rAAV vector production according to another embodiment of the present disclosure is shown. DETAILED DESCRIPTION

[0035] The following examples are used to illustrate the technical solutions of the present invention, and the following examples should not be considered as limiting the scope and spirit of the present invention.

[0036] Example 1: Plasmid construction method

[0037] The molecular cloning techniques used in the following examples, such as amplification of DNA fragments, restriction endonuclease digestion of DNA fragments, gel recovery of DNA fragments, connection of two DNA fragments, transformation of competent cells of the connection product, plasmid extraction, preparation and identification, etc., are all mature and known techniques in the art. The following reagents are involved in the following examples: high-fidelity DNA polymerase (Acori); restriction endonuclease (NEB); DNA fragment gel recovery kit (Omega, D2500-02); plasmid mini-extraction kit (OMEGA, D6943-02); chemical competent cells (XL-10gold); sequence synthesis was completed by Nanjing GenScript Biotechnology Co., Ltd.; plasmid sequencing was completed by Qingke Biotechnology Co., Ltd. Table 1 is a description of SEQ ID NO: 1-SEQ ID NO: 13. The element sequence information used in each plasmid involved in the following embodiments is an example for realizing the present disclosure. Those skilled in the art can expect that the effects described in the present disclosure can be achieved by replacing each element sequence on the plasmid used in the following embodiments with other element sequences with similar biological functions, including but not limited to the backbone sequence of the plasmid (such as replication origin, resistance gene, etc.), restriction site sequence, transposon repeat sequence, inducible system response element sequence, insulator sequence, promoter sequence, intron sequence, polyadenylation signal (PolyA) sequence, gene sequences optimized with different codons, mutants of the above functional element sequences and gene sequences, and the cloning position, cloning order and cloning direction of each functional element sequence and gene sequence.

[0038] The specific plasmid construction method is as follows:

[0039] 1. Construction of plasmid pCMV-L4-22K: Using pAdEasy-1 (Addgene, #16400) as a template, primers were used to amplify the adenovirus L4-22K encoding nucleic acid sequence (SEQ ID NO: 1), and the amplified fragment was ligated to plasmid pCMV-M1 (Addgene, #23007) to construct plasmid pCMV-L4-22K.

[0040] 2. Construction of plasmid pCMV-L4-33K: Using pAdEasy-1 (Addgene, #16400) as a template, primers were used to amplify the adenovirus L4-33K encoding nucleic acid sequence (SEQ ID NO: 3), and the amplified fragment was ligated to plasmid pCMV-M1 (Addgene, #23007) to construct plasmid pCMV-L4-33K.

[0041] 3. Construction of plasmid pCMV-L4-22_33K: Using pAdEasy-1 (Addgene, #16400) as a template, primers were used to amplify the adenovirus L4-22_33K encoding nucleic acid sequence (wherein the L4-22K gene sequence overlaps with the L4-33K gene sequence, SEQ ID NO: 5), and the amplified fragment was connected to the plasmid pCMV-M1 (Addgene, #23007) to construct the plasmid pCMV-L4-22_33K.

[0042] 4. Construction of plasmid pCMV-UL5: The synthetic fragment of the HSV-1 virus UL5 encoding nucleic acid sequence (SEQ ID NO: 6) was ligated into the plasmid pCMV-M1 (Addgene, #23007) to construct the plasmid pCMV-UL5.

[0043] 5. Construction of plasmid pCMV-UL8: The synthetic fragment of the HSV-1 virus UL8 encoding nucleic acid sequence (SEQ ID NO: 8) was ligated into the plasmid pCMV-M1 (Addgene, #23007) to construct the plasmid pCMV-UL8.

[0044] 6. Construction of plasmid pCMV-UL29: The synthetic fragment of the HSV-1 virus UL29 encoding nucleic acid sequence (SEQ ID NO: 10) was ligated into the plasmid pCMV-M1 (Addgene, #23007) to construct the plasmid pCMV-UL29.

[0045] 7. Construction of plasmid pCMV-UL52: The synthetic fragment of the HSV-1 virus UL52 encoding nucleic acid sequence (SEQ ID NO: 12) was ligated into the plasmid pCMV-M1 (Addgene, #23007) to construct the plasmid pCMV-UL52.

[0046] Table 1. Sequence description

[0047] SEQ ID NO: describe 1 L4-22K nucleic acid sequence (585bp) 2 L4-22K protein amino acid sequence (194aa) 3 L4-33K nucleic acid sequence (684bp) 4 L4-33K protein amino acid sequence (227aa) 5 L4-22_33K nucleic acid sequence (886bp) 6 UL5 nucleic acid sequence (2649 bp) 7 UL5 amino acid sequence (882aa) 8 UL8 nucleic acid sequence (2253 bp) 9 UL8 amino acid sequence (750aa) 10 UL29 nucleic acid sequence (3591 bp) 11 UL29 amino acid sequence (1196aa) 12 UL52 nucleic acid sequence (3177 bp) 13 UL52 amino acid sequence (1058aa)

[0048] Example 2: Effect of adding L4-22K and / or L4-33K genes as helper genes on rAAV vector production when using commonly used adenovirus helper genes

[0049] 293T cells (ATCC, CRL-3216) were seeded in a 6-well plate (Corning 3516) at 8E+05 cells per well, and the culture medium was 2 ml of DMEM complete medium. After culturing for 24 hours, transfection was performed according to the PEI method. During transfection, 200 μl of transfection reagent was added to each well, in which the total amount of plasmid was 5 ug, of which pAAV-EF1a-FLuc-WPRE-HGHpA plasmid (AAV transfer vector, which carries AAV on both sides) ITR sequence of firefly luciferase coding sequence under the control of EF1a promoter and WPRE detection sequence, Addgene #87951, hereinafter referred to as "pAAV-Fluc plasmid") 0.5 μg, pAAV-RC plasmid (carrying adenovirus Rep and Cap gene sequences, Novopro, V009666#) 0.5 μg, pHelper plasmid (carrying adenovirus E4, E2A and VARNA gene sequences, Novopro, V005569#) 35 ng; in addition, E The mpty group added 0.6 μg of empty vector (pCMV-M1 plasmid), the L4-22K group added 0.6 μg of pCMV-L4-22K plasmid, the L4-33K group added 0.6 μg of pCMV-L4-33K plasmid, the L4-22K+L4-33K group added 0.6 μg of pCMV-L4-22K and pCMV-L4-33K plasmids (0.3 μg each), the L4-22_33K group added 0.3 μg of pCMV-L4-22_33K plasmid, and the remaining plasmid amount was filled with the above empty vector. The mass ratio of total plasmid amount to PEI was 1:4, and repeated 2 times independently. After 48 hours of cell transfection, an equal volume of 1% Tween20 was added for cell lysis. The cell lysate was treated with universal nuclease (Yisheng Biotechnology (Shanghai) Co., Ltd., 20156ES60), and then adeno-associated virus genome copies (vg) were detected using ABIQuantStudio 5 real-time fluorescence quantitative PCR system.

[0050] Figure 1Empty group: transient co-transfection of pAAV-RC plasmid, pAAV-Fluc plasmid and pHelper plasmid; L4-22K group: transient co-transfection of pAAV-RC plasmid, pAAV-Fluc plasmid, pHelper plasmid and pCMV-22K plasmid; L4-33K group: transient co-transfection of pAAV-RC plasmid, pAAV-Fluc plasmid, pHelper plasmid and pCMV-33K plasmid ; L4-22K+L4-33K group: transient co-transfection of pAAV-RC plasmid, pAAV-Fluc plasmid, pHelper plasmid, pCMV-22K plasmid and pCMV-33K plasmid; L4-22_33K group: transient co-transfection of pAAV-RC plasmid, pAAV-Fluc plasmid, pHelper plasmid and pCMV-22_33k plasmid; NC group (negative control): only transient transfection of empty vector plasmid.

[0051] The results are as follows Figure 1 As shown, the titer value of the L4-22K group was 8.4E9 vg / ml, which was significantly higher than the titer value of 4.1E9 vg / ml in the Empty group (P-value=0.0114). The titer value of the L4-33K group was 1.1E10 vg / ml, which was significantly higher than the titer value of the Empty group (P-value=0.0029). The titer value of the L4-22K+L4-33K group was 1.8E10 vg / ml, and the titer value of the L4-22_33K group was 2.1E10 vg / ml. There was no significant difference in the titer values ​​between the two groups (P-value=0.2690). The titer value of the L4-22K+L4-33K group was significantly higher than that of the L4-22K group (P-value=0.0308), and the titer value of the L4-33K group was also significantly higher than that of the L4-33K group (P-value=0.0478). The titer value of L4-22_33K group was significantly higher than that of L4-22K group (P-value=0.0068), and the titer value of NC group was also significantly higher than that of L4-33K group (P-value=0.0090). The titer value of NC group was lower than the detection limit (5E7vg / ml).

[0052] Based on the above results, it can be determined that when using commonly used adenovirus-derived auxiliary genes (pHelper plasmids carrying adenovirus E4, E2A and VA RNA gene sequences) to produce rAAV vectors, adding L4-22K gene or L4-33K gene as additional auxiliary gene can significantly improve the yield of rAAV vectors. At the same time, it is more surprising that compared with adding L4-22K gene or L4-33K gene as additional auxiliary gene alone, adding L4-22K gene and L4-33K gene together (regardless of whether the two gene sequences are provided in a separate form (L4-22K+L4-33K group) or in an overlapping form as in the L4 region of Ad5 (L4-22_33K group)) can further significantly improve the yield of rAAV vectors, proving that adding L4-22K gene and L4-33K gene together can produce obvious synergistic effect.

[0053] Example 3: Effect of adding L4-22K and / or L4-33K genes as helper genes on rAAV vector production when using common herpes simplex virus helper genes

[0054] 293T cells (ATCC, CRL-3216) were seeded in a 6-well plate (Corning 3516) at 8E+05 cells per well, and the culture medium was 2 ml of DMEM complete medium. After culturing for 24 hours, transfection was performed according to the PEI method. During transfection, 200 μl of transfection reagent was added to each well, and the total amount of plasmid was 5 ug, of which pAAV-EF1a-FLuc-WPRE-HGHpA plasmid (AAV transfer vector, which carries AAV on both sides) ITR sequence of firefly luciferase coding sequence under the control of EF1a promoter and WPRE detection sequence, Addgene #87951, hereinafter referred to as "pAAV-Fluc plasmid") 0.5 μg, pAAV-RC plasmid (carrying adenovirus Rep and Cap gene sequences, Novopro, V009666 #) 0.5 μg, pCMV-UL5 plasmid 0.3 μg, pCMV-UL8 plasmid 0.3 μg, pCMV-UL29 plasmid 0.3 μg, pCMV-UL52 plasmid 0.3 μg; in addition, 0.6 μg of empty vector (pCMV-M1 plasmid) was added to the Empty group, 0.6 μg of pCMV-L4-22K plasmid was added to the L4-22K group, 0.6 μg of pCMV-L4-33K plasmid was added to the L4-33K group, 0.6 μg of pCMV-L4-22K and pCMV-L4-33K plasmid (0.3 μg each) was added to the L4-22K+L4-33K group, 0.3 μg of pCMV-L4-22_33K plasmid was added to the L4-22_33K group, and the remaining plasmid amount was filled with empty vector. The mass ratio of total plasmid amount to PEI was 1:4, and repeated 2 times independently. After 48 hours of cell transfection, an equal volume of 1% Tween20 was added for cell lysis. The cell lysate was treated with universal nuclease (Yisheng Biotechnology (Shanghai) Co., Ltd., 20156ES60), and then adeno-associated virus genome copies (vg) were detected using ABIQuantStudio5 real-time fluorescence quantitative PCR system.

[0055] Figure 2Empty group: transient co-transfection of pAAV-RC plasmid, pAAV-Fluc plasmid, pCMV-UL5 plasmid, pCMV-UL8 plasmid, pCMV-UL29 plasmid and pCMV-UL52 plasmid; L4-22K group: transient co-transfection of pAAV-RC plasmid, pAAV-Fluc plasmid, pCMV-UL5 plasmid, pCMV-UL8 plasmid, pCMV-UL29 plasmid, pCMV-UL52 plasmid and pCMV-22K plasmid; L4-33K group: transient co-transfection of pAAV-RC plasmid, pAAV-Fluc plasmid, pCMV-UL5 plasmid, pCMV-UL8 plasmid, pCMV-UL29 plasmid, pCMV-UL52 plasmid and pCMV-22K plasmid. V-UL52 plasmid and pCMV-33K plasmid; L4-22K+L4-33K group: transient co-transfection of pAAV-RC plasmid, pAAV-Fluc plasmid, pCMV-UL5 plasmid, pCMV-UL8 plasmid, pCMV-UL29 plasmid, pCMV-UL52 plasmid, pCMV-22K and pCMV-33K plasmid; L4-22_33K group: transient co-transfection of pAAV-RC plasmid, pAAV-Fluc plasmid, pCMV-UL5 plasmid, pCMV-UL8 plasmid, pCMV-UL29 plasmid, pCMV-UL52 plasmid and pCMV-22_33k plasmid; NC group (negative control): only transient transfection of empty vector plasmid.

[0056] The results are as follows Figure 2 As shown, the titer value of the L4-22K group was 1.3E10 vg / ml, which was significantly higher than the titer value of 1.8E9 vg / ml of the Empty group (P-value=0.0093). The titer value of the L4-33K group was 8.9E9 vg / ml, which was significantly higher than the titer value of the Empty group (P-value=0.0088). The titer value of the L4-22K+L4-33K group was 2.0E10 vg / ml, and the titer value of the L4-22_33K group was 2.4E10 vg / ml. There was no significant difference in the titer values ​​between the two groups (P-value=0.1558). The titer value of the L4-22K+L4-33K group was significantly higher than that of the L4-22K group (P-value=0.0228), and the titer value of the L4-33K group was also significantly higher than that of the L4-33K group (P-value=0.0037). The titer value of L4-22_33K group was significantly higher than that of L4-22K group (P-value=0.0394), and was also significantly higher than that of L4-33K group (P-value=0.0201). The titer value of NC group was below the detection limit (5E7 vg / ml).

[0057] Based on the above results, it can be determined that when using commonly used auxiliary genes (UL5, UL8, UL29, UL52) from herpes simplex virus to produce rAAV vectors, adding L4-22K gene or L4-33K gene as additional auxiliary gene can significantly improve the yield of rAAV vectors. At the same time, it is more surprising that compared with adding L4-22K gene or L4-33K gene as additional auxiliary gene alone, adding L4-22K gene and L4-33K gene together (regardless of whether the two gene sequences are provided in a separate form (L4-22K+L4-33K group) or in an overlapping form as in the L4 region of Ad5 (L4-22_33K group)) can further significantly improve the yield of rAAV vectors, proving that adding L4-22K gene and L4-33K gene together can produce obvious synergistic effect.

Claims

1. A method for producing a recombinant adeno-associated virus vector, characterized in that: In the method, adenovirus is not used as a helper virus, and the method includes using a nucleic acid sequence encoding adenovirus L4-22K protein and / or a nucleic acid sequence encoding adenovirus L4-33K protein as helper gene sequences for the production of recombinant adeno-associated virus vectors.

2. The method according to claim 1, characterized in that The method also includes the use of a variety of additional helper gene sequences.

3. The method according to claim 2, characterized in that The plurality of additional auxiliary gene sequences include one or more nucleic acid sequences derived from adenovirus, herpes simplex virus, hepatitis C virus, influenza virus, human herpes virus, boca virus or human papillomavirus.

4. The method according to claim 2, characterized in that: The plurality of additional auxiliary gene sequences include nucleic acid sequences derived from adenovirus E1A, E1B, E2A, E4 or VA RNA genes or a combination thereof.

5. The method according to claim 2, characterized in that: The multiple additional auxiliary gene sequences include nucleic acid sequences derived from herpes simplex virus UL5, UL8, UL12, UL29, UL30, UL42, UL52, ICP0, ICP4, ICP22, ICP34.5 or US11 genes or combinations thereof.

6. The method according to claim 1 or 2, characterized in that: The method comprises transferring the nucleic acid sequence encoding adenovirus L4-22K protein and / or the nucleic acid sequence encoding adenovirus L4-33K protein into a host cell.

7. The method according to claim 6, characterized in that The host cell is a mammalian cell and can be selected from HEK293 cells, A549 cells, Hela cells, HepG2 cells, BHK cells, COS cells, Vero cells, MDCK cells, Per.C6 cells, CAP cells, and cells derived therefrom.

8. A recombinant adeno-associated virus vector packaging / production cell for producing a recombinant adeno-associated virus vector without using adenovirus as a helper virus, characterized in that: The packaging / production cells contain adenovirus L4-22K protein or its encoding nucleic acid and / or adenovirus L4-33K protein or its encoding nucleic acid.

9. The packaging / production cell according to claim 8, characterized in that The packaging / production cell further comprises a nucleic acid sequence derived from an adenovirus E1A, E1B, E2A, E4 or VA RNA gene or a combination thereof.

10. The packaging / production cell according to claim 8, characterized in that The packaging / production cell further comprises a nucleic acid sequence derived from the herpes simplex virus UL5, UL8, UL12, UL29, UL30, UL42, UL52, ICP0, ICP4, ICP22, ICP34.5 or US11 gene or a combination thereof.

11. Use of adenovirus L4-22K protein or nucleic acid encoding it and / or adenovirus L4-33K protein or nucleic acid encoding it for producing a recombinant adeno-associated virus vector without using adenovirus as a helper virus.

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