Adeno-associated virus production platform

Abstract

Provided herein is a novel rAAV-based HSV production method that includes two engineered cell lines: engineered CHO cells for the production of multiple AAV serotypes and engineered BHK-21 for the production of rHSV-1 stocks used in the production of rAAV in CHO cells. The developed method provides a scalable, serum-free manufacturing platform.

Description

[Technical Field]

[0001] Recombinant adeno-associated viral vectors (rAAVs) are the leading platform for gene delivery, with three licensed products currently approved as of December 2021 (Bulcha et al., 2021). rAAVs have several advantages as gene delivery vectors, including their ability to transduce a variety of proliferating and non-proliferating cells, harbor cell / tissue-specific promoters, and induce a reduced immune response compared to other viral vectors (Kang et al., 2009). AAVs are small, non-enveloped viruses in the genus Dependovirus in the family Parvoviridae (Srivastava et al., 1983; Daya and Berns, 2008). The 4.6 kb single-stranded DNA genome of AAV contains two viral genes, rep and cap. These genes can be removed and replaced with a cassette expressing a therapeutic transgene with the necessary rep and cap genes provided in trans (Becerra et al., 1988). The AAV capsid is icosahedral and assembled from 60 viral protein (VP) monomers, with approximately 5 copies of VP1, 5 copies of VP2, and 50 copies of VP3 (Van Vliet et al., Methods Mol. Biol. 2008;437:51-91).

[0002] Currently, different cell culture expression platforms for rAAV exist, including stable packaging cell lines expressing the rep and cap genes of the desired rAAV serotype, stable proviral cell lines stably expressing Rep, Cap, and a transgene, and triple transient transfection (Clark et al., 1995; Clark et al., 2002; Qiao et al., 2002). Triple transient transfection is the most common method for rAAV production and uses three plasmids: one encoding the gene of interest (GOI) flanked by AAV inverted terminal repeats (ITRs), a second encoding the AAV rep and cap genes, and a third encoding adenovirus helper function genes. This three-plasmid system can be simplified by including the helper genes in the rep-cap plasmid, allowing for co-transfection (Grimm et al., 1998; Clark KR, 2002). However, the triple transient transfection method is difficult to scale up due to the very high doses required, which often results in low specific yields of infectious particles (ip), resulting in high DNase-resistant particle (DRP / ip) ratios (50-100) (Grimm et al., Hum Gene Ther;9:2745-2760 (1998) and Zolotukhin et al., 2002 Methods;28:158-167).

[0003] Similar to adenovirus, recombinant HSV-1 (rHSV-1) has also been reported to be capable of supporting rAAV replication as part of a helper virus system (Buller et al., 1981 J Virol;40:241-247). The minimal HSV helper genes required for rAAV replication are the HSV helicase-primase complex (UL5, UL8, UL52), HSV DNA polymerase, and HSV DNA-binding protein (UL29) genes (Weindler and Heilbronn, 1991 J Virol;65:2476-2483). Furthermore, it has been reported that rHSV coinfection using two HSV-1 vectors (one encoding the desired rAAV rep / cap serotype and the second encoding the GOI) produced rAAV of multiple different serotypes in various cell lines with very high specific yields and very low (DRP / ip) ratios (Kang et al., 2009). Another advantage of the HSV helper system is that HSV can replicate and provide helper functions in different mammalian cells, which means that HSV helper functions circumvent the host range restriction for rAAV production, whereas rAAV production with adenovirus helpers requires human cell lines for success (Buller et al., 1970 J Gen Virol;43:663-672).

[0004] Chinese hamster ovary (CHO) cells are primarily used as expression hosts for the production of recombinant monoclonal antibodies (mAbs) and other therapeutic proteins, comprising the fastest-growing segment of the biopharmaceutical industry (Walsh G., 2018 Nat. Biotechnol;36:1136-1145). Due to their regulatory tolerance and lower manufacturing costs compared to human cells, using CHO cells for virus production is desirable. However, literature experience with the use of CHO cells for rAAV production is limited, if at all, likely due to cellular restriction factors in these cells that can affect rAAV production and interfere with viral packaging. For example, it has been shown that CHO cells do not support vaccinia virus replication at the stage of viral intermediate protein synthesis (Ramsey-Ewing and Moss. 1995 Virology. 1995 Feb 1;206(2):984-93). Furthermore, CHO cells are inherently resistant to HSV-1 productive infection because they lack key receptors for HSV-1 entry and infection (Montgomery et al., 1996). Thus, there is a need in the art to engineer serum-free adapted suspension CHO CAT-S cells to be permissive for infection with inactive HSV-1 vectors encoding the necessary elements for diverse rAAV production. Summary of the Invention

[0005] The present disclosure is directed to baby hamster kidney (BHK) cells adapted to grow in serum-free conditions, which stably express a hamster codon-optimized herpes simplex virus 1 (HSV-1) ICP27 open reading frame containing a deletion of a non-essential element of infected cell protein 27 (ICP27). In one embodiment, the cells are grown in suspension. In another embodiment, the non-essential element of ICP27 is the 5' and 3' untranslated regions (UTRs).

[0006] The present disclosure also relates to cell lines comprising the BHK cells described herein.

[0007] The present disclosure also relates to a method of producing a recombinant adeno-associated virus (rAAV) vector, comprising introducing a recombinant herpesvirus (rHSV) vector containing AAV rep and cap sequences and a sequence encoding a gene of interest into a BHK cell or cell line described herein, and culturing the cell or cell line under conditions to produce the rAAV vector.

[0008] The present disclosure is also directed to Chinese hamster ovary (CHO) cells adapted to grow in serum-free conditions, which stably express one or more polypeptides required for herpes simplex virus-1 (HSV-1) entry and infection. In one aspect, the CHO cells stably express herpesvirus entry mediator (HVEM) and / or nectin-1. In another aspect, the HVEM and / or nectin-1 sequences are codon-optimized for expression in CHO cells.

[0009] The present disclosure is also directed to the CHO cells described herein.

[0010] The present disclosure also relates to a method for producing a recombinant adeno-associated virus (rAAV) vector, comprising introducing a recombinant herpesvirus (rHSV) vector containing AAV rep and cap sequences and a sequence encoding a gene of interest (GOI) into a CHO cell or cell line described herein and culturing the cell or cell line under conditions to produce the rAAV vector. In another embodiment, the rHSV vector is introduced at a multiplicity of infection of about 4:1, 6:1, 8:1, or 10:1 rHSV-rep / cap:rHSV-GOI. In another embodiment, the AAV serotype is AAV6, AAV8, or AAV9. In another embodiment, the gene of interest encodes any therapeutic biological compound. In another embodiment, the therapeutic biological compound is an antibody or a chimeric antigen receptor. [Brief explanation of the drawings]

[0011] [Figure 1A]The plasmid constructs used for stable transfection and generation of eight pools are shown. Four single cassettes are shown. In pool 1 (CMV-HVEM), the HVEM ORF was constructed under the CMV promoter and an upstream SV40 polyA. In pool 2 (CMV-HVEM-CO), the codon-optimized HVEM ORF was constructed under the CMV promoter and an upstream SV40 polyA. In pool 3 (CMV-nectin-1), the nectin-1 ORF was constructed under the CMV promoter and a BGH polyA. In pool 4 (CMV-nectin-1-CO), the codon-optimized nectin-1 ORF was constructed under the CMV promoter and a BGH polyA. [Figure 1B] The plasmid constructs used for stable transfection and generation of eight pools are shown. Four dual cassettes are shown. In pool 5 (CMV-HVEM-nectin-1), HVEM and nectin-1 ORFs were constructed flanked by a CMV promoter and SV40 polyA and a CMV promoter and BGH polyA, respectively. In pool 6 (CMV-HVEM-nectin-1-CO), codon-optimized HVEM and nectin-1 ORFs were constructed under a CMV promoter and upstream SV40 polyA and a CMV promoter and upstream BGH polyA, respectively. In pool 7 (Spro-HVEM-nectin-1), HVEM and nectin-1 ORFs were constructed under a synthetic promoter (Spro) and upstream SV40 polyA and Spro and BGH polyA, respectively. In pool 8 (Spro-HVEM-Nectin-1-CO), codon-optimized HVEM and Nectin-1 ORFs were constructed and flanked by Spro and SV40 polyA and Spro and BGH polyA, respectively. [Figure 1C]Plasmid constructs used for stable transfection and generation of eight pools are shown, showing that high levels (levelS1) of HVEM surface expression were detected from the pools (CMV-HVEM, CMV-HVEM-CO, CMV-HVEM-nectin-1, CMV-HVEM-nectin-1-CO, Spro-HVEM-nectin-1, and Spro-HVEM-nectin-1-CO), expressed as mean fluorescence intensity (MFI). [Figure 1d] Plasmid constructs used for stable transfection and generation of eight pools are shown. High levels of nectin-1 surface expression, expressed as MFI, were detected from the pools (CMV-nectin-1, CMV-nectin-1-CO, CMV-HVEM-nectin-1, CMV-HVEM-nectin-1-CO, Spro-HVEM-nectin-1, and Spro-HVEM-nectin-1-CO). Green fluorescent protein (GFP) expression and rAAV9-GFP production from stable CHO cell pools are shown. [Figure 2A] Green fluorescent protein (GFP) expression and rAAV9-GFP production from stable CHO cell pools are shown. The average GFP expression from rHSV-nols-AAV-GFP-infected stable cell pools (MOI = 10) at a total of six time points (12, 24, 36, 48, 72, and 96) postinfection is shown. Pools 1 and 2 outperformed all other pools and infected hosts. All pools were superior to wild-type CHO host infection (p < 0.0001). [Figure 2B]Green fluorescent protein (GFP) expression and rAAV9-GFP production from stable CHO cell pools are shown. qPCR rAAV9-GFP titers (vg / mL) from the supernatants of stable cell pools infected at 24 hpi with rHSV-nols-AAV-GFP and rHSV-AAV9 vectors at an MOI of 1:1 are shown. No significant differences were observed in the physical titers of rAAV9-GFP produced from the eight tested pools. All coinfected cell pools yielded higher rAAV9-GFP physical titers compared to wild-type host-infected cells. No significant differences in rAAV9-GFP titers were detected among the tested pools (p=0.0976). All samples were tested in duplicate, and all data are presented as the mean ± SD. [Figure 3] 1 shows generation of high- and intermediate-level CHO-HVEM-expressing clones upon rHSV-GFP infection by flow cytometry analysis. [Figure 4A] Characterization and testing of CHO-HVEM-expressing clones selected for rHSV-1-GFP infection are shown. The final 24 selected recovered clones with high HVEM expression (expressed as MFI) are shown. Ten of these clones were high-level HVEM-expressing clones (clones 1, 7, 21, 23, 24, 33, 36, 40, 63, and 64). The remaining 14 clones were intermediate-level HVEM-expressing clones (clones 9, 11, 13, 14, 15, 16, 23, 28, 29, 42, 46, 51, 54, and 62). [Figure 4B] Characterization and testing of selected CHO-HVEM-expressing clones for rHSV-1-GFP infection is shown. All 24 clones were retested for rHSV-1 GFP vector entry and infection using an IncuCyte (two time points were recorded). All infected clones showed high GFP expression after infection. However, no significant differences in GFP expression were observed among the clones tested (p=0.5251). [Figure 5A] Figure 1 shows the trial production of rAAV6.2-GFP in eight selected CHO-HVEM clones, demonstrating decreased cell viability after co-infection with rHSV-1 vector. [Figure 5B] Figure 1 shows the trial production of rAAV6.2-GFP in eight selected CHO-HVEM clones, demonstrating a decrease in viable cell density after co-infection with rHSV-1 vectors. [Figure 5C] Figure 1 shows the trial production of rAAV6.2-GFP in eight selected CHO-HVEM clones. The rAAV6.2-GFP titers of eight CHO-HVEM expression clones tested at an MOI of 1:1 are shown. [Figure 5D] Figure 1 shows the test production of rAAV6.2-GFP in eight selected CHO-HVEM clones. Figure 2 shows the test of rAAV6.2-GFP production in CHO-HV-C1 and CHO-HV-C62 clones using different MOIs. [Figure 5E] Figure 1 shows the trial production of rAAV6.2-GFP in eight selected CHO-HVEM clones. Figure 2 shows the test of rAAV8-GFP and rAAV9-GFP production in CHO-HV-C1 using an MOI of 4:1. [Figure 6] There is a significant improvement in both cell viability (FIG. 6a) and rAAV6.2 GFP vector production (FIG. 6b) after co-infection with CHO-HV-C1. [Figure 7] A schematic diagram of the process for rAAV vector recovery and purification using the PEG-chloroform method is shown. [Figure 8A] Analytical characteristics of rAAV produced in the CHO-HV-C1 clone show good expression of VP1, VP2, and VP3 capsid proteins from either purified rAAV6.2-GFP (produced at different MOIs of 4:2, 6:2, 8:2, and 10:2) and rAAV9-GFP vectors (produced at different MOIs of 4:2, 6:2, and 8:2). [Figure 8B] Figure 1 shows analytical characteristics of rAAV produced in the CHO-HV-C1 clone. Figure 2 shows a mini-transmission electron micrograph (miniTEM) of purified rAAV6.2-GFP showing 91% intact capsids free of aggregates and cellular debris. [Figure 8C]Analytical characteristics of rAAV produced in the CHO-HV-C1 clone are shown. A miniTEM of purified rAAV9-GFP is shown, showing 79.5% complete capsids. White arrows are used to indicate complete capsids, while black arrows are used to indicate incomplete (empty) capsids. [Figure 8D] 1 shows analytical characteristics of rAAV produced in the CHO-HV-C1 clone. rAAV capsid ratio detection using the CE-SDS method is shown, along with the detection of impurities, and absorbance values ​​for VP1, VP2, and VP3 from the tested rAAV6.2-GFP and rAAV9-GFP produced in the CHO-HV-C1 clone are shown. [Figure 9] Testing for infectious rHSV-1 residues in purified rAAV on V27 cells is shown. Complemented V27 cells stably expressing the HSV-1 ICP27 protein were tested for any infectious residues of the rHSV-1 vector used to produce rAAV in the CHO-HV-C1 clone. Two to four days after infection, no cytopathic effect (CPE) was observed in wells inoculated with either PEG-chloroform-purified rAAV6.2-GFP and / or rAAV9-GFP vectors, indicating complete inactivation of the rHSV-1 vectors used for co-infection. In contrast, wells inoculated with the rHSV-AAV-GFP vector showed clear CPE, indicated by cell rounding and detachment of the cell sheet. [Figure 10A] 1 shows the infectivity of rAAV produced in CHO-HV-C1 cells. The infectivity of the purified rAAV6.2-GFP vector produced in the CHO-HV-C1 clone, purified by the PEG-chloroform method, and tested in Ad293 cells is compared to the infectivity of the rAAV6.2-GFP vector produced in HEK293 cells and purified by chromatography. [Figure 10B]Figure 1 shows the infectivity of rAAV produced in CHO-HV-C1 cells. The infectivity of the PEG-chloroform-purified rAAV9-GFP vector produced in the CHO-HV-C1 clone and tested in Ad293 cells is compared to the infectivity of the chromatographically purified rAAV9-ZsGreen vector produced in HEK293 cells. [Figure 10C] Figure 1 shows the infectivity of rAAV produced in CHO-HV-C1 cells. Figure 2 shows in vitro transduction of rAAV produced in CHO-HV-C1 cells. Transduction of PEG-chloroform-purified rAAV6.2 and rAAV9-GFP produced in CHO cells was compared with transduction of rAAV6.2-GFP and rAAV9-ZsGreen produced in HEK293 cells and purified by chromatography. [Figure 11A] The biodistribution of CHO cell-derived rAAV is shown. The study design is shown. Mice were divided into five groups. G1, G2, G3, and G4 were inoculated with 10 vg / 100 μL of rAAV6.2-GFP-CHO, rAAV9-GFP-CHO, triple transient transfection (TTT)-derived rAAV6.2-GFP, or TTT-derived rAAV9-ZsGreen per mouse, respectively. G5 was inoculated with sterile PBS. Three weeks after tail vein injection, all mice were euthanized, and tissues (liver, heart, lung, kidney, and skeletal muscle) were harvested and tested for GFP copies by qPCR and expression by confocal microscopy. [Figure 11B-01] Figure 1 shows the biodistribution of CHO cell-derived rAAV. GFP titers in G1 and G3 tissues using qPCR. [Figure 11B-02] Figure 1 shows the biodistribution of CHO cell-derived rAAV. GFP titers in G1 and G3 tissues using qPCR. [Figure 11C-01] Figure 1 shows the biodistribution of CHO cell-derived rAAV. Figure 2 shows GFP / ZsGreen titers in G2 and G4 tissues using qPCR. [Figure 11C-02] Figure 1 shows the biodistribution of CHO cell-derived rAAV. Figure 2 shows GFP / ZsGreen titers in G2 and G4 tissues using qPCR. [Figure 11D] Figure 1 shows the biodistribution of CHO cell-derived rAAV. GFP expression from liver sections of all groups was observed using confocal microscopy. [Figure 12A] Development and selection of HSV-1-producing CHO-ICP27 pools and clones are shown. Development of a stable CHO-HV1-ICP27 pool using random integration is shown. The Chinese hamster codon-optimized HSV-1 ICP27 ORF was subcloned into an in-house developed plasmid under a CMV promoter and upstream SV40 polyA for cell line development. The same plasmid encoded a puromycin cassette consisting of a CMV promoter and BGH polyA downstream of the puromycin ORF. [Figure 12B] Development and selection of HSV-1-producing CHO-ICP27 pools and clones are shown. Development of a stable CHO-HV1-ICP27 pool using CRISPR / Cas9 technology is shown. The donor plasmid was constructed using an in-house developed pCLD plasmid backbone and a puromycin cassette flanked by a CMV promoter downstream and an SV40 polyA upstream, encoding a codon-optimized Chinese hamster ICP27 ORF, and an SV40 promoter and SV40 polyA upstream. The two cassettes were flanked by right and left homology arms (750 base pairs each). The total length of the two cassettes is 4.1 kb. [Figure 12C] Development and selection of HSV-1-producing CHO-ICP27 pools and clones are shown. The mean fluorescence intensity (MFI) of ICP27 expression from the final selected clones is shown. Seven clones, such as C6-S, were site-specifically integrated, while 17 clones, such as C-11R, were randomly integrated. [Figure 13] Figure 1 shows the production of rHSV-AAV9 in CHO-HV1-ICP27-C11. rHSV-AAV9 production was tested in selected CHO-HV1-ICP27-C11 clones at 33°C using three different MOIs and in-house developed medium. The recovered rHSV-AAV9 was titrated by plaque assay on V27 cells. [Figure 14]Construction of BHK-21-ICP27 expression pools is shown. (Pool 1) Pool 1 expresses the codon-optimized ICP27 ORF under the HSV-1 ICP27 endogenous promoter with puromycin as the selectable marker. (Pool 2) Pool 2 expresses the codon-optimized ICP27 ORF under the CMV promoter with puromycin as the selectable marker. (Pool 3) Pool 3 expresses the non-codon-optimized ICP27 ORF under the CMV promoter with neomycin as the selectable marker. [Figure 15] This figure shows the production of HSV-AAV6.2 in the BHK-21-ICP27 pool. The recovered BHK21-ICP27 pool was tested for rHSV-AAV6.2 production (MOI 0.15 PFU / mL) using Xell HEK TF in the presence of serum or 4% FBS. Three days after infection, purified virus was titrated by plaque assay on V27 cells. [Figure 16] 1 shows silver staining of impurities from PEG-chloroform purification of rAAV samples. DETAILED DESCRIPTION OF THE INVENTION

[0012] Recent studies have shown that the production of different recombinant adeno-associated viruses (rAAVs) using recombinant herpes simplex virus 1 (rHSV-1) vectors has produced rAAVs with higher physical and transduction titers compared to those produced by commonly used triple transient transfection methods and / or baculovirus-based systems. However, rAAV-based HSV production platforms currently face two major challenges: (1) high productivity relies on commercially available serum-supplemented media, which poses costly large-scale production and a high risk of introducing adventitious factors into the final product; and (2) the difficulty of scaling up rHSV-1 vectors in adherent Vero cells (such as the V27 cell line) that express the HSV-1 ICP27 protein. To address the first challenge, the present disclosure provides eight serum-free, adapted CHO cell pools expressing receptors (HVEM and / or nectin-1) essential for HSV-1 entry and infection. Using high-throughput methods, the present disclosure provides a clone (designated CHO-HV-C1) with the highest HVEM receptor expression. Interestingly, higher yields of rAAV6.2-GFP, rAAV8-GFP, and rAAV9-GFP vectors were produced within 24 hours of coinfection in the CHO-HV-C1 clone at lower multiplicities of infection (MOIs), ~9.21 log , respectively. 10 , 9.40 log 10 and 9.61 log 10 This was achieved with titers of viral genomes / mL (vg / mL) compared to 10 log in non-CHO-based platforms at 48–72 hours post-coinfection at higher MOIs. 10(Kang et al., 2009. Gene Therapy 16, 229-239) reported rAAV titers of 1000 vg / mL. Furthermore, rAAV produced in the CHO-HV-C1 clone had in vitro and in vivo transduction efficacies comparable to those produced by triple transient transfection. To address the second challenge, the present disclosure provides a proprietary serum-free suspension BHK-21 cell pool engineered to express the HSV-1 ICP27 protein. Interestingly, the BHK-21-ICP27-expressing pool grown in serum-free medium produced rHSV-1 titers comparable to those of V27 cells.

[0013] Gene therapy holds great promise for treating many different diseases for which no treatments are currently available, such as cystic fibrosis, heart failure, and Duchenne muscular dystrophy. In recent years, gene therapy has focused on the use of rAAV vectors due to their long-term sustained expression after delivery to target organs and their non-pathogenic profile in humans. Current methods for producing rAAV for clinical trials include triple transient transfection (the most common method), packaging or producer cell lines, and helper virus systems, such as HSV-1, baculovirus, and / or human adenovirus-5. Transfection-based methods, such as triple transient transfection, are difficult to scale up and result in low specific yields of infectious particles (ip) and DNase-resistant particles (Grimm et al., 1998. Hum Gene Ther;9:2745-2760; Zolotukhin et al., 2002. Methods;28:158-167). On the other hand, packaging or producer cell lines are limited to the production of one rAAV serotype / product. Recent studies have shown that rHSV-1-assisted rAAV production provides a highly efficient production method (Kang et al., 2009). However, typical rAAV production using the rHSV-1 system involves infecting virus-producing cells, such as BHK-21 cells, with two replication-deficient rHSV-1 vectors in serum-supplemented medium, which is very expensive for large-scale production and may introduce adventitious viral agents and / or prions into the final product. Another drawback of rAAV production using the rHSV-1 system is that scaling up rHSV-1 vector stocks is challenging because current rHSV-1 vector production relies on adherent Vero cells (called V27 cells, Rice and Knipe J Virol. 1990 Apr;64(4):1704-15) that express the HSV-1 ICP27 protein.

[0014] CHO cells are the primary mammalian cell type used for the production of recombinant protein biologics due to their ability to accurately fold, assemble, and modify recombinant proteins (Aggarwal, 2014. Nat. Biotechnol. 32, 32-39; Jayapal et al., 2007. Cell Engineering Progress, 103, 40-47; and Walsh, 2018. Nat. Biotechnol; 36:1136-1145). Certain animal cell types, such as porcine testis (ST) and CHO cells, can efficiently bind the HSV-1 virus but limit viral entry (Shieh et al., 1992. J. Cell Biol. 116, 1273-1281; Subramanian et al., 1994. J. Virol. 68, 5667-5676). Furthermore, it has been reported that porcine and CHO cells become susceptible to HSV-1 entry upon expression of a human cDNA encoding HVEM (Montgomery et al., 1996. Cell Vol. 87, 427-436). The present disclosure aimed to engineer suspension serum-free adapted CHO cells to produce different rAAVs using the HSV-1 helper system, a virus that does not naturally infect wild-type CHO cells.

[0015] rAAV is the leading platform for gene delivery, with three licensed products approved by the end of 2021 (Bulcha et al., 2021). rAAV has several advantages as a gene delivery vector, including the ability to transduce a variety of proliferating and non-proliferating cells, harbor cell / tissue-specific promoters, and induce a reduced immune response compared to other viral vectors (Kang et al., 2009).

[0016] Currently, different cell culture expression platforms for rAAV exist, including stable packaging cell lines expressing the rep and cap genes of the desired rAAV serotype, stable proviral cell lines stably expressing Rep, Cap, and a transgene, and triple transient transfection (Clark et al., 1995. Hum Gene Ther;6:1329-1341; Clark et al., 2002. Kidney Int;61s:9-15; Qiao et al., 2002. Kidney Int;61s:9-15). Triple transient transfection is the most common method for rAAV production and uses three plasmids: one encoding the gene of interest (GOI) flanked by AAV inverted terminal repeats (ITRs), a second encoding the AAV rep and cap genes, and a third encoding adenovirus helper function genes. This three-plasmid system can be simplified by including helper genes in the rep-cap plasmid, allowing for co-transfection (Grimm et al., 1998 and Clark KR, 2002). However, the triple transient transfection method is difficult to scale up due to the very high doses required, resulting in low specific yields of infectious particles (ip), and often resulting in high DNase-resistant particle (DRP / ip) ratios (50-100) (Grimm et al., 1998. Hum Gene Ther;9:2745-2760; Zolotukhin et al., 2002. Methods;28:158-167).

[0017] Similar to adenovirus, recombinant HSV-1 (rHSV-1) has also been reported to be capable of supporting rAAV replication as part of a helper virus system (Buller et al., 1981). The minimal HSV helper genes required for rAAV replication are the HSV helicase-primase complex (UL5, UL8, UL52), HSV DNA polymerase, and HSV DNA-binding protein (UL29) genes (Weindler and Heilbronn, 1991. J Virol; 65:2476-2483). Furthermore, it has been reported that rHSV coinfection using two HSV-1 vectors (one encoding the desired AAV2 rep and cap, and the second encoding the GOI) produced rAAVs of multiple different serotypes in various cell lines with very high specific yields and very low (DRP / ip) ratios (Kang et al., 2009). Another advantage of the HSV helper system is that HSV can replicate and provide helper functions in different mammalian cells, which means that HSV helper functions circumvent the host range restriction for rAAV production, whereas rAAV production with adenovirus helpers requires human cell lines (Buller et al., 1970. J Gen Virol; 43:663-672).

[0018] Chinese hamster ovary (CHO) cells are primarily used as expression hosts for the production of recombinant monoclonal antibodies (mAbs) and therapeutic proteins, comprising the fastest-growing segment of the biopharmaceutical industry (Walsh G., 2018). Due to their regulatory tolerance and lower manufacturing costs compared to human cells, using CHO cells for virus production is desirable. However, literature experience with the use of CHO cells for rAAV production is limited, if at all, likely due to cellular restriction factors in these cells that can affect rAAV production and interfere with viral packaging. For example, it has been shown that CHO cells do not support vaccinia virus replication at the stage of viral intermediate protein synthesis (Ramsey-Ewing and Moss. Virology. 1995 Feb 1;206(2):984-93). Furthermore, CHO cells are inherently resistant to HSV-1 productive infection because they lack a key receptor for infection by non-replicating HSV-1 (Montgomery et al., 1996. Cell Vol. 87, 427-436). The present disclosure provides serum-free, adapted suspension CHO-CAT-S cells engineered to be permissive for HSV-1 entry and infection for the production of diverse rAAVs using the HSV-1 system.

[0019] definition In order that this disclosure may be more readily understood, certain terms are first defined. As used herein, unless expressly provided otherwise herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the specification.

[0020] It should be noted that terms such as "a" or "an" refer to one or more of that entity. For example, a "feed medium" is understood to represent one or more feed media. Thus, terms such as "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0021] The term "and / or," as used herein, should be construed as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" used herein in a phrase such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0022] Whenever an embodiment is described herein with the word "comprising," it is understood that other similar embodiments described with the words "consisting of" and / or "consisting essentially of" are also provided.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0024] Units, prefixes, and symbols are denoted in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not intended to limit the various aspects of this disclosure, which may be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0025] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any list or listed members.

[0026] Terms such as "about" or "essentially comprising" refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially comprising" can mean within or more than one standard deviation, per the practice in the art. Alternatively, "about" or "essentially comprising" can mean a range of up to 20%. Moreover, particularly with respect to biological systems or processes, these terms can mean up to an order of magnitude or up to five times the value. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range for that particular value or composition.

[0027] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof (such as integer tenths and hundredths), where appropriate, unless otherwise indicated.

[0028] The terms "adeno-associated virus," "AAV virus," "AAV virion," "AAV virus particle," and "AAV particle," used synonymously herein, refer to a viral particle composed of at least one capsid protein of AAV and an enclosed polynucleotide corresponding to the AAV genome. Wild-type AAV refers to a virus belonging to the genus Dependovirus and family Parvoviridae. The wild-type AAV genome is approximately 4.7 kb in length and consists of single-stranded deoxyribonucleic acid (ssDNA) that can be positive- or negative-sense. The wild-type genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand and three open reading frames (ORFs). ORFrep encodes the four Rep proteins required for the AAV life cycle. ORFcap contains nucleotide sequences encoding the capsid proteins VP1, VP2, and VP3, which interact to form a capsid with icosahedral symmetry. Finally, the assembly activating protein (aap) ORF overlaps with the cap ORF and encodes the AAP protein, which is thought to promote capsid assembly. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide different from the wild-type AAV genome, such as a transgene delivered to a mammalian cell) flanked by AAV ITRs, it is typically known as an "AAV vector particle" or "AAV viral vector" or "AAV vector" or "recombinant AAV vector." The present invention also encompasses the use of double-stranded or self-complementary AAV (also called dsAAV or scAAV).

[0029] "AAV virus" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein (preferably from all of the capsid proteins of wild-type AAV) and an encapsidated polynucleotide. When the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as a "rAAV vector particle" or simply an "rAAV vector."

[0030] "Packaging" refers to a series of intracellular events that result in assembly of capsid proteins and encapsidation of the vector genome to form an AAV particle.

[0031] The AAV "rep" and "cap" genes refer to polynucleotide sequences encoding the adeno-associated virus replication and encapsidation proteins, respectively. They are found in all AAV serotypes tested and are described below and in the art. AAV rep and cap are referred to herein as AAV "packaging genes."

[0032] As used herein, the term "hybrid AAV" refers to an AAV that contains capsid proteins of one AAV serotype and genomic material from another AAV serotype.

[0033] As used herein, the term "chimeric AAV" refers to an AAV that comprises gene and / or protein sequences derived from two or more AAV serotypes, and may include mutations made to the gene sequences of the two or more AAV serotypes. An exemplary chimeric AAV may comprise a chimeric AAV capsid, e.g., a capsid protein having one or more regions of amino acids derived from two or more AAV serotypes.

[0034] As used herein, the term "AAV variant" refers to an AAV that contains one or more amino acid mutations in its genome or proteins compared to a parent AAV, for example, one or more amino acid mutations in its capsid protein compared to a parent AAV.

[0035] The term "viral vector" refers to a gene transfer vector or gene delivery system derived from a virus. Such vectors can be constructed using recombinant techniques known in the art. In some respects, the virus used to derive such vectors is selected from AAV, helper-dependent adenovirus, hybrid adenovirus, Epstein-Barr virus, retrovirus, lentivirus, herpes simplex virus, Sendai virus (HVJ), Moloney murine leukemia virus, poxvirus, and HIV-related viruses.

[0036] As used herein, the term "AAV virion" or "AAV particle" refers to a viral particle comprising a capsid that includes at least one AAV capsid protein that encapsidates an AAV vector described herein, which in some embodiments may further comprise a heterologous polynucleotide sequence or transgene.

[0037] As used herein, the term "engineered cell" and its grammatical equivalents refer to a cell that contains at least one alteration of a nucleic acid within the genome of the cell or that contains at least one exogenous nucleic acid or protein. Alterations include additions, deletions, and / or substitutions within a nucleic acid sequence. Thus, engineered cells include cells that contain added, deleted, and / or altered genes.

[0038] Various aspects of the disclosure are described in further detail in the following subsections.

[0039] Adeno-associated virus (AAV) Adeno-associated virus (AAV) is a nonpathogenic, single-stranded DNA parvovirus. AAV has a capsid diameter of approximately 20 nm. Each end of the single-stranded DNA genome contains inverted terminal repeats (ITRs), the only cis-acting elements required for genome replication and packaging. The AAV genome contains two viral genes, rep and cap. The virus uses two promoters and alternative splicing to generate four proteins required for replication (Rep 78, Rep 68, Rep 52, and Rep 40). A third promoter generates transcripts for the three structural viral capsid proteins 1, 2, and 3 (VP1, VP2, and VP3) through alternative splicing and alternative translation initiation codon combinations (Berns KI et al., Bioessays. 1995;17:237-45). The three capsid proteins share the same C-terminal 533 amino acids, but VP2 and VP1 contain additional N-terminal sequences of 65 and 202 amino acids, respectively. AAV virions contain a total of 60 copies of VP1, VP2, and VP3 found in crude extracts at a ratio of 1:1:5 (Aucoin MG et al., Biotechnol Adv. 2008;26(1):73-88), or in a range of 1:1:8 to 1:1:20 by densitometry (Grimm D et al., Gene Ther. 1999;6(7):1322-1330; Kronenberg S et al., EMBO Rep. 2001;2(11):997-1002), arranged in a T=1 icosahedral symmetry (Rose JA et al., J Virol. 1971;8:766-70). AAV requires adenovirus (Ad), herpes simplex virus (HSV), or other viruses as helper viruses to complete its lytic life cycle (Atchison RW et al., Science. 1965;149:754-6; Hoggan MD et al., Proc Natl Acad Sci USA. 1966;55:1467-74). Furthermore, in the absence of helper virus, wild-type (wt) AAV establishes latent infection by Rep protein-assisted integration through the interaction of the ITRs with the chromosome (Berns et al., 1995).

[0040] AAV serotype There are several different AAV serotypes, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and rh-AAV-10. In vivo studies have shown that various AAV serotypes exhibit different tissue or cell tropism. For example, AAV1 and AAV6 are two serotypes that are efficient for transducing skeletal muscle (Gao GP et al., Proc Natl Acad Sci USA. 2002;99:11854-11859; Xiao W et al., J Virol. 1999;73:3994-4003; Chao H et al., Mol Ther. 2000;2:619-623).

[0041] Since the development of naturally occurring AAV serotypes into gene therapy vectors, much effort has been focused on understanding the tropism of each serotype so that further modifications to the virus can be implemented to enhance the efficiency of gene transfer. One approach is to create hybrid vectors with desired qualities from each parent by swapping domains from one serotype capsid to another. Because the viral capsid is involved in cellular receptor binding, understanding the viral capsid domains important for binding is important. Mutational studies on viral capsids (primarily AAV2) conducted before the availability of crystal structures were primarily based on functionalizing the capsid surface by adsorption of exogenous moieties, insertion of peptides at random positions, or comprehensive mutagenesis at the amino acid level (Choi et al. Curr Gene Ther. 2005 June;5(3):299-310).

[0042] In some aspects, the present disclosure provides methods for producing rAAV particles having capsid proteins expressed by multiple AAV serotypes. This is achieved by co-infecting producer cells with an rHSV-expressing virus and an rHSV-rep2capX helper virus whose cap gene product is derived from an AAV serotype other than, or in addition to, AAV2. Recombinant AAV vectors are generally based on the AAV2 capsid. It has recently been demonstrated that rAAV vectors based on capsids from the AAV1, AAV3, AAV4, AAV5, AAV8, or AAV9 serotypes differ from AAV2 in their tropism.

[0043] Capsids derived from other AAV serotypes offer advantages over rAAV vectors based on AAV2 capsids in certain in vivo applications. First, the appropriate use of rAAV vectors with specific serotypes can increase the efficiency of in vivo gene delivery to specific target cells that are poorly or not infected at all by AAV2-based vectors. Second, when re-administration of rAAV vectors becomes clinically necessary, it may be advantageous to use rAAV vectors based on other AAV serotypes. It has been demonstrated that re-administration of rAAV vectors with the same capsid may be ineffective, possibly due to the generation of neutralizing antibodies against the vector. This problem can be avoided by administering rAAV particles whose capsids are composed of proteins from different AAV serotypes that are not affected by the presence of neutralizing antibodies against the first rAAV vector. It will be appreciated that construction of recombinant HSV vectors similar to rHSV but encoding cap genes from other AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV5-AAV9) can be accomplished using the methods described herein for producing rHSV. In certain embodiments, recombinant AAV vectors constructed using cap genes from different AAVs are implemented. [Example]

[0044] Experimental Method Generation of stable CHO pools The open reading frames (ORFs) of human HVEM and nectin-1 were downloaded from the NCBI database (GenBank U70321.1 and AF060231.1, respectively). Both the HVEM and nectin-1 ORFs were codon-optimized for expression in hamster cells using online tools (https: / / www.idtdna.com / CodonOpt and https: / / www.thermofisher.com / us / en / home / life-science / cloning / gene-synthesis / geneart-gene-synthesis / geneoptimizer.html, respectively). The delivered plasmids encoding either HVEM or nectin-1 were subcloned into in-house developed plasmids downstream of an enhanced human cytomegalovirus (CMV) promoter and / or a synthetic promoter (Brown et al., 2017) to generate eight different constructs. All constructed plasmids encoded glutathione synthetase (GS) under the control of the SV40 promoter, allowing selection of transfected cells in methionine sulfoximine (Bebbington et al., 1992. Bio / technology (Nature Publishing Company), 10(2), 169-175). All final plasmids were verified by whole-plasmid sequencing (Macrogen). In-house developed suspension serum-free adapted CHO cells were thawed in 125 mL shake flasks and cultured in 30 mL of CD-CHO medium (Thermo Fisher) supplemented with 6 mM L-glutamine (Gibco) and dextran sulfate (50 mg / mL, Sigma-Aldrich). The cells were incubated at 37°C with 120 rpm agitation in a 6% CO2 humidified incubator. Viable cell density (VCD) and viability were measured daily using a Vi-Cell automated cell counter (Beckman Coulter).

[0045] Stable CHO cell pools were generated according to standard in-house protocols. Briefly, eight aliquots of CHO CAT-S cells (1 × 10 per aliquot) were cultured. 7Viable cells (1 x 10 per pool) were pelleted at 200 x g for 5 minutes. The cell pellets were mixed with 7 µg of each purified linearized in-house developed pCLD plasmid and then transfected using the Amaxa cell line nucleofector Kit V (Lonza) according to the manufacturer's instructions. 24 hours after transfection, cell viability was measured, and 75 µM / mL MSX (Sigma-Aldrich) was added to each pool for recombinant cell selection. Aliquots from the collected cell pools (1 x 10 per pool) were collected. 6 Viable cells (100% viable cells) were tested for either HVEM and / or nectin-1 receptor expression using FACS staining. Briefly, cells were incubated with 150 μL of either a 1:200 dilution of anti-CD270 (HVEM) eBioscience PE clone eBioHVEM-122 (Invitrogen) and / or a 1:200 dilution of nectin-1 monoclonal antibody clone R1.302-PE (Invitrogen) in PBS (Gibco) supplemented with 1% bovine serum albumin (BSA) (Invitrogen) for 15 minutes at room temperature in the dark. After incubation, the diluted antibodies were discarded, and the cells were then fixed with Fix and Perm medium A (Life Technologies) and incubated for 15 minutes at room temperature in the dark. The stained cells were then washed twice with PBS and resuspended in FACS buffer (PBS supplemented with 0.1% BSA) for flow cytometry using an LSR II instrument (BD Biosciences). Flow data were analyzed with FlowJo v10.0 software (Tree Star, Inc.).

[0046] rHSV-1 infection in stable CHO pools Aliquots (1 x 10) from each harvested cell pool and CHO CAT-S host cells 6Viable cells (number of cells) were infected with rHSV-GFP (MOI = 10) in a 6-well cell culture plate (Corning) for 1 hour at 37°C in a humidified 5% CO2 incubator for virus adsorption. After 1 hour, the infected cell pool was centrifuged at 1200 rpm for 5 minutes, and excess viral supernatant was discarded. The infected cell pellet was resuspended in CD-CHO medium and incubated at 37°C in a humidified 5% CO2 incubator connected to an IncuCyte (Sartorius). Cell imaging of the infected pool for GFP expression was determined using the default settings of the IncuCyte, and GFP expression was captured every 12 hours from each infected cell pool for a total of five time points.

[0047] rAAV9-GFP vector production in stable CHO pools Aliquot (1 x 10) of each cell pool 6Viable cells (number of cells) were infected with rHSV-AAV9 rep / cap:rHSV-AAV-GFP vectors at an MOI of 1:1 in 6-well culture plates (Corning). The infected cell pool was incubated at 37°C in a 5% CO2 humidified incubator for 24 hours. After 24 hours, infected cell supernatants were harvested and tested for rAAV9-GFP titer using qPCR. Briefly, harvested supernatants were digested with DNase I (200 U / µL, Invitrogen) for 1 hour at 37°C, followed by incubation at 95°C for 10 minutes for enzyme inactivation. The digested samples were then incubated with an equal volume of proteinase K digestion mix (200 mM NaCl, 20 mM Tris-HCl, pH 8.0, 2 mM ethylenediaminetetraacetic acid, pH 8.0, 0.5% sodium dodecyl sulfate, and proteinase K (20 mg / mL)) at 55°C for 1 hour, followed by 95°C for 10 minutes for enzyme inactivation. Reactions for absolute qPCR quantification were performed using 5 μL of diluted template plus TaqMan Fast Universal PCR 2x Master Mix (Applied Microbiology). PCR was performed using a standard linearized AAV plasmid developed in-house in a 20-μL reaction containing 20 μM of each CMV forward primer (5′-TTCCTACTTGGCAGTACATCTACG′-3), CMV reverse primer (5′-GTCAATGGGGTGGAGACTTGG-′3), and CMV probe (5′-FAM-TGAGTCAAACCGCTATCCACGCCCA-NFQ-′3) using a PCR thermocycler (QuantaBio Q, Qiagen). The PCR cycle profile was 95°C for 2 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s.

[0048] Generation and testing of rHSV-GFP infection in CHO-HVEM-expressing clones Single cell deposition cloning was performed using a BD Influx cell sorter (BD Biosciences) according to Evans et al., 2015 (Biotechnology Progress, 31(5), 1172-1178). Briefly, 3 × 106 Aliquots containing viable HVEM-CHO-expressing cells were stained with anti-human CD270 (HVEM)-PE (Invitrogen) antibody diluted 1:200 in PBS for 15 minutes at room temperature in the dark. Stained cells were washed twice with sterile PBS, pelleted at 200 × g for 5 minutes, and then resuspended in 1 mL of sorting buffer. Cells were sorted from the PE-gated fraction and plated into two 384-well plates (Agilent) containing in-house conditioned medium supplemented with 50 μM MSX / mL. Selected recovered clones were further tested for rHSV-GFP infection, and mean GFP expression was calculated from two time points (24 and 48 hpi) using IncuCyte as previously described.

[0049] Production of rAAV6.2-GFP vector in final CHO-HVEM clones Selected clones were also further tested for rAAV6.2-GFP vector production using a 1:1 MOI of rHSV-AAV6.2 rep / cap:rHSVGFP. All infected clones were incubated at 37°C in a 5% CO2 humidified incubator with agitation at 120 rpm for 3 days. Aliquots (1 mL) from each coinfected clone were collected 24 and 48 hours after coinfection and centrifuged at 200 × g for 5 minutes. The collected samples (cell pellets) were then prepared for rAAV titration using qPCR. Briefly, the infected cell pellets were collected, mixed with AAV lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl), and subjected to three cycles of freeze / thaw in an isopropanol dry ice bath, followed by centrifugation at 12,000 rpm for 30 minutes at 4°C. After centrifugation, the supernatant was collected, and the rAAV6.2-GFP titer was determined using qPCR as previously described. In subsequent experiments, two of the final selected clones were further tested for rAAV6.2-GFP vector production using different MOIs and incubation temperatures. rAAV6.2-GFP titers were determined 24 hours after co-infection from the collected cell pellets and titrated using qPCR as previously described. Purification of rAAV produced in the final selected CHO clones was performed according to (Negrini et al., Curr Protoc Neurosci. 2020 Sep;93(1):e103). The rAAV produced in the final selected CHO clones was purified using the polyethylene glycol (PEG) chloroform method according to Negrini et al. (Negrini et al., 2020) with some modifications, including the use of 0.5% Triton X-100 (v / v) for cell lysis and rHSV-1 inactivation, followed by precipitation with 8% polyethylene glycol 8000 and 150 mM NaCl. The viral pellet was further treated with Benzonase (Sigma, 50 U / mL) and RNase (Invitrogen, 10 μg / mL) for 1 hour at 37°C, followed by a 1:1 chloroform (Sigma) treatment.The aqueous layer after chloroform treatment was collected and concentrated using an AMICON filter (Sigma). The concentrated virus was stored at -80°C until use.

[0050] Analytical characterization of CHO-derived rAAV The purified rAAV was tested for capsid protein expression using Western blotting. Briefly, purified rAAV6.2-GFP and rAAV9-GFP vectors produced in the final selected clones using different MOIs of rHSV-1 vector were prepared for SDS-PAGE gels by adding appropriate volumes of 4x NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Life Technologies) and 10x NuPAGE sample reducing agent (Thermo Fisher). The samples were then incubated at 70°C for 10 minutes. For each serotype, equal volumes of rAAV were loaded onto Bolt 4-12% Bis-Tris plus 12-well gels (Invitrogen) and run using 1x NuPAGE running buffer (Thermo Fisher). After electrophoresis, the gel was subjected to dry transfer using an iBolt 2 NC mini stack (Invitrogen) and subsequently blocked for 1 hour at room temperature in 5% nonfat milk (Amresco) diluted in TBS (Bio-Rad). After blocking, the membrane was incubated overnight at 4°C with 1:200 AAV VP1, VP2, and VP3 antibodies (Genentech, USA) diluted in 5% nonfat milk with gentle shaking. After incubation, the blotted membrane was washed three times with TBS supplemented with 0.1% Tween (Life Technologies), then incubated for 1 hour with 1:100 goat anti-mouse IgG (Thermo Scientific), followed by washing. The membrane was then incubated with Supersignal West Pico Plus Substrate (Thermo Scientific) and imaged using an Amersham Imager 680 (GE Healthcare). In subsequent experiments, purified rAAV from the final selected CHO clone was visualized using a mini-transmission electron microscope (Mini-TEM, Vironova).Briefly, PEG-chloroform-purified rAAV6.2-GFP and rAAV9-GFP samples were placed on a 400-mesh glow-discharged carbon grid by first inverting the grid and placing it on a 10 μL droplet of rAAV deposited on Parafilm for 30 seconds. Excess sample was blotted by gently touching the edge of the grid to Whatman filter paper. The grid was then washed twice with two 20 μL droplets of double-distilled water. The grid containing the sample was then stained with a 20 μL droplet of 1.5% uranyl acetate for 10 seconds. Excess dye was blotted by gently touching the edge of the grid to Whatman filter paper. The rAAV samples were then visualized using a Mini-TEM instrument. In a separate, independent experiment, analysis of rAAV capsid protein (VP1:VP2:VP3) ratios was performed using a self-developed capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) method based on Kurosawa et al. (Mol Ther Methods Clin Dev. 2020 Oct 4;19:330-340).

[0051] Infectious rHSV-1 residues in purified CHO-derived rAAV V27 cells expressing a stable copy of the rHSV-1 ICP27 protein were cultured at 0.5 × 10 in six-well culture plates in DMEM supplemented with 10% FBS (Gibco) and 500 μg / mL Geneticin (Gibco). 6 Viable cells / well were seeded overnight. After 16 hours, cells were washed twice with sterile PBS and then diluted 1:100 (~10 10 Plates were infected with purified rAAV6.2-GFP and rAAV9-GFP vectors at a concentration of 0.15 pfu / mL. The rHSV-AAV-GFP vector was used as a positive control (MOI = 0.15 pfu / cell). Infected cells were incubated at 37°C for 2 hours for virus adsorption. After incubation, excess virus was removed, infection medium (DMEM supplemented with 2% FBS) was added, and plates were incubated and monitored on an IncuCyte for 4 days to capture any cytopathic effects.

[0052] Infectivity and in vitro transduction of CHO-derived rAAV For infectivity, Ad293 cells were seeded into 96-well culture plates (2 × 10 cells / well) and incubated overnight at 37°C in a humidified 5% CO2 incubator. Tenfold dilutions of rAAV6.2-GFP, rAAV9-GFP, and / or rAAV9-ZsGreen were prepared. These were either produced in CHO clones and purified with PEG-chloroform, and / or produced in HEK293 cells using triple transfection (Kimura et al., Sci Rep 9, 13601, 2019) and purified by affinity chromatography. Each virus dilution was used to infect four wells, and the infected cells were incubated at 37°C in a 5% CO2 incubator for 5 days. On day 5 post-infection, infected cells were imaged for GFP / Zs-Green expression using the default settings of the IncuCyte, and viral titers were calculated using the Reed-Muench method (Reed and Muench, American Journal of Epidemiology;27;3;493-497, 1938). For transduction analysis, Ad293 cells were cultured as described above and then infected at different multiplicities of transduction (MOT) with CHO-derived rAAV and / or similar vectors produced in HEK293 cells using triple transfection as described above.

[0053] Biodistribution of CHO-derived rAAV All animal experiments were approved by the Institutional Animal Care and Use Committee of AstraZeneca (Gaithersburg, MD, USA). Eight-week-old male C57bl / 6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were divided into five groups (n = 5 per group). All mice were inoculated with 1 × 10 insulin via the tail vein using a BD insulin syringe (Becton Dickinson). 11Each mouse was inoculated with the appropriate rAAV vector (or saline) at 100 μg / 100 μL. Inoculated mice were monitored daily for any clinical symptoms of disease. Three weeks after injection, mice were euthanized by CO2 and organs were harvested. Half of the harvested tissue sections were frozen on dry ice in microcentrifuge tubes for qPCR analysis, and the other half was fixed in 10% neutral-buffered formalin for histological examination. DNA extraction from the harvested tissues was performed using the All-Prep DNA / RNA Mini Kit (Qiagen) according to the manufacturer's instructions. qPCR reactions were performed on a QuantStudio 7 Flex using in-house developed linearized plasmids (pAAV-GFP for groups 1, 2, and 3, and pAAV-ZsGreen for group 4). Extracted genomic DNA (100 ng) was used as a template, and specific GFP primers and probes (forward 5'-GAACCGCATCGAGCTGAA-'3, reverse 5'-TGCTTGTCGGCCATGATATAG-'3 and probe The primers used were 5' / 56-FAM / ATCGACTTC / ZEN / AAGGAGGACGGCAAC / 3IABkFQ'3, and ZsGreen primers and probes, forward 5'-GTACCACGAGTCCAAGTTCTAC-'3 and reverse 5'-CACGTCGCCCTTCAAGAT-'3, and probe 5' / 56-FAM / CCCGTGATG / ZEN / AAGAAGATGACCGACAA / 3IABkFQ / '3. The cycling conditions were an initial denaturation at 95°C for 3 seconds, 40 cycles of 95°C for 10 seconds, and annealing / extension at 60°C for 20 seconds. For histological examination, liver tissue slices from harvested tissues were prepared using a Leica 3050s microtome (Germany), stained, mounted, and evaluated for GFP detection using a confocal microscope. GFP quantification of liver slides was performed using an axioscanner.

[0054] Engineering rHSV-1 vector producer cell pools Two proprietary suspension cell lines, CHO-K1 and BHK-21, were selected for engineering experiments. For suspension CHO cells, two strategies were used: (1) random integration. Here, the HSV-1 ICP27 ORF (GenBank AB235845.1) sequence was codon-optimized for hamster cell expression by IDT, chemically synthesized, and then subcloned downstream of a CMV promoter in a proprietary plasmid encoding a puromycin cassette for selection. For site-specific integration, exon 1 of the C12Orf35 locus (GenBank XM_027430029) from the CHO genome was selected as one of the transcription hotspots (Zhao et al., Appl Microbiol Biotechnol. 2018 Jul;102(14):6105-6117). The CRISPy bioinformatics tool with default parameters was used to select sgRNA target sequences according to (Ronda et al., 2014). The selected gRNA target (5'GGACTTAACCACTCGATGGC-'3) was synthesized by IDT, delivered as gblocks, annealed, and subcloned into a linearized CRISPR nuclease expression vector (GeneArt CRISPR CD4) backbone to generate the sgRNA expression vector using the GeneArt CRISPR CD4 kit (Invitrogen) according to the manufacturer's instructions. A donor DNA plasmid was constructed using a proprietary plasmid backbone, avoiding any protospacer adjacent motif (PAM) sites identical to the gRNA target. The 5' and 3' homology arms (750 base pairs each) flanking the sgRNA target sequence were chemically synthesized (IDT) and contained 110 nucleotides as gene linkers, with distinct restriction sites between them for cloning. Transfection of the final AAV CHO clone followed the process previously described. The transfected cell pool was selected using 5 μg puromycin / mL for 2 weeks.Clones were generated by single-cell deposition using anti-HU CD270 (HVEM) eBioscience PE clone eBioHVEM-122 in 384-well plates using a BD Influx cell sorter (BD Biosciences) as previously described (Evans et al., 2015). (2) For BHK-21 suspension cells, proprietary, in-house developed serum-free suspension-adapted BHK-21 cells were maintained in Xell HEK TF medium (Xell AG, Germany). Cells were cultured at 0.3 × 10 cells / well. 6 Cells were seeded at a density of 1000 cells / mL in 30 mL of medium in a 125 mL shake flask (Nunc, Denmark) and incubated in a shaking incubator with agitation at 120 rpm for 3 days at 5% CO2. The ICP27 (ORF) from the HSV-1 genome (GenBank KM222723.1) was downloaded, codon-optimized using the online IDT tool (https: / / www.idtdna.com / CodonOpt), synthesized, and delivered into a commercially available plasmid. The codon-optimized ICP27 gene was subcloned into an in-house developed plasmid downstream of either the CMV promoter or the endogenous ICP27 promoter, generating pCLD-CMV-ICP27 and pCLD-EN-ICP27, respectively, using restriction enzyme cloning. The constructed pCLD plasmids contained a puromycin cassette for selection. Another version of the ICP27 (non-codon-optimized) ORF was synthesized by GeneArt (Thermo Fisher, USA) and subcloned into a commercially available expression plasmid downstream of a CMV promoter using BamHI-NotI restriction enzyme cloning to generate a third plasmid (designated pCDNA-ICP27). pCDNA-ICP27 contained a neomycin cassette for selection. The three plasmids were amplified in competent DH5α and purified using maxiprep (Qiagen, USA). The final plasmids were verified by Sanger sequencing (Macrogen, USA). Aliquots of BHK-21 cells (7 × 10 each) were transfected with 1000 kJ / ml of 1000 kcal ... 6) were transfected with linearized plasmids (2.5 μg each) using the Amaxa nucleofection Kit L (Lonza, USA) in a nucleofector II according to the manufacturer's instructions. 48 hours after transfection, the transfected cells were placed under 400 μg / mL Geneticin (Gibco, USA) and / or 10 μg / mL Puromycin (Gibco, USA) selection for 3 weeks.

[0055] rHSV-1 infection in selected CHO-ICP27 clones The final selected clones were tested for rHSV-1 infection. Briefly, an aliquot (1 × 10 6 Viable cells (number of cells) were infected with rHSV-AAV9 at an MOI of 10 in antibiotic-free DMEM, then incubated on ice for 20 minutes, followed by incubation at 37°C for 2 hours. After incubation, excess virus was decanted, and the infected cells were washed twice with sterile PBS to remove residual rHSV-AAV9 vector. After washing, the infected cells were overlaid with in-house developed medium (2 mL / well) in a 6-well plate and incubated at 37°C in a 5% CO2 humidified incubator for 24 hours. After 24 hours, 1 mL of supernatant was collected from the infected cells and inoculated into V27 cells to determine cytopathic effects. Virus recovered from the inoculated V27 cells was purified by ultracentrifugation and subjected to Western blot detection using an anti-HSV-1 glycoprotein D antibody (EMD Millipore Corp). In separate experiments, the final CHO clones and / or V27 cells were infected with rHSV-AAV9 at different MOIs using the same approach as above. The infected cells were then incubated in 6-well plates containing in-house developed medium (for CHO) or DMEM-2% FBS for V27 at 33°C in a 5% CO2 humidified incubator for 2 days. Clarified virus was collected from the lysed culture and titered on V27 cells by plaque assay (Kang et al., 2009. Gene Therapy 16, 229-239).

[0056] Production of rHSV-1 in the BHK-21-ICP27 pool The recovered BHK-ICP27 pool was tested for rHSV-1 production. Briefly, cells were infected with the rHSV-AAV6.2 vector (MOI = 0.15 PFU) by direct inoculation in Xell HEK TF medium supplemented with 4% FBS or under serum-free conditions. Infected cells were incubated at 37°C, 120 rpm, and 5% CO2 in a humidified incubator for 3–4 days. Cell viability and density were measured daily postinfection using a Vi-Cell™. After 3–4 days, infected cultures were subjected to three cycles of freezing and thawing in an isopropanol-dry ice bath, followed by centrifugation at 4500 rpm for 15 minutes at 4°C. After centrifugation, the supernatant was subjected to ultracentrifugation at 10,000 rpm for 75 minutes at 4°C using a JA 20 rotor. The resuspended virus was titrated on V27 cells using a plaque assay according to (Kang et al., 2009).

[0057] statistical analysis One-way ANOVA with the Tukey-Kramer method (post-hoc test) was used to compare GFP expression after rHSV-1 infection in CHO cell pools and to compare rAAV9-GFP titers produced from different cell pools. Two-way ANOVA was used to compare rAAV6.2-GFP titers produced from different clones at two different time points. A two-tailed Mann-Whitney test was used to compare the infectious titers of rAAV6.2-GFP and rAAV9-GFP produced in CHO cells, or rAAV6.2-GFP and rAAV9-ZsGreen produced by triple transient transfection. A Kruskal-Wallis test with a Dunn test for correction was used to compare rAAV6.2-GFP, rAAV9-GFP, and / or rAAV9-ZsGreen titers between collected mouse tissues. GraphPad Prism version 9.1.2 (GraphPad Software Inc.) was used for all tests, and a p value of 0.05 or less was considered significant.

[0058] result Generation of stable CHO pools We successfully constructed eight different vectors using our proprietary plasmid backbone: (1) two vectors encoding either a codon-optimized or non-codon-optimized HVEM ORF flanked by a CMV promoter and SV40 poly(A) tail; (2) two vectors encoding either a codon-optimized or non-codon-optimized Nectin-1 ORF flanked by a CMV promoter and BGH poly(A) tail; (3) two vectors encoding either a codon-optimized or non-codon-optimized HVEM and Nectin-1 ORF flanked by a CMV promoter, SV40 poly(A) tail, and BGH poly(A) tail, respectively; and (4) two vectors encoding either a codon-optimized or non-codon-optimized HVEM and Nectin-1 ORF flanked by a synthetic promoter (Spro), SV40 poly(A) tail, and BGH poly(A) tail, respectively (Figures 1a and 1b). After 10 days of MSX selection, aliquots of each stable cell pool (1 × 10 viable cells / pool) were harvested and tested for HVEM and / or Nectin-1 receptor expression using FACS staining. Pools 1 (CMV-HVEM), 2 (CMV-HVEM-Co), 5 (CMV-HVEM-Nectin-1), 6 (CMV-HVEM-Co-Nectin-1-Co), 7 (Spro-HVEM-Nectin-1), and 8 (Spro-HVEM-Co-Nectin-1-Co) showed 32.35%, 65%, 60.9%, 51%, 60%, and 53.8% HVEM-expressing cells, respectively (Fig. 1c), whereas pool 3 (C The pools containing the CMV-nectin-1, 4 (CMV-nectin-1-Co), 5 (CMV-HVEM-nectin-1), 6 (CMV-HVEM-Co-nectin-1-Co), 7 (Spro-HVEM-nectin-1), and 8 (Spro-HVEM-Co-nectin-1-Co) showed 69.75%, 53.45%, 61.3%, 49.3%, 63.7%, and 53.8% nectin-1-expressing cells, respectively (Figure 1d). Interestingly, duplicate pools such as pools 5, 6, 7, and 8 showed good expression of HVEM and nectin-1, indicating that the CMV and synthetic promoters were equivalent in conferring high levels of expression of HVEM and nectin-1 proteins. Furthermore, no significant differences in HVEM and nectin-1 receptor expression were observed using either the non-codon-optimized and / or codon-optimized protein versions.

[0059] rHSV-1 infection and rAAV9-GFP production in stable CHO pools Using the receptor stably expressed in these CHO pools, it was important to understand which construct provided the best rAAV expression after rHSV-1 infection and subsequent co-infection with the rHSV-1 vector. Each of the stable pools generated above was infected with rHSV-GFP at an MOI of 10, and host CHO cells were infected as a negative control. GFP expression data were collected from all infected stable cell pools (n = 8) every 12 hours post-infection for a total of up to 60 hours. All eight infected cell pools showed significant mean GFP expression starting at 12 hours post-infection (hpi), whereas infected host CHO cells showed the lowest GFP expression (Figure 2a). The highest levels of GFP expression were observed at 12 hpi in pools expressing HVEM alone, with pools 1 and 2 showing the highest GFP expression (p-value < 0.0001 compared to infected host CHO cells), followed by pools 4 and 3 (codon-optimized, p-value < 0.01 compared to infected host CHO cells). Additionally, stable cell pools 1 and 2 showed the highest overall GFP expression, with pool 1 outperforming pool 2 in overall mean GFP expression.

[0060] One day after co-infection, cell supernatants were collected and the rAAV9-GFP vectors were titrated using qPCR. Pool 1 showed an average of 8.2 log 10 Pools 2, 3, 4, 5, 6, 7, and 8 exhibited the highest rAAV9-GFP physical titers in vg / mL, averaging 7.97, 7.76, 7.99, 8.07, 7.68, 7.71, and 7.99 log 10 vg / mL. However, the difference in qPCR titers of the rAAV9-GFP vector produced in pool 1 compared to the titers produced in the other pools was not significant. The rAAV9-GFP titers produced from all pools were significantly higher than the rAAV9-GFP titer obtained from infected host CHO cells (6.84 log 10The mean GFP expression and rAAV9-GFP titer after rHSV-GFP vector infection were significantly higher than those of the control group (p<0.0001) (Fig. 2b). These data indicate that pool 1 exhibited the highest mean GFP expression and the highest rAAV9-GFP titer after rHSV-GFP vector infection, and therefore this pool was selected for single-cell cloning.

[0061] Generation and testing of CHO-HVEM-expressing clones for rHSV-GFP infection Single high- and medium-HVEM-expressing CHO clones (Figure 3) were selected and plated in two 384-well plates using our in-house conditioned medium for two weeks. Deposition of single cells per well was confirmed by imaging using a Cellavista (Evans et al., 2015). After two weeks, 64 clones were recovered, showing high viability (90-95%) and favorable growth profiles. The selected clones were passaged three more times in deep 96-well plates containing our in-house medium supplemented with MSX. After three passages, 24 of the original 64 clones showed good HVEM expression by FACS staining (Figure 4a). These clones were expanded and further tested for rHSV1-GFP vector infection. Average GFP expression was calculated from two time points (24 and 48 hpi). No statistically significant differences in average GFP expression levels were detected between the clones tested (Figure 4b).

[0062] rAAV production in CHO-HVEM-expressing clones Eight clones (designated CHO-HV-C1, CHO-HV-C13, CHO-HV-C15, CHO-HV-C23, CHO-HV-C24, CHO-HV-C46, CHO-HV-C62, and CHO-HV-C64) that exhibited the highest average GFP expression after rHSV-GFP vector infection were selected and evaluated for their ability to produce rAAV by coinfecting them with two rHSV-1 vectors (one containing the AAV2rep and AAV6.2cap genes and the other containing the GFP gene) at an MOI of 1:1. Following infection, the viability of the eight clones rapidly declined over several days, whereas the infected host CHO cells showed only a slight decline (3–7%) over the course of coinfection when incubated at 37°C (Figure 5a). Therefore, the viable cell density (VCD) of all coinfected clones also showed a slight decline (0.3 × 10) in infected host CHO cells. 6 The AAV rep protein appears to have a detrimental effect on the metabolism of infected engineered CHO cells, as indicated by the sharp decline in cell viability and VCD in co-infected cells incubated at 37°C compared to host CHO cells, where the rHSV-1 vector undergoes degradation after cell entry (Figure 5b). Therefore, we sought to minimize this detrimental effect by lowering the incubation temperature.

[0063] Interestingly, clone #1 (designated CHO-HV-C1) produced the highest rAAV6.2-GFP vector titer per mL of cell lysate at 24 hpi (~8.83 log 10 vg / mL, 1 × 10 6 cells), whereas the other clones showed a log 10 vg / mL. However, the difference in rAAV6.2-GFP titers produced in the CHO-HV-C1 clone compared to those produced by the other tested clones was not significant (p=0.89). However, they all showed a 6.24 log increase at 24 hpi. 10The rAAV6.2-GFP titers of all eight co-infected clones decreased slightly at 48 hpi (Fig. 5c).

[0064] Clones CHO-HV-C1 and CHO-HV-C62 produced the highest titers of rAAV6.2-GFP vector. Clone CHO-HV-C1 is a high HVEM-expressing clone, while clone CHO-HV-C62 is a medium HVEM-expressing clone. In subsequent experiments, the final selected clones (CHO-HV-C1 and CHO-HV-C62) were tested for rAAV6.2-GFP vector production using rHSV-AAV6.2 and rHSV-GFP at MOIs of 2:1, 3:1, and 4:1, respectively.

[0065] Interestingly, MOIs of 2:1 and 3:1 significantly reduced the 10 sucrose concentration at 24 hpi compared to that obtained with an MOI of 1:1. 6 There was no significant improvement in the rAAV6.2-GFP titer per cell lysate (data not shown). On the other hand, an MOI of 4:1 significantly improved the physical titer of rAAV6.2-GFP produced in the CHO-HV-C1 and CHO-HV-C62 clones (p=0.0211). Furthermore, the CHO-HV-C1 clone outperformed CHO-HV-C62 in producing rAAV6.2-GFP vectors at an MOI of 4:1, achieving a titer of 10 at 24 hpi. 6 9.89 and 9.37 log per cell lysate, respectively 10 vg / mL (p=0.0261) (FIG. 5d), indicating that the MOI of rHSV-1 has a significant effect on rAAV6.2-GFP in CHO cells.

[0066] Because CHO-HV-C1 was the best-growing clone for rAAV6.2-GFP production, its ability to produce the other AAV serotypes 8 and 9 expressing the GFP transgene was further tested. Using the same optimal infection parameters described in the previous experiment, 1 × 10 cells were obtained for the rAAV8-GFP and rAAV9-GFP vectors, respectively. 6 9.21 and 9.4 log per cell lysate 10 Mean titers of 10 ... 10 The results showed a decrease in titer to 1000 vg / mL (Figure 5e). These data indicate that the above infection parameters work across the serotypes tested. Furthermore, cell lysates yielded the highest physical titers of the AAV serotypes tested at 24 hpi compared to titers at 30 and 48 hpi.

[0067] The cell viability of CHO-HV-C1 cells co-infected with either rAAV8-GFP and / or rAAV9-GFP vectors for production rapidly decreased at 24 hpi, similar to that observed for rAAV6.2-GFP vector production, indicating that the decrease in cell viability was not AAV serotype-specific. Because the rapid decrease in cell viability after co-infection with rHSV-1 vectors may affect the final rAAV titer, we tested the effect of a temperature shift from 37°C to 33°C, which showed a significant improvement in both cell viability and rAAV6.2-GFP titer after co-infection (Figure 6). Therefore, we tested co-infection at an MOI of 4:1 (rHSV-1 AAV6.2:rHSV-1-GFP) at 33°C. There was only a slight decrease in cell viability (5-7%) for coinfected cultures with a slight improvement in rAAV6.2-GFP titer in the lysates, and a ~2-fold increase in titer in the medium at 24 hpi compared to those incubated at 37°C (data not shown). This finding indicates that the incubation temperature after coinfection is an essential factor for either cell viability and / or rAAV production in the CHO platform.

[0068] Analytical characterization of CHO-derived rAAV The entire process of recovering and purifying rAAV vectors takes one day to perform using our in-house developed PEG-chloroform method (Figure 7). Briefly, the infected whole culture is lysed with 0.5% Triton X-100 (v / v) for 1–3 hours with gentle shaking. The Triton-treated culture is then centrifuged at 1200 rpm for 5 minutes, and the supernatant is filtered through a 0.2 μm PES filter. The filtrate is mixed with 1 / 4 volume of 40% PEG 8000 / 5M NaCl on ice for 1 hour, followed by centrifugation at 4500 rpm for 40 minutes at 4°C. The PEG-virus pellet is resuspended in resuspension buffer and subjected to Benzonase (50 U / mL), RNase A (20 μg / mL) treatment at 37°C for 1 hour, mixing the tube every 15 minutes. After Benzonase treatment, the mixture is mixed with chloroform in a 1:1 ratio and centrifuged at 12,000 rpm for 5 minutes. After centrifugation, the chloroform is evaporated under a biological safety cabinet, and the aqueous layer is collected and concentrated. The final purified rAAV is stored at -80°C.

[0069] Western blotting of equal volumes of purified rAAV produced in the CHO-HV-C1 clone using different MOIs of HSV-1 demonstrated comparable expression of VP1, VP2, and VP3 capsid proteins (Figure 8a). Furthermore, examination of purified rAAV6.2-GFP and rAAV9-GFP vectors using mini-TEM revealed 91 and 79.5% complete capsids for rAAV6.2-GFP and rAAV9-GFP, respectively (Figures 8b and 8c). Furthermore, analysis of the VP1:VP2:VP3 molar ratios of these rAAVs was consistent with that reported in the literature (Figure 8d). These data indicate that rAAV vectors produced in CHO cells express capsid proteins well, with a high percentage of AAV complete capsids. However, the high percentage of complete capsids obtained may be related to the purification method used; therefore, it is worthwhile to reexamine the percentage of complete capsids after using other purification methods, such as chromatography.

[0070] Residual infectious HSV-1 in CHO-derived rAAV Purified rAAV6.2-GFP and rAAV9-GFP vectors were transfected into an HSV-1 complementing cell line (V27) at a 10 log transfection rate, along with rHSV-GFP vector (MOI 0.15 PFU / cell) as a positive control. 10 The purified drug substance was tested for any residual infectious HSV-1 vector by inoculating it with 1000 μg / mL of each vector. No cytopathic effect was observed in wells inoculated with either the purified rAAV6.2-GFP or rAAV9-GFP vectors at day 4 postinfection. However, typical cytopathic effects in the form of infected cell rounding and cell sheet detachment began to appear in wells inoculated with the rHSV-GFP vector at day 2 postinfection, with complete cell sheet detachment occurring at day 3 postinfection (Figure 9). These data indicate that our in-house developed purification method is highly efficient in inactivating rHSV-1 vectors, and no residual infectious rHSV-1 was detected in the purified rAAV.

[0071] Infectivity and in vitro transduction of CHO-derived rAAV The infectivity and in vitro transduction efficacy of rAAV produced in CHO cells and purified using PEG-chloroform were tested and compared with that produced using a standard triple transient transfection method and purified by affinity chromatography. For infectivity, GFP expression in infected wells was recorded 5 days post-infection using the default settings of the IncuCyte. The rAAV6.2-GFP vector produced in the CHO-HV-C1 clone (referred to as rAAV6.2-CHO) and the rAAV6.2-GFP produced in HEK293 cells (referred to as rAAV6.2-GFP TTT) had comparable infectious titers of 1.65 x 10, respectively. 7 and 1.1 × 10 7 50% tissue culture infection rate (TCID 50 / mL) (Figure 10a). On the other hand, the rAAV9-GFP vector produced in the CHO-HV-C1 clone (referred to as rAAV9-GFP CHO) showed a 6 × 10 6 TCID 50 / mL, whereas the rAAV9-ZsGreen vector produced in HEK293 cells using triple transient transfection, designated (rAAV9-Zs-Green-TTT), showed 6 × 10 5 TCID 50 / mL (Fig. 10b).

[0072] 2 x 10 of the four aforementioned vector preparations 5 From 1×10 3 Different multiplicities of transfection (MOTs), ranging from 100 to 1000 vg / cell, were tested in Ad293 cells for transduction comparison, and average GFP expression was recorded at day 3 postinfection using the default settings of the IncuCyte. rAAV6.2-GFP-CHO and rAAV6.2-GFP-TTT demonstrated robust transduction at all MOTs tested. Meanwhile, rAAV9-GFP-CHO showed higher transduction efficiency compared to rAAV9-ZsGreen-TTT (Figure 10c), consistent with the observed infectivity data. These data indicate that rAAV6.2-GFP-CHO and rAAV9-GFP-CHO have good in vitro infectivity and transduction activity. Furthermore, the higher infectivity and transduction of rAAV9-GFP-CHO relative to rAAV9-ZsGreen-TTT observed here may be related to differences in the percentage of intact capsids, purification method, and formulation buffer used for each of the different sample preparations.

[0073] Biodistribution of CHO-derived AAV The biodistribution of rAAV6.2-GFP-CHO and rAAV9-GFP-CHO was evaluated in parallel with rAAV6.2-GFP-TTT and rAAV9-Zs-Green-TTT to determine whether in vivo behavior mimicked the in vitro data. Twenty-five 3-week-old mice were divided into five groups (n = 5 per group). Mice were inoculated with either rAAV6.2-GFP-CHO (G1), rAAV9-GFP-CHO (G2), rAAV6.2-GFP-TTT (G3), rAAV9-Zs-Green-TTT (G4), or PBS (G5). All mice were inoculated with 100% PBS according to their grouping. 11 G1 mice were inoculated intravenously into the tail vein with either 100 μg of rAAV or 100 μL of PBS. Three weeks after inoculation, inoculated mice were euthanized, and tissues with high tropism (heart, liver, lung, kidney, and skeletal muscle) were harvested from each inoculated animal (Figure 11a). These tissues were assessed for rAAV titer in homogenized tissues using qPCR (targeting GFP and / or Zs-Green genes) and histopathological examination using confocal microscopy. For qPCR, G1 mice showed lower GFP copy numbers than G3 mice in all harvested tissues except the kidney, but at this time, G1 mice had a higher average GFP copies / mg, ~5.38 × 10 4 AAV genomes / mg DNA, whereas ~3.89 × 10 4 Although the AAV genomes / mg DNA were significantly higher in G1 and G3 livers than in G4, the difference was not statistically significant. Furthermore, the rAAV6.2-GFP copy numbers in G1 and G3 livers were the highest among all inoculated mice compared with the titers from other tissues in both groups. Interestingly, G2 mice showed higher average GFP copies / mg DNA in the heart, lung, kidney, and skeletal muscle than G4 mice. However, G4 livers showed higher average GFP copies / mg DNA than G2 livers, with average titers of 3.79 × 10 6 and 2.11 × 10 6vg / mg DNA. Furthermore, GFP titers from the liver of either G2 and / or G4 showed the highest GFP copies in all inoculated mice in both groups compared with titers from other tissues (Fig. 11c).

[0074] Liver tissue slices were prepared from the five groups and examined for GFP expression using a confocal microscope. As expected from the qPCR data, clear GFP and / or Zs-Green signals were observed in the livers of all inoculated groups except G5 (Figure 11d). Furthermore, quantification of GFP and Zs-Green in liver slides using a slide scanner with a self-developed script showed that liver sections from G3 showed significant biodistribution compared to those from G1, correlating with the qPCR data. Furthermore, liver sections from G4 also showed higher Zs-Green signals than those from G2; however, the difference was not significant. No GFP signal was detected in the mock-infected group (G5) inoculated with sterile PBS. These data indicate that rAAV produced in CHO cells exhibits good in vivo transduction after tail vein injection. Furthermore, the lower GFP levels in liver sections, especially those from G1, may be related to several factors, such as the presence of impurities that cannot be completely removed by the PEG-chloroform method.

[0075] Engineering CHO-HV-C1 cells for rHSV-1 production For random integration, a linearized plasmid developed and constructed in-house containing a synthetic Chinese hamster codon-optimized HSV-1 ICP27 ORF downstream of the CMV promoter and upstream of the SV40 polyA, and a puromycin ORF downstream of the CMV promoter and upstream of the BGH polyA, was used for transfection (Fig. 12a).

[0076] Two plasmids were constructed for site-specific integration. The first contained the codon-optimized ICP27 ORF downstream of a CMV promoter and upstream of an SV40 polyA, followed by a puromycin cassette flanked by an SV40 promoter and SV40 polyA. The total length of the two cassettes was 4.1 kb, flanked by right and left homology arms (750 bp each) (Figure 12b). The second contained a synthetic sgRNA of CHO exon 1 C12orf35. Pools of either random and / or site-specific integrations were recovered after three weeks of double selection using 5 μg / mL puromycin and 50 μM MSX / mL. After three passages in our proprietary medium using double selection, 24 clones, including seven site-edited clones (clones 1–7) and 17 randomly integrated clones, were selected, demonstrating high growth viability and ICP27 expression (Figure 12c). Random integration clone #11 (designated CHO-HV-ICP27-C11), which showed the highest growth profile and ICP27 expression, was selected for further testing for rHSV-1 vector production.

[0077] Infection and production of rHSV-1 vectors in CHO-HV-ICP27-C11 cells The CHO-HV-ICP27-C11 clone was infected with rHSV-AAV9 (MOI = 10) and incubated at 37°C for 2 hours for virus adsorption. After 2 hours, infected cells were washed twice with sterile 1x PBS to remove viral residue. The infected cells were then incubated at 37°C for 24 hours in a 5% CO2 static humidified incubator. On day 2, 1 mL of clarified supernatant from infected cells was passaged onto V27 cells. Two days after infection, cell rounding and detachment of infected cell sheets were observed. Furthermore, expression of HSV-1 glycoprotein D (gD) was observed in V27 cell lysates after infection with rHSV-1 virus propagated in the CHO-HV-ICP27-C11 clone (data not shown). This result indicates that the CHO-HV-ICP27-C11 clone supports productive infection of the rHSV-1 vector.

[0078] Therefore, we compared the productivity of the CHO-HV-ICP27-C11 clone in V27 cells. rHSV-AAV9 was infected into either CHO-HV-ICP27-C11 or V27 cells at different MOIs (0.2, 0.5, and 1 PFU / cell) using serum-free proprietary medium or DMEM supplemented with 2% (v / v) FBS, respectively. The infected cell cultures were incubated at 33°C in a humidified incubator with 5% CO for 4–5 days. rHSV-AAV9 from the infected cell cultures was released by three freeze-thaw cycles and titrated by plaque assay on V27 cells. CHO-HV-ICP27-C11 cells produced significantly lower rHSV-AAV9 titers than V27 cells, with the CHO-HV-ICP27-C11 clone producing 5 × 10 rHSV-AAV9 titers at MOIs of 0.2, 0.5, and 1 PFU / cell on day 2 post-infection. 3 , 4×10 4 , and 3.2 × 10 5 V27 cells produced 1 × 10 PFU / mL, whereas V27 cells produced 1 × 10 PFU / mL, respectively. 6 , 6.75 x 10 6 and 2 x 10 6 PFU / mL (Figure 13).

[0079] Production of HSV-AAV6.2 vectors in the BHK-21-ICP27 pool The three recovered BHK21-ICP27 pools (Figure 14) were expanded and stored. Aliquots of the pools (1 x 10) were cultured in 30 mL shake flasks. 6 Viable cells / mL were tested for HSV-AAV6.2 vector production either in the presence of 4% FBS or under serum-free conditions. The viability of infected cultures slightly decreased from 87% to 85.5% and 84.4% for cultures supplemented with 4% FBS and 0% FBS, respectively, on day 1 post-infection. On day 2 post-infection, cell viability decreased to 71.6% and 65.1%, respectively, for cultures supplemented with 4% FBS and 0% FBS. On day 3 post-infection, cell viability decreased significantly to 45.1% and 44%, respectively, for cultures supplemented with 4% FBS and 0% FBS. Furthermore, viable cell densities averaged 1 × 10 for both infected cultures on each day post-infection.5 cells / mL decreased (data not shown).

[0080] The recovered virus was titrated on V27 cells using a plaque assay. The infected BHK-21-CMV-ICP27 pool supplemented with 4% FBS yielded 3.5 × 10 6 PFU / mL, whereas 1.2 × 10 6 PFU / mL produced by serum-free infected BHK-21-CMV-ICP27 pools (Figure 15).

[0081] We successfully engineered serum-free suspension CHO cell pools expressing either HVEM and / or nectin-1, which are required for rHSV-1 entry and infection. All engineered stable CHO pools showed significant susceptibility to rHSV-1-GFP vector entry and infection, as evidenced by GFP expression, compared with wild-type CHO cells. The CMV-HVEM and CMV-HVEM-CO pools, expressing non-codon-optimized and CHO-codon-optimized HVEM genes, respectively, outperformed all other pools in GFP expression at five different time points post-infection with the rHSV-GFP vector, compared with wild-type infected CHO cells. These data are consistent with a previous report that showed that engineered CHO cells expressing the HVEM receptor became permissive for HSV-1 entry and infection (Montgomery et al., Cell Vol. 87, 427-436).

[0082] Interestingly, pool #1 (designated CMV-HVEM) also outperformed all other stable CHO cell pools in producing high physical titers of rAAV9-GFP vector from cell lysates at 24 hpi, leading to the generation of monoclonal cells from this pool; however, the difference in titer was not significant compared to the other pools. Using a high-throughput method, we selected the top 24 CHO-HVEM-expressing clones that exhibited high growth profiles and HVEM expression. These were narrowed down to eight clones (C#1, C#13, C#15, C#23, C#24, C#62, and C#64) that showed the highest GFP expression after rHSV-nols-AAV-GFP infection at different time points. These final eight clones were further tested for rAAV6.2-GFP vector production, starting with an MOI of 1:1 (rHSV-AAV6.2:rHSV-nols-AAV-GFP) in our proprietary medium at 37°C for 3 days. A rapid decline in both cell viability and viable cell density was observed beginning at 24 hpi. Both cell medium and cell lysates were harvested at 24 hpi and 48 hpi and then tested for rAAV6.2-GFP physical titers using Q-PCR. The highest rAAV6.2-GFP physical titers were detected in cell lysates of all co-infected clones at 24 hpi, after which the titers slightly decreased at 48 hpi. Lower rAAV6.2-GFP physical titers were detected in the medium of all co-infected clones at different time points (data not shown), indicating that the rAAV6.2-GFP vector primarily engages cells in the CHO platform. Furthermore, harvesting at different time points, such as 30 hpi, did not show any improvement in the final rAAV6.2-GFP physical titers compared to those obtained at 24 hpi. These findings differ from other systems that have used AAV-HSV-1-based production, such as HEK293, where peak rAAV production was achieved 52 hours post-coinfection (Kang et al., 2009). Interestingly, the final eight clones tested varied in HVEM expression.For example, clone numbers (C#1, C#23, and C#24) were high HVEM-expressing clones, while clone numbers (C#13, C#15, C#46, C#62, and C#64) were medium HVEM-expressing clones that may have an effect on rAAV production.

[0083] Because clones C#1 (referred to as CHO-HV-C1) and C#62 (referred to as CHO-HV-C62) produced the highest physical titers for the rAAV6.2-GFP vector at 24 hpi, different MOIs were tested for these two clones to improve the final rAAV6.2-GFP titer. MOIs (2:1, 3:1, and 4:1, 6:1, 8:1, and 10:1 from rHSV-AAV6.2 and rHSV-GFP, respectively) were tested. MOIs of 2:1 and 3:1 did not produce significantly improved titers compared to an MOI of 1:1. Interestingly, an MOI of 4:1 yielded ~×10 rAAV6.2-GFP from clones CHO-HV-C1 (high HVEM expression) and CHO-HV-C62 (intermediate HVEM expression), respectively. 10 vg / mL(10 13 vg / L) and ~10 9.37 vg / mL(10 12.37 vg / L), indicating a positive correlation between HVEM expression and rAAV production. Interestingly, no significant increase was detected in the titer of rAAV6.2-GFP produced using an MOI of 6:1, 8:1, and 10:1 compared to an MOI of 4:1 (data not shown). Therefore, CHO-HV-C1 was further selected to test the production of other AAV serotypes, such as AAV8 and AAV9, using different MOIs, such as 1:1, 2:1, 3:1, and 4:1. MOIs of 1:1, 2:1, and 3:1 resulted in a ~10 8 vg / mL(10 11 vg / L), whereas an MOI of 4:1 resulted in a final titer of 10 for rAVV8-GFP and rAAV9-GFP, respectively. 12.21 and 10 12.40The titers improved to 1000 vg / L. Interestingly, good rAAV8 and rAAV9 titers were detected in the cell culture medium at 24 hpi using an MOI of 4:1 (data not shown), indicating that rAAV8-GFP and rAAV9-GFP are not cell-associated like rAAV6.2-GFP in the CHO platform. Therefore, for the CHO platform, only a low MOI is required for the production of rAAV6.2-GFP, rAAV8-GFP, and rAAV9-GFP, in contrast to other studies that reported a high MOI, such as 12:2, as the optimal MOI for rAAV production (Kang et al., 2009).

[0084] The cell viability of CHO-HV-C1 co-infected cells for both rAAV8-GFP and rAAV9-GFP vector production rapidly decreased at 24 hpi, similar to that for rAAV6.2-GFP vector production, indicating that this decrease was not AAV serotype-specific. Because the rapid decrease in cell viability after rHSV-1 vector co-infection might affect the final rAAV titer, we tested the effect of a temperature shift from 37°C to 33°C, which showed significant improvements in both cell viability and rAAV6.2-GFP titer after co-infection (data not shown). It has been reported that HSV-1 vector stability is 2.5-fold higher at 33°C than at 37°C, and that synchronous HSV-1 infections incubated at 33°C produced twofold higher amounts of vector than those incubated at 37°C (Wechuck et al., 2002). Therefore, we tested coinfection at an MOI of 4:1 (rHSV-1 AAV6.2:rHSV-1-nols-AAV-GFP, respectively) at 33°C with shaking at 120 rpm. Interestingly, at 33°C, a slight decrease (5-7%) in cell viability of coinfected cultures was found, along with a slight improvement in the physical titer of rAAV6.2-GFP in the lysates, and the rAAV6.2-GFP titer in the medium produced at 24 hpi was increased by ~2-fold compared to that incubated at 37°C (data not shown). This finding indicates that shifting the temperature to 33°C after coinfection enhances cell viability and final rAAV6.2-GFP production in the CHO platform.

[0085] Using our in-house developed PEG-chloroform purification method followed by Amicon concentration, we generated purified rAAV6.2-GFP and rAAV9-GFP vectors that demonstrated high potency in both in vitro and in vivo studies. This purification method is simple, inexpensive, and rapid, particularly for producing rAAV with titers suitable for preclinical studies, and is comparable to the more time-consuming iodixanol ultracentrifugation method, consistent with other studies (Wu et al., 2001. Chin. Sci. Bull. 46, 485-488; Negrini et al., Curr Protoc Neurosci. 2020 Sep;93(1):e103). However, impurities were discovered within the purified samples during CE-SDS testing. These impurities were confirmed by silver staining of the same samples (Figure 16), indicating that PEG-chloroform is ideal for preparing rAAV samples for in vitro testing but not for in vivo or clinical trials. Our observations are consistent with a recent study (Kimura et al., Sci Rep. 2019 Sep 19;9(1):13601).

[0086] Analysis of the capsid protein ratios (VP1:VP2:VP3) of different rAAVs, such as rAAV6.2-GFP and rAAV9-GFP, produced in CHO-HV-C1 cells using our in-house developed CE-SDS method showed that the CHO-derived rAAVs had highly comparable VP1:VP2:VP3 capsid ratios compared to the AAV6.2 positive control produced by a triple transient transfection system. This indicates that the rAAVs produced in CHO-HV-C1 cells are fully packaged, compared to other platforms that report the need for genetic manipulation to enhance VP expression for some AAV serotypes produced in insect cells using the baculovirus system. For example, early attempts to adapt AAV-5 for production in insect cells using a baculovirus system showed low levels of VP1 incorporation into capsids (Urabe et al., J Virol. 2006;80:1874-85; Mietzsch et al., Hum Gene Ther. 2014;25:212-22). Interestingly, examination of full / empty capsids for rAAV produced in the CHO-HV-C1 clone showed a higher percentage of full capsids than previously reported (Small et al., Mol Ther Methods Clin Dev. 2016 May 11;3:16031). Furthermore, when tested in vitro, rAAV derived from CHO cells showed higher infectious titers compared to those produced by triple transient transfection. Furthermore, CHO-derived rAAV showed highly comparable biodistribution in mice compared to that produced by triple transient transfection, especially for rAAV9-GFP.

[0087] To address the second challenge in scaling up rHSV1 vector stocks, we reengineered CHO-HV-C1 cells to express the rHSV-1 Chinese hamster codon-optimized ICP27 protein using both random integration and / or CRISPR / Cas9 technology. Using proprietary media, the final selected clone (designated CHO-HV1-ICP27-C11) demonstrated productive infection with rHSV-1, as indicated by viral replication propagated on the CHO-HV1-ICP27-C11 clone in V27 cells. However, the rHSV-1 vector production capacity of CHO-HV1-ICP27-C11 was lower than that of V27 cells. Expression of late HSV-1 viral proteins in infected CHO-HV1-ICP27 C11 cells appeared to be negligible or below normal levels compared with expression of early and mid-stage viral genes (data not shown). Furthermore, CHO cells may not provide the elements necessary for optimal expression of many HSV genes, especially the late genes, because CHO cells may express several inhibitory factors that interfere with or block HSV-1 late viral gene expression, consistent with our results (Shieh et al., J Cell Biol. 1992 Mar;116(5):1273-81). Other factors may be related to the proprietary medium used, which may have some inhibitory effect on rHSV-1 vector production. Therefore, we engineered our own serum-free adapted BHK-21 cells to express the HSV-1 ICP27 protein for the production of rHSV-1 vectors. Interestingly, the stably transfected BHK-21-ICP27 pools produced comparable rHSV-1 titers in Xell medium in the presence or absence of FBS.

[0088] Thus, the present disclosure provides an rAAV-based HSV production platform in engineered CHO cells that provides a scalable, serum-free manufacturing platform that will facilitate the production of future rAAV-based biotherapeutics in a low-cost manner.

Claims

1. Baby hamster kidney cells adapted to grow in serum-free conditions and stably expressing a hamster codon-optimized herpes simplex virus 1 (HSV-1) ICP27 open reading frame containing a deletion of a non-essential element of infected cell protein 27 (ICP27).

2. The cells of claim 1 , wherein the cells are grown in suspension.

3. The cell of claim 1 or 2, wherein the non-essential elements of ICP27 are the 5' and 3' untranslated regions (UTRs).

4. A cell line comprising the cells according to any one of claims 1 to 3.

5. 1. A method for producing a recombinant adeno-associated virus (rAAV) vector, comprising:

10. A method comprising introducing a recombinant herpesvirus (rHSV) vector comprising AAV rep and cap sequences and a sequence encoding a gene of interest into a cell of any one of claims 1 to 3 or a cell line of claim 4, and culturing the cell or cell line under conditions for producing the rAAV vector.

6. Chinese hamster ovary (CHO) cells adapted to grow in serum-free conditions, stably expressing one or more polypeptides required for herpes simplex virus-1 (HSV-1) entry and infection.

7. The CHO cell according to claim 5, wherein the CHO cell stably expresses herpesvirus entry mediator (HVEM) and / or nectin-1.

8. 7. The CHO cell of claim 6, wherein the HVEM and / or Nectin-1 sequences are codon-optimized for expression in the CHO cell.

9. A cell line comprising the CHO cell of claim 5 or 6.

10. 1. A method for producing a recombinant adeno-associated virus (rAAV) vector, comprising:

10. A method comprising introducing a recombinant herpesvirus (rHSV) vector comprising AAV rep and cap sequences and a sequence encoding a gene of interest (GOI) into a cell of any one of claims 5 to 7 or a cell line of claim 8, and culturing the cell or cell line under conditions to produce the rAAV vector.

11. The method of claim 5 or 9, wherein the rHSV vector is introduced at a multiplicity of infection of rHSV-rep / cap:rHSV-GOI of about 4:1, 6:1, 8:1, or 10:

1.

12. 11. The method of any one of claims 5, 9, or 10, wherein the AAV serotype is AAV6, AAV8, or AAV9.

13. The method of claim 5 or 9, wherein the gene of interest encodes any therapeutic biological compound.

14. 13. The method of claim 12, wherein the biological compound is an antibody or a chimeric antigen receptor.

Images