NaCl and KCl mediate log increase in AAV vector particles and infectious titers in a specific/timely manner with the HSV platform

Abstract

The increasing demand for adeno-associated virus (AAV) vectors, a result from the surging interest for their potential to cure human genetic diseases by gene transfer, tumbled on low-performing production systems. Innovative improvements to increase both yield and quality of the vector produced have become a priority undertaking in the field. In a previous study, we showed that adding a specific concentration of sodium chloride (NaCl) to the production medium resulted in a dramatic increase of AAV vector particle and infectious titers when using the herpes simplex virus (HSV) production system, both in adherent or suspension platforms. In this work, we studied additional salts and their impact on AAV vector production. We found that potassium chloride (KCl), or a combination of KCl and NaCl, resulted in the highest increase in AAV vector production. We determined that the salt-mediated effect was the most impactful when the salt was present between 8 and approximately 16 h post-infection, with the highest rate increase occurring within the first 24 h of the production cycle. We showed that the AAV vector yield increase did not result from an increase in cell growth, size, or viability. Furthermore, we demonstrated that the impact on AAV vector production was specifically mediated by NaCl and KCl independently of their impact on the osmolality of the production media. Our findings convincingly showed that NaCl and KCl were uniquely efficacious to promote up to a 10-fold increase in the production of highly infectious AAV vectors when produced in the presence of HSV. We think that this study will provide unique and important new insights in AAV biology toward the establishment of more successful production protocols.

Comparison of highly pure rAAV9 vector stocks produced in suspension by PEI transfection or HSV infection reveals striking quantitative and qualitative differences

“…In earlier works, we and others have shown that the HSV platform generated rAAV stocks of similar or higher infectivity than transient transfection. [21][22][23]36,37 In the current work, the infectious titers reported for the PEI method were similar or higher than the ones found with the HSV method, which was a great improvement from our previous calcium phosphate transfection data. This suggest that perhaps the suspension format, combined with a different transfection reagent, is more favorable to rAAV production and potency.…”

“…Previously, we had reported side-byside data comparing the calcium phosphate transfection in adherent HEK293 21 and the HSV production system in adherent or suspension-adapted HEK293. 22,23 From both studies, the HSV platform in suspension was found superior and resulted in higher yields. Paulk et al 28 recently presented a side-by-side comparison of the PEI-transfection in HEK293 platform with the Baculovirus expression system (BEV) in insect cells, and demonstrated significant differences in the post-translational modifications of the AAV capsid, the percentage of full capsids, and the virus potency.…”

“…Cells were co-infected at a MOI of 2:4 with rHSV-GFP and rHSV-AAV9, respectively, in the presence of 60 mM NaCl (run # 3) or 40 mM KCl supplementation (run # 1 and 2). 23 Cells were harvested after 2 (HSV infection) or 3 (PEI transfection) days incubation at 37 C, 5% CO 2 , 75% RH, and 100-125 rpm. The 3 L harvest was transferred to 6 Â 500 mL Conical tubes (Corning) and cell were spun down at 2,000 rpm, 4 C, for 20 min (Sorvall ST-40R, Thermo Fisher Scientific), rinsed in 500 mL 1Â DPBS (HyClone, Cat # SH3002802), combined into one or two tubes and spun down again.…”

“…2,3 In our previous work, we demonstrated the superiority of the HSV method when used in suspension in HEK293 cells with respect to rAAV titers and quality. [21][22][23] In the current work, we developed a PEI-based production method for rAAV in a suspension-based format using the same commercial Expi293F cell line used successfully with our HSV platform. The current study presents a detailed side-by-side comparison of a PEI transfection and an HSV infection platform in suspension.…”

Increased recombinant adeno‐associated virus production by HEK293 cells using small molecule chemical additives

“…[5][6][7] Small molecule enhancers of recombinant protein production have demonstrated efficacy in a wide variety of mammalian cell lines [3,8,9] and the addition of chemicals as diverse as sodium chloride, sodium butyrate (NaBu) and soy peptones have been shown in previous studies to improve rAAV production yields. [9][10][11] Targeting of discrete pathways involved in the replication, packaging and trafficking of viral particles by bioactive small molecule cell culture additives offers a simple and cost-effective way of increasing viral titre and reducing overall production costs.…”