Department of Bioengineering, McGill University, Montreal, Canada.
Department of Chemical Engineering, Polytechnique Montréal, Montreal, Canada.
Biotechnol J. 2023 Sep;18(9):e2300051. doi: 10.1002/biot.202300051. Epub 2023 Jul 2.
In recent years, the use of adeno-associated viruses (AAVs) as vectors for gene and cell therapy has increased, leading to a rise in the amount of AAV vectors required during pre-clinical and clinical trials. AAV serotype 6 (AAV6) has been found to be efficient in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 10 viral genomes (VG), making large-scale production of AAV6 necessary. Suspension cell-based platforms are currently limited to low cell density productions due to the widely reported cell density effect (CDE), which results in diminished production at high cell densities and decreased cell-specific productivity. This limitation hinders the potential of the suspension cell-based production process to increase yields. In this study, we investigated the improvement of the production of AAV6 at higher cell densities by transiently transfecting HEK293SF cells. The results showed that when the plasmid DNA was provided on a cell basis, the production could be carried out at medium cell density (MCD, 4 × 10 cells mL ) resulting in titers above 10 VG mL . No detrimental effects on cell-specific virus yield or cell-specific functional titer were observed at MCD production. Furthermore, while medium supplementation alleviated the CDE in terms of VG/cell at high cell density (HCD, 10 × 10 cells mL ) productions, the cell-specific functional titer was not maintained, and further studies are necessary to understand the observed limitations for AAV production in HCD processes. The MCD production method reported here lays the foundation for large-scale process operations, potentially solving the current vector shortage in AAV manufacturing.
近年来，腺相关病毒（AAV）作为基因和细胞治疗的载体得到了广泛应用，这导致在临床前和临床试验中所需的 AAV 载体数量增加。已经发现 AAV 血清型 6（AAV6）在转导不同细胞类型方面非常有效，并已成功用于基因和细胞治疗方案。然而，为了有效地将转基因递送到单个细胞中，需要估计需要 10 个病毒基因组（VG）的载体，这使得大规模生产 AAV6 成为必要。基于悬浮细胞的平台由于广泛报道的细胞密度效应（CDE），目前仅限于低细胞密度生产，这会导致在高细胞密度下生产减少和细胞特异性生产力下降。这种限制阻碍了悬浮细胞生产工艺提高产量的潜力。在这项研究中，我们通过瞬时转染 HEK293SF 细胞来研究提高 AAV6 在更高细胞密度下的生产。结果表明，当以细胞为基础提供质粒 DNA 时，可以在中等细胞密度（MCD，4×10 细胞 mL ）下进行生产，导致滴度超过 10 VG mL 。在 MCD 生产中，没有观察到对细胞特异性病毒产量或细胞特异性功能滴度的有害影响。此外，虽然培养基补充在高细胞密度（HCD，10×10 细胞 mL ）生产中在 VG/细胞方面缓解了 CDE，但细胞特异性功能滴度无法维持，需要进一步研究以了解在 HCD 过程中观察到的 AAV 生产限制。这里报道的 MCD 生产方法为大规模工艺操作奠定了基础，可能解决 AAV 制造中的当前载体短缺问题。
