Lakhasly

AAV6 Vector Design, Production, and Purification

Adeno-associated virus serotype 6 (AAV6) vectors were constructed using the pAAV-MCS plasmid, which contains inverted terminal repeats (ITRs) from AAV2.Additionally, all AAV6 vectors underwent ddPCR for precise quantification of vector genomes.Additionally, a tracking vector for targeted hematopoietic stem/progenitor cells (HSPCs) post-transplantation was developed, featuring a bi-cistronic Nluc cDNA-T2A-TurboGFP cassette driven by the SFFV promoter, with 400 bp homology arms to HBB.The gcHBB-SCD vector was assembled with Gibson Assembly Mastermix and features 2.4 kb of homologous sequence to HBB, including specific SNP variations.In-process testing included silver stain SDS-PAGE analysis, bioburden assessment, and viral genomic titer determination via qPCR.The cells were expanded to a final volume of 3L in spinner flasks and co-transfected with the plasmids using a polyethylenimine (PEI) transfection method.

AAV6 Vector Design, Production, and Purification

Adeno-associated virus serotype 6 (AAV6) vectors were constructed using the pAAV-MCS plasmid, which contains inverted terminal repeats (ITRs) from AAV2. The gcHBB-SCD vector was assembled with Gibson Assembly Mastermix and features 2.4 kb of homologous sequence to HBB, including specific SNP variations. Additionally, a tracking vector for targeted hematopoietic stem/progenitor cells (HSPCs) post-transplantation was developed, featuring a bi-cistronic Nluc cDNA-T2A-TurboGFP cassette driven by the SFFV promoter, with 400 bp homology arms to HBB. AAV6 vectors were produced according to established protocols and stored at −80 °C with 0.001% F-68 pluronic acid. Purified gcHBB-SCD AAV6 vectors were also obtained from Vigene Biosciences.

For mid-scale production, HEK293T cells were cultured in suspension using Freestyle F17 media supplemented with 5 mM GlutaMAX and 0.2% (w/v) Pluronic F68. The cells were expanded to a final volume of 3L in spinner flasks and co-transfected with the plasmids using a polyethylenimine (PEI) transfection method. Two days post-transfection, cells were harvested by centrifugation, resuspended in Dulbecco’s phosphate-buffered saline (DPBS), and stored at −80 ± 20 °C. Total yields of gcSCD-AAV6 were quantified using qPCR, pooling four 3L replicates before lysis. Chemical lysis was performed with Triton™ X-100, followed by clarification through depth filtration.

Purification involved affinity chromatography with AVB Sepharose resin, followed by ultrafiltration using tangential flow filtration. The final product was formulated in DPBS with 0.001% Pluronic F68 and filtered through a sterilizing-grade filter. In-process testing included silver stain SDS-PAGE analysis, bioburden assessment, and viral genomic titer determination via qPCR. The final gcSCD-AAV6 preparations achieved an average titer of 8 × 10^11 vg/mL and were stored at −80 ± 20 °C until needed. Additionally, all AAV6 vectors underwent ddPCR for precise quantification of vector genomes.