92. Evaluation of Producer Cell Line Platform for Production of Oversized AAV-FVIII Vectors

Recombinant adeno-associated virus (rAAV) vectors are being evaluated as gene delivery vehicles in several clinical trials. The 4.7 kb wild-type (WT) size genome of AAV presents a challenge for incorporating larger transgenes with incomplete vector genome (vg) packaging being a frequent outcome. To test the feasibility of producing oversized rAAV production using the producer cell line (PCL) method, we generated slightly oversized rAAV vectors (harboring 5.1 or 5.4 kb sized vgs) containing a liver-restricted promoter (mTTR) and a codon-optimized cDNA encoding human B-domain deleted FVIII (FVIIIco). Genomes were packaged into the AAVrh8R serotype vector using the PCL process and compared to matched vectors generated via the triple transfection (TXN) method. Vectors were then characterized for production yields, integrity of packaged genomes and homogeneity. The data showed that the PCL platform was able to produce oversized AAV vectors at levels that were 10- to 100-fold higher than the TXN process with yields greater than 100,000 vg/cell. The PCLs were stable with consistent production maintained up to 60 passages. Southern and dot blot analyses of the packaged genomes demonstrated encapsidation of genomes larger than 4.7 kb in the PCL generated vector while the majority of genomes packaged via the TXN method were 4.7 kb in size. Furthermore, the PCL process generated more vector DNA-containing particles and less packaging of non-vector DNA. Testing the PCL generated vectors in the hemophilia A knock-out (KO) mouse model showed a 2-fold higher plasma FVIII activity (Coatest) and vg copies in the livers than obtained with the vectors made by the TXN process. In summary, the PCL production process generated higher yields of oversized rAAV/FVIIIco vectors as well as higher quality vectors than the TXN method. Hence, the PCL platform may be used for producing greater quality oversized rAAV vectors at levels that can meet the needs for clinical studies.