AAV hFIX(REGENXBIO, Inc.)

Adeno-associated virus (AAV) vectors are increasingly popular in gene therapy because they are unassociated with human disease, replication dependent, and less immunogenic than other viral vectors and can infect a variety of cell types. These vectors have been used in over 130 clinical trials, and one AAV product has been approved for treatment of lipoprotein lipase deficiency in Europe. To meet the demand for the increasing quantities of AAV required for clinical trials and treatment, a scalable high-capacity technology is required. Bioreactors meet these requirements but limited options are available for adherent HEK 293T/17 cells. Here we optimize the transient transfection of HEK293T/17 cells for the production of AAV human factor IX in a disposable fixed-bed bioreactor, the iCELLis(®) Nano (PALL Corporation). A fixed bed in the center of the iCELLis bioreactor is surrounded by culture medium that is pumped through the bed from the bottom of the bioreactor so that a thin film of the medium overflows the bed and is replenished with oxygen and depleted of CO2 as it returns to the surrounding medium reservoir. We show that this fixed-bed bioreactor can support as many as 2.5 × 10(8) cells/ml of fixed bed (1.9 × 10(6) cells/cm(2)). By optimizing culture and transfection parameters such as the concentration of DNA for transfection, day of harvest, size of PEI/DNA particles, and transfection medium, and adding an additional medium change to the process, we increased our yield to as high as 9.0 × 10(14) viral particles per square meter of fixed bed. We also show an average GFP transfection of 97% of cells throughout the fixed bed. These yields make the iCELLis a promising scalable technology for the clinical production of AAV gene therapy products.

### Study Hypothesis The initial hope that gene therapies might treat many diseases has not been largely realized, in part, owing to limitations of delivery mechanisms, adverse effects of therapies, and failure to produce results superior to standards of care. Hemophilia B, a known X-linked deficiency of the factor IX gene (FIX), results from low FIX levels and leads to increased mortality and substantial morbidity, including crippling chronic joint disease, requiring frequent and expensive prophylactic or acute protein therapy. This study hypothesized that the use of a single injection of a modified adenovirus-associated virus (AAV) vector expressing FIX could establish long-term therapeutically relevant levels of this blood protein in men affected with hemophilia B. ### How Was the Hypothesis Tested? AAV vectors allow for the delivery of transgenes and may have superior profiles to other targeting strategies, particularly, owing to muted immune responses, though it can still be challenging to obtain therapeutic doses without triggering host immune responses. The authors previously designed a FIX AAV vector (scAAV2/8-LP1-hFIXco) with several features, including a liver-specific promoter, designed self-complementary in a tail-to-tail dimer, to increase transcriptional activation …