682. Applied In Silico Engineering of Factor VIII-AAV Transgene Cassettes

Efforts to develop a clinical adeno-associated virus (AAV) vector product for hemophilia A have been hampered by many factors including low-level factor VIII (FVIII) expression, inefficient vector manufacture, and dose limiting vector toxicity. We recently described pre-clinical testing of a 5.86kb AAV2/8 vector encoding a bioengineered FVIII transgene (ET3) that confers enhanced FVIII secretion efficiency. FVIII-deficient (hemophilia A) mice treated at vector doses ranging from 5e11-2e13 vp/kg were corrected of their FVIII deficiency and bleeding phenotype. However due its oversized genome, the vector suffered from low titer manufacture and substantial inter-particle heterogeneity. Although the packaging capacity of AAV vectors is debated, vector genome sizes 4.7-5.0kb are manufactured to higher yield and consistency than those exceeding 5.0kb. The B-domain deleted FVIII coding sequence is 4.4kb, and with the addition of necessary viral and regulatory control elements, FVIII-AAV genomes exceed the packaging capacity of the virus by 10-25%. We therefore sought to engineer a FVIII-AAV genome <5.0kb in length allowing for both enhanced FVIII expression and efficient packaging. Although ET3 already exhibits enhanced secretion over human FVIII due to the incorporation of specific non-native amino acids, others have shown synonymous codon optimization to benefit hepatic FVIII-AAV expression as well. Therefore, we examined the codon usage of 43 highly expressed, liver-specific genes and used the resulting information to generate a novel codon usage table. Using this table, we synthesized codon-optimized versions of both BDD human FVIII and ET3, the former of which demonstrated 3-fold superior expression in vitro. Likewise, this novel codon optimization algorithm provided the ET3-AAV transgene cassette with a 4-fold in vitro and 14-fold in vivo enhancement of expression. However, the large size of the codon optimized ET3-AAV genome remained incompatible with efficient viral vector packaging. To address this design limitation, we used a combinatorial transcription factor binding site assembly approach to create a panel of liver-specific promoters ranging in size from 62-172 bases. These promoters represent a 30-90% size reduction over currently utilized liver specific promoters such as HLP and HCR-hAAT, which range in size from 250 to over 700 bases. Critically, a small subset of these promoters drive comparable transgene expression levels and specificity to that observed with HLP and HCR-hAAT. As a last design change, we eliminated nonessential viral genomic DNA and cloning remnants that are present in many earlier AAV designs. This removed an additional 80 bases. Collectively, these approaches enabled the development of a high expression FVIII-AAV genome of only 4.8kb in length. The exceptionally small size of this FVIII transgene may allow for the addition of desirable regulatory control features such as efficient introns or scaffold matrix attachment regions that could provide additional benefit. Furthermore, these technologies should be generalizable and likely enabling to other liver-directed AAV gene therapies.