Capsid- and genome-modified AAV3 vectors for hemophilia gene therapy.

PROJECT SUMMARY/ABSTRACT: Remarkable progress has been made in gene therapy of hemophilia B as well as hemophilia A using recombinant adeno-associated virus (AAV) vectors. The United States Food and Drug Administration (USFDA) granted approval Hemgenix for gene therapy of hemophilia B in November 2022, and Roctavian for gene therapy of hemophilia A in June, 2023. Both Hemgenix and Roctavian are based on recombinant AAV5 serotype vectors. However, AAV5 vectors are less than optimal since they do not transduce human hepatocytes efficiently. Thus, extremely high vector doses are needed to achieve clinical efficacy, which necessitates the use of prophylactic immune-suppression. Of the 10 most commonly used AAV serotype vectors, we have identified AAV3 as the most efficient in transducing primary human hepatocytes. In addition to capsid-modified AAV3 vectors that are ~10-15-fold more efficient than their wild-type counterpart, we have also generated genome-modified AAV vectors in which we have engineered the AAV inverted terminal repeats (ITRs) with which up to ~22-fold improved transgene expression can be achieved. In the current application, we wish to combine both capsid- and genome-modifications to develop optimized AAV3 vectors to pursue the following three Specific Aims: Specific Aim 1: To evaluate the underlying molecular mechanism of enhanced transgene expression from ITR- engineered AAV vectors. Specific Aim 2: To evaluate the efficacy of ITR-engineered AAV3 vectors in primary human hepatocytes using humanized mice in vivo. Specific Aim 3: To evaluate the efficacy of ITR-engineered AAV8 vectors for phenotypic correction of murine hemophilia A and B, and evaluate the immunogenicity of these vectors. The capsid+genome-modified optimized AAV vectors are likely to be more efficacious at lower doses, thus being less immunogenic, thereby further increasing the safety as well as reducing vector production costs, ensuring translation to the clinic with higher probability of success for gene therapy of human liver diseases in general, and hemophilia B and hemophilia A in particular.