Adeno-associated virus (AAV) vectors have been successfully used in phase I clinical trials in patients with hemophilia B. However, one of the obstacles encountered in clinica trial for hemophilia gene therapy is humoral immunity to AAV, a consequence of the fact that AAV vectors have been engineered from a wild-type AAV naturally infecting humans. Several approaches have been considered to design neutralizing antibody (Nab)-evading AAV vectors, including chemical modification, rational design and combinatorial mutagenesis of the capsid as well as biological depletion of Nab titer. These approaches have only been tested on cell lines or in animal models, it has been demonstrated that the result from mouse experiments does not represent that from big animals such as primates and dogs, so the data for AAV Nab escape mutants generated in mice tissues may not always translated into that in human. Further, the dearth of information on AAV transduction in human liver hinders the application of AAV vector in hemophilia gene therapy. Recently mouse model xenografted with human hepatocytes has been used to develop AAV vector for human liver targeting gene therapy. However, it is also unknown whether the result generated from this model will be directly translated into human clinical trial. To address these outstanding concerns (development of human liver tropic AAV with Nab escape activity), we will establish a mouse xenografted model with canine hepatocytes and examine the transduction efficiency of AAV serotypes in canine hepatocytes in both canine hepatocyte xenografted mice and normal dogs to validate the model feasibility (Aim1a). Next we will apply the AAV directed evolution strategy to select AAV variants which can evade AAV Nab generated in dogs and have canine liver tropism (Aim 1b and 1c). Next we will use these canine Nab evasion mutants to deliver optimized canine FIX into hemophilia B dogs pre-immunized with AAV and to examine the correction of hemophilia phenotype (Aim 2). Finally, we will utilize human IVIG as the source of Nabs to develop clinical human liver-tropic AAV mutants with the capacity of evade AAV Nabs in mouse model xenografted with human hepatocytes (Aim 3).
Approximately half of the human population have antibodies in their blood that block adeno- associated virus (AAV) infection. For systemic delivery of AAV for hemophilia gene therapy, it is necessary to develop AAV variants that can escape pre-existing antibodies elicited by the natural AAVs. Recently we have successfully established a mouse model xenografted with human liver cells. In this proposal, we will use the valuable mouse model to develop AAV mutants capable of antibody escape and with human liver tropism. It is thought that these novel escape mutants will overcome the primary hurdles currently facing AAV gene therapy for the treatment of hemophilia in the clinic.
