Ultrasound Cavitation for Facilitated Cardiac Transduction of AAV - SUMMARY There is intense interest in gene therapy for the treatment of a wide variety of inherited and acquired cardiovascular diseases. Yet, progress in this field has been limited because of the low efficiency in transfection for naked DNA vectors such as plasmids, and the adverse effects associated with more efficient viral vectors. A promising solution to these problems has been the development of bioengineered adeno-associated virus (AAV) vectors, which are members of the parvovirus family that are maintained as nuclear episomes. Despite the success of AAVs in preclinical studies, early clinical trials indicate that the high doses of AAVs required for therapeutically-sufficient myocyte transduction are associated with dose-related serious and sometimes fatal immune reactions. This dose-related safety issue could be overcome by increasing the efficiency of cardiomyocyte-specific AAV transduction. Our laboratories originally pioneered the use of ultrasound (US) inertial cavitation of cationic microbubble (MB) vectors to augment delivery of plasmid DNA, which is confined predominately to perivascular cells. More recently, our pilot experiments indicate that MB cavitation with US within the diagnostic frequency and power range can markedly augment transduction of AAV9, which has myocyte tropism, and that transduction occurs almost exclusively in cardiomyocytes. Importantly, enhanced transduction of AAV does not require coupling of the vector to the MB surface. The overall goal of this proposal is to better understand the mechanisms and ideal conditions for cavitation-facilitated AAV transduction (CFAT) in preparation for translation into humans. In Aim 1, murine studies will be used to evaluate the dose-related efficacy, safety, and mechanisms responsible for CFAT using recombinant AAV9 optical reporter systems. In this Aim, we will also optimize the acoustic condition focusing on key US variables such as acoustic pressure, pulse duration, and line density. Mechanisms for enhanced transduction with CFAT will be studied with a focus on cell responses that occur with inertial cavitation of MBs that are likely to increase AAV transport from the blood pool including glycocalyx modification, caveolin-mediated transcytosis, altered cell junction permeability, and sialidasae activity. In Aim 2, we will perform proof-of-concept studies demonstrating enhanced therapeutic effect and safety of CFAT compared to conventional AAV9 therapy in a murine cardiomyopathy model. For these studies, we will use mice with a known human mutation in the myosin binding protein-C3 gene that produces a haploinsufficiency model of cardiac hypertrophy and reduced systolic function. As a key translational step, in Aim 3 we will evaluate efficacy and safety of CFAT in non-human primates using an AAV9 encoding a Na/I symporter to allow spatial and temporal assessment of transduction efficiency with positron emission tomography imaging and 18F-tetrafluoroborate. Completion of these Aims will form the basis for testing CFAT in patients with cardiovascular disease where AAV gene therapy bears great promise, but trials have been delayed based on concerns regarding therapeutic index.
