Enhanced gene delivery for CNS and sensory disorders

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PROJECT SUMMARY/ABSTRACT. Gene therapy using adeno-associated virus (AAV) vectors has shown great promise in human clinical trials for diseases such as blindness and hemophilia. However, gene therapy is in its early phases to address inherited hearing loss (IHL). Sensory cells called “hair cells”, reside in the inner ear and mediate hearing and balance, and represent a major target for gene therapy to correct IHL. AAV vectors are currently the most promising vector for gene therapy to the inner ear, although gene delivery efficiency to hair cells is suboptimal. The major long- term goal of this proposal is to develop an effective gene delivery platform for therapy of hearing and balance disorders. We have developed a hybrid gene delivery vector comprised of AAV vectors associated with endogenous nanoparticles called exosomes, which we have termed exo-AAV. Our recent manuscript using gene addition therapy with exo-AAV1 achieved unprecedented gene delivery to inner ear hair cells and partial rescue of hearing and balance in a mouse model of deafness involving the tetraspan membrane protein of hair cell stereocilia (Tmhs) gene. The current proposal is aimed at enhancing our gene delivery efficiency and translation towards clinically relevant large animal models. We have recently discovered that an AAV9 variant, AAV9-PHP.B, robustly transduces hair cells after injection into neonatal mice and non-human primates (NHP). Gene delivery to the inner ear of NHP by AAV has never be demonstrated before and will be a crucial step towards clinical trials. We will test whether we can improve AAV9-PHP.B’s transduction efficiency even further via its incorporation into exosomes (exo-AAV9-PHP.B). Our specific aims are (1) To improve gene delivery to cochlear hair cells using AAV9-PHP.B and exo-AAV9-PHP.B; (2) Investigate mechanisms of enhanced exo-AAV transduction of cells and AAV9-PHP.B transduction of hair cells; (3) To develop the exo-AAV platform to allow industrial scale-up/manufacturing for clinical use. For aim 1, we have preliminary data showing that AAV9-PHP.B mediates efficient gene delivery to hair cells of the cochlea in vivo in rodents and NHP. In Aim 1a we will directly compare exo- AAV9-PHP.B and standard AAV9-PHP.B at several doses for delivery to hair cells in mice and in NHPs. In Aim 1b we test the ability of exo-AAV9-PHP.B and AAV9-PHP.B encoding Tmhs to rescue hearing and balance dysfunction in the Tmhs knockout mouse. In Aim 1 c we will compare exo-AAV9-PHP.B and AAV9-PHP.B at gene delivery to hair cells in NHPs. In Aim 2, we will investigate mechanism of transduction of hair cells by exo-AAV using blockade of specific cellular processes as well as AAV capsid mutants to discern specific contributions to gene transfer by the AAV capsid or exosome components. In aim 3, we will use properties of the exosome membrane to allow anion-exchange column chromatography, an industrial scalable approach, for the purification of exo-AAV. We are also collaborating with a cell and gene therapy contract research organization to develop scalable production of exo-AAV in suspension cells. These are important steps towards clinical translation of the technology. This research is important as it addresses an unmet medical need as well as financial burden to society, treatment of inherited hearing loss. Furthermore, the data obtained during the work will be important for other diseases amenable to treatment with the exo-AAV gene therapy system.