Optimized Production and Validation of Rationally Designed AAV Vectors for Cockayne Syndrome Gene Therapy

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Project Summary- Abstract. Our current proposal is focusing on the production of novel AAV-based gene therapy for ultra-rare Cockayne Syndrome (CS). CS is affected both males and females in equal numbers with the incidence of less than 1 case per 250,000 live births in the U.S. The existing therapies are manly supportive rather than curative, and in severe cases, patients usually do not survive past childhood. The mutations in the ERCC8 (CSA)/ERCC6 (CSB) genes causing disease have been already identified however no disease altering gene therapies are available. On the other hand, Adeno-associated virus (AAV) has been widely acknowledged as a safe and effective clinical-stage vector for gene therapy for a broad spectrum of inherited diseases, and most recently, AAV-based treatments for inherited retinal dystrophy and spinal muscular atrophy (SMA) were approved by the US FDA and became available for patients. Thus our interdisciplinary team of basic, translational and clinical scientists propose to design a continuous AAV production and purification process prototype to address the needs for novel CS gene therapy. To achieve that goal we, first, will collect clinical data and confirmation of mutations. We also will evaluation of the prevalence of serotype-specific anti-AAV neutralizing antibodies (NA) to identify AAV serotype(s) with the potential to be used in the vast majority of CS patients. Second, optimize AAV capsid and expression cassette to ensure robust therapeutic C S A gene expression and test novel vectors on an animal model of CS in vivo. Finally, we will test centrifugation-free continuous AAV production protocol based on c ross-flow filtration followed by dual- chromatography purification. Our robust quality control procedures included state-of-art methods such as cryo- EM discrimination of full and empty AAV capsids with high accuracy and novel noninvasive in vivo tracking of AAV distribution. In summary, our proposal will result in a reliable protocol for AAV purification and quality control which can be easily implemented in GMP applications and ultimately used for AAV-mediated CS gene therapies in the clinic.