The available data of AAV vectors in the clinic emphasize the importance of continued optimization efforts at the levels of the AAV capsid, genome and transgenic cassette. A focus of this proposal is to derive clinical AAV vector best suited for systemic disorders (MPS, Hurlers, etc.). At the capsid level, it is apparent that animal models do not always predict the human outcome and that more efficient human specific capsids are required to achieve a lower administered dose.
In Aim 1, we seek to create a new paradigm of AAV vector selection for human transduction by generating the first AAV receptor expression map on tissues of mouse, primate and human origin. This tissue specific AAV receptor Atlas will be overlaid with AAV binding and transduction data in an effort to tease out regions of the capsid important for tissue specific interactions in varied backgrounds. In addition novel chimeric capsids isolated from a directed evolution strategy on primate and human livers established in a mouse model will be triaged against our receptor/binding atlas to determine if in vitro binding correlates to in vivo results. Then, capsid isolates from a primatized-liver mouse model will be investigated for primate liver transduction in vivo to determine if this strategy represents a valid method to derive primate (human &non human) liver specific AAV capsids. At the level of the AAV genome, we have assembled a panel of DNA repair dependent AAV substrates that report critical aspects of genome persistence including circularization, concatemerization and homology directed annealing. Investigations of these reagents in mutant backgrounds defective in different DNA repair pathways will offer insights into the preferred reliance on homologous recombination and non-homologous end joining mechanisms in vitro and in vivo providing a better prediction of vector performance in diseased settings (Aim 2). At the level of the vector transgene, we demonstrate in mouse liver, heart and eye a novel method to induce transgene synthesis using the IVS2- 654 intron and an anti-sense oligonucleotide. The work herein seeks to generate smaller synthetic variants that exhibit tighter control as well as altered transgene expression levels, thus providing a panel of regulatory switches which can be tailored for specific applications. Finally, a strategy is proposed to engineer an "off" switch for the induced transgene synthesis from IVS2-654, which may also allow the precise tuning of transgene synthesis at a fixed vector dose. Collectively, the results of the proposed experiments seek to address the observed clinical deficiencies in AAV gene therapy applications for diseases of systemic nature by our continued optimization efforts at the levels of the capsid and genome as well as the transgenic DNA cassette.

Public Health Relevance

The proposed experiments seek to enhance viral based DNA delivery vectors for the treatment of human disease. To do this we will establish a new paradigm in which the observed variations in delivery efficiency between humans and animal models are better understood. In addition we seek to enhance AAV vectors at the DNA level for enhanced delivery efficiency, by further understanding the mechanism of genome persistence and expression. Finally, a molecular on- off switch will be engineered and incorporated into our viral delivery format to control therapeutic protein expression in the clinical setting.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Gene and Drug Delivery Systems Study Section (GDD)
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Park, Eun-Chung
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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Nicolson, Sarah C; Samulski, R Jude (2014) Recombinant adeno-associated virus utilizes host cell nuclear import machinery to enter the nucleus. J Virol 88:4132-44
Bartus, Raymond T; Weinberg, Marc S; Samulski, R Jude (2014) Parkinson's disease gene therapy: success by design meets failure by efficacy. Mol Ther 22:487-97
Hirsch, Matthew L; Samulski, R Jude (2014) AAV-mediated gene editing via double-strand break repair. Methods Mol Biol 1114:291-307
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Gray, S J; Nagabhushan Kalburgi, S; McCown, T J et al. (2013) Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates. Gene Ther 20:450-9
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Asokan, Aravind; Samulski, R Jude (2013) An emerging adeno-associated viral vector pipeline for cardiac gene therapy. Hum Gene Ther 24:906-13
Mitchell, Angela M; Li, Chengwen; Samulski, R Jude (2013) Arsenic trioxide stabilizes accumulations of adeno-associated virus virions at the perinuclear region, increasing transduction in vitro and in vivo. J Virol 87:4571-83

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