237. In Silico, Ancestral Reconstruction of AAV Particles Circumvents Pre-Existing Immunity in Humans

AAV VECTOR DEVELOPMENT infecting human polarized airway epithelia from apical membrane at multiplicity of infection (MOI) as low as 10-3 genome copies (gc)/cell. Using trans-complementation with HBoV1 expressed viral proteins, we successfully generated a rHBoV-luciferase vector from an ORFdisrupted rHBoV1 genome and demonstrated this vector efficiently transduces human airway epithelia (HAE) from the apical surface. However, the rHBoV1 vector stock was found to be contaminated with a significant level of replication competent virus generated during the production procedure. To develop a safer vector for the application in gene therapy, we explored the possibility of generating a chimeric vector by parvovirus cross genus pseudotyping and found that HBoV1 capsid can efficiently encapisdate a recombinant adeno-associated virus genome. This property allowed us to create an AAV2/HBoV1 chimeric viral vector retaining the safety of a rAAV genome and the tropism of a HBoV1 capsid. Importantly, rAAV/HBoV1 vector was capable of delivering an oversized rAAV genome of 5.5kb to HAE. In an HEK293 cell production system, the yields of rAAV2/HBoV1 vectors were 20% to 30% of that of rAAV2 for a number of transgene cassettes including firefly luciferase, eGFP, mCherry and human CFTR. rAAV2/HBoV1 vectors also effectively packaged 4.6kb and 5.5kb genomes at similar efficiencies. With a luciferase reporter, we found that rAAV2/HBoV1 infection of HAE from the apical membrane gives 70-fold greater transgene expression than rAAV2 and 5.5-fold greater than rAAV1. Molecular studies demonstrated that viral uptake from the apical surface was significantly greater for AAV2/HBoV1 than for rAAV2 and rAAV1. Although rAAV2/ HBoV1 is effectively endocytosed by non-differentiated airway cells in monolayer culture, the polarization of airway epithelial cells is required for HBoV1 capsid-mediated gene transfer, suggesting polarization influences entry pathways of the virus. rAAV/HBoV1 transduction was also limited by the ubiquitin-proteasome pathway, inhibition of proteasome activity during the infection period dramatically enhanced rAAV/HBoV1 transduction by >1000-fold. Furthermore, the 0.9kb increased package capacity of the rAAV genome makes rAAV2/HBoV1 a prime candidate vector for cystic fibrosis gene therapy. A rAAV2/HBoV1-CFTR virus with a 5.5kb genome containing the full-length CFTR coding sequence and a strong CBA promoter corrected CFTR-dependent chloride transport in CF HAE significantly greater than rAAV vectors. In summary, utilizing the combined advantages of AAV and HBoV1, we have developed a novel promising chimeric viral vector for the application of human gene therapy for cystic fibrosis and other pulmonary diseases, as well as vaccines against wild type HBoV1 infections.

237. In Silico, Ancestral Reconstruction of AAV Particles Circumvents Pre-Existing Immunity in Humans

Eric Zinn,1 Eva Plovie,1 Debalina Sarkar,1 Vadim Khaychuk,1 Livia Carvalho,1 Samiksha Shah,1 Luk H. Vandenberghe.1 1 Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA. Recent successes in early stage clinical trials for hemophilia and inherited blindness have begun to suggest that AAV could become a platform for in vivo mediated gene transfer. A critical obstacle for broad use of this technology however remains the substantial level of pre-existing immunity in humans. This currently excludes large patient populations from enrolling in clinical trials, and later on from reaping the benefits from a licensed gene therapy drug. While many approaches have been proposed to overcome this issue, most elevate the complexity and safety of the clinical paradigm. Our goal in this project was to derive a novel AAV vector disrupted in as many putative epitopes as possible while retaining structural integrity and the desirable functional properties of AAV. S90

Under the assumption that AAV evolved primarily under selective pressure from host humoral and cellular immune responses, our group hypothesized that ancestral AAV capsids would be more refractory to pre-existing immunity in contemporary human populations. Using maximum-likelihood methods, we reconstructed an ancestral Cap gene. In order to compensate for uncertainty inherent to the reconstruction process, we constructed a probabilistic space around that ancestral node. This space was synthesized as an AAV DNA library for AAV packaging. Each members of this library is over 8% different from any contemporary counterparts and disrupted in more than 60 putative epitopes. Approximately 33% of AAV variants in this space were found to form infectious particles. Viral yields of lead candidates of our screen were similar to those of AAV2. In vitro per particle infectivity varied between that of AAV2 and AAV8. Structurally stable and infectious lead candidates were shown to transduce murine retina and liver, equal to and sometimes exceeding the activity of AAV8 . Finally, these vectors were evaluated in a neutralizing antibody assay against pooled human IVIg and demonstrated to be 8-64-fold less susceptible to neutralization than any of its descendants including AAV8 . Similar challenge against individual human sera corroborated these results, suggesting that ancestral AAVs may circumvent pre-existing immunity, enabling broader application of AAV gene therapies.

238. Highly Efficient Motor Neuron Transduction in Adult Mouse Spinal Cord by Continuous Intrathecal Infusion of rAAV9 Vectors Using a Slow-Releasing Osmotic Pump Dan Wang,1,2 Jia Li,1 Dan Burt,1 Guangping Gao.1,2 Gene Therapy Center, University of Massachusetts, Worcester, MA; 2Department of Microbiology & Physiology System, University of Massachusetts, Worcester, MA.

1

Some neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) primarily affect motor neurons, causing rapid loss of mobility and high mortality in adult patients. Currently there is no effective treatment available for ALS. Recombinant adeno-associated virus (rAAV)-delivered shRNAs to silence causative toxic mutant gene such as SOD1 in motor neurons is an attractive approach for gene therapy of familiar ALS. Nonetheless, a major barrier for rAAV-mediated gene silencing is to achieve highly efficient and widespread motor neuron transduction in the spinal cord. Recent studies have documented that rAAV9 and some other AAV serotypes can transduce motor neurons via either intravenous (i.v.) or intrathecal (i.t.) injections in mouse and large animal models. However, the efficiency of global motor neuron transduction is limited by rAAV titer as well as the volume of a single rAAV bolus that can be injected in a short period of time. Here, using C57B6/J adult mice (6 wks old) as a model and rAAV9FFluc or scAAV9EGFP, we attempted to develop a clinically compatible method to deliver high doses of rAAVs to the cerebrospincal fluid (CSF) space for efficient and global motor neuron transduction. We compared 200 ul (2x1012 GCs in total) single bolus i.v. injections, 8 ul (8x1010 GCs in total) single bolus i.t. (lumbar) injections and the continuous infusions of up to 200 ul (2x1012 GCs in total and up to 8 ul/hour) of the same vectors in 1-3 days into the spinal cord with a slow-releasing osmotic pump for spread (rAAV9FFluc) and neuronal (scAAV9EGFP) transduction at 3 wks after vector delivery. We found that, among 3 delivery methods compared, continuous pump infusions produced much wider spread FFluc transduction throughout the whole spinal cord. Using EGFP vector, we further demonstrated that pump infusions evoked much more efficient neuronal transduction (e.g. nearly 100% of NeuN+/ EGFP+ neurons in the ventral horn) but less astrocytic transduction. We also characterized the stability of rAAV9 in the pump at 37°C, revealing a very minor loss of infectious virions after 24 hours but up to 30% loss after 72 hours. Our study establishes a robust means Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy