Scholarly article on topic '238. Highly Efficient Motor Neuron Transduction in Adult Mouse Spinal Cord by Continuous Intrathecal Infusion of rAAV9 Vectors Using a Slow-Releasing Osmotic Pump'

238. Highly Efficient Motor Neuron Transduction in Adult Mouse Spinal Cord by Continuous Intrathecal Infusion of rAAV9 Vectors Using a Slow-Releasing Osmotic Pump Academic research paper on "Biological sciences"

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Academic research paper on topic "238. Highly Efficient Motor Neuron Transduction in Adult Mouse Spinal Cord by Continuous Intrathecal Infusion of rAAV9 Vectors Using a Slow-Releasing Osmotic Pump"

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 ORF-disrupted 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 '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

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 ofAAV8 . 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,12 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.

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

to transduce motor neurons in adult spinal cord, which will greatly facilitate the evaluation of treatment strategies targeting this hard-to-reach population of cells in adult mice, which can be readily translated into clinical application. Currently, we are evaluating intracerebroventricular infusion of rAAV vectors using osmotic pumps, aiming to achieve enhanced global CNS transduction by delivering rAAV into CSF space.

239. Enteric Nervous System Transduction Following Intravascular Delivery of AAV9

S. E. Gombash Lampe,1 C. J. Cowley,1 F. L. Christofi,1 J. A.

Fitzgerald,1 K. D. Foust.1

'The Ohio State University, Columbus.

Gastrointestinal (GI) dysfunction is frequently reported in patients with neurologic and neuromuscular diseases. Symptoms related to GI dysmotility, such as constipation, gastroparesis and gastroesophageal reflux, impair the quality of life for patients with Parkinson's disease, multiple sclerosis, spinal muscular atrophy, spinal cord injury, autism spectrum disorders, and dementia. Peristalsis is highly regulated by the enteric nervous system, composed of the myenteric and submucosal nerve plexuses. Therefore, development of novel therapeutics that target enteric neurons may be of great benefit in treating GI symptoms. The purpose of the current study was to characterize transduction efficiency and transgene distribution in the myenteric plexus of the complete GI tract following intravenous injection of adeno-associated virus serotype 9 expressing green fluorescent protein (scAAV9-CB-GFP) in neonatal (P1) and juvenile (P21) mice. Neonatal mice were intravenously injected with scAAV9-CB-GFP (3 x 1011 vg) in the temporal face vein on P1 and juvenile mice received tail vein injections on P21 (2 x 1012 vg). Mice were euthanized at 60-90 days of age and the complete GI tract was dissected to expose the myenteric plexus. Immunohistochemical labeling of the myenteric plexus revealed GFP immunoreactivity in the esophagus, stomach, small intestine, cecum and colon in mice injected at each age. Neuronal transduction ranged from 21-57% depending on GI region analyzed. GFP colocalized with HuD positive myenteric neurons and S100 positive glial cells. Characterization of specific neuronal subtypes is ongoing and co-labeling studies thus far are consistent with multiple types of neurons in the ENS. Quantification of GFP immunoreactive neurons throughout the GI tract revealed increased transduction in P21 injected mice compared to P1 injected mice in most regions. This is in contrast to CNS data where neuronal transduction is more prominent in neonate injected animals. Segmental examinations of myenteric neurons in the colon showed no oral-aboral bias. These findings are significant because it shows AAV9 can be used as a tool to study intestinal function and for the development of GI therapeutics. Comparisons with other AAV serotypes are ongoing.

240. Efficient and Targeted Transduction of Nonhuman Primate Liver With Optimized AAV3B Vectors Through Systemic Delivery

Li Zhong,1,2 Shaoyong Li,1,3 Mengxin Li,1,3 Chen Ling,4 Qin Su,1 Ran He,1 Arun Srivastava,4 Guangping Gao.1,3 'Gene Therapy Center, UMMass Medical School, Worcester, MA; 2Pediatrics, UMass Medical School, Worcester, MA; Microbio & Physiol Systems, UMass Medical School, Worcester, MA; 4Pediatrics, University of Florida, Gainesville, FL.

Recent studies have demonstrated that rAAV3B vectors transduce human liver cancer cells as well as primary human hepatocytes efficiently in vitro. The combinations of modifications of specific surface-exposed serine (S) and threonine (T) residues on rAAV3 capsids further augment transduction efficiency in human liver cancer cells. Moreover these optimized rAAV3B vectors can achieve targeted delivery in a human liver cancer xenograft model after systemic

administration, suggesting that AAV3B and their derivative vectors are ideal for liver-directed gene therapy in humans. Here, we used the nonhuman primate model for liver-directed gene transfer to examine the safety and efficacy of these vectors which is critically important for their further clinical development in liver gene therapy in humans. To this end, we first investigated sero-epidemiology ofAAV3B in a rhesus monkey colony and found that the pre-existing neutralized antibodies (Nab) against AAV3B are relatively low in rhesus macaques (i.e. 52% of animals with no detectable Nab at a sensitivity of 1:5). To study liver tropism of optimized AAV3B vectors in NHP liver, we dosed three young (3-4 months) male rhesus macaques with S663V+T492V modified AAV3B (AAV3B.ST)-CB-EGFP vector at 1x1013 GC/kg intravenously and analysed EGFP expression and histopathology in the liver 7 days later by fluorescence microscopy. The results revealed that the optimized AAV3B.ST vector led to efficient and specific EGFP expression in the liver (36% hepatocytes in average as compared to 25% achieved by AAV7 in NHP liver) but not in other tissues, and without apparent hepatotoxicity. To quantitatively assess transduction efficiency of AAV3B and optimized AAV3B.ST vectors in NHPs, we i.v. injected three young male rhesus macaques with wild type (WT) and ST modified AAV3B-CB-rhCG vectors at 1x1013 GC/kg respectively and monitored serum rhCG levels for 3 months. Both WT and ST modified AAV3B vectors led to efficient and sustained rhCG expression in 104 - 105 rU/ml range up to 91 days, but ST modification enhanced rhCG expression by 2 (at late stage) to 5 (at early stage) folds as compared to WT AAV3B vector. This was confimed with qRT-PCR analysis of liver rhCG mRNA levels at the 91 day end point, showing again a 2-fold increased transcription by ST modification. The biodistribution analysis of the persisting vector genomes indicated a predominant liver targeting. Clinical chemistry and histopathology examinations of the study animals showed no apparent vector-related toxicity. In summary, the optimal AAV3B.ST vector can safely and specifically delivery transgene to primate liver and resulted in a further increased transduction efficiency as compared to WT AAV3B vectors after systemic administration. Our studies should be important and informative for clinical development of optimized AAV3B vectors for liver-directed gene therapy in humans.

* Equally contributed authors

# Co-corresponding authors.

241. Vector Design Tour De Force: Integrating Combinatorial and Rational Approaches To Derive Novel Adeno-Associated Virus (AAV) Variants

Damien Marsic,1 Lakshmanan Govindasamy,2 Seth Currlin,1 David Markusic,1 Yu-Shan Tseng,2 Roland W. Herzog,1 Mavis Agbandje-McKenna,2 Yuan Lu,3 Steve Ghivizzani,3 Sergei Zolotukhin.1 'Pediatrics, University of Florida, Gainesville, FL; 2Biochemistry and Molecular Biology, University of Florida, Gainesville, FL; 3Orthopaedics & Rehabilitation, University of Florida, Gainesville, FL.

AAV-derived vectors are promising tools for human gene therapy applications because of their absence of pathogenicity, episomal localization and stable transgene expression. However, significant limitations to their clinical use are their lack of specificity and their susceptibility to neutralization by human antibodies. Both of these limitations are determined by the nature of amino acid residues exposed at the surface ofthe capsid. Methodologies to improve existing AAV vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Although both methodologies have been successful in creating improved vectors, their utility is limited by our understanding of the AAV life cycle and by the technical boundaries of the protocols for directed evolution. The strategy adopted in this project integrates both approaches in one unified design. We have conducted a process of "virtual family

Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy