287 Fusion of Either Gap 43 or Tau to the Clostridial Tetanus Toxin Light Chain Improves Synaptic Inhibition in the Rat Spinal Cord Following Adenoviral Vector Gene Delivery Molecular Therapy Volume 2[.]
NEUROLOGICAL THERAPY: NEURODEGENERATION AND IMAGING are assessing HTT mRNA expression in other regions of the brain and in peripheral tissues Vascular AAV2/9-mi2.1-GFP treatment significantly prevented weight loss in N171-82Q mice compared to controls (p0.05) Together, these results demonstrate that AAV2/9 is an exciting gene therapy tool that can be used to deliver HD-specific RNAi constructs to many regions of the brain and peripheral tissues following a single injection to the vasculature 285 Transcriptional Targeting of HSV VectorMediated Transgene Expression to Specific Sensory Neuron Subtypes Mark F Doyal,1 Mingdi Zhang,1 George Huang,1 Justus Cohen,1 Nicole Scheff,2 Michael Gold,2 William F Goins,1 Joseph C Glorioso.1 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, PA Peripheral injection of replication defective HSV-1 vectors results in vector uptake at sensory nerve terminals and retrograde delivery of the genome to the neuronal nuclei within dorsal root (DRG) and trigeminal ganglia (TG) These genomes enter a latency-like state and can serve as a persistent platform for expression of antinociceptive products that reduce pain signaling in animal models of acute and chronic pain We have used the transiently active HCMV and persistently active viral latency (LAT) promoter to drive transgene expression; however neither promoter has specificity for subpopulations of sensory neurons which mediate the development of chronic pain Furthermore, in the case of the CMV promoter, nonspecific expression may result in unwanted off-target effects Here we describe the use of neuronal promoters to target transgene expression to specific sensory neuron subpopulations Using these transcriptionally targeted vectors we hope to identify sensory neuron subtypes that contribute to chronic pain signaling and identify those populations of cells where disruption of the pain response will be most effective In a proof of principle experiment we tested the promoters of several neuronal subtype marker genes to target the expression of the fluorescent marker mCherry to the neuronal population in which the promoter is endogenously active Primary rat sensory neurons were transduced with HSV vectors expressing mCherry under the control of the promoters for the heavy neurofilament (highly expressed in A fibers), calcitonin gene related peptide (CGRP, expressed in a population of nociceptive, peptidergic C fibers), transient receptor potential vanilloid (TRPV1, heat sensitive nociceptors), neuropeptide Y (upregulated in sensory neurons after nerve injury), or CMV and HSV LAT promoter controls Promoter targeting efficiency was determined by immunofluorescence using antibodies to the targeted neuronal marker within a larger population of pan-neuronal antibody NeuN reactive neurons In vitro experiments in cultured rat DRG and TG neurons have shown; (i) the CMV promoter drives transgene expression in both neuronal and non-neuronal cells, whereas each of the neuronal promoters tested as well as the LAT promoter restrict expression to neuronal cells only; and (ii) expression from neuronal promoters is targeted to the appropriate neuronal population with a high degree of specificity (70-80%) and sensitivity (80-90%) In vivo studies to evaluate vector transgene performance by injection of vectors into the hindpaw of rats corroborated our in vitro model with neuronal subtype-specific expression from neuronal promoters and nonspecific expression from the CMV promoter This will ultimately be used to target analgesic transgene products in order to precisely determine the sensory populations contributing to a pain phenotype, and generate therapeutics with minimal off-target effects (e.g on non-nociceptive neurons) Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy 286 Mechanisms of Caspase-1 Mediated Schwannoma Regression Mehran Taherian,1 Shilpa Prabhakar,2,5 Giulia Fulci,3 Miguel SenaEsteves,4 Xandra O Breakefield,2,5 Gary J Brenner.1 Anesthesiology, Massachusetts General Hospital, Boston, MA; Neurology and Radiology, Massachusetts General Hospital, Boston, MA; 3Neurosurgery, Massachusetts General Hospital, Boston, MA; 4Neurology, U Mass Medical School, Worcester, MA; Program in Neuroscience, Harvard Medical School, Boston, MA Schwannoma tumors are composed of Schwann-lineage cells and form along peripheral, spinal and cranial nerves These tumors can cause pain, sensory/motor dysfunction, and death through compression of peripheral nerves, the spinal cord, and/or the brain stem We have previously shown gene therapy for these tumors using a xenograft model in which immortalized human schwannoma cells (HEI-193) expressing a fluorescent protein and luciferase are implanted in the sciatic nerve of nude mice Our strategy has been to express the apoptotic protein, caspase-1 (ICE, IL1-converting enzyme) under the P0 promoter, selectively active in Schwann cells during development, using an adeno-associated vector (AAV1) injected directly into the tumors Caspase-3 is the most common pro-apoptotic target for therapeutic modulation However, unlike caspase-3, caspase-1 has a strong pro-inflammatory component in addition to induction of apoptosis Caspase-1 activates pro-IL-1b and pro-IL-18, which in turn trigger immune responses mediated by neutrophils and monocytes, or NK cells; it has been associated with both innate and adaptive immunity In addition, caspase-1 is activated by several chemotherapeutic drugs and can sensitize tumor cells to chemotherapy and radiation Our recent data indicate that AAV1P0-ICE not only induces apoptosis of infected caspase-1-expressing HEI-193 schwannoma cells, but also kills non-infected tumor cells through a bystander effect mediated by both tumor- and host-specific mechanisms Supernatants derived from HEI-193 cells transfected with AAV1-P0-ICE DNA kill non-transfected tumor cells in vitro Similarly, peripheral nerve implantation of a 1:1 mixture of AAV-P0ICE DNA-transfected and non-transfected HEI-193 cells prevented tumor growth This bystander killing effect induced by AAV1-P0-ICE was also observed when established intra-sciatic HEI-193 tumors were injected with AAV1-P0-ICE transfected cells Interestingly, intra-sciatic implantation of HEI-193 cells weeks after injection at the same location of AAV1-P0-ICE DNA-transfected cells, i.e long after all of the originally implanted caspase-1expressing HEI-193 cells should be dead, also prevents formation of a tumor These data suggest that caspase-1-induced death of HEI-193 cells in vivo leads to local changes in the region of the tumor capable of preventing formation of a subsequent tumor This investigation of AAV1-P0-ICE induced bystander killing of tumor cells has the potential to generate new therapeutics for the neurofibromatoses, as well as, for other benign neoplasms Not only can caspase-1 induce apoptosis, but the resulting tumor killing and associated activation of host immune responses may generate a vaccination effect that could control the development and growth of subsequent schwannomas 287 Fusion of Either Gap-43 or Tau to the Clostridial Tetanus Toxin Light Chain Improves Synaptic Inhibition in the Rat Spinal Cord Following Adenoviral Vector Gene Delivery Eleanor M Donnelly,1 Chalonda R Handy,2 Marie Kim,1 Jeremiah Huang,1 Thais Federici,1 Nicholas M Boulis.1 Neurosurgery, Emory University, Atlanta, GA; 2Nationwide Children’s Hospital, Columbus, OH Clostridial bacterial toxins, are assuming increased importance as therapeutic agents for modulating neural transmission in refractory conditions The similarity between the functional components of S109 NEUROLOGICAL THERAPY: NEURODEGENERATION AND IMAGING the Clostridial toxins presents an opportunity for the development of therapies involving the use of the tetanus light chain (LC) As an alternative to the commonly used botulinum treatment approach which requires repeated protein injections, viral vector delivery of LC can give stable LC expression Indeed, in earlier studies we have demonstrated effective synaptic inhibition using adenoviral directedLC expression LC proteases targets proteins in the SNARE complex responsible for synaptic vesicle docking at the synaptic terminal In order to being about a more potent inhibition we are investigating methods of transporting the LC protein from the cell body to synaptic terminal via fusing the LC protein with sequences of proteins which are natively transported to the synaptic terminal We have evaluated the benefits of fusing the LC with either the 3’ untranslated region of the microtubule-associated protein tau or the N-terminus region of the cytoplasmic protein neuromodulin (Gap-43) for axonal targeting For our research purposes, four recombinant vectors were produced: Ad.LC, Ad.LC.Gap-43, Ad.LC.tau and Ad-Green Fluorescent Protein (GFP) Each vector contained the cytomegalovirus (CMV) promoter and was constructed to express GFP In all studies, adult rats received ipsilateral spinal cord injections into the lumbar region and motor function was assessed by measures of gait and strength We found that all LC expressing vectors resulted in inhibition of motor activity without pathological effects as assessed by cresyl violent staining Importantly, rats treated with Ad.GFP did not demonstrate changes in locomotor activity at any time during the course of the study Whereas Ad.LC treated animals showed impairments in gait and ipsilateral hindlimb strength approximately days after vector delivery; interestingly, animals that received Ad.LC.Gap-43 demonstrated declines in ipsilateral motor function 1-2 days post vector administration Moreover, Ad.LC.tau vector delivery resulted in ipsilateral locomotor inhibition occurring 3-4 days post vector administration This data demonstrates that gene transfer of LC can be achieved without cell loss and axonal targeting can result in enhanced transgene potency and activity We are currently determining the relative level of GFP expression in each animal group using protein assays such as immunohistochemisty and western blotting techniques 288 MR-Guided Delivery of AAV2-hAADC into the Midbrain of Macaques Results in a Safe and Efficient Axonal Transport of AADC into the Striatum Krystof S Bankiewicz,1 Adrian P Kells,1 Lluis Samaranch,1 Waldy San Sebastian,1 Nitasha Sharma,1 John Bringas,1 Phillip Pivirotto,1 John Forsayeth.1 Department of Neurological Surgery, University of California, San Francisco, CA Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare and debilitating recessive genetic disorder in which mutations in the AADC gene result in a reduced activity of the AADC enzyme and lead to an inability to synthesize catecholamines [norepinephrine (NE), epinephrine (EP), dopamine (DA)] and serotonin As a result, afflicted children suffer severe movement and affective disorders with a consequently curtailed and difficult life We plan on using AAV2hAADC gene transfer to restore AADC levels in the brains of these affected children An IND-enabling safety study was performed in 18 non-human prmates that evaluated dose levels of AAV2-hAADC and a saline (control) group Infusate was delivered bilaterally into the midbrain under MR-guidance using our clinical brain delivery platform The MRI tracer, gadolinium, was used to visualize AAV vector administration in real time into the substantia nigra and ventral tegmental area In all cases, the precise, complete and reflux-free delivery of AAV was observed with no clinical adverse effects In animals that were euthanized at weeks, histological analysis showed efficient anterograde axonal transport of AADC to the caudate and putamen Animals scheduled for euthanasia at and months will S110 be processed and the results will be presented This data supports the safety and efficacy of MR-guided real time delivery of AAV2-hAADC into the midbrain of juvenile monkeys and justifies the use of axonal transport to achieve widespread delivery of AADC in the striatum of AADC-deficient children 289 Noninvasive, Quantitative PET Imaging of AAV-Mediated D2R80A Reporter Gene Expression in Live Animal Brains Sea Young Yoon,1 Datta Ponde,2 Harish Poptani,2 Jessica Bagel,3 Patricia A O’Donnell,3 Charles H Vite,3 John H Wolfe.1,3 Research Institute of the Children’s Hospital of Philadelphia, Philadelphia; 2Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia; 3W.F Goodman Center for Comparative Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia In vivo imaging of vector gene expression is crucial for evaluating the efficacy and response of gene therapy, particularly when traditional methods for evaluating gene expression that require invasive tissue sampling would not be ideal or provide only limited information A positron emission tomography (PET)-based reporter gene imaging system enables noninvasive, repetitive monitoring of transgene expression levels and spatial distribution in the living brain following vector gene delivery Several PET reporter genes have been developed for monitoring gene activity D2R80A is a dopamine-2 receptor mutant that binds radioactive analogs of dopamine but has been inactivated for intracellular signaling (Liang et al., Gene Ther 2001); and has been used to monitor peripheral organ transduction Since radioactive dopamine analogs cross the blood brain barrier, D2R80A can be used to monitor the distribution, magnitude and duration of AAV-mediated expression in the mouse brain The dopamine analogs bind to cells in the striatum where D2R is normally expressed, providing a well defined internal control The PET-imaging signals correlated well with the reporter gene expression levels and distribution as assessed post-mortem by immunofluorescence, in situ hybridization, and real time PCR In the current study, two separate AAV1 vectors, one to express D2R80A and the other to express a therapeutic gene, feline -mannosidase were constructed under the control of the human GUSB gene promoter Both vectors were coinjected in the significantly larger cat brain into two injection tracks unilaterally into the cortex, hippocampus and thalamus, and at 9, 16, 24 weeks post-injection, in vivo PET imaging was performed for longitudinal monitoring following intravenous administration of [18F]-fallypride Reporter gene activity (bound to [18F]-fallypride) was quantified in the injected brain regions relative to the striatal binding No significant decrease in signal was observed over time indicating continued D2R80A production and no adverse immune response against the reporter gene Post-mortem brain analyses are underway to correlate the PET signal in the cats with the expression of both vectors in tissues by immunofluorescence, in situ hybridization, and real time PCR to assess D2R80A as a surrogate marker for the therapeutic gene [18F]-fallypride PET imaging of D2R80A provides a noninvasive, quantitative and sensitive method to monitor the vector gene expression, which could facilitate the development of enhanced vector delivery and potentially be used clinically in gene therapy of neurogenetic diseases Funding source: NIH R01-DK063973 and R01-NS038690 Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy ... THERAPY: NEURODEGENERATION AND IMAGING the Clostridial toxins presents an opportunity for the development of therapies involving the use of the tetanus light chain (LC) As an alternative to the. .. after vector delivery; interestingly, animals that received Ad.LC .Gap- 43 demonstrated declines in ipsilateral motor function 1-2 days post vector administration Moreover, Ad.LC .tau vector delivery. .. transported to the synaptic terminal We have evaluated the benefits of fusing the LC with either the 3’ untranslated region of the microtubule-associated protein tau or the N-terminus region of the