Cocaine Conjugate-Based Anti-Cocaine Vaccine in Shielding the Brain from Systemically Administered Cocaine

OPHTHALMIC DISORDERS AND TECHNOLOGICAL ADVANCES 451. Efcient Expression of Smn from Integration-Decient Lentiviral Vectors

Sherif O. Ahmed,1 Hugo Peluffo,1 Edmund Foster,2 Natalia Lago,2 Lawrence Moon,2 Thomas Hutson,2 Ping Yip,2 Klaus Wanisch,1 Stephen B. McMahon,2 Rafael J. Yáñez-Muñoz.1 1 School of Biological Sciences, Royal Holloway-University of London, Egham, United Kingdom; 2Wolfson Centre for AgeRelated Diseases, King’s College London, London, United Kingdom. Spinal muscular atrophy (SMA) is an inherited disease in which spinal cord motor neurons are progressively lost. We are pursuing novel therapeutic avenues to improve survival and preserve motor neuron function in SMA, using integration-decient lentiviral vectors (IDLVs) based on HIV-1. Our previous work has shown that IDLVs are as efcient as standard integrating lentivectors for gene expression in quiescent tissues in vivo, including eye, brain and muscle, and more recently we have shown similar efcacy in the spinal cord. Following intra-spinal injection in the ventral horn, IDLVs transduced motor neurons efciently, reaching frequencies of eGFP expression around 20-40%, which were stable for the 2-month period under study. When overall eGFP expression in white and grey matter was measured by semi-quantitative densitometrical analysis of eGFP immunoreactivity, no signicant differences were observed between vectors or time points. The same was true for the quantication of the total area of eGFP immunoreactivity, and for the percentages of transduced neurons (double staining for NeuN and eGFP) and motor neurons (double staining with ChAT and eGFP). Thus, integrating vectors and IDLVs demonstrate similar transduction efciency in the spinal cord in vivo. We have developed mouse Smn (Survival of Motor Neuron) and growth factor-expressing lentivectors to test the efciency of expression of these potentially therapeutic transgenes in a model of SMA. Expression of Smn in cultured dorsal root ganglia neurons was similarly efcient from IDLVs and integrating lentivectors, judging by Western blot. Similarly, rescue of motor neurons from death in culture upon withdrawal of exogenous growth factor was achieved with equal efciency by integrating and non-integrating lentivectors expressing GDNF. These results warrant further in vivo testing of therapeutic efcacy of IDLVs in animal models of SMA, which are currently in progress.

452. Efcacy of an Adenovirus Vector/ Cocaine Conjugate-Based Anti-Cocaine Vaccine in Shielding the Brain from Systemically Administered Cocaine

Martin J. Hicks,1 Bishnu P. De,1 Jonathan B. Rosenberg,1 Jesse T. Davidson,1 Amira Y. Moreno,2 Kim D. Janda,2 Neil R. Hackett,1 Stephen M. Kaminsky,1 Stefan Worgall,1 Ronald G. Crystal.1 1 Genetic Medicine, Weill Cornell Medical College, New York, NY; 2 The Scripps Research Institute, La Jolla, CA. Cocaine addiction is a worldwide problem for which there is no effective therapy. Although cocaine is a small molecule not recognized by the immune system, based on the knowledge that adenoviruses (Ad) elicit robust anti-humoral responses against the Ad capsid, we hypothesized that if we could link cocaine to the Ad capsid, it may be possible to use the resulting Ad/Cocaine conjugate as an anti-cocaine vaccine that would prevent cocaine from reaching the brain after inhalation or intravenous administration. To assess this hypothesis, a cocaine analog GNC (6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo[3.2.1]octane2-carbonyloxy hexanoic acid) was conjugated to the capsid of an intact E1-E3- Ad5 vector using 1-(3-dimethylaminopropyl)-3ethylcarbodiimide and N-hydroxy-sulphosuccinimide to create an AdGNC vaccine. Studies assessing the coupling reaction demonstrated that a ratio of 10 µg GNC to 0.5x1012 particle units Ad elicited the Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy

highest anti-cocaine antibody titers (ELISA) at 10 wk post-AdGNC administration to BALB/c mice. Western analysis demonstrated the GNC was bound to the capsid proteins hexon, ber, and penton base. To assess the effectiveness of the AdGNC vaccine in shielding the brain from cocaine, a model was established where 3H-cocaine (1.5 µg total cocaine) was administered intravenously to BALB/c mice, a challenge that results in a high proportion of the administered cocaine bound to the brain within 1 min after administration. Administration of AdGNC to the mice resulted in anti-cocaine antibody titers of 3.0x103 reciprocal units at 2 wk, 5.5x104 reciprocal units at 5 wk, and 2.0x105 reciprocal units at 10 wk. Intravenous administration of the 3H-cocaine to naive mice (n=4) resulted at 1 min in 181 ± 18 ng cocaine/g brain and 57 ± 2 ng cocaine/ml serum (ratio brain to serum 3.2). In contrast, intravenous administration of the 3H-cocaine 10 wk after administration of the AdGNC vaccine signicantly (p<0.01) reduced the amounts of cocaine reaching the brain. One min after cocaine administration, there was only 119 ± 5 ng cocaine/g brain but 190 ± 26 ng cocaine/ml serum (ratio brain to serum 0.6), i.e., the vaccine sequestered cocaine in the blood, resulting in a 34% reduction in the amount of cocaine reaching the brain. Assessment of the cocaine in the serum showed that the AdGNC vaccine resulted in 68% of the cocaine to be bound to serum IgG. These observations demonstrate that it is possible to couple an addictive small molecule like cocaine onto the capsid of Ad vectors and the resulting Ad/cocaine conjugate can elicit high titer anti-cocaine antibodies, shielding the brain from systemically administered cocaine. It may be possible to harness this transfer technology to develop an effective anti-cocaine vaccine.

453. Genetically Engineered Suppressor Cells Target CNS and Inhibit Activated Effector T-Cells

Moa Fransson,1 Elena Piras,2 Hao Wang,1 Ida Rasmusson,1 Katarina LeBlanc,3,4 Eva Brittebo,2 Angelica S. I. Loskog.1 1 Division of Clinical Immunology, Uppsala University, Uppsala, Sweden; 2Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden; 3Karolonska Institue, Division of Clinical Immunology, Stockholm, Sweden; 4Karolinska University Hospital, Hematology Center, Stockholm, Sweden. Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the mouse model of MS (EAE) treatments with either T regulatory (Treg) cells or multipotent mesenchymal stromal cells (MSC) have proven benecial. However, systemic administration of such cells may cause problems with infections due to a general immunosuppression. We hypothesized that Treg cells and MSCs can be directed to the CNS using gene transfer of CNSdirected chimeric articial receptors (CARs). A single chain variable fragment (scFv) was cloned from the hybridoma (8.18C5) producing anti-human myelin oligodendrocyte glycoprotein (MOG) antibodies. The scFv was cloned into a CAR and transferred into a lentiviral vector. The nal CAR construct consisted of a scFv linked via a hinge region to the transmembrane and intracellular part of a CD3 zeta chain. The zeta chain was further coupled to a CD28 domain. CD4+ T-cells were transduced with the MOG-directed CAR expressed in trans with murine FoxP3 which promotes Treg cell differentiation while MSC were modied using the MOG-directed CAR only. Engineered Tregs and MSCs were analyzed for their ability to suppress polyclonally stimulated T-cells in thymidine proliferation assays with or without MOG+ cells. Further, engineered suppressive cells were stained with a uorescent dye and injected into naïve mice i.p. Mice were sacriced at day 1 and day 10 and whole body sectioning was performed to trace the engineered suppressive cells in vivo. Antibodies from the MOG hybridoma were cross reactive to mouse MOG as shown by immunohistochemistry of mouse brain tissue. By inserting the genes for FoxP3 and MOG-directed CAR we developed CNS-specic Tregs. The FoxP3 mRNA levels were two-fold greater than that of Mock transduced cells and the engineered Tregs potently suppressed S175