146. Alternative Methods for Ex Vivo Gene Transfer to Primary Murine B Cell

GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY I for podocalyxin, and immunohistochemistry using the lectins helix pomatia agglutinin and peanut agglutinin demonstrated restoration of sialylation in the embryonic kidneys of one of two mutant mice. Additional animal studies are underway to optimize hGNE-Lipoplex delivery (route of administration, dose, and timing) and to conrm the ndings of these pilot studies.

145. Lentivirally Induced Over-Expression in the Mouse Hypothalamus; a Study of the Effects of Melanin Stimulating Hormones (MSH) in Obesity and Diabetes

Kim Eerola,1,2 Siru Virtanen,1,2 Eriika Savontaus,1 Mikko Savontaus.2 1 Department of Pharmacology, Drug Development and Therapeutics, University of Turku, Turku, Finland; 2Turku Centre for Biotechnology, Univesity of Turku, Turku, Finland.

Obesity and Type II Diabetes are an increasing health issue in the world and the negative effects of these conditions on health and life expectancy are evident. The pro-opiomelanocortin pathway is involved in metabolic regulation, mediating the effects of leptin in the hypothalamus. The melanocortin stimulating hormones; α-MSH, β-MSH and γ-MSH stimulate body homeostasis and decrease feeding behavior centrally. MSH peptides have been shown to have anti-inammatory effects and are also associated in the regulation of blood pressure. The three MSH peptides are derived from proopiomelanocortin (POMC) and are agonists of melanocortin receptors (MCR1-5). The MC-receptors can be found in different tissues such as melanocytes (MCR1) and in the arcuate and paraventricular nucleus of the hypothalamus. Investigations of the effects of native MSH peptides in the hypothalamus have proved to be difcult due to their short half-lives. In order to study the long term effects of MSH peptides, an accurate delivery and a stable expression system of the target gene is needed. The aim of this study was to deliver the lentiviral vectors carrying α-MSH, β-MSH and γ-MSH into the arcuate nucleus of mice to produce a stable gene expression and to analyze the effects of this intervention on body weight, glucose and insulin metabolism. Second generation lentiviruses were produced in HEK293T cells and the viral particles were collected from cell growth medium by ultra-centrifugation. The MSH construct used are unique, all containing a signal-sorting element from the N’terminus of the POMC sequence. The biological effects of the MSH vectors were shown by infection of melanocytes to produce melanin. Melanin concentration was measured by absorbance of the growth medium at 405 nm. Over-expression induced by these viruses was shown to be stable for over six months in vitro. The viral vectors were delivered into the arcuate nucleus using stereotaxic injection. The animals were perfused 16 days after treatment and the tissues were collected and placed in sucrose solution. The brains were snap-freezed and sectioned to 10 µm thick slides using a cryostat. The sections were stained using immunohistochemistry (IHC) to detect the induced overexpression. We have shown that MSH vector constructs/lentiviral vectors containing the N’terminal POMC signal-sorting followed by the three MSH peptides leads to production of bioactive MSH in vitro. We have also shown that lentiviral vectors can accurately be delivered into the arcuate nucleus in the mouse brain using a GFP control virus and that these vectors produce a controlled overexpression in order to study the effects of MSH peptides in obesity and type II diabetes.

146. Alternative Methods for Ex Vivo Gene Transfer to Primary Murine B Cell

Babak Moghimi, Ou Cao. Pediatrics, University of Florida, Gainesville, FL; Pediatrics, University of Florida, Gainesville, FL. Primary autologous B lymphocytes, following ex vivo gene transfer and re-implantation, can present the transgene product in a tolerogenic fashion to CD4+ T cells, thereby promoting antigen-specic immune tolerance. This approach has been successfully used to prevent autoimmune disease and adaptive responses to therapeutic proteins in several animal models. Furthermore, gene modied autologous lymphoma B cells, e.g. expressing certain cytokine genes, can augment cancer therapy. However, efcient gene transfer to primary B cells required use of retroviral vectors, which increase the risk of insertional mutagenesis. Here, we evaluated several viral and nonviral gene transfer approaches using primary murine B cells. Resting splenic B cells were isolated using magnetic activated cell sorting. Negative selection resulted in 98% purity as determined by staining for CD19 and ow cytometry. Puried B cells were activated with LPS (20 ug/ml) for 24hrs. Ex vivo GFP gene transfer was performed by means of nucleofection, lipofectamine, adenoviral infection, or murine retroviral infection. In the latter case, murine stem cell virus with an ecotropic envelope (kindly provided by Dr. David Scott) was used. Adenoviral gene transfer was performed with Ad5/11 or Ad5/35 vectors with tropism for hematopoietic cells, kindly provided by Dr. Andre Lieber. The Ad vectors were added to B cell cultures with and without calcium phosphate precipitation (using an MOI of 100 viral particles per cell). For transfections, naked plasmid DNA was utilized (1 ug/10^6 cells). The Amaxa nucleofection technology represents a modied electroporation technique for effective transfer of nucleic acids to the nucleus and thus enhances the efciency in particular for primary cells. A protocol and reagents for use with primary B cells have only recently become available, which allowed us to include this method in our comparison. Efciency of ex vivo gene transfer was determined by ow cytometry using GFP, CD19, B220, and 7-AAD (vital dye) as markers. Nucleofection yielded the highest level of gene transfer with 60-65% of B cells being GFP+. Efciencies were 30-35% for retrovirus, ∼20% for Ad5/11, ∼15% for Ad5/35, and ∼10% for lipofectamine-mediated transfection. Calcium phosphate precipitation increase efciencies for Ad vectors to ∼30% (Ad5/11) and ∼25% (Ad5/35). The gene transfer procedure caused the most cell death for the lipofectamine method (∼45%), followed by nucleofection (∼35%), and viral vector (5-10% in each case). For all methods, gene transfer efciencies were nearly identical for B cells from C57BL/6 or C3H/OuJ mice. We are currently investigating the distribution of transferred gene modied B cells to peripheral blood and lymphoid organs for non-viral and viral gene transfer. In conclusion, recent advances in gene transfer technologies provide alternatives to retroviral vectors for primary B cells. If stable gene transfer is desired, non-integrating vector systems may be combined with transposon- or phage integrase-based systems or future sitespecic systems to achieve integration into the host B cell genome.

147. A Full Characterization of the CAG 140 Huntington’s Disease Mouse Model

Aaron C. Rising,1 Eileen Denovan-Wright,2 Ron Mandel.1 Department of Neuroscience Powell Gene Therapy Center, McKnight Brain Institute University of Florida, Gainesville, FL; 2 Department of Pharmacology, Dalhousie University, Halifax, NS, Canada.

1

Huntington’s Disease (HD) is a neurodegenerative disorder whose behavioral, motor and cognitive symptoms progress as the individual ages. HD is caused by an expansion of a tract of CAG codons in the rst exon of the huntingtin gene. The CAG codon codes for glutamine and the expansion translates to an excess of these glutamines in the S56

Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy