696. Carboxymethylcellulose Increases Non-Viral Gene Transfer in Mouse Airways

Inborn Errors of Lysosomal Metabolism in both cell lines. PEI-PLA2 conjugates lead to ~3000-fold higher luciferase expression than PEI alone in HepG2 cells. In HEK293 cells, the improvement was ~1000-fold. Higher N/P ratios lead to better enhancement using the conjugates with N/P ratios 20 and 40 providing maximum improvement. At N/P ratios higher than 40, transfection efficiencies using the conjugate dropped but were still higher than PEI. In case of GFP-transfected cells, PEI-PLA2 conjugates resulted in ~ 80% transfection in HEK293 cells, which was higher than with PEI alone. Visual inspection of the cells also indicated lower toxicity with the conjugates. Conclusion. PEI-PLA2 conjugates had significantly higher transfection efficiencies and lower toxicities compared to PEI-DNA polyplexes. These results clearly indicate the potential for further exploring this approach to obtain improved non-viral gene delivery vectors using this approach. Further characterization of the conjugates in terms of physicochemical properties of polyplexes, DNA condensation, and effect of different PEI to PLA molar ratios are currently underway.

696. Carboxymethylcellulose Increases NonViral Gene Transfer in Mouse Airways

Uta Griesenbach,1,2 Cuixiang Meng,1,2 Raymond Farley,1,2 Patrick L. Sinn,3 Paul B. McCray, Jr,3 Seng H. Cheng,4 Ronald K. Scheule,4 Eric W. F. W. Alton.1,2 1 Department of Gene Therapy, Imperial College at the National Heart & Lung Institute, London, United Kingdom; 2The UK Cystic Fibrosis Gene Therapy Consortium, I, London, United Kingdom; 3 Program in Gene Therapy, Department of Pediatrics, University of Iowa, Iowa City, IA; 4Genzyme Corporation, Framingham, MA. In an attempt to increase non-viral gene transfer to the lung we are assessing various adjuncts. Derivatives of methylcellulose (MC) are widely used in food and drug manufacturing. It has previously been shown that MC and carboxymethylcellulose (CMC) increase viral gene transfer to the airways (Sinn P 2005). The proposed mode of action of these viscoelastic gels is inhibition of mucociliary clearance (MCC), thereby increasing contact time between the gene transfer agent and the target cell. Here, we assessed if CMC and MC increase Genzyme lipid 67 (GL67A)-mediated gene transfer to the airways of mice. We first determined that the addition of these substances to GL67A/pCIKLux complexes (0.1-1.5%) caused no visible precipitation over a 2 hr period, although viscosity of the solutions increased. We next determined if mice tolerated nasal perfusion of GL67A/pCIKLux complexes (80 mg DNA in 400 ml total volume) containing CMC or MC. Survival after perfusion with 1% CMC and MC was 90 and 100%, respectively (n=8), but mortality increased to 100% (n=3) when mice were perfused with 1.5% CMC, likely due to the viscous solution blocking the airways (mice are obligate nose breathers). The addition of MC (0.5% + 1%) decreased gene transfer in the mouse nose, however perfusion with 0.5% CMC (but not 0.25% or 1%) containing lipid/DNA complexes reproducibly increased gene expression by 3-fold (n= 16, p<0.05). We also assessed the effects of prolonged contact time of liposome/DNA complexes with the cells by comparing our standard 150 µl dose with a more dilute 400 µl dose both containing 80 µg DNA requiring 22 and 60 min perfusion, respectively. This prolonged perfusion alone increased gene transfer by 6-fold (n=8, p<0.05) and was further enhanced by the addition of 0.5% CMC leading to an overall 25-fold enhancement (n=8, p<0.001) in gene expression compared to our standard formulation. We next assessed if CMC increased gene transfer in the mouse lung using whole body nebulisation chambers. 0.5% CMC was either nebulised for 1 hr immediately before or simultaneously with GL67A/ pCIKLux. The former did not increase gene transfer, whereas the latter significantly increased gene transfer by 4-fold (p<0.0001, n=18). We also tried to increase the concentration of CMC from 0.5% to 1%, but due to the high viscosity of the solution, this material could not be nebulised using the Pari LC+ nebuliser. This study suggests that the S260

inhibition of MCC may improve non-viral gene transfer efficiency. Administration of CMC to the bacteria-infected cystic fibrosis lung may be problematic, because CMC may be utilised as an additional carbon source by the bacteria and it is unlikely that the modest 4-fold CMC-mediated increase warrants a change in the formulation of the GL67A/pCIKLux complexes. However, the study may promote assessments of other cilia static agents in the context of non-viral gene transfer.

Inborn Errors of Lysosomal Metabolism 697. GALC Over-Expression Toxicity in Hematopoietic Stem Cells Is Rescued by microRNA Regulation: New Perspectives for Gene Therapy of Globoid Leukodystrophy

Ilaria Visigalli,1,2 Bernhard Gentner,1 Silvia Ungari,2 Martina Cesani,1 Sabata Martino,3 Serge Rivest,4 Manuel Grez,5 Didier Trono,6 Angela Gritti,1 Alfred Merrill,7 Luigi Naldini,1,2 Alessandra Biffi.1,2 1 HSR-TIGET, San Raffaele Scientific Institute, Milan, Italy; 2VitaSalute University, San Raffaele Scientific Institute, Milan, Italy; 3 University of Perugia, Perugia, Italy; 4Universitè Laval RobertGiffard, Quèbec, Canada; 5University Medical School Frankfurt, Frankfurt, Germany; 6Ecole Polytechnique Fèdèrale de Lausanne, Lausanne, Switzerland; 7Georgia Institute of Technology, Atlanta. *Equal contribution Globoid leukodystrophy (GLD) is a lysosomal storage disease (LSD) due to the deficiency of galactocerebrosidase (GALC). Pathology is characterized by accumulation of nonmetabolized substrates leading to severe dysmyelination. We are developing a gene therapy strategy for GLD based on hematopoietic stem/progenitor cells (HSPC) and lentiviral vectors (LV). The efficiency of LV gene transfer and the possibility of multiple vector integrations in HSPC might allow obtaining GALC expression levels largely above the physiological ones. Upon LV gene transfer in HSPC, we observed an overt toxicity related to GALC over-expression, which determined the inability of transduced cells to proliferate and differentiate in vitro, the occurrence of apoptosis, and negative selection of highly transduced cells. When GALC-transduced HSPC were transplanted into lethally conditioned mice, either homo- or heterozygous, they failed to repopulate the hosts, demonstrating a functional impairment in vivo. The accumulation of pro-apoptotic molecules synthesized by the functional GALC, such as ceramide or sphingosine, could explain the apoptosis of transduced HSPC. Differentiated cells of the myeloid lineage (monocytes and microglia), which constitute the effector population in HSPC transplantation for the treatment of LSD, are not affected by GALC over-expression. The same results were obtained in human B and T lymphocytes, confirming the unique sensitivity of HSPC to the toxicity of the enzyme. To overcome this limitation, we developed strategies allowing to de-target vector expression from HSPC while permitting GALC over-expression in effector cells. We tested myeloid-specific promoters and microRNA regulation. Despite allowing some degree of lineage specificity, myeloid promoters only allowed reaching low GALC activity levels in differentiated cells. We thus generated a LV carrying GALC with the tagged sequence of a microRNA specifically expressed in HSPC, under the control of the PGK promoter (PGK. GALC.mirT). PGK.GALC.mirT transduced HSPC were able to proliferate and form colonies in vitro at the same extent as GFP controls, without undergoing apoptosis or negative selection. Their in vitro differentiated progeny expressed the transgene at the same level as cells transduced by PGK.GALC without mirT. Remarkably, transplantation of PGK.GALC.mirT transduced HSC into lethally conditioned heterozygous mice allowed repopulation of the hosts and long-term survival. Therefore, microRNA regulation allows rescue of the functional impairment due to GALC over-expression Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy