683. Cationic Mitochondriotropic Vesicles for DNA

LIPID MEDIATED GENE TRANSFER of these cationic amphiphiles. A better efficiency was obtained with Chol/lipid 2 lipoplexes than with Chol/lipid 1 lipoplexes when using the cytosolic luciferase expression vector. As anticipated, transfection efficiency of Lipid 3 was greatly inhibited in the presence of Bafilomycin A1. By contrast, endosome escape of DNA with a new cholesterol based cationic lipid containing no histidine head-group (Alanine-Cholesteryl-Ethylamide; lipid 4) seemed to be independent of endosome acidification. However, transfection efficacy of lipids 3 & 4 was similar. In conclusion, we show that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds or cholesterol is sufficient to impart remarkable transfection properties on the resulting cationic amphiphile via endosome-disrupting characteristics of the histidine functionalities.

683. Cationic Mitochondriotropic Vesicles for DNA Delivery to Mitochondria Volkmar Weissig, Gerard G. M. D’Souza. The number of diseases found to be associated with defects of the mitochondrial genome has grown significantly since 1988. Despite major advances in understanding mtDNA defects at the genetic and biochemical level, there is no satisfactory treatment available for a vast majority of patients. Objective limitations of conventional biochemical treatment for patients with defects of mtDNA warrant the exploration of gene therapeutic approaches. Two different strategies for mitochondrial gene therapy are imaginable [1] The first involves expressing a wild-type copy of the defective gene in the nucleus, with cytoplasmic synthesis and subsequent targeting of the gene product to the mitochondria (“allotopic expression”). Besides the different codon usage in mitochondria, however, there are possibly four major difficulties in adapting this nuclear-cytosolic approach for mitochondrial gene therapy to mammalian cells [2]. First, the majority of mtDNA defects involve tRNAs and to date, no natural mechanism has been reported for the mitochondrial uptake of cytosolic tRNAs in mammalian cells. Second, it is generally agreed that the thirteen proteins encoded for by mtDNA are very hydrophobic peptides, which would not be readily imported by the mitochondrial protein import machinery. However, since the 13 mitochondrial coded proteins are not equally hydrophobic, the allotopic expression of at least some of the peptides appears as possible [3]. Third, it has been hypothesized that some of the proteins encoded by the mitochondrion may potentially be toxic if synthesized in the cytosol [4]. Fourth, according to a hypothesis termed co-location for redox regulation [5], the co-location of mtDNA and its products may be essential for the rapid control of gene expression by the redox state in the mitochondrial matrix. Considering all problems associated with the nuclear-cytosolic approach the development of methods for the direct transfection of mitochondria [6] as an alternative approach towards mitochondrial gene therapy seems highly warranted. We have developed a strategy for Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright © The American Society of Gene Therapy

mitochondrial gene therapy which involves the transport of a DNAmitochondrial leader sequence peptide conjugate to mitochondria using cationic mitochondriotropic vesicles, the liberation of this conjugate from the cationic vector upon contact with the mitochondrial outer membrane followed by DNA uptake via the mitochondrial protein import machinery. For the design of cationic mitochondriotropic vesicles we have utilized the self-assembly behavior of dequalinium, a cationic single-chain bola-amphiphile which is known to selectively accumulate in mitochondria. We found that such bola-amphiphiles are able to form liposome-like cationic vesicles (“bolasomes”), which we termed “DQAsomes” when prepared from dequalinium [7, 8]. Data will be presented showing that DQAsomes fulfill all essential prerequisites for a mitochondriaspecific DNA delivery system. [1] Z. M. Chrzanowska, et al. Gene Ther 2 (1995) 311 [2] G. G. M. D’Souza, V. Weissig. Current Gene Therapy (2004) in print [3] G. Manfredi et al. Nat Genet (2002) 394 [4] H. T. Jacobs. J Mol Evol 32 (1991) 333 [5] J. F. Allen. Phil Trans R Soc Lond 358 (2003) 19 [6] V. Weissig, V. P. Torchilin. Curr Pharm Biotechnol 1 (2000) 325 [7] V. Weissig et al. Pharm Res 15 (1998) 334 [8] V. Weissig et al. S.T.P. Pharma Sciences 11 (2001) 91 Dr. Weissig is the Chairman of the Scientific Advisory Board of MitoVec, Inc. and has financial interest in this company. Dr. Weissig’s patents pertaining to the DNA delivery to mitochondria have been licensed by MitoVec, Inc.

684. Expanded Parvoviral Replicons as Novel Nonviral Vectors for Cancer Gene Therapy Boris R. A. Blechacz,1 Jean Rommelaere,2 Christiane Dinsart,2 Brandenburger Annick,3 Russell J. Stephen.1 1 Molecular Medicine Program, Mayo Clinic, Rochester, MN; 2 Applied Tumor Virology Program, German Cancer Research Center, Heidelberg, Germany; 3IRIBHN-IBMM, Universite Libre de Bruxelles, Gosselies, Belgium. Gene therapy is an appealing approach to the treatment of human malignancies but significant barriers exist. The use of viral gene therapy vectors is limited due to immunogenicity and toxicity. Nonviral gene transfer systems achieve only low transduction efficiencies. Autonomous parvoviruses are known for their oncotropism and oncosuppressive potential. In addition, recombinant parvoviruses provide high level transgene expression. We therefore developed and optimized parvoviral replicon vectors based on the autonomous parvovirus Minute Virus of Mice (MVMp) and MVMp/EBV (Epstein Barr virus) hybrid vectors. The use of parvoviral replicon vectors will take advantage of the tumor cellspecific cytotoxicity of parvoviruses. Gene delivery of these replicons as SPLP-liposomes will be nontoxic and non-immunogenic allowing repetitive administration. Transgene size, normally limited by inefficient encapsidation of recombinant parvoviruses exceeding a total size of 106% of the wildtype, is not limited in these parvoviral replicon vectors. The parvoviral genome is excised, replicated and expressed in tumor cells resulting in high plasmid copy numbers and high-level transgene expression. Comparison to other highly potent expression vectors, replicative and non-replicative, showed the desirable characteristics of parvoviral replicon vectors. We expect high level cytotoxicity on tumor cells with major bystander effects by using these MVMp-based replicon vectors for expression of the human sodium iodide symporter gene (hNIS) and the Gibbon ape leukaemia virus fusogenic membrane glycoprotein gene (Galv-FMG). Both of these therapeutic genes have proved their strong antitumoral potential in different tumor-models in the past. By packaging these therapeutic MVMp-replicons in SPLP-liposomes we expect a highly S259

WHAT’S HOT IN THE LUNG: RECENT ADVANCES IN LUNG GENE THERAPY efficient vector for cytoreductive cancer gene therapy. Results: MVMp and MVMp/EBV-hybrid replicon vectors containing the marker genes CEA, luciferase and GFP and the therapeutic transgenes Galv-FMG and hNIS have been cloned. Excision and replication of parvoviral DNA has been shown as early as 24 h after transfection. High-level transgene expression in different cell lines was achieved. Excision, replication and transgene expression has been shown up to an increase of the parvoviral genome size of 355% of the wildtype. Comparison to CMV-promoter driven replicative and non-replicative expression vectors showed higher and longer transgene expression levels by the parvoviral replicon vectors. Transgene expression by MVMp and MVMp/EBV-hybrid vectors increased over time in dependence of the susceptibility of the cell line towards MVMp and EBV. Parvoviral replicons containing Galv-FMG were shown to have a very strong cytotoxic potential resulting in an almost complete destruction of susceptible cell layers. Transduction of cells with hNIS expressing parvoviral replicon vectors resulted in high level 125I-uptake. Altogether these results show the high potential of parvoviral replicons as vectors for cancer gene therapy.

WHAT’S HOT IN THE LUNG: RECENT ADVANCES IN LUNG GENE THERAPY 685. Epithelium-Specific Gene Therapy Vector Protects Cftr Knockout Mice from Acute Lung Infection David R. Koehler,1 Umadevi Sajjan,1 McKerlie Colin,2 Allan Coates,1 Keith Tanswell,1 Forstner F. Janet,2 Jim Hu.1 1 Programme in Lung Biology Research, Hospital for Sick Children, Toronto, ON, Canada; 2Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, ON, Canada. Development of safe and effective vectors remains a major challenge for lung gene therapy. We developed a helper-dependent adenoviral vector for cystic fibrosis (CF) lung gene therapy, using epithelium-specific control elements to drive expression of the CF transmembrane conductance regulator (Cftr) gene. The vector expressed properly localized CFTR in human CF bronchial epithelial cells cultured at an air-liquid interface. Following a single intranasal dose, the vector expressed human CFTR specifically in the airway epithelium of mice, for up to 3 months. Acute inflammation was minimal to moderate. CFTR protein was also detected in the airway epithelium of 2 month-old mice that received one vector dose postnatally. Delivery of the vector to rabbit lung, in an aerosol containing surfactant, yielded strong gene expression (lacZ reporter) specifically in the airway epithelium, including trachea, bronchi, and bronchioles. To test the therapeutic potential of the vector, we challenged adult mice with a clinical strain of the CF-associated pathogen Burkholderia cepacia complex (Bcc). Cftr knockout mice, but not wild-type littermates, challenged with aspirated Bcc developed severe lung histopathology and had high lung bacteria counts. Cftr knockout mice receiving the Cftr gene therapy vector 1 week before Bcc challenge had less severe histopathology, and the number of Bcc in their lungs was reduced to the level seen in wild-type littermates. These results suggest Cftr gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens.

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686. CAR-Expression Is Essential for Tumor Inititation in Lung Cancer Xenografts Min Qin,1 Brian Escuadro,1 Ling Zhang,1 Sherven Sharma,1 Raj K. Batra.1 1 UCLA Lung Cancer Research Program, UCLA/WLA-VAMC, Los Angeles, CA. Rationale: CAR, the Coxsackievirus Adenovirus Receptor, has primarily been studied in its role as the initial cell surface attachment receptor for Coxsackie and group C-Adenoviruses (Ad). Recent studies indicate that CAR serves to mediate homotypic cell-cell adhesion as a component of the tight junction and/or adherens junction. CAR’s role in tumorigenesis has been considered, but no mechanistic studies probing this role have been reported. Experimental Design: Non-small cell lung cancer (NSCLC) cells with exuberant CAR expression (FACS: 99.9% positive w/ MCF 55.2) were stably transfected with control and AS-CAR vectors, and CAR(-)clones isolated (FACS: 1.9% positive w/ MCF 9.6). Cells were phenotypically characterized and tumorigenesis was compared in xenograft models. In separate experiments, CAR was functionally blocked using a monoclonal antibody and a specific ligand (Ad fiber knob), and the impact on tumor formation assessed. Results: Expectedly, CAR (-) cells were refractory to Adtransduction and demonstrated marked reductions in CAR-mRNA by RT-PCR. Silencing CAR expression in cells that exhibited relatively “high” surface expression of this molecule profoundly inhibited their ability to develop xenografts in host mice (Day 40 tumor volume: CAR (+) cells 574 + 304 mm3, CAR (-) cells 0 mm3; n = 10 scid/scid mice, p