422 Development of HIV 1 Based Lentiviral Vectors Expressing Pre Trans Splicing Molecules (PTMs) for Liver Directed Therapies Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The Americ[.]
GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY II by fasting blood glucose levels at 3, 6, and 10 weeks following AAV administration Moreover, a glucose tolerance test at months post administration also showed no effect of PGC-1α expression Western blotting indicates that PGC-1α was increased about 2-3 fold in the high dose group versus control animals RT-PCR of DNA isolated from the liver of the AAV-treated animals indicate that our gene was delivered in a dose-dependent fashion and liver histology will also be examined for any signs of immune response to transgene expression or other abnormalities These results suggest that PGC1α over-expression in the liver may not result in disregulation of gluconeogenesis and a diabetic phenotype 421 Promoter Selection for AAV VectorMediated Factor IX Expression in Skeletal Muscle Akihiro Kume,1 Hiroya Yagi,1 Hiroaki Mizukami,1 Masashi Urabe,1 Tomonori Tsukahara,1 Akira Ishiwata,2 Jun Mimuro,2 Seiji Madoiwa,2 Tsukasa Ohmori,2 Yoichi Sakata,2 Keiya Ozawa.1 Genetic Therapeutics, Jichi Medical University, Shimotsuke, Tochigi, Japan; 2Cell and Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan Hemophilia is an X-linked bleeding diathesis caused by a deciency of either coagulation factor VIII (FVIII; hemophilia A) or factor IX (FIX; hemophilia B) While rened management with coagulation factor products has signicantly improved the prognosis, regular supplementation of FVIII or FIX would be preferable for prophylaxis Gene therapy is an ideal modality for this purpose, and adeno-associated virus (AAV) vectors are promising vehicles for gene delivery to target tissues such as liver and muscle In developing AAV vectors for hemophilia, efcient promoters are required to achieve therapeutic levels of FVIII or FIX, while tissue-specic expression is desirable for safety reasons These tasks are particularly challenging with AAV vectors, because the size of their genome is strictly limited Considering the accessibility and safety, skeletal muscle is a superb target tissue for hemophilia gene therapy Therefore we investigated the efcacy of several small-sized promoters including cytomegalovirus promoter (CMV), truncated form of elongation factor-1 promoter (EFS), muscle creatine kinase minimal promoter linked with one (MCK) or two (dMCK) enhancer core elements In our reporter constructs, these promoters were placed to drive the human FIX (hFIX) gene Human embryonic kidney 293 cells, Huh7 human hepatoma cells, C2C12 mouse myoblasts were transfected with the expression plasmids, and the hFIX in the culture medium was examined by ELISA and immunoblot As expected, CMV promoter was strongest in 293, Huh7 and undifferentiated C2C12 cells while MCK and dMCK promoters had trace activity During C2C12 differentiation into myotubes, however, MCK and dMCK promoters demonstrated greater activity than other promoters Based on this result, we constructed serotype 1-pseudotyped self-complementary AAV vectors with CMV-hFIX (AAV1/CMV-hFIX) and dMCK-hFIX (AAV1/dMCK-hFIX) for in vivo evaluation × 1011 genome copies of the AAV vectors were injected to the hindlimb muscle of C57BL/6 mice and plasma hFIX was measured by ELISA The hFIX levels were gradually increased and reached to 4.9 ± 3.7 % with AAV1/ CMV-hFIX (n = 4) and 2.1 ± 2.9 % with AAV1/dMCK-hFIX (n = 4) at 11 weeks postinjection The hFIX expression was maintained at these levels up to 25 weeks The reason for the discrepancy between in vitro and in vivo hFIX expression, i.e CMV promoter worked better than dMCK in vivo, is currently under investigation Since the plasma hFIX level was generally low and signicantly deviated even in each cohort, intramuscular gene delivery might not be successful The animals were sacriced and tissue DNA analysis is ongoing Much more potent promoter may be required for in vivo expression following muscle-directed gene transfer using AAV vectors.α Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy 422 Development of HIV-1 Based Lentiviral Vectors Expressing Pre-Trans-Splicing Molecules (PTMs) for Liver-Directed Therapies Madaiah Puttaraju,1 S Gary Manseld,1 Jun Wang,1 Nikolay Korokhov,1 Jenice D’Costa,1 Ziping C Chen,1 Katherine Radd,1 Gerard J McGarrity,1 Laurent M Humeau.1 VIRxSYS Corporation, Gaithersburg, MD Spliceosome mediated RNA trans-splicing (SMaRT) is one of the few RNA-based technologies that can potentially restrict the production of a protein of therapeutic interest to a specic cell type or organ We combined this RNA trans-splicing technology with the company’s proven lentiviral vector (LVs) delivery system to maximize PTM delivery to target cells and achieve persistent gene expression We generated VSV-G pseudotyped HIV-1 based LVs encoding a PTM that trans-splices human apolipoprotein A-I (hapoA-I) into highly abundant human albumin pre-mRNAs in hepatocytes to increase blood levels of this protein and ultimately correct familial apolipoprotein A-I deciency This PTM cassette contains a binding domain targeted to intron of human albumin, splice acceptor sequences and hapoA-I coding sequence Initial testing of these LV-hapoA-I PTMs were performed in HepG2, and human primary hepatocytes These cell types produce and process albumin and apoA-I, and therefore are ideal cell models for pre-testing clinical candidate LV-hapoA-I PTMs The low conservation of intron sequences among species precludes the use of most animal models for pre-testing our clinical candidate human PTMs HepG2 cells and hepatocytes were transduced efciently by LV-PTMs and analysis of RNA from these cells by RT-PCR for transcripts containing albumin exon fused to hapoA-I coding sequences conrmed accurate transsplicing into human albumin pre-mRNAs We also quantied levels of hapoA-I PTM RNAs, albumin pre-mRNAs, trans-spliced mRNA, and LV copies per cell by qRT-PCR and qPCR, respectively; all four parameters showed clear dose response relationships Increased MOIs produced a linear increase in the number of LV copies per cell, and cell samples with higher copy values had higher levels of unspliced PTM RNA Similarly, analysis of trans-splicing level showed a close linear relationship with PTM RNA (Figure) and produced in the range of 5-20 copies of trans-spliced chimeric mRNA per cell in HepG2 and 1-5 copies in human primary hepatocytes In all samples analyzed a higher level of PTM RNA or target pre-mRNA resulted in higher levels of trans-splicing These results demonstrate: successful integration of the company’s LV delivery platform with SMaRT technology, trans-splicing to a true endogenous pre-mRNA target in human primary hepatocytes, and conrm and extend our previous observations of the intimate relationship between PTM RNA, target pre-mRNA and trans-splicing Currently, experiments are in progress to assess the therapeutic potential of these LV-PTMs in engineered animal models Results from these studies will serve as a foundation for the future development of liver directed RNA-based therapies S163 CARDIOVASCULAR GENE & CELL THERAPY GSK-3β and FoxO3a, was reduced in the control and normalized in the AAV-VEGF-B animals Cardiac VEGFR-1 expression was reduced four-fold in all paced dogs, suggesting that exogenous VEGF-B exerted a compensatory receptor stimulation Together, these results point toward VEGFR-1 signalling in the heart as an important mediator of cardiomyocyte function, with clear therapeutic implications 424 Osteopontin Enhances the Angiogenic Potential of Endothelial Progenitor Cells Erin E Vaughan,1 Angela Duffy,2 Timothy O’Brien.1 Regenerative Medicine Institute, National University of Ireland, Galway, Ireland; 2Medtronic, Galway, Ireland Cardiovascular Gene & Cell Therapy 423 AAV-Mediated Intramyocardial VEGF-B167 Expression Exerts a Potent Cardioprotective Effect in Small and Large Animal Models of Myocardial Infarction and Heart Failure Lorena Zentilin,1 Vincenzo Lionetti,2 Uday Puligadda,1 Silvia Moimas,1 Serena Zacchigna,1 Martino Pepe,3 Mohammed Mamdani,3 Khaled Qanud,3 Xiabin Xu,3 Thomas H Hintze,3 Fabio A Recchia,3 Mauro Giacca.1 International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy; 2Scuola Superiore Sant’Anna, Pisa, Italy; New York Medical College, Valhalla, NY VEGF-B, a member of the vascular endothelial growth factor family, is emerging as a major cardiovascular protecting factor We have recently observed, in a rat model of acute myocardial infarction, that the intramyocardial injection of AAV2-VEGF-B167, which selectively binds VEGFR-1, signicantly improves both regional and global cardiac contractility and maintains favorable tissue remodeling over time in the absence of a signicant angiogenic response (Zentilin et al 2010 FASEB J., in press) We found that cardiomyocytes expressed VEGFR-1, VEGFR-2 and Neuropilin-1 and that, in particular, VEGFR-1 was specically upregulated upon exposure of cardiomyocytes to hypoxia/reoxygenation or cardiotoxic drugs Histological and molecular analysis highlighted a marked antiapoptotic effect exerted both in vitro on isolated neonatal cardiomyocytes and in vivo after induction of ischemia We now show that VEGF-B is capable to elicit a compensatory, hypertrophic response mediated by the up-regulation of genes involved in intracellular calcium transients such as ryanodyne receptors and SERCA2a, as well as of PGC-1a, a powerful regulator of mitochondrial metabolism and cardiac energetics Consistent with this conclusion, we found that VEGF-B exerts a similarly striking, angiogenesis-unrelated cardioprotective effect in a pacing-induced model of non-ischemic heart failure in chronically instrumented dogs Control animals subjected to weeks of continuous pacing suffered from an overt congestive heart failure; on the contrary, at the same time point, intramyocardial AAV9-mediated VEGF-B expression maintained heart contractility and prevented LV wall thinning Also in this model, apoptosis, revealed as a number of TUNEL positive cardiomyocytes and cleaved caspase 3, was signicantly reduced in myocardium expressing VEGF-B In a consistent manner, phosphorylation of Akt, a major negative regulator of apoptosis, was superphysiological in AAV-VEGF-B treated tissue, while the inhibitory phosphorylation of the pro-apoptotic intracellular mediators S164 Patients with Type Diabetes Mellitus (T1DM) often demonstrate an inability to generate an appropriate angiogenic response to ischemia One possible reason for this may be due to a decrease in both the number and function of endothelial progenitor cells (EPCs) We have previously shown that EPCs cultured from patients with T1DM are impaired in their ability to adhere to activated endothelial cells as well as their ability to induce tubule formation in a matrigel assay Further, we observed a down- regulation of the angiogenic protein osteopontin (OPN) in diabetic EPCs We hypothesized that the down regulation of OPN may contribute to the impaired function of diabetic EPCs Interestingly, when diabetic EPCs were pre-incubated with recombinant OPN for 24 hours their ability to induce tubule formation was restored to levels observed from healthly control EPCs In vitro, this suggests that OPN plays an important role in EPC-mediated angiogenesis To further explore the role of OPN in EPC mediated angiogenesis, we isolated EPCs from OPN knockout mice and found they too were impaired in their ability to bind activated endothelial cells and form tubules in a matrigel tubule assay Further, this impairment was reversed upon exposure to OPN We went on to determine that conditioned media (CM) from wild-type (WT) EPCs was sufcient to enhance tubule formation, suggesting that secreted factors are responsible for the EPC-mediated angiogenic response Interestingly, we found that incubation of the CM with an osteopontin neutralizing antibody did not abrogate tubule formation Thus, it does not appear that OPN acts directly on the endothelial cells to induce tubule formation Therefore, we hypothesized that OPN may be acting in an autocrine manner on EPCs to induce the secretion of additional angiogenic proteins Indeed, using a chemi-array, we found that exposure of KO EPCs to OPN induced the release of IL-6, TGF alpha, and FGF alpha to levels secreted by WT cells To assess the role of OPN in vivo, a murine model of hind-limb ischemia was used Ischemia was induced in the left limb of OPN KO animals and laser doppler blood ow measurements were obtained pre- and post-operatively as well as at days and 14 post surgery At the time of surgery an intramuscular injection of: diluent (control);1 million OPN KO EPCs; or million KO EPCs pre-incubated with recombinant OPN was performed At day 7, signicant improvement was noted in the mice injected with KO EPCs pre-incubated with OPN and this improvement was enhanced at day 14 There was no signicant improvement noted with the injection of KO EPCs alone Taken together this data demonstrates that the pre-treatment of OPN KO EPCs with recombinant OPN results in an improved response to induced hind limb ischemia Our data suggests this may be due to the induced secretion of angiogenic proteins Further, we have shown that the treatment of diabetic EPCs with OPN improves their angiogenic potential in vitro Although further research is needed, this is a promising step for the future of autologus cell therapy in the treatment of ischemia related conditions Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ... (Zentilin et al 2 010 FASEB J., in press) We found that cardiomyocytes expressed VEGFR -1, VEGFR-2 and Neuropilin -1 and that, in particular, VEGFR -1 was specically upregulated upon exposure of cardiomyocytes... days and 14 post surgery At the time of surgery an intramuscular injection of: diluent (control) ;1 million OPN KO EPCs; or million KO EPCs pre- incubated with recombinant OPN was performed At... autologus cell therapy in the treatment of ischemia related conditions Molecular Therapy Volume 18 , Supplement 1, May 2 010 Copyright © The American Society of Gene & Cell Therapy