- Email: [email protected]
113. Transduction of the Vasculature Using rAAV6
inhibiting its pathway; we have generated a soluble form of the Hip protein lacking its transmembrane domain (HIP-d-22) which may be used to block Shh extracellular diffusion , thus preventing its binding to the Patched receptor. We have demonstrated that recombinant HIP-d-22, secreted in the medium oftransfected 293 cells , is able to bind Shh. In addition , HIP-d-22 expression in the mesenchymal cell line C3H IOTI/2 inhibits Shh-induced osteogenic differentiation. We have tested HIP-d-22 expression in the murine retina following administration of AAV vectors which were then injected subretinally in a mouse model of retinopathy of prematurity (ROP). This resulted in reduction of retinal neovascularization, suggesting the ability of the HIP-d-22 decoy receptor to block the Shh pathway in this model. The second approach we are testing is to inhibit Shh via short interfering RNAs (siRNA) complementary to Shh. We have designed five different siRNA oligos in regions of identity between human and murine Shh mRNA. An oligo resulting in >70% reduction ofShh expression in 293 transfected cells have been selected. Periocular injection of the selected oligo results in reduction ofShh expression in the retina of both adult C57B16 and ROP mice. The effect of periocular injections of the SI-II-1 siRNA on retinal neovaseularization development in ROP mice is being tested. Our results confirm that the Shh pathway plays a key role in the induction of ocular ncovascularization. Importantly, such data suggest two novel strategies for short and long term inhibition of this pathway to be exploited for the treatment ofocular neovascular diseases.
113. Transduction of the Vasculature Using rAAV6 Akshay Krishnamurty,' Brian Schultz,' James MiAllen,' Eric Finn,' Caitlin Doremus,' Luke M. Judge,' Morayma Reyes,' Jeffrey S. Chamberlain.'
[Neurology, University ofWashington, Seattle, W:4 ; 2patllOlogy, University ofWashington, Seattle, H~ .
Our lab has previously reported that intravascular delivery of rAAV6 vectors can transduce nearly 100% of the striated muscle cells in adult mice (Gregorevic et al, Nat Med 2004). These studies raise the question ofwhether rAAV6 is also able to transduce vascular cells. As vasculature endothelial cells (EC) and smooth muscle cells can both proliferate in vivo and since AAV does not integrate in the genome efficiently, we used cre-loxP technology to study transduction of vasculature in vivo. AAV6 constructs expressing Cre recombinase were injected into the tail veins ofloxP·ROSA26 mice. Cre expression in cells of these mice results in excision of a loxls-flankcd DNA segment that normally prevents expression of a lacZ gene, thus allowing LacZ expression. Three viral titers were used, 10"1 I, 10"12, 10"13 vector genomes (vg). Four weeks after injection, muscle mononuclear cells were harvested from tibialis anterior, quadriceps and gastrocnemius muscles for culture in endothelial cell growth medium. Staining of muscle EC cells for beta-Gal expression (at multiple times during a 10 week culture) revealed 35-40% positive cells. In addition, tissue sections of the heart, liver, GI, spleen, esophagus and multiple skeletal muscles were analyzed for LaeZ staining. We used multiple EC (vWF, CD3 I, CD34, Sca-I) and smooth muscle (smooth muscle actin and smooth muscle myosin) markers to detennineAAV transduction cfficiency ofthese two types of vascular cells in these tissues. Highest transduction was observed in the vessels of the heart and skeletal muscles obtaining approximately 50% at the lowest dose but nearly 100% at the highest dose (10"13). Vessels ofthe liver, GI and spleen displayed variable transduction at the lowest and intermediate dose, but more than 50% at the highest dose. TheAAV6-CMV-Cre vector was also injected into ROSA2610xP-GFP.These mice constitutively express lacZ. In the presence ofCre recombinase, lacZ expression is replaced with eGFP expression . Mice were injected with 10"13 Molecular Therapy Volume 15 ~ Supplement I. May 2007 Copyright © Th e AmericanSociety of Gf,.'Jl1; Therapy
vg prior to analysis of the transduction efficiency of cells from bone marrow and skeletal muscles. Whereas very poor transduction was observed in bone marrow cells «1% CD45+ cells, <0.5% of Sca-I+/CD34+/CD45+ cells were GFP+) higher transduction was observed in muscle tissue (10% of all muscle mononuclear cells were GFP+ , and ofthose 30% expressed endothelial markers such as Sea-I+/34+/31+). These cells were sorted and cultured in endothelial medium to demonstrate that they were bona fide ECs. In addition, freshly sorted GFP+ ECs were 1M transplanted into wt mice. Two weeks later GFP+ cells were observed in multiple vessels and perivascular tissues of the injected muscle. The ability of rAAV6 to efficiently transduce the vasculature when administered systemically suggests a potential application to diseases of the vasculature such as ischemia and atherosclerosis.
114. AAV-Mediated Delivery of Paraoxonase 1 (PON1) to the ApoE-J- Mouse as Gene Therapy for Atherosclerosis Aideen O 'Doherty,' Mira Rosenblat.' Michael Aviram,' Timothy O'Brien.'
[Regenerative Medicine Institute, National University ofIreland, Galway, Ireland; "I he Lipid Research Laboratory; Rambam Medical Center, Haifa, Israel.
High-density lipoproteins (HDL) possess a protective effect aga inst LDL oxidation and the enzyme Paraoxonase I (PONI) is thought to stabilise HDL and enhance its effects. This makes it an ideal candidate for gene therapy for atherosclerosis and coronary heart disease, among the primary causes of death in the Western world. ApoE knockout (apoli-z-) mice are an established model of atherosclerotic disease, displaying advanced atherosclerotic aortic lesions and a marked increase in total plasma cholesterol levels. We generated high titre pseudotyped adenoassociated virus (AAV) serotype 2/5 stock using a plasmid construct for human PON I under a CMV promoter within AAV2 ITRs and a plasmid for AAV5 (pDF5), co-transfected in 2931' cells. We treated 8-10 week old apoE-/- mice with 2.5xI0'" DNAse-resistant particles (drp) of pscudotypcd Aavd-Pobll or AAV5-GFPand fed them a " Western" diet with 42% fat and 0.15% cholesterol, Expression ofPONI was observed in the injected muscle. We found atheroprotective effects at 5 weeks post-PON I treatment, relative to GFP-treated controls, with an approximately 3-fold difference in atherosclerotic lesion coverage between the groups (relative lesion area values 440.49 ± 349.08 versus 1274.77 ± 436.57; *p = 0.0245). These data suggest that systemic expression ofPONl , delivered by AAV, may be therapeutically beneficial in reducing atherosclerotic burden.
115. Adeno-Associated Virus 2-Based Gene Delivery of the Angiotensin II Type 2 Receptor Reduces Atherosclerosis and Associated Markers in LDLR Knockout Mice
Changping Hu,' Abhijit Dandapat,' Paul L. Hermonat,' Yong Liu,' Jawahar L. Mehta.'
[InternalMedicine, University ofArkansasfor Medical Sciences, Little Rock, AR.
Angiotensin II (Ang II), via type I receptor (AT IR), activation exerts a significant role in atherogenesis and collagen synthesis, while the angiotensin II type 2 receptor (AT2R) antagonizesATIR. We hypothesized that AT2R gene delivery by adeno-associatcd virus type 2 (AAV/AT2R) may inhibit arterial atherosclerosis and collagen synthesis. To test the hypothesis , LDLR knockout (KO) mice were tail vein injected withAAV/Ar2R and fed a4%cholesterol diet for 18 weeks. AAV/AT2R delivery into LDLR KO mice reduced atherosclerosis in the aorta (P
113. Transduction of the Vasculature Using rAAV6 Akshay Krishnamurty,' Brian Schultz,' James MiAllen,' Eric Finn,' Caitlin Doremus,' Luke M. Judge,' Morayma Reyes,' Jeffrey S. Chamberlain.'
[Neurology, University ofWashington, Seattle, W:4 ; 2patllOlogy, University ofWashington, Seattle, H~ .
Our lab has previously reported that intravascular delivery of rAAV6 vectors can transduce nearly 100% of the striated muscle cells in adult mice (Gregorevic et al, Nat Med 2004). These studies raise the question ofwhether rAAV6 is also able to transduce vascular cells. As vasculature endothelial cells (EC) and smooth muscle cells can both proliferate in vivo and since AAV does not integrate in the genome efficiently, we used cre-loxP technology to study transduction of vasculature in vivo. AAV6 constructs expressing Cre recombinase were injected into the tail veins ofloxP·ROSA26 mice. Cre expression in cells of these mice results in excision of a loxls-flankcd DNA segment that normally prevents expression of a lacZ gene, thus allowing LacZ expression. Three viral titers were used, 10"1 I, 10"12, 10"13 vector genomes (vg). Four weeks after injection, muscle mononuclear cells were harvested from tibialis anterior, quadriceps and gastrocnemius muscles for culture in endothelial cell growth medium. Staining of muscle EC cells for beta-Gal expression (at multiple times during a 10 week culture) revealed 35-40% positive cells. In addition, tissue sections of the heart, liver, GI, spleen, esophagus and multiple skeletal muscles were analyzed for LaeZ staining. We used multiple EC (vWF, CD3 I, CD34, Sca-I) and smooth muscle (smooth muscle actin and smooth muscle myosin) markers to detennineAAV transduction cfficiency ofthese two types of vascular cells in these tissues. Highest transduction was observed in the vessels of the heart and skeletal muscles obtaining approximately 50% at the lowest dose but nearly 100% at the highest dose (10"13). Vessels ofthe liver, GI and spleen displayed variable transduction at the lowest and intermediate dose, but more than 50% at the highest dose. TheAAV6-CMV-Cre vector was also injected into ROSA2610xP-GFP.These mice constitutively express lacZ. In the presence ofCre recombinase, lacZ expression is replaced with eGFP expression . Mice were injected with 10"13 Molecular Therapy Volume 15 ~ Supplement I. May 2007 Copyright © Th e AmericanSociety of Gf,.'Jl1; Therapy
vg prior to analysis of the transduction efficiency of cells from bone marrow and skeletal muscles. Whereas very poor transduction was observed in bone marrow cells «1% CD45+ cells, <0.5% of Sca-I+/CD34+/CD45+ cells were GFP+) higher transduction was observed in muscle tissue (10% of all muscle mononuclear cells were GFP+ , and ofthose 30% expressed endothelial markers such as Sea-I+/34+/31+). These cells were sorted and cultured in endothelial medium to demonstrate that they were bona fide ECs. In addition, freshly sorted GFP+ ECs were 1M transplanted into wt mice. Two weeks later GFP+ cells were observed in multiple vessels and perivascular tissues of the injected muscle. The ability of rAAV6 to efficiently transduce the vasculature when administered systemically suggests a potential application to diseases of the vasculature such as ischemia and atherosclerosis.
114. AAV-Mediated Delivery of Paraoxonase 1 (PON1) to the ApoE-J- Mouse as Gene Therapy for Atherosclerosis Aideen O 'Doherty,' Mira Rosenblat.' Michael Aviram,' Timothy O'Brien.'
[Regenerative Medicine Institute, National University ofIreland, Galway, Ireland; "I he Lipid Research Laboratory; Rambam Medical Center, Haifa, Israel.
High-density lipoproteins (HDL) possess a protective effect aga inst LDL oxidation and the enzyme Paraoxonase I (PONI) is thought to stabilise HDL and enhance its effects. This makes it an ideal candidate for gene therapy for atherosclerosis and coronary heart disease, among the primary causes of death in the Western world. ApoE knockout (apoli-z-) mice are an established model of atherosclerotic disease, displaying advanced atherosclerotic aortic lesions and a marked increase in total plasma cholesterol levels. We generated high titre pseudotyped adenoassociated virus (AAV) serotype 2/5 stock using a plasmid construct for human PON I under a CMV promoter within AAV2 ITRs and a plasmid for AAV5 (pDF5), co-transfected in 2931' cells. We treated 8-10 week old apoE-/- mice with 2.5xI0'" DNAse-resistant particles (drp) of pscudotypcd Aavd-Pobll or AAV5-GFPand fed them a " Western" diet with 42% fat and 0.15% cholesterol, Expression ofPONI was observed in the injected muscle. We found atheroprotective effects at 5 weeks post-PON I treatment, relative to GFP-treated controls, with an approximately 3-fold difference in atherosclerotic lesion coverage between the groups (relative lesion area values 440.49 ± 349.08 versus 1274.77 ± 436.57; *p = 0.0245). These data suggest that systemic expression ofPONl , delivered by AAV, may be therapeutically beneficial in reducing atherosclerotic burden.
115. Adeno-Associated Virus 2-Based Gene Delivery of the Angiotensin II Type 2 Receptor Reduces Atherosclerosis and Associated Markers in LDLR Knockout Mice
Changping Hu,' Abhijit Dandapat,' Paul L. Hermonat,' Yong Liu,' Jawahar L. Mehta.'
[InternalMedicine, University ofArkansasfor Medical Sciences, Little Rock, AR.
Angiotensin II (Ang II), via type I receptor (AT IR), activation exerts a significant role in atherogenesis and collagen synthesis, while the angiotensin II type 2 receptor (AT2R) antagonizesATIR. We hypothesized that AT2R gene delivery by adeno-associatcd virus type 2 (AAV/AT2R) may inhibit arterial atherosclerosis and collagen synthesis. To test the hypothesis , LDLR knockout (KO) mice were tail vein injected withAAV/Ar2R and fed a4%cholesterol diet for 18 weeks. AAV/AT2R delivery into LDLR KO mice reduced atherosclerosis in the aorta (P