- Email: [email protected]
557. Distinct Integration Profiles after Sleeping Beauty Transposase-Mediated Somatic Integration from AAV and Adenoviral Vectors
ADENOVIRUS AND OTHER DNA VIRUS VECTORS II 555. Liver-Detargeted Ad5/48 Chimaeric Hexon Based Oncolytic Adenoviruses Targeting TGFβ Signaling: A Safe and Effective Approach for the Treatment of Prostate Cancer Bone Metastases
Weidong Xu, Zhenwei Zhang, Zebin Hu, Stuart Stock, Charles Brendler,1 Prem Seth.1 1 Medicine and Surgery, NorthShore Res Inst, University of Chicago, Evanston, IL; 2Molcular Pharmacology, Northwestern University, Chicago, IL. 1
1
1
2
Background: There is a tremendous need to develop novel approaches for the treatment of prostate cancer bone metastases. We have previously shown that systemic delivery of oncolytic Adenovirus 5 (Ad5) expressing soluble transforming growth factor receptorII-Fc protein (Ad.sTRFc) inhibited bone metastases in a mouse prostate tumor model. However, the majority of the Ad.sTRFc was taken up by the liver causing hepatic and systemic toxicities. Since Ad5 uptake in the liver is primarily due to the binding of hexon with blood factor X (FX), and that Ad48 hexon has reduced binding with FX, to prevent the adenoviral-induced liver and systemic toxicity, a chimaeric Ad5/48 adenovirus mHAd.sTRFc was created. Our goal is to examine the safety and efficacy of mHAd.sTRFc in a mouse model of prostate cancer bone metastases. Methods: mHAd.sTβRFc construction. Seven hypervariable regions of Ad48 hexon were genetically inserted in Ad5 expressing sTGFRIIFc gene. Liver toxicity studies. Following intravenous delivery of adenoviruses, HE staining of the liver tissues were performed to evaluate liver necrosis. Plasma liver alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured. Bone metastasis model. PC3-luc2 cells were injected in left heart ventricle of nude mice. In mice bearing skeletal tumors, vectors were injected via tail vein. Bone metastases were monitored real time by bioluminescence imaging (BLI) and X-ray radiography. Tumor burden was analyzed by HE staining; ostoclasts were measured by tartarate resistant acid phosphates (TRAP) staining, plasma calcium levels were determined, and microCT analyses of the bones were performed. Results: Systemic delivery of a low dose (2.5x1010 viral particles (VPs)/mouse or a high dose (1011 VPs/ mouse ) of Ad5 based Ad.sTRFc produced significant liver necrosis and a transient increase in ALT and AST levels; the high dose (1011 VPs/mouse) killed all the mice by day 3. On the other hand, either low or high dose of Ad5/48 chimaeric mHAd.sTRFc virus did not produce liver damage or increased plasma ALT and AST levels; a dose (up to 2x1011 VPs/mouse tested thus far) did not cause animal deaths. There was a much reduced liver uptake of mHAd.sTRFc compared to Ad.sTRFc. Intravenous delivery of Ad.sTRFc and mHAd.sTRFc (5x1010 VPs/mouse) exhibited a significant inhibition of bone metastases as revealed by BLI and X-ray analyses. The higher dose of mHAd.sTRFc (2x1011 VPs/mouse or 4x1011 VPs/mouse) were more effective than the lower dose (5x1010 VPs/mouse) of Ad.sTRFc or mHAd.sTRFc, and produced a significant reduction in the tumor burden, hypercalcemia and osteoclasts, and produced normal bone architect as revealed by the microCT scan analyses. Conclusion: Ad5/48 chimaeric hexon based oncolytic adenovirus mHAd.sTRFc produce much reduced liver and systemic toxicity and larger and safe dose can be administered to produce superior anti-tumor responses. Thus, mHAd.sTRFc should be tested in the clinical trials in prostate cancer patients.
S214
556. Enhanced Anti-Tumor Efficacy by Oncolytic Adenovirus Using a Stimuli Responsive Block Copolymer mPEG-b-Poly(His)
Joung-Woo Choi,1 You Han Bae,2 Chae-Ok Yun.3 Graduate Program for Nanomedical Science and Technology, Yonsei University, Seoul, Korea; 2Department of Pharmaceutics and Pharmaceutical Chemistry, The University of Utah, Salt lake city, Korea; 3Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Korea. 1
Adenovirus (Ad) has shown great promise in cancer gene therapy. Ad coated with cationic polymers have already been shown to enhance transduction, but it showed nonspecific binding with cells, resulted in evoked side effect. To overcome these hurdles, in this study adenovirus coated with pH-sensitive poly(His) polymer through ionic interaction for passive tumor targeting. We have characterized the effect of polymer concentration on particle size distribution, zeta potential, transduction efficiency, and structural properties. Remarkable enhanced transduction efficiency was observed in cells treated with Ad/poly(His) nanocomplex compared to naked Ad at low pH, which is hallmark of cancer. In addition, cellular trafficking using time lapse total internal reflection fluorescence showed that the Ad/poly(His) nanocomplex significantly enhanced cellular uptake at lower pH, in contrast to neutral pH. The biodistribution study confirmed that the Ad/poly(His) nanocomplex can effectively accumulate at the tumor site by the EPR effect and exhibited 3.3-fold higher luciferase expressions in tumor than the naked Ad. Additionally, MTT assay and cancer cell xenografts studies showed that polymercoated nanocomplex exhibited potent cancer cell killing effect and also inhibited the tumor growth. Furthermore, poly(His)-coated Ad complex was not significantly inhibited by serum, in contrast to naked Ad. Moreover, Ad/poly(His) nanocomplex significantly reduced the innate immune response in contrast to naked Ad, as assessed by interleukin-6 (IL-6) cytokine release from macrophage cells. These results suggest that the stimuli-responsive Ad/poly(His) nanocomplex has potential to targeting tumors.
557. Distinct Integration Profiles after Sleeping Beauty Transposase-Mediated Somatic Integration from AAV and Adenoviral Vectors
Wenli Zhang,1 Christina Rauschhuber,2 Martin Haeusl,2 Nadine Muether,2 Anja Ehrhardt.1 1 Institute for Virology and Microbiology, Private University Witten/Herdecke, Witten, Germany; 2Max von Pettenkofer-Institut, Ludwig-MAximilians-University Munich, Munich, Germany; 3Max Delbrück Center for Molecular Medicine, Berlin, Germany. Hyperactive Sleeping Beauty (SB) transposase systems for somatic integration hold great promise for gene therapeutic applications. For efficient delivery of hyperactive transposase versions such as HSB5 and SB100X, we have focused on the development of viral hybrid-vectors based on high-capacity adenoviral vectors (HC-AdVs) and recombinant adeno-associated virus (rAAV). Both vector types represent promising tools for therapeutic gene delivery because they result in efficient transduction and long-term transgene expression in non-dividing cells. However, as HC-AdV and rAAV mostly remain episomal, vector genomes and transgene expression are lost over time in dividing cells. Therefore, to stably transduce tissues and cells undergoing many cell divisions, we developed HC-AdV/ transposase and AAV/transposase hybrid-vectors for stable transgene expression. The AAV/transposase hybrid-vector system utilizes the hyperactive SB transposase SB100X for somatic integration and was tested in HeLa cells. After performing colony forming assays we observed that in contrast to the inactive mSB control group, the SB100X group showed 10-fold increased numbers of colonies. Based on a plasmid rescue strategy we analyzed the SB100X-mediated Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
ADENOVIRUS AND OTHER DNA VIRUS VECTORS II integration profile after transposition from the AAV vector and of the 91 integration events identified, 50% were located in genes. For the HC-AdV/transposase hybrid-vector we identified 163 independent transposition events from liver of HSB5 treated mice based on a PCR strategy. Of these events 77% were found in non-gene areas and 23% in genes indicating a random integration pattern. In addition, 27 extra-chromosomal integration events were observed, most likely caused by transposon excision and subsequent transposition into the delivered vectors. Furthermore, the chromosomal integration site analysis revealed a strong bias towards integration into the X-chromosome. In summary this study demonstrates for the first time that different integration profiles can be observed dependent on the method and viral vector used for delivery of the transposon. The AAV/transposase hybrid-vector shows a bias towards integration into genes with efficiencies of up to 50%. In contrast, integration site analysis of the HC-AdV/transposase hybrid-vector revealed a random integration profile with respect to integration efficiencies into genes and a strong bias towards integration into the X-chromosome. Notably, the latter phenomenon was not observed when using rAAV. For high resolution of integration events after transposition form HC-AdV and rAAV, a large number of integration sites is currently being analysed by deep sequencing using a LAM-PCR method for both hybrid-vector systems.
558. Screening and Characterization of Alternative Adenovirus Serotypes with Enhanced Virus-Lateralization Ability In Vitro for Improved Antitumor Efficacy
Junji Uchino,1 David T. Curiel,2 Hideyo Ugai.1 Cancer Biology Division, Department of Radiation Oncology, School of Medicine, Washington University in St. Louis, St. Louis, MO; 2Cancer Biology Division, Biologic Therapeutics Center, Department of Radiation Oncology, School of Medicine, Washington University in St. Louis, St. Louis, MO. 1
Human adenovirus serotype 5 (HAdV-C5) is the most widely employed as adenovirus serotype for cancer gene therapy. However, therapeutic efficacy of HAdV-C5 is limited, as its virus progeny are not sufficiently released from infected cells. Therefore, successful cancer gene therapy using HAdV-C5-based vectors requires efficient virus lateralization from an infected tumor cell to surrounding tumors. To overcome this limitation, we sought to develop novel oncolytic adenoviruses with enhanced virus-spread ability. Since there are at least 52 serotypes of human adenoviruses, we characterized the virus-spread ability of other adenovirus serotypes as compared with that of HAdV-C5. We first examined a host range of 16 serotypes selected from species A to F in the human lung carcinoma A549 cells. As a result, 10 serotypes of adenoviruses (HAdV-C2, B3, E4, C5, D9, D10, A31, B34, B35 and D51) were propagated in A549 cells, but 6 serotypes (HAdV-B14, B16, D20, B21, F40 and F41) were not. Therefore, we suggest that the former 10 serotypes of adenoviruses may be suitable for oncolytic adenovirus vectors. We next compared virus-spread ability of these 10 serotypes, along with that of HAdV-C5, by plaque assay. The plaque sizes of HAdV-D9 and HAdV-D51 were larger than those of other serotypes including HAdV-C5. Since the nucleotide sequence of the HAdV-D9 genome is reported, we further characterized the biological function of HAdV-D9. We confirmed that HAdV-D9 was efficiently released from infected cells to culture medium at an earlier time point as compared with HAdV-C5 by one-step growth curve analysis. Also, HAdV-D9 replicated well in MCF-7 breast cancer cells which are CAR-negative. Moreover, we measured cell killing activity of HAdV-D9 along with HAdV-C5 in a broad range of human breast cancer and malignant mesothelioma cell lines by MTS assay. Our results showed that HAdV-D9 more effectively killed 4 of 8 cell lines than HAdV-C5 and similarly induced cytopathic effect in the other cell lines. In order to Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
understand target cells of HAdV-D9, we investigated the HAdV-D9 receptor. The primary receptors of species D has been suggested to be the coxsackie B virus and HAdV receptor (CAR), CD46, sialic acid, or integrins in previous reports. In this study, we assessed human CAR (hCAR) and hCD46 by quantitatively measuring cellular uptake of the HAdV-D9 genome in CHO, CHO-hCAR, and CHO-hCD46. Interestingly, CHO cells as well as CHO-hCAR and CHO-hCD46 were efficiently infected with HAdV-D9 as compared with HAdV-C5. Therefore, we suggest that HAdV-D9 binds to other receptors rather than hCAR and hCD46. Collectively, HAdV-D9 has enhanced virusspread ability, improves cell killing, and targets cells independently of hCAR and hCD46. Therefore, this work supports the consideration of HAdV-D9-based oncolytic therapies for cancer treatment.
559. Improved Animal Models for Testing Gene or Cell Therapies for Atherosclerosis
Liang Du,1 Jingwan Zhang,1 David Dichek.1 Department of Cardiology, Internal Medicine, University of Washington, Seattle, WA. 1
Background: Gene therapy delivered to the blood vessel wall could augment current therapies for atherosclerosis. We reported previously that gene therapy with a helper-dependent adenovirus expressing apo A-I (HDAdapoAI) significantly decreased atherosclerosis 4 wk after infusion into arteries of cholesterol (chol)-fed rabbits. However, lesions in this model (Fig 1: ApoAI study) are small, almost exclusively foam cells, and regress by 8 wk. We therefore developed animal models that would allow testing vascular wall gene therapy on larger, more complex lesions, and for longer periods. Methods: 20 rabbits were allocated into 4 groups with each group receiving a unique combination of diet, vector, and surgical protocol (Fig 1). Group I rabbits were initially fed a very high chol diet, after which their carotids were infused with HDAdNull, then continued on sliding-scale chol for 24 wk. Group II rabbits were fed a high-chol diet, then received HDAdNull along with placement of periarterial collars. Group III rabbits were fed a high-chol diet, then received both HDAdNull and a first-generation adenovirus (FGAdNull). Group IV rabbits were fed a high-chol diet, then continued on sliding scale chol for 24 wk, with no surgery, collar, or vector. Segments of infused carotids were analyzed for intimal lesion area and content and for presence of vector DNA. Results: A high-chol diet alone (Group IV) did not cause intimal growth. Additional “insults” [very high chol diet, surgery, and HDAdNull (Group I), high-chol diet, surgery, HDAdNull, and collar (Group II), or high-chol diet, surgery, HDAdNull, and FGAdNull (Group III)] all caused significant intimal growth. Compared to our original model, intimal lesions in each of Groups I, II, and III were all larger: 6.2×105 ± 5.0 ×105 μm2; 3.1×105 ± 1.7 ×105 μm2; and 1.4×105 ± 5.5 ×104 μm2 for Groups I – III, respectively, compared to 1.5×104 ± 4.3 ×103μm2 in our original model (P < 0.0002 for all). No vector was detectable in Group III arteries whereas substantial vector was present in Group I and II arteries (approx 1 copy per endothelial cell). Group I lesions were more eccentric than Group II lesions, with more macrophages (46 ± 4.6 vs 5.3 ± 1.3% macrophage area; P < 0.0001), more lipid (18 ± 0.90 vs 6 ± 1.7% lipid area; P < 0.0001), and fewer smooth muscle cells (6.3 ± 1.4 vs 39 ± 2.8% SMC area; P < 0.0001). Conclusions: We developed 2 new animal models for testing the efficacy of gene-therapy vectors such as HDAd in preventing or reversing intimal growth and decreasing lipid, SMC, and macrophage accumulation. The model used for Group II is more suitable for testing efficacy in lesions with a predominant SMC component; the Group I model is better suited for testing efficacy in lesions rich in lipid and macrophages. These new models will allow testing of the efficacy of HDAdapoAI on larger lesions and at later time points.
S215
Weidong Xu, Zhenwei Zhang, Zebin Hu, Stuart Stock, Charles Brendler,1 Prem Seth.1 1 Medicine and Surgery, NorthShore Res Inst, University of Chicago, Evanston, IL; 2Molcular Pharmacology, Northwestern University, Chicago, IL. 1
1
1
2
Background: There is a tremendous need to develop novel approaches for the treatment of prostate cancer bone metastases. We have previously shown that systemic delivery of oncolytic Adenovirus 5 (Ad5) expressing soluble transforming growth factor receptorII-Fc protein (Ad.sTRFc) inhibited bone metastases in a mouse prostate tumor model. However, the majority of the Ad.sTRFc was taken up by the liver causing hepatic and systemic toxicities. Since Ad5 uptake in the liver is primarily due to the binding of hexon with blood factor X (FX), and that Ad48 hexon has reduced binding with FX, to prevent the adenoviral-induced liver and systemic toxicity, a chimaeric Ad5/48 adenovirus mHAd.sTRFc was created. Our goal is to examine the safety and efficacy of mHAd.sTRFc in a mouse model of prostate cancer bone metastases. Methods: mHAd.sTβRFc construction. Seven hypervariable regions of Ad48 hexon were genetically inserted in Ad5 expressing sTGFRIIFc gene. Liver toxicity studies. Following intravenous delivery of adenoviruses, HE staining of the liver tissues were performed to evaluate liver necrosis. Plasma liver alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured. Bone metastasis model. PC3-luc2 cells were injected in left heart ventricle of nude mice. In mice bearing skeletal tumors, vectors were injected via tail vein. Bone metastases were monitored real time by bioluminescence imaging (BLI) and X-ray radiography. Tumor burden was analyzed by HE staining; ostoclasts were measured by tartarate resistant acid phosphates (TRAP) staining, plasma calcium levels were determined, and microCT analyses of the bones were performed. Results: Systemic delivery of a low dose (2.5x1010 viral particles (VPs)/mouse or a high dose (1011 VPs/ mouse ) of Ad5 based Ad.sTRFc produced significant liver necrosis and a transient increase in ALT and AST levels; the high dose (1011 VPs/mouse) killed all the mice by day 3. On the other hand, either low or high dose of Ad5/48 chimaeric mHAd.sTRFc virus did not produce liver damage or increased plasma ALT and AST levels; a dose (up to 2x1011 VPs/mouse tested thus far) did not cause animal deaths. There was a much reduced liver uptake of mHAd.sTRFc compared to Ad.sTRFc. Intravenous delivery of Ad.sTRFc and mHAd.sTRFc (5x1010 VPs/mouse) exhibited a significant inhibition of bone metastases as revealed by BLI and X-ray analyses. The higher dose of mHAd.sTRFc (2x1011 VPs/mouse or 4x1011 VPs/mouse) were more effective than the lower dose (5x1010 VPs/mouse) of Ad.sTRFc or mHAd.sTRFc, and produced a significant reduction in the tumor burden, hypercalcemia and osteoclasts, and produced normal bone architect as revealed by the microCT scan analyses. Conclusion: Ad5/48 chimaeric hexon based oncolytic adenovirus mHAd.sTRFc produce much reduced liver and systemic toxicity and larger and safe dose can be administered to produce superior anti-tumor responses. Thus, mHAd.sTRFc should be tested in the clinical trials in prostate cancer patients.
S214
556. Enhanced Anti-Tumor Efficacy by Oncolytic Adenovirus Using a Stimuli Responsive Block Copolymer mPEG-b-Poly(His)
Joung-Woo Choi,1 You Han Bae,2 Chae-Ok Yun.3 Graduate Program for Nanomedical Science and Technology, Yonsei University, Seoul, Korea; 2Department of Pharmaceutics and Pharmaceutical Chemistry, The University of Utah, Salt lake city, Korea; 3Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Korea. 1
Adenovirus (Ad) has shown great promise in cancer gene therapy. Ad coated with cationic polymers have already been shown to enhance transduction, but it showed nonspecific binding with cells, resulted in evoked side effect. To overcome these hurdles, in this study adenovirus coated with pH-sensitive poly(His) polymer through ionic interaction for passive tumor targeting. We have characterized the effect of polymer concentration on particle size distribution, zeta potential, transduction efficiency, and structural properties. Remarkable enhanced transduction efficiency was observed in cells treated with Ad/poly(His) nanocomplex compared to naked Ad at low pH, which is hallmark of cancer. In addition, cellular trafficking using time lapse total internal reflection fluorescence showed that the Ad/poly(His) nanocomplex significantly enhanced cellular uptake at lower pH, in contrast to neutral pH. The biodistribution study confirmed that the Ad/poly(His) nanocomplex can effectively accumulate at the tumor site by the EPR effect and exhibited 3.3-fold higher luciferase expressions in tumor than the naked Ad. Additionally, MTT assay and cancer cell xenografts studies showed that polymercoated nanocomplex exhibited potent cancer cell killing effect and also inhibited the tumor growth. Furthermore, poly(His)-coated Ad complex was not significantly inhibited by serum, in contrast to naked Ad. Moreover, Ad/poly(His) nanocomplex significantly reduced the innate immune response in contrast to naked Ad, as assessed by interleukin-6 (IL-6) cytokine release from macrophage cells. These results suggest that the stimuli-responsive Ad/poly(His) nanocomplex has potential to targeting tumors.
557. Distinct Integration Profiles after Sleeping Beauty Transposase-Mediated Somatic Integration from AAV and Adenoviral Vectors
Wenli Zhang,1 Christina Rauschhuber,2 Martin Haeusl,2 Nadine Muether,2 Anja Ehrhardt.1 1 Institute for Virology and Microbiology, Private University Witten/Herdecke, Witten, Germany; 2Max von Pettenkofer-Institut, Ludwig-MAximilians-University Munich, Munich, Germany; 3Max Delbrück Center for Molecular Medicine, Berlin, Germany. Hyperactive Sleeping Beauty (SB) transposase systems for somatic integration hold great promise for gene therapeutic applications. For efficient delivery of hyperactive transposase versions such as HSB5 and SB100X, we have focused on the development of viral hybrid-vectors based on high-capacity adenoviral vectors (HC-AdVs) and recombinant adeno-associated virus (rAAV). Both vector types represent promising tools for therapeutic gene delivery because they result in efficient transduction and long-term transgene expression in non-dividing cells. However, as HC-AdV and rAAV mostly remain episomal, vector genomes and transgene expression are lost over time in dividing cells. Therefore, to stably transduce tissues and cells undergoing many cell divisions, we developed HC-AdV/ transposase and AAV/transposase hybrid-vectors for stable transgene expression. The AAV/transposase hybrid-vector system utilizes the hyperactive SB transposase SB100X for somatic integration and was tested in HeLa cells. After performing colony forming assays we observed that in contrast to the inactive mSB control group, the SB100X group showed 10-fold increased numbers of colonies. Based on a plasmid rescue strategy we analyzed the SB100X-mediated Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
ADENOVIRUS AND OTHER DNA VIRUS VECTORS II integration profile after transposition from the AAV vector and of the 91 integration events identified, 50% were located in genes. For the HC-AdV/transposase hybrid-vector we identified 163 independent transposition events from liver of HSB5 treated mice based on a PCR strategy. Of these events 77% were found in non-gene areas and 23% in genes indicating a random integration pattern. In addition, 27 extra-chromosomal integration events were observed, most likely caused by transposon excision and subsequent transposition into the delivered vectors. Furthermore, the chromosomal integration site analysis revealed a strong bias towards integration into the X-chromosome. In summary this study demonstrates for the first time that different integration profiles can be observed dependent on the method and viral vector used for delivery of the transposon. The AAV/transposase hybrid-vector shows a bias towards integration into genes with efficiencies of up to 50%. In contrast, integration site analysis of the HC-AdV/transposase hybrid-vector revealed a random integration profile with respect to integration efficiencies into genes and a strong bias towards integration into the X-chromosome. Notably, the latter phenomenon was not observed when using rAAV. For high resolution of integration events after transposition form HC-AdV and rAAV, a large number of integration sites is currently being analysed by deep sequencing using a LAM-PCR method for both hybrid-vector systems.
558. Screening and Characterization of Alternative Adenovirus Serotypes with Enhanced Virus-Lateralization Ability In Vitro for Improved Antitumor Efficacy
Junji Uchino,1 David T. Curiel,2 Hideyo Ugai.1 Cancer Biology Division, Department of Radiation Oncology, School of Medicine, Washington University in St. Louis, St. Louis, MO; 2Cancer Biology Division, Biologic Therapeutics Center, Department of Radiation Oncology, School of Medicine, Washington University in St. Louis, St. Louis, MO. 1
Human adenovirus serotype 5 (HAdV-C5) is the most widely employed as adenovirus serotype for cancer gene therapy. However, therapeutic efficacy of HAdV-C5 is limited, as its virus progeny are not sufficiently released from infected cells. Therefore, successful cancer gene therapy using HAdV-C5-based vectors requires efficient virus lateralization from an infected tumor cell to surrounding tumors. To overcome this limitation, we sought to develop novel oncolytic adenoviruses with enhanced virus-spread ability. Since there are at least 52 serotypes of human adenoviruses, we characterized the virus-spread ability of other adenovirus serotypes as compared with that of HAdV-C5. We first examined a host range of 16 serotypes selected from species A to F in the human lung carcinoma A549 cells. As a result, 10 serotypes of adenoviruses (HAdV-C2, B3, E4, C5, D9, D10, A31, B34, B35 and D51) were propagated in A549 cells, but 6 serotypes (HAdV-B14, B16, D20, B21, F40 and F41) were not. Therefore, we suggest that the former 10 serotypes of adenoviruses may be suitable for oncolytic adenovirus vectors. We next compared virus-spread ability of these 10 serotypes, along with that of HAdV-C5, by plaque assay. The plaque sizes of HAdV-D9 and HAdV-D51 were larger than those of other serotypes including HAdV-C5. Since the nucleotide sequence of the HAdV-D9 genome is reported, we further characterized the biological function of HAdV-D9. We confirmed that HAdV-D9 was efficiently released from infected cells to culture medium at an earlier time point as compared with HAdV-C5 by one-step growth curve analysis. Also, HAdV-D9 replicated well in MCF-7 breast cancer cells which are CAR-negative. Moreover, we measured cell killing activity of HAdV-D9 along with HAdV-C5 in a broad range of human breast cancer and malignant mesothelioma cell lines by MTS assay. Our results showed that HAdV-D9 more effectively killed 4 of 8 cell lines than HAdV-C5 and similarly induced cytopathic effect in the other cell lines. In order to Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
understand target cells of HAdV-D9, we investigated the HAdV-D9 receptor. The primary receptors of species D has been suggested to be the coxsackie B virus and HAdV receptor (CAR), CD46, sialic acid, or integrins in previous reports. In this study, we assessed human CAR (hCAR) and hCD46 by quantitatively measuring cellular uptake of the HAdV-D9 genome in CHO, CHO-hCAR, and CHO-hCD46. Interestingly, CHO cells as well as CHO-hCAR and CHO-hCD46 were efficiently infected with HAdV-D9 as compared with HAdV-C5. Therefore, we suggest that HAdV-D9 binds to other receptors rather than hCAR and hCD46. Collectively, HAdV-D9 has enhanced virusspread ability, improves cell killing, and targets cells independently of hCAR and hCD46. Therefore, this work supports the consideration of HAdV-D9-based oncolytic therapies for cancer treatment.
559. Improved Animal Models for Testing Gene or Cell Therapies for Atherosclerosis
Liang Du,1 Jingwan Zhang,1 David Dichek.1 Department of Cardiology, Internal Medicine, University of Washington, Seattle, WA. 1
Background: Gene therapy delivered to the blood vessel wall could augment current therapies for atherosclerosis. We reported previously that gene therapy with a helper-dependent adenovirus expressing apo A-I (HDAdapoAI) significantly decreased atherosclerosis 4 wk after infusion into arteries of cholesterol (chol)-fed rabbits. However, lesions in this model (Fig 1: ApoAI study) are small, almost exclusively foam cells, and regress by 8 wk. We therefore developed animal models that would allow testing vascular wall gene therapy on larger, more complex lesions, and for longer periods. Methods: 20 rabbits were allocated into 4 groups with each group receiving a unique combination of diet, vector, and surgical protocol (Fig 1). Group I rabbits were initially fed a very high chol diet, after which their carotids were infused with HDAdNull, then continued on sliding-scale chol for 24 wk. Group II rabbits were fed a high-chol diet, then received HDAdNull along with placement of periarterial collars. Group III rabbits were fed a high-chol diet, then received both HDAdNull and a first-generation adenovirus (FGAdNull). Group IV rabbits were fed a high-chol diet, then continued on sliding scale chol for 24 wk, with no surgery, collar, or vector. Segments of infused carotids were analyzed for intimal lesion area and content and for presence of vector DNA. Results: A high-chol diet alone (Group IV) did not cause intimal growth. Additional “insults” [very high chol diet, surgery, and HDAdNull (Group I), high-chol diet, surgery, HDAdNull, and collar (Group II), or high-chol diet, surgery, HDAdNull, and FGAdNull (Group III)] all caused significant intimal growth. Compared to our original model, intimal lesions in each of Groups I, II, and III were all larger: 6.2×105 ± 5.0 ×105 μm2; 3.1×105 ± 1.7 ×105 μm2; and 1.4×105 ± 5.5 ×104 μm2 for Groups I – III, respectively, compared to 1.5×104 ± 4.3 ×103μm2 in our original model (P < 0.0002 for all). No vector was detectable in Group III arteries whereas substantial vector was present in Group I and II arteries (approx 1 copy per endothelial cell). Group I lesions were more eccentric than Group II lesions, with more macrophages (46 ± 4.6 vs 5.3 ± 1.3% macrophage area; P < 0.0001), more lipid (18 ± 0.90 vs 6 ± 1.7% lipid area; P < 0.0001), and fewer smooth muscle cells (6.3 ± 1.4 vs 39 ± 2.8% SMC area; P < 0.0001). Conclusions: We developed 2 new animal models for testing the efficacy of gene-therapy vectors such as HDAd in preventing or reversing intimal growth and decreasing lipid, SMC, and macrophage accumulation. The model used for Group II is more suitable for testing efficacy in lesions with a predominant SMC component; the Group I model is better suited for testing efficacy in lesions rich in lipid and macrophages. These new models will allow testing of the efficacy of HDAdapoAI on larger lesions and at later time points.
S215