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876. Microtubule Acetylation Enhances Binding of Plasmid DNA in Gene Transfer
CHEMICAL AND MOLECULAR CONJUGATES III immunoprecipitate assay. The TTRAP region for ΦC31 integrase binding was roughly located on 62-180aa. Interesting, we also defined TTRAP as a novel ND10-associated protein, as we identified strong interaction between TTRAP and PML by co-immunoprecipitate and co-localization assay. Furthermore, the functional interaction between ΦC31 integrase and TTRAP was also studied in ΦC31 integrase activity reporter cell line, and the result suggested that endogenous TTRAP may cause a mild inhibition in ΦC31 integration efficiency. Taken together, our results not only enriched the story about ΦC31 integrase activity in host cells, but also gave new insight on the popular opinion that ND10 is important as nuclear defense against external insults.
Chemical and Molecular Conjugates III 876. Microtubule Acetylation Enhances Binding of Plasmid DNA in Gene Transfer
Melissa A. Badding,1 David A. Dean.1 Pediatrics, University of Rochester School of Medicine, Rochester, NY.
1
For non-viral gene therapy to be successful, plasmid DNA must not only cross multiple extracellular and intracellular barriers, but also exhibit sufficiently high expression levels. One of these barriers is the cytoskeletal meshwork within the cytoplasm of the cell. Our lab and others have demonstrated that plasmids traffic through the cytoplasm by directed movement along microtubules. We have shown that there is a subset of modified microtubules that remain intact even after cytoskeletal disruption by drugs or mechanical strain and are post-translationally acetylated. Experiments in which microtubule acetylation is increased by inhibition of the tubulin deacetylase HDAC6 show greater transfection efficiency and more rapid localization of DNA to the nucleus. Based on these findings, we hypothesize that acetylated microtubules bind more DNA through adapter proteins than do unacetylated microtubules, resulting in more rapid movement of DNA along these microtubules to the nucleus. To test this, two variations of an in vitro microtubule-binding spin-down assay were employed, using purified tubulin, HeLa cell extract, and plasmid DNA. Real time PCR was used to quantify the percent of pelleted DNA with either unmodified or acetylated microtubules in the first set of experiments. The resulting data showed that more DNA pelleted with acetylated microtubules than with unacetylated microtubules. In the second set of experiments, immunofluorescence was used to visualize the co-localization of both microtubule-binding motor proteins and plasmid DNA to unmodified or acetylated microtubules, which were spun onto coverslips. The immunofluorescence results show cytoplasmic motor proteins and DNA preferentially co-localize with acetylated microtubules more so than with unacetylated microtubules. Current studies are underway to evaluate movement in real-time with both in vitro microtubule gliding experiments and in vivo injection studies. Taken together, these findings will provide a foundation for determining how modulation of microtubule acetylation can be used as a means to increase intracellular trafficking of plasmid DNA and enhance gene therapy.
S334
877. Non-Viral Gene Therapy Using Arginine Modified Polyamidoamine Dendrimer (PAMAMRG4) and Minicircle VEGF DNA into the Skin Wound of Diabetic Mice Successfully Improved the Healing and Microarchitecture
Jeong H. Park,1,3 Ki H. Nam,2 Chang S. Yoon,1 Hye S. Jung,1 Tae K. Kim,1,3 Min J. Kwon,1,3 Soon H. Lee,1,3 Soo J. Chung,1,4 Mi K. Kim,1,5 Jong S. Park.2 1 Molecular Therapy Lab, Paik Memorial Institute for Clinical Research, Inje University, Busan, Korea, Republic of; 2School of Chemistry and Molecular Engineering, Seoul National University, Seoul, Korea, Republic of; 3Internal Medicine, College of Medicine, Inje University, Busan, Korea, Republic of; 4Pathology, College of Medicine, Inje University, Busan, Korea, Republic of; 5 Internal Medicine, Maryknoll Medical Center, Busan, Korea, Republic of.
Chronic un-healing foot wound is a serious problem in the complicated diabetic patients. To accelerate the healing of diabetic skin wounds, various kinds of growth factors have been employed with limited success. The short half-life of administered growth factors onto the chronic un-healing wounds with over-expressed proteinases might have limited wide-spread clinical usage. To overcome this, growth factor gene therapy might be an attractive alternative rather than direct application of factors on the wounds. We previously reported that PAMAM-RG4, which is composed of a polyamidoamine (PAMAM) dendrimer (generation 4) grafted with basic L-arginine residues, showed nearly equal gene delivery efficacies in in-vitro experiments with BPEI(25kDa) without serious cytotoxicity. This study was performed to see that the non-viral gene therapy using PAMAM-RG4 complexed with minicircle human VEGF(vascular endothelial growth factor) DNA could improve the healing of skin wound of streptozotocin induced diabetic C57BL/6J mice. Total 60 C57BL/6J mice (8 week-old) were used. Diabetes mellitus was induced by the single intra-peritoneal injection of streptozotocin. Minicircle human VEGF DNA was generated by the method using bacteriophage ϕC31 integrase previously described by Dr. Mark A Kay. After making 6mm size skin wound by biopsy punch, 20µg of minicircle VEGF DNA complexed with PAMAM-RG4 (N/P ratio=4:1) was injected subcutaneously. Serial measurements of wound sizes and expressed human VEGF in the wound tissues (ELISA) were performed. H/E and Masson-Trichrome stains were done to see the soundness of the tissue microarchitectures. At the 4th days after injection, human VEGF was detected at the wound tissues of the animals with gene therapy, and then gradually increasing till the day 12. The sizes of the wounds were significantly smaller in the mice with gene therapy after the day 6, and the difference persisted till the day 12. H/E and Masson-Trichrome stains of the wound tissues revealed that the healing wounds of the diabetic mice with the gene therapy showed much improved well-organized microarchitectures with the abundant collagen deposition, which could not be seen in the control diabetic mice. No adverse effects were observed in the healing tissues of the diabetic mice with the gene therapy using PAMAM-RG4 and human VEGF minicircle DNA. In conclusion, PAMAM-RG4 was very effective in the gene therapy for the skin wounds of diabetic mice without any adverse effects. KEY WORDS: PAMAM-RG4, VEGF, Skin Wound, Diabetes Mellitus.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Chemical and Molecular Conjugates III 876. Microtubule Acetylation Enhances Binding of Plasmid DNA in Gene Transfer
Melissa A. Badding,1 David A. Dean.1 Pediatrics, University of Rochester School of Medicine, Rochester, NY.
1
For non-viral gene therapy to be successful, plasmid DNA must not only cross multiple extracellular and intracellular barriers, but also exhibit sufficiently high expression levels. One of these barriers is the cytoskeletal meshwork within the cytoplasm of the cell. Our lab and others have demonstrated that plasmids traffic through the cytoplasm by directed movement along microtubules. We have shown that there is a subset of modified microtubules that remain intact even after cytoskeletal disruption by drugs or mechanical strain and are post-translationally acetylated. Experiments in which microtubule acetylation is increased by inhibition of the tubulin deacetylase HDAC6 show greater transfection efficiency and more rapid localization of DNA to the nucleus. Based on these findings, we hypothesize that acetylated microtubules bind more DNA through adapter proteins than do unacetylated microtubules, resulting in more rapid movement of DNA along these microtubules to the nucleus. To test this, two variations of an in vitro microtubule-binding spin-down assay were employed, using purified tubulin, HeLa cell extract, and plasmid DNA. Real time PCR was used to quantify the percent of pelleted DNA with either unmodified or acetylated microtubules in the first set of experiments. The resulting data showed that more DNA pelleted with acetylated microtubules than with unacetylated microtubules. In the second set of experiments, immunofluorescence was used to visualize the co-localization of both microtubule-binding motor proteins and plasmid DNA to unmodified or acetylated microtubules, which were spun onto coverslips. The immunofluorescence results show cytoplasmic motor proteins and DNA preferentially co-localize with acetylated microtubules more so than with unacetylated microtubules. Current studies are underway to evaluate movement in real-time with both in vitro microtubule gliding experiments and in vivo injection studies. Taken together, these findings will provide a foundation for determining how modulation of microtubule acetylation can be used as a means to increase intracellular trafficking of plasmid DNA and enhance gene therapy.
S334
877. Non-Viral Gene Therapy Using Arginine Modified Polyamidoamine Dendrimer (PAMAMRG4) and Minicircle VEGF DNA into the Skin Wound of Diabetic Mice Successfully Improved the Healing and Microarchitecture
Jeong H. Park,1,3 Ki H. Nam,2 Chang S. Yoon,1 Hye S. Jung,1 Tae K. Kim,1,3 Min J. Kwon,1,3 Soon H. Lee,1,3 Soo J. Chung,1,4 Mi K. Kim,1,5 Jong S. Park.2 1 Molecular Therapy Lab, Paik Memorial Institute for Clinical Research, Inje University, Busan, Korea, Republic of; 2School of Chemistry and Molecular Engineering, Seoul National University, Seoul, Korea, Republic of; 3Internal Medicine, College of Medicine, Inje University, Busan, Korea, Republic of; 4Pathology, College of Medicine, Inje University, Busan, Korea, Republic of; 5 Internal Medicine, Maryknoll Medical Center, Busan, Korea, Republic of.
Chronic un-healing foot wound is a serious problem in the complicated diabetic patients. To accelerate the healing of diabetic skin wounds, various kinds of growth factors have been employed with limited success. The short half-life of administered growth factors onto the chronic un-healing wounds with over-expressed proteinases might have limited wide-spread clinical usage. To overcome this, growth factor gene therapy might be an attractive alternative rather than direct application of factors on the wounds. We previously reported that PAMAM-RG4, which is composed of a polyamidoamine (PAMAM) dendrimer (generation 4) grafted with basic L-arginine residues, showed nearly equal gene delivery efficacies in in-vitro experiments with BPEI(25kDa) without serious cytotoxicity. This study was performed to see that the non-viral gene therapy using PAMAM-RG4 complexed with minicircle human VEGF(vascular endothelial growth factor) DNA could improve the healing of skin wound of streptozotocin induced diabetic C57BL/6J mice. Total 60 C57BL/6J mice (8 week-old) were used. Diabetes mellitus was induced by the single intra-peritoneal injection of streptozotocin. Minicircle human VEGF DNA was generated by the method using bacteriophage ϕC31 integrase previously described by Dr. Mark A Kay. After making 6mm size skin wound by biopsy punch, 20µg of minicircle VEGF DNA complexed with PAMAM-RG4 (N/P ratio=4:1) was injected subcutaneously. Serial measurements of wound sizes and expressed human VEGF in the wound tissues (ELISA) were performed. H/E and Masson-Trichrome stains were done to see the soundness of the tissue microarchitectures. At the 4th days after injection, human VEGF was detected at the wound tissues of the animals with gene therapy, and then gradually increasing till the day 12. The sizes of the wounds were significantly smaller in the mice with gene therapy after the day 6, and the difference persisted till the day 12. H/E and Masson-Trichrome stains of the wound tissues revealed that the healing wounds of the diabetic mice with the gene therapy showed much improved well-organized microarchitectures with the abundant collagen deposition, which could not be seen in the control diabetic mice. No adverse effects were observed in the healing tissues of the diabetic mice with the gene therapy using PAMAM-RG4 and human VEGF minicircle DNA. In conclusion, PAMAM-RG4 was very effective in the gene therapy for the skin wounds of diabetic mice without any adverse effects. KEY WORDS: PAMAM-RG4, VEGF, Skin Wound, Diabetes Mellitus.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy