- Email: [email protected]
81. Survivin-Targeting Artificial MicroRNAs Mediated by Adenovirus Devitalize Tumor Activity in Cancer Cells and Xenograft Models
ADENOVIRUS VECTORS AND OTHER DNA VIRUS VECTORS I as low as 40 nM, significantly enhances rAAV-mediated (both singleand double-stranded) and rAdv-mediated transgene expression in different cell lines. Furthermore, combination therapies of bufalin and rAdv-p53 strongly inhibited the proliferation of different malignant cell lines, including human cervical cancer and HCC cells. The therapeutic effect was more efficient than either treatment alone, and in a time- and dose-dependent manner. It was evident that the percentage of cells undergone apoptosis increased significantly after treatment. These studies suggested a potential combinatorial therapy of bufalin and bufalin-containing medicine in future malignant celltargeted, viral vector-based gene therapy.
79. Generation of a Glioma-Targeted Janus Conditional Replicating Adenoviral Vector With CD133FF-Pseudotyped Fiber
Julius W. Kim,1 Joshua R. Kane,1 Alan L. Chang,1 Jacob Young,1 Jian Qian,1 Atique U. Ahmed,1 Maciej S. Lesniak.1 1 Surgery, Univerisity of Chicago, Chicago, IL. The dismal clinical context of advanced-grade glioma warrants the development of novel strategies with the potential for direct patient impact. In this regard, conditionally replicating adenoviruses (CRAds) represent a potentially effective, novel approach for glioma therapy Furthermore, the CRAd-loaded tumor-tropic stem cell carrier approach has shown appreciable preclinical efficacy. However, several improvements can be achieved in regards to the critical functions of CRAds: more efficient targeting, directed tumor-specific replication, and restricted lateral spread of glioma. The development of an adenoviral vector capable of targeting, being initially “loaded on a cell carrier,” and “specifically targeted to glioma”, respectively, represents a critical field challenge to allow for the development of tumor targeted therapies for treating gliomas. The initiation point for demonstrating this novel approach is the development of specifically targeted adenovirus fibers, which dictate the attachment of an adenoviral vector to a target cell. For this, we have generated a set of a specific cell targeting fibers through the incorporation of phage panned peptides specific for glioma cells and for cells expressing CD133, a marker for stem cell carriers, on a Fiber Fibritin-based Ad5 fiber, designated as MGFF and CD133FF. Then, we generated Ad5MGFF-CMV-GFP (and Survivin-E1) and HEK293 stably expressing CD133FF. Ad5MGFF infects glioma cell lines with high specificity, exhibiting drastically lower infectivity in non-cancer cell lines (including stem cell carrier cell lines) and other types of tumor cell lines. To load this glioma specific Ad5MGFF, we propagated this virus in HEK293 stably expressing CD133FF, generating pseudo-typed CD133FF expressing Ad5. The CD133FF fiber pseudo-typed Ad5MGFF Janus virus was efficiently loaded on a neural stem cell carrier, replicated inside the cell carrier, and was released as highly glioma-specific Ad5MGFF. We will show corroborating data regarding efficient serial targeting–cell carrier and glioma cell, glioma-specific replication, and restricted lateral spread of glioma. By maximizing advantages of cell carrier based therapy through efficient natural cell tropism and protection from immune surveillance, this novel Janus viral vector generation technique we describe herein has markedly profound potential as a cell carrier-based cancer therapy with systemic administration of adenoviral vector for many different types of cancer therapy.
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80. Biodistribution of Oncolytic Adenovirus After the Injection of Carrier Cells Infected With Oncolytic Adenovirus
Katsuyuki Hamada,1 Kazuko Takagi,1 Wenlin Huang,2 Hiroshi Itoh,3 Kenzabro Tani,4 Akihiro Nawa.1 1 Obstetrics and Gynecology, Ehime University, Toon, Ehime, Japan; 2Cancer Center, Su Yat-sen University, Guangzhou, Guangdong, China; 3Animal Medical Center, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan; 4Advanced Molecular and Cell Therapy, Kyusyu University, Higashi-ku, Fukuoka, Japan. Although replication-competent viruses have been developed to treat cancers, their cytotoxic effects are insufficient, since infection with them is inhibited by generation of neutralizing antibodies. To address this limitation, we developed a carrier cell system to deliver a replication-competent adenovirus. Carrier cells infected with oncolytic adenovirus were injected into syngeneic subcutaneous ovarian tumors after immunization with adenovirus and induced complete tumor regression by the induction of antiadenoviral and antitumoral CTL responses. To start clinical trial, toxicity and biodistribution study were carried out in nude mice, rabbits and beagle dogs. Acute toxicity and distribution test were carried out after the single injection of carrier cells into ovarian tumor in nude mice. Chronic toxicity test was carried out by 8 injections of carrier cells into rabbits for 4 weeks. Excretion study was carried out to determine whether oncolytic adenovirus was excreted from the beagle dogs after the single injection of carrier cells. Acute toxicity test did not show any lethal side effects in nude mice. In biodistribution test, single injection of carrier cells into ovarian tumor induced the peak levels of oncolytic adenovirus the next day but did not show any significant levels of that in nude mice 14 days after injection. In chronic toxicity test, 8 injections of 1.25×107 cells/kg or less did not show any significant toxicity in rabbits. In excretion test, oncolytic adenovirus was not excreted into the urine and the stool of beagle dogs. This oncolytic adenovirus-infected carrier cell system might prove effective and safe in the preclinical efficacy and the biosafety test, respectively. Clinical trial is being scheduled to treat recurrent solid cancers.
81. Survivin-Targeting Artificial MicroRNAs Mediated by Adenovirus Devitalize Tumor Activity in Cancer Cells and Xenograft Models
Yudan Chi,1 Xiang Wang,1 Yong Yang,1 Chao Zhang.1 Anti-Infection Immunity and Vaccine Research, Institut Pasteur of Shanghai, Shanghai, China.
1
As an inhibitor of apoptosis, survivin undertakes constitutively active in a variety of tumors for negatively regulation of cell cycle progressivity. It highly expresses in most human tumors and fetal tissue while being completely absent in terminally differentiated cells, which suggests survivin might be a target for cancer therapy that would distinguish transformed and normal cells. Here we demonstrate a promising therapeutic strategy against cancer via artificial microRNAs (amiRNAs) targeting survivin. After screening, two effective amiRNAs with the potential of suppressing survivin in cancer cells were obtained and then cloned into adenoviral vector. With dose-dependent recombinant viruses infecting cancer cells, downregulation of survivin in cancer cells could dramatically inhibit cancer cell growth and impair cell survival. Mechanistic investigations indicated survivin-targeting amiRNAs inducing cell apoptosis were associated with triggering the downstream effector caspase-3 and cleaved parp, and induction of P53 signaling pathway including the regulation of total P53 and phosphorylation level of P53. Furthermore, amiRNAs treatment contributed to the incomplete mitosis, corresponding to the cell cycle arrested at G2/M phase. Cancer cell Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
ADENOVIRUS VECTORS AND OTHER DNA VIRUS VECTORS I death by lack of survivin in turn mediated survival signaling such as phosphorylated Erk. In vivo study, survivin-targeting amiRNAs based on adenoviral transduction effectively hindered tumor growth and caused tumor regression in mouse xenograft models. Taken together, a newly therapeutic method based on adenovirus-mediated amiRNA is established with the potential of clinical application in cancer treatment. *Correspondence: Dongming Zhou, E-mail: [email protected]
82. Molecular Characterization of a Rare Species D Adenovirus-Derived Vector With Low Off-Target Transduction and Rapid Clearance
Natalya Belousova,1 Galina Mikheeva,1 Chiyi Xiong,1 Loren J. Stagg,1 Mihai Gagea,1 Patricia S. Fox,1 Roland L. Bassett,1 John E. Ladbury,1 Chun Li,1 Victor Krasnykh.1 1 University of Texas MD Anderson Cancer Center, Houston, TX. Unique molecular properties of various adenovirus (Ad) serotypes have been essential to the development of gene vectors with improved infectivity, lower off-target in vivo transduction, toxicity, and altered immunogenicity. Many of these accomplishments resulted from studies of species D adenoviruses—the most diverse yet underexplored group of Ads. Previously reported low seroprevalence in humans of Ad43, an otherwise unstudied member of species D, identified this rare serotype as an attractive prototype of a novel platform for human gene therapy vectors. This study is the first to focus on assessment of biological properties of Ad43 essential to its vectorization. We found that Ad43 virions do not interact with blood coagulation factor X and cause low random transduction upon systemic delivery. Intravenously injected Ad43 clears host tissues more quickly than do traditionally used Ad5 vectors. We identified two receptors that Ad43 uses for infection in vitro and demonstrated that the tropism of Ad43 can be altered genetically to achieve gene delivery via a cancer-associated receptor.
83. Decorin-Expressing Adenovirus Decreases Collagen Synthesis and Upregulates the MMP Expression in Keloids; Implication for Reversing Pathologic Fibrosis
Won Jai Lee,1 Hyo-Min Ahn,3 Hyun Roh,1 Ju Hee Lee,2 Il-Kyu Choi,3 Yong Oock Kim,1 Dae Hyun Lew,1 Chae-Ok Yun.3 1 Plastic & Reconstructive Surgery, Human Tissue Restoration, Yonsei University College of Medicine, Seoul, Korea; 2 Dermatology and Cutaneous Biology Research Institute, Human Tissue Restoration, Yonsei University college of Medicine, Seoul, Korea; 3Bioengineering, College of Engineering, Hanyang University, 17 Haengdang-Dong, Seondgdong-Gu, Seoul, Korea.
Background Decorin is a natural neutralizer of TGF-β1 and reduced synthesis of decorin has been associated with the dermal scars, and therefore an increase of decorin expression appears to reduce scar tissue. To investigate the therapeutic potentials of decorin-expressing replication-incompetent adenovirus (dE1-RGD/GFP/DCN) for keloid and hypertrophic scars, we examined the effects of dE1-RGD/GFP/ DCN on the expression levels of type I and III collagen as well as MMP-1 and -3 in human dermal fibroblast (HDFs) and keloid-derived fibroblast (KFs) cell lines. Methods Decorin expressions were examined by immunofluorescence assay in keloid tissues. HDFs and KFs were infected with dE1-RGD/GFP/DCN or control virus, and protein levels of decorin and secreted TGF-ß1 were assessed by ELISA, while mRNA levels of collagen type I, collagen III, MMP-1, MMP-3 by real time RT-PCR. Additionally, the expression levels of major extracellular matrix (ECM) were investigated by immunohistochemistry in keloid spheroids transduced with dE1-RGD/GFP/DCN. Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Results Lower expression of decorin was examined in the keloid region compared to the adjacent normal tissues. Treatment with dE1-RGD/DFP/DCN decreased mRNA levels of type I collagen in the TGF-β (10 ng/ml)-treated HDFs and KFs and TGF-β protein expression in the HDFs. In addition, the expression of MMP-1 and-3 mRNA was strongly upregulated in KFs and TGF-β-treated HDFs after treatment with dE1-RGD/DFP/DCN. Moreover, expression of major ECMs such as type I collagen, fibronectin, and elastin were significantly reduced in keloid spheroids transduced with dE1-RGD/ DFP/DCN. Conclusion These results support the use of decorin-expressing adenovirus in gene therapy protocols to lower collagen synthesis in KFs, as this decrease is a highly desirable effect for the treatment of keloids.
84. Effects of Alginate Gel-Encapsulated Adenoviruses Expressing Relaxin on Scar Remodeling in a Pig Model
In Sik Yun,1 Won Jai Lee,1 Yong Oock Kim,1 Eun Hye Kang,1 IlKyu Choi,2 Hoguen Kim,3 Kee Yang Chung,4 Seung-Kyu Han,5 Dae Hyun Lew,1 Dong Kyun Rah,1 Chae-Ok Yun.2 1 Department of Plastic & Reconstructive Surgery, Yonsei University College of Medicine, Seoul, Korea; 2Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Korea; 3Department of Pathology, Yonsei University College of Medicine, Seoul, Korea; 4Department of Dermatology and Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, Korea; 5Department of Plastic & Reconstructive Surgery, Korea University College of Medicine, Seoul, Korea. Adenoviruses (Ad) have been well documented as vectors in the context of cancer gene therapy. However, repetitive treatments of Ad therapies have shown to be limited due to their short maintenance of biological activity in vivo. In this study, we investigated the efficacy of sustained Ad delivery using an injectable alginate gel matrix system containing Ad expressing Relaxin (RLX), a transforming growth factor-b1 (TGF-b1) antagonist implicated as a potent collagen rearranger and major extracellular matrix components reducer. Additionally, Yorkshire pigs scar tissues were used as our animal models for their similarities in dermal composition and repair mechanisms of human skin. Relaxin-expressing replicationincompetent Ad loaded in alginate gel (gel/Ad-RLX) decreased scar size, color index and pliability in a pig scar tissues. These results were observed through expression of relaxin, which was assessed by immunofluorescence staining. Immunohistochemical investigation of gel/Ad-RLX-treated groups revealed decreased major extracellular matrix (ECM) protein expression levels in scar tissues. Furthermore, treatment with gel/Ad-RLX showed decreased tissue inhibitors of metalloproteinases-1 (TIMP-1) and alpha-smooth muscle actin (α-SMA) proteins while showing markedly increased expression levels of matrix metalloproteinases-1 (MMP-1) in a pig scar tissues. Moreover, gel/Ad-RLX significantly downregulated TGF-b1 and upregulated TGF-b3 mRNA expression in a pig scar tissues. Together, these results support relaxin’s prominent role in scar remodeling and suggest that relaxin-expressing adenovirus loaded in alginate gel may have therapeutic effects on scar formation.
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79. Generation of a Glioma-Targeted Janus Conditional Replicating Adenoviral Vector With CD133FF-Pseudotyped Fiber
Julius W. Kim,1 Joshua R. Kane,1 Alan L. Chang,1 Jacob Young,1 Jian Qian,1 Atique U. Ahmed,1 Maciej S. Lesniak.1 1 Surgery, Univerisity of Chicago, Chicago, IL. The dismal clinical context of advanced-grade glioma warrants the development of novel strategies with the potential for direct patient impact. In this regard, conditionally replicating adenoviruses (CRAds) represent a potentially effective, novel approach for glioma therapy Furthermore, the CRAd-loaded tumor-tropic stem cell carrier approach has shown appreciable preclinical efficacy. However, several improvements can be achieved in regards to the critical functions of CRAds: more efficient targeting, directed tumor-specific replication, and restricted lateral spread of glioma. The development of an adenoviral vector capable of targeting, being initially “loaded on a cell carrier,” and “specifically targeted to glioma”, respectively, represents a critical field challenge to allow for the development of tumor targeted therapies for treating gliomas. The initiation point for demonstrating this novel approach is the development of specifically targeted adenovirus fibers, which dictate the attachment of an adenoviral vector to a target cell. For this, we have generated a set of a specific cell targeting fibers through the incorporation of phage panned peptides specific for glioma cells and for cells expressing CD133, a marker for stem cell carriers, on a Fiber Fibritin-based Ad5 fiber, designated as MGFF and CD133FF. Then, we generated Ad5MGFF-CMV-GFP (and Survivin-E1) and HEK293 stably expressing CD133FF. Ad5MGFF infects glioma cell lines with high specificity, exhibiting drastically lower infectivity in non-cancer cell lines (including stem cell carrier cell lines) and other types of tumor cell lines. To load this glioma specific Ad5MGFF, we propagated this virus in HEK293 stably expressing CD133FF, generating pseudo-typed CD133FF expressing Ad5. The CD133FF fiber pseudo-typed Ad5MGFF Janus virus was efficiently loaded on a neural stem cell carrier, replicated inside the cell carrier, and was released as highly glioma-specific Ad5MGFF. We will show corroborating data regarding efficient serial targeting–cell carrier and glioma cell, glioma-specific replication, and restricted lateral spread of glioma. By maximizing advantages of cell carrier based therapy through efficient natural cell tropism and protection from immune surveillance, this novel Janus viral vector generation technique we describe herein has markedly profound potential as a cell carrier-based cancer therapy with systemic administration of adenoviral vector for many different types of cancer therapy.
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80. Biodistribution of Oncolytic Adenovirus After the Injection of Carrier Cells Infected With Oncolytic Adenovirus
Katsuyuki Hamada,1 Kazuko Takagi,1 Wenlin Huang,2 Hiroshi Itoh,3 Kenzabro Tani,4 Akihiro Nawa.1 1 Obstetrics and Gynecology, Ehime University, Toon, Ehime, Japan; 2Cancer Center, Su Yat-sen University, Guangzhou, Guangdong, China; 3Animal Medical Center, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan; 4Advanced Molecular and Cell Therapy, Kyusyu University, Higashi-ku, Fukuoka, Japan. Although replication-competent viruses have been developed to treat cancers, their cytotoxic effects are insufficient, since infection with them is inhibited by generation of neutralizing antibodies. To address this limitation, we developed a carrier cell system to deliver a replication-competent adenovirus. Carrier cells infected with oncolytic adenovirus were injected into syngeneic subcutaneous ovarian tumors after immunization with adenovirus and induced complete tumor regression by the induction of antiadenoviral and antitumoral CTL responses. To start clinical trial, toxicity and biodistribution study were carried out in nude mice, rabbits and beagle dogs. Acute toxicity and distribution test were carried out after the single injection of carrier cells into ovarian tumor in nude mice. Chronic toxicity test was carried out by 8 injections of carrier cells into rabbits for 4 weeks. Excretion study was carried out to determine whether oncolytic adenovirus was excreted from the beagle dogs after the single injection of carrier cells. Acute toxicity test did not show any lethal side effects in nude mice. In biodistribution test, single injection of carrier cells into ovarian tumor induced the peak levels of oncolytic adenovirus the next day but did not show any significant levels of that in nude mice 14 days after injection. In chronic toxicity test, 8 injections of 1.25×107 cells/kg or less did not show any significant toxicity in rabbits. In excretion test, oncolytic adenovirus was not excreted into the urine and the stool of beagle dogs. This oncolytic adenovirus-infected carrier cell system might prove effective and safe in the preclinical efficacy and the biosafety test, respectively. Clinical trial is being scheduled to treat recurrent solid cancers.
81. Survivin-Targeting Artificial MicroRNAs Mediated by Adenovirus Devitalize Tumor Activity in Cancer Cells and Xenograft Models
Yudan Chi,1 Xiang Wang,1 Yong Yang,1 Chao Zhang.1 Anti-Infection Immunity and Vaccine Research, Institut Pasteur of Shanghai, Shanghai, China.
1
As an inhibitor of apoptosis, survivin undertakes constitutively active in a variety of tumors for negatively regulation of cell cycle progressivity. It highly expresses in most human tumors and fetal tissue while being completely absent in terminally differentiated cells, which suggests survivin might be a target for cancer therapy that would distinguish transformed and normal cells. Here we demonstrate a promising therapeutic strategy against cancer via artificial microRNAs (amiRNAs) targeting survivin. After screening, two effective amiRNAs with the potential of suppressing survivin in cancer cells were obtained and then cloned into adenoviral vector. With dose-dependent recombinant viruses infecting cancer cells, downregulation of survivin in cancer cells could dramatically inhibit cancer cell growth and impair cell survival. Mechanistic investigations indicated survivin-targeting amiRNAs inducing cell apoptosis were associated with triggering the downstream effector caspase-3 and cleaved parp, and induction of P53 signaling pathway including the regulation of total P53 and phosphorylation level of P53. Furthermore, amiRNAs treatment contributed to the incomplete mitosis, corresponding to the cell cycle arrested at G2/M phase. Cancer cell Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
ADENOVIRUS VECTORS AND OTHER DNA VIRUS VECTORS I death by lack of survivin in turn mediated survival signaling such as phosphorylated Erk. In vivo study, survivin-targeting amiRNAs based on adenoviral transduction effectively hindered tumor growth and caused tumor regression in mouse xenograft models. Taken together, a newly therapeutic method based on adenovirus-mediated amiRNA is established with the potential of clinical application in cancer treatment. *Correspondence: Dongming Zhou, E-mail: [email protected]
82. Molecular Characterization of a Rare Species D Adenovirus-Derived Vector With Low Off-Target Transduction and Rapid Clearance
Natalya Belousova,1 Galina Mikheeva,1 Chiyi Xiong,1 Loren J. Stagg,1 Mihai Gagea,1 Patricia S. Fox,1 Roland L. Bassett,1 John E. Ladbury,1 Chun Li,1 Victor Krasnykh.1 1 University of Texas MD Anderson Cancer Center, Houston, TX. Unique molecular properties of various adenovirus (Ad) serotypes have been essential to the development of gene vectors with improved infectivity, lower off-target in vivo transduction, toxicity, and altered immunogenicity. Many of these accomplishments resulted from studies of species D adenoviruses—the most diverse yet underexplored group of Ads. Previously reported low seroprevalence in humans of Ad43, an otherwise unstudied member of species D, identified this rare serotype as an attractive prototype of a novel platform for human gene therapy vectors. This study is the first to focus on assessment of biological properties of Ad43 essential to its vectorization. We found that Ad43 virions do not interact with blood coagulation factor X and cause low random transduction upon systemic delivery. Intravenously injected Ad43 clears host tissues more quickly than do traditionally used Ad5 vectors. We identified two receptors that Ad43 uses for infection in vitro and demonstrated that the tropism of Ad43 can be altered genetically to achieve gene delivery via a cancer-associated receptor.
83. Decorin-Expressing Adenovirus Decreases Collagen Synthesis and Upregulates the MMP Expression in Keloids; Implication for Reversing Pathologic Fibrosis
Won Jai Lee,1 Hyo-Min Ahn,3 Hyun Roh,1 Ju Hee Lee,2 Il-Kyu Choi,3 Yong Oock Kim,1 Dae Hyun Lew,1 Chae-Ok Yun.3 1 Plastic & Reconstructive Surgery, Human Tissue Restoration, Yonsei University College of Medicine, Seoul, Korea; 2 Dermatology and Cutaneous Biology Research Institute, Human Tissue Restoration, Yonsei University college of Medicine, Seoul, Korea; 3Bioengineering, College of Engineering, Hanyang University, 17 Haengdang-Dong, Seondgdong-Gu, Seoul, Korea.
Background Decorin is a natural neutralizer of TGF-β1 and reduced synthesis of decorin has been associated with the dermal scars, and therefore an increase of decorin expression appears to reduce scar tissue. To investigate the therapeutic potentials of decorin-expressing replication-incompetent adenovirus (dE1-RGD/GFP/DCN) for keloid and hypertrophic scars, we examined the effects of dE1-RGD/GFP/ DCN on the expression levels of type I and III collagen as well as MMP-1 and -3 in human dermal fibroblast (HDFs) and keloid-derived fibroblast (KFs) cell lines. Methods Decorin expressions were examined by immunofluorescence assay in keloid tissues. HDFs and KFs were infected with dE1-RGD/GFP/DCN or control virus, and protein levels of decorin and secreted TGF-ß1 were assessed by ELISA, while mRNA levels of collagen type I, collagen III, MMP-1, MMP-3 by real time RT-PCR. Additionally, the expression levels of major extracellular matrix (ECM) were investigated by immunohistochemistry in keloid spheroids transduced with dE1-RGD/GFP/DCN. Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Results Lower expression of decorin was examined in the keloid region compared to the adjacent normal tissues. Treatment with dE1-RGD/DFP/DCN decreased mRNA levels of type I collagen in the TGF-β (10 ng/ml)-treated HDFs and KFs and TGF-β protein expression in the HDFs. In addition, the expression of MMP-1 and-3 mRNA was strongly upregulated in KFs and TGF-β-treated HDFs after treatment with dE1-RGD/DFP/DCN. Moreover, expression of major ECMs such as type I collagen, fibronectin, and elastin were significantly reduced in keloid spheroids transduced with dE1-RGD/ DFP/DCN. Conclusion These results support the use of decorin-expressing adenovirus in gene therapy protocols to lower collagen synthesis in KFs, as this decrease is a highly desirable effect for the treatment of keloids.
84. Effects of Alginate Gel-Encapsulated Adenoviruses Expressing Relaxin on Scar Remodeling in a Pig Model
In Sik Yun,1 Won Jai Lee,1 Yong Oock Kim,1 Eun Hye Kang,1 IlKyu Choi,2 Hoguen Kim,3 Kee Yang Chung,4 Seung-Kyu Han,5 Dae Hyun Lew,1 Dong Kyun Rah,1 Chae-Ok Yun.2 1 Department of Plastic & Reconstructive Surgery, Yonsei University College of Medicine, Seoul, Korea; 2Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Korea; 3Department of Pathology, Yonsei University College of Medicine, Seoul, Korea; 4Department of Dermatology and Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, Korea; 5Department of Plastic & Reconstructive Surgery, Korea University College of Medicine, Seoul, Korea. Adenoviruses (Ad) have been well documented as vectors in the context of cancer gene therapy. However, repetitive treatments of Ad therapies have shown to be limited due to their short maintenance of biological activity in vivo. In this study, we investigated the efficacy of sustained Ad delivery using an injectable alginate gel matrix system containing Ad expressing Relaxin (RLX), a transforming growth factor-b1 (TGF-b1) antagonist implicated as a potent collagen rearranger and major extracellular matrix components reducer. Additionally, Yorkshire pigs scar tissues were used as our animal models for their similarities in dermal composition and repair mechanisms of human skin. Relaxin-expressing replicationincompetent Ad loaded in alginate gel (gel/Ad-RLX) decreased scar size, color index and pliability in a pig scar tissues. These results were observed through expression of relaxin, which was assessed by immunofluorescence staining. Immunohistochemical investigation of gel/Ad-RLX-treated groups revealed decreased major extracellular matrix (ECM) protein expression levels in scar tissues. Furthermore, treatment with gel/Ad-RLX showed decreased tissue inhibitors of metalloproteinases-1 (TIMP-1) and alpha-smooth muscle actin (α-SMA) proteins while showing markedly increased expression levels of matrix metalloproteinases-1 (MMP-1) in a pig scar tissues. Moreover, gel/Ad-RLX significantly downregulated TGF-b1 and upregulated TGF-b3 mRNA expression in a pig scar tissues. Together, these results support relaxin’s prominent role in scar remodeling and suggest that relaxin-expressing adenovirus loaded in alginate gel may have therapeutic effects on scar formation.
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