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Topically delivered siRNA for cutaneous gene suppression
Vol. 207, No. 3S, September 2008
Surgical Forum Abstracts
S103
mRNA stability. The clinical relevance of the observed 3’UTR mutant is supported by our observation that it is also associated with both a growth advantage and an increased sensitivity to EGFR targeted therapy in LIM1215, a human MSI CRC cell line.
in gestation the neural ectoderm is exposed to the amniotic cavity via the open neural tube. At this developmental stage, nascent CNS stem cells are theoretically accessible. Based on these developmental events, we hypothesized that intraamniotic delivery of lentiviral vector would result in relatively efficient gene transfer to CNS progenitor cells.
Topically delivered siRNA for cutaneous gene suppression
METHODS: Time-dated Balb/c mice underwent ultrasound guided intraamniotic injection at post coital day 8 (E8) using a VisualSonics Biomicroscopy system. Each fetus received 109 infectious particles of a self inactivating HIV-1 based vector, encoding GFP, driven by the CMV promoter. The CNS was analyzed by fluorescent stereoscopic microscopy and by immunohistochemistry and confocal microscopy of brain sections for co-localization of GFP and neural, glial or neural progenitor markers.
Alexander M Sailon BA, Vishal D Thanik MD, Richard A Zoumalan MD, Christopher C Chang BA, Jamie P Levine MD, FACS, Stephen M Warren MD, Pierre B Saadeh MD New York University School of Medicine, New York, NY INTRODUCTION: Cutaneous diseases are a large source of morbidity. Ultimately, aberrant local gene signaling is responsible for most of these derangements, including neoplasms and autoimmune skin disorders. Targeted modulation of gene signaling may provide an attractive alternative to current therapeutic modalities. METHODS: A ten-millimeter diameter area on depilitated dorsum of wild-type (C57/BLK6) mice was pretreated with 1% triton-x solution (6 hours). The solution was removed and small interfering RNA (siRNA) against mitogen-activated protein kinase-1 (MAPK-1) (selected as proof of principle due to its ubiquity) was incorporated into a cooling agarose matrix, applied to pre-treated skin, and covered with an occlusive dressing. Skin was harvested (day 5; non-treated skin⫽control). Skin was evaluated (immunohistochemistry, Western blot, rtPCR) for MAPK-1 expression. Lung, liver, and spleen homogenates were also evaluated. RESULTS: All mice tolerated the triton-x and gel-based siRNA without obvious toxicity or gross skin changes. Histologic analysis of the treated areas demonstrated near complete knockdown of MAPK-1 through the full-thickness epidermis and dermis. Control skin demonstrated normal MAPK-1 expression. Western blot and rtPCR demonstrated near-complete knockdown of MAPK-1 expression in treated skin compared to controls. Whole organ homogenate showed no systemic knockdown of MAPK-1 expression. CONCLUSIONS: We have developed a novel method to transcutaneously modulate local gene expression in intact skin. Only the local, treated area was affected, with surrounding skin exhibiting normal gene expression. Knockdown was near-complete with no systemic effects/delivery. By selecting specific siRNA(s) of interest, targeted therapy of gene dysregulation in cutaneous disease states is possible.
Gene transfer to progenitor cells of the central nervous system by early intraamniotic delivery of lentiviral vector David Stitelman MD, Endo Masayuki MD, PhD, Philip Zoltick MD, Timothy Brazelton MD, PhD, Alan W Flake MD, FACS Children’s Hospital of Philadelphia, Philadelphia, PA INTRODUCTION: Efficient gene transfer to central nervous system (CNS) progenitor cells remains an elusive goal in gene therapy. Early
RESULTS: Evidence of diffuse CNS GFP expression was seen at birth and all ages thereafter up to 1 year of age by fluorescent stereoscopic microscopy. Immunohistochemistry confirmed persistent expression of GFP by neurons, glial cells, and by neurons in the olfactory bulb and hippocampus, which are known to be derived from neural progenitor division. CONCLUSIONS: These data confirm that early intraamniotic injection of lentiviral vector can result in efficient transduction of CNS progenitors that ultimately form the brain with associated long term expression of transgene. This relatively simple approach, has potential biological application toward understanding of CNS gene function, and ultimately may have therapeutic application toward several devastating congenital neurologic disorders.
Tautomycetin suppresses carcinoid tumor growth and bioactive hormone production through inactivation of glycogen synthase kinase-3 beta Scott N Pinchot MD, Joel T Adler BA, Yinggang Luo PhD, Jianhua Ju PhD, Wenli Li PhD, Ben Shen PhD, Muthusamy Kunnimalaiyaan PhD, Herbert Chen MD, FACS University of Wisconsin, Madison, WI INTRODUCTION: Carcinoids are neuroendocrine (NE) tumors that produce bioactive substances such as 5-hydroxytryptamine (5HT) and chromogranin A (CgA), which can cause the carcinoid syndrome. Besides surgery, limited curative and palliative treatments are available to patients with carcinoid tumors. Glycogen synthase kinase-3 beta (GSK-3 beta) is an important regulator of cell proliferation and survival. In this study, we investigated a novel treatment for carcinoid cell growth based on pharmacologic inactivation of GSK-3 beta using the natural compound tautomycetin (TTN), an antifungal antibiotic isolated from Streptomyces griseochromogens. METHODS: Human pancreatic (BON) and pulmonary (H727) carcinoid cells were treated with TTN (1 to 5 micromolar) or control (DMSO) for 48 hours. To measure for inactivation of GSK-3 beta, western analysis was performed for phospho-GSK-3 beta and total GSK-3 beta. To measure bioactive substances, western analysis was performed for the NE tumor markers CgA and human achaete-scute complex-like 1 (ASCL1). Cellular growth was measured by MTT cell-proliferation assay.
Surgical Forum Abstracts
S103
mRNA stability. The clinical relevance of the observed 3’UTR mutant is supported by our observation that it is also associated with both a growth advantage and an increased sensitivity to EGFR targeted therapy in LIM1215, a human MSI CRC cell line.
in gestation the neural ectoderm is exposed to the amniotic cavity via the open neural tube. At this developmental stage, nascent CNS stem cells are theoretically accessible. Based on these developmental events, we hypothesized that intraamniotic delivery of lentiviral vector would result in relatively efficient gene transfer to CNS progenitor cells.
Topically delivered siRNA for cutaneous gene suppression
METHODS: Time-dated Balb/c mice underwent ultrasound guided intraamniotic injection at post coital day 8 (E8) using a VisualSonics Biomicroscopy system. Each fetus received 109 infectious particles of a self inactivating HIV-1 based vector, encoding GFP, driven by the CMV promoter. The CNS was analyzed by fluorescent stereoscopic microscopy and by immunohistochemistry and confocal microscopy of brain sections for co-localization of GFP and neural, glial or neural progenitor markers.
Alexander M Sailon BA, Vishal D Thanik MD, Richard A Zoumalan MD, Christopher C Chang BA, Jamie P Levine MD, FACS, Stephen M Warren MD, Pierre B Saadeh MD New York University School of Medicine, New York, NY INTRODUCTION: Cutaneous diseases are a large source of morbidity. Ultimately, aberrant local gene signaling is responsible for most of these derangements, including neoplasms and autoimmune skin disorders. Targeted modulation of gene signaling may provide an attractive alternative to current therapeutic modalities. METHODS: A ten-millimeter diameter area on depilitated dorsum of wild-type (C57/BLK6) mice was pretreated with 1% triton-x solution (6 hours). The solution was removed and small interfering RNA (siRNA) against mitogen-activated protein kinase-1 (MAPK-1) (selected as proof of principle due to its ubiquity) was incorporated into a cooling agarose matrix, applied to pre-treated skin, and covered with an occlusive dressing. Skin was harvested (day 5; non-treated skin⫽control). Skin was evaluated (immunohistochemistry, Western blot, rtPCR) for MAPK-1 expression. Lung, liver, and spleen homogenates were also evaluated. RESULTS: All mice tolerated the triton-x and gel-based siRNA without obvious toxicity or gross skin changes. Histologic analysis of the treated areas demonstrated near complete knockdown of MAPK-1 through the full-thickness epidermis and dermis. Control skin demonstrated normal MAPK-1 expression. Western blot and rtPCR demonstrated near-complete knockdown of MAPK-1 expression in treated skin compared to controls. Whole organ homogenate showed no systemic knockdown of MAPK-1 expression. CONCLUSIONS: We have developed a novel method to transcutaneously modulate local gene expression in intact skin. Only the local, treated area was affected, with surrounding skin exhibiting normal gene expression. Knockdown was near-complete with no systemic effects/delivery. By selecting specific siRNA(s) of interest, targeted therapy of gene dysregulation in cutaneous disease states is possible.
Gene transfer to progenitor cells of the central nervous system by early intraamniotic delivery of lentiviral vector David Stitelman MD, Endo Masayuki MD, PhD, Philip Zoltick MD, Timothy Brazelton MD, PhD, Alan W Flake MD, FACS Children’s Hospital of Philadelphia, Philadelphia, PA INTRODUCTION: Efficient gene transfer to central nervous system (CNS) progenitor cells remains an elusive goal in gene therapy. Early
RESULTS: Evidence of diffuse CNS GFP expression was seen at birth and all ages thereafter up to 1 year of age by fluorescent stereoscopic microscopy. Immunohistochemistry confirmed persistent expression of GFP by neurons, glial cells, and by neurons in the olfactory bulb and hippocampus, which are known to be derived from neural progenitor division. CONCLUSIONS: These data confirm that early intraamniotic injection of lentiviral vector can result in efficient transduction of CNS progenitors that ultimately form the brain with associated long term expression of transgene. This relatively simple approach, has potential biological application toward understanding of CNS gene function, and ultimately may have therapeutic application toward several devastating congenital neurologic disorders.
Tautomycetin suppresses carcinoid tumor growth and bioactive hormone production through inactivation of glycogen synthase kinase-3 beta Scott N Pinchot MD, Joel T Adler BA, Yinggang Luo PhD, Jianhua Ju PhD, Wenli Li PhD, Ben Shen PhD, Muthusamy Kunnimalaiyaan PhD, Herbert Chen MD, FACS University of Wisconsin, Madison, WI INTRODUCTION: Carcinoids are neuroendocrine (NE) tumors that produce bioactive substances such as 5-hydroxytryptamine (5HT) and chromogranin A (CgA), which can cause the carcinoid syndrome. Besides surgery, limited curative and palliative treatments are available to patients with carcinoid tumors. Glycogen synthase kinase-3 beta (GSK-3 beta) is an important regulator of cell proliferation and survival. In this study, we investigated a novel treatment for carcinoid cell growth based on pharmacologic inactivation of GSK-3 beta using the natural compound tautomycetin (TTN), an antifungal antibiotic isolated from Streptomyces griseochromogens. METHODS: Human pancreatic (BON) and pulmonary (H727) carcinoid cells were treated with TTN (1 to 5 micromolar) or control (DMSO) for 48 hours. To measure for inactivation of GSK-3 beta, western analysis was performed for phospho-GSK-3 beta and total GSK-3 beta. To measure bioactive substances, western analysis was performed for the NE tumor markers CgA and human achaete-scute complex-like 1 (ASCL1). Cellular growth was measured by MTT cell-proliferation assay.