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505 Novel therapeutic approach using cell-mediated IL-12 gene transfer with adipose-derived mesenchymal stem cells for melanoma treatment
Genetic Disease, Gene Regulation and Gene Therapy | ABSTRACTS 504
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Identification of a novel mevalonate kinase missense mutation in a case of disseminated superficial actinic porokeratosis R Stone1, G Glinos2, A Giubellino2, M Tomic-Canic1 and M Miteva2 1 University of Miami, Miami, FL and 2 Department of Dermatology and Cutaneous Surgery, University of MiamiMiller School of Medicine, Miami, FL Keratinocyte differentiation is a tightly controlled process that is essential for survival. The mevalonate/isoprenoid biosynthesis pathway has a central role in protein isoprenylation as well as in cholesterol synthesis by the epidermis, contributing to the processes of terminal differentiation and barrier formation in the skin. Disseminated superficial actinic porokeratosis (DSAP) is the most common form of porokeratosis, a group of inherited keratinization disorders characterized by lesions containing hyperkeratotic ridges that correlate histopathologically with the presence of a cornoid lamella. Several causal DSAP mutations have previously been identified in members of the mevalonate pathway, including mevalonate kinase (MVK). In this study, a 50 year old woman with a 15-year history of non-pruritic 3-5 mm pink papules with scaly rims on her arms and legs presented to the clinic. The diagnosis of DSAP was made based upon family history, characteristic clinical appearance of the lesions, and histologic findings on skin biopsy. Sanger sequencing of the MVK exons was performed on genomic DNA extracted from a paraffin-embedded biopsy specimen. A novel heterozygous missense mutation was identified in exon 5 (c.455:G>A), resulting in the substitution of tyrosine for cysteine (p.C152Y) in a predicted functional domain of the MVK enzyme. Impaired keratinocyte differentiation and apoptosis and defective sterol-mediated barrier formation resulting from mevalonate pathway mutations can account for the clinical DSAP phenotype.
Novel therapeutic approach using cell-mediated IL-12 gene transfer with adipose-derived mesenchymal stem cells for melanoma treatment T Arita1, T Kishida2, N Katoh1, O Mazda3 and J Asai1 1 Dermatology, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2 Immunology, Kyoto Prefectural University of Medicine, Kyoto, Japan and 3 Immunology, Kyoto Prefectural University of Medicine, Kyoto, Japan Recent studies have revealed that adipose-derived mesenchymal stem cells (AD-MSC) have the property of migrating toward tumor site, and therefore, could act as a vehicle for an antitumor gene therapy. IL-12 is one of the most potent cytokines having strong anti-tumor effects, however, systemic application is problematic due to its toxicity. Efficient local administration of IL-12 is thought to be promising and desirable approach to reduce its toxicity. The purpose of this study is to engineer IL-12 overexpressing AD-MSC and to evaluate therapeutic potential to melanoma. IL-12 overexpressing AD-MSC was generated with gene transfer using a retroviral vector. Successful transduction of the IL-12 in AD-MSC was confirmed by PCR and ELISA. Migration capacity and efficacy of treatment are under consideration by further experiments with melanoma mouse model. These data suggest that AD-MSC mediated gene delivery shows therapeutic potential for the treatment of melanoma.
A case of recessive dystrophic epidermolysis bullosa with a novel c.6885_6898del14 mutation S Shinkukma1, T Masunaga2, S Miyawaki2, S Takashima1, T Nomura1, Y Fujita1, H Nakamura1 and H Shimizu1 1 Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan and 2 Department of Dermatology, Ehime University, Ehime, Japan Recessive dystrophic epidermolysis bullosa (RDEB), an inherited blistering skin disorder, is caused by mutations in the COL7A1 gene encoding type VII collagen (COL7). Gene analysis in RDEB has so far disclosed certain genotype-phenotype correlations, and, therefore, it is important to understand the molecular mechanism of each mutation. A 2-day-old girl was referred to our department with extensive blistering and erosions on the whole body. Immunofluorescence mapping showed that COL7 at the dermal-epidermal junction was markedly reduced. Transmission electron microscopy showed clefts within the sublamina densa region and a few thin, poorly formed anchoring fibrils. These results led us to diagnose RDEB, generalized severe. Direct sequencing analysis revealed that the patient was compound heterozygous for the mutations, c.6573+1G>C and c.6885_6898del14. c.6573+1G>C is a well-known splice site mutation, resulting in aberrant splicing leading to a frameshift. On the other hand, pathogenic mechanism of the novel mutation (c.6885_6898del14) which is located at exon-intron boundary region is unclear. To identify the molecular mechanism of the mutation, we performed reverse transcriptase-PCR and sequencing of cDNA from fibroblasts that showed two splicing variants leading to a frameshift and a downstream premature termination codon.
Understanding mechanisms that govern premature termination codon read-through in human skin diseases V Atanasova1, Q Jiang1, J Uitto2 and A South2 1 Thomas Jefferson University, Philadelphia, PA and 2 TJU, Philadelphia, PA Up to 30% of mutant alleles contributing to genetic diseases harbor a premature termination codon (PTC). As result there is lack of functional protein synthesized and this often leads to severe clinical consequences, as is the case with recessive dystrophic epidermolysis bullosa (RDEB) where approximately 43% of patients harbor PTC mutations in COL7A1. RDEB is a rare genetic skin disease characterized by trauma induced skin blistering and extra-cutaneous complications. The aim of this study is to understand mechanisms determining endogenous and pharmacologically enhanced read-through of PTC mutations in RDEB patient derived skin cells. Unraveling the mechanisms which determine and allow for read-through induced protein synthesis would be a valuable tool in designing therapies for genetic diseases caused by PTC mutations. We evaluated two compounds which were shown to induce read-through e amlexanox and gentamicin e in RDEB patient skin cells harboring PTCs. We show by Western blotting that increase in full length type VII collagen is only evident in cells which initially exhibit endogenous read-through, detected as trace amount of protein. These data suggest that endogenous read-through, even at low level, is a pre-requisite for amlexanox or gentamicin induced read-through of PTCs. Furthermore, the data show that efficiency of readthrough protein synthesis correlates with increased phosphorylation of up-frameshift protein 1 (UPF1). UPF1 is an ATP driven RNA helicase critical for initiation of non-sense mediated mRNA decay (NMD). Read-through efficiency also correlated with an increase in PTC containing transcripts after treatment. This study is the first to elucidate mechanisms that govern read-through of PTC in human skin cells and forms the basis for development of future therapeutic approaches for genetic diseases caused by PTC mutations.
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Reciprocal regulation of lysyl hydroxylase 3 and type VII collagen secretion: Pathomechanistic link in recessive dystrophic epidermolysis bullosa M Farshchian1, J Salas Alanis2 and A South3 1 Thomas Jefferson University, Philadelphia, PA, 2 RDEB SCC Sequencing Consortium, Monterrey, Mexico and 3 TJU, Philadelphia, PA We previously showed that the collagen modifying enzyme, lysyl hydroxylase 3 (LH3) localized to the basement membrane and is reduced in recessive dystrophic epidermolysis bullosa (RDEB) patient skin. RDEB is caused by loss of function mutations in COL7A1, the gene encoding type VII collagen (C7), and is characterized by trauma induced skin and mucosal membrane blistering. LH3 is the only lysyl hydroxylase (LH) to be secreted and is the only LH capable of attaching carbohydrates to hydroxylysine residues of collagen. However, the exact role of extracellular LH3 remains unclear. The aim of this study was to understand the relationship between C7 and LH3 expression and to determine the impact of reduced LH3 in RDEB patient skin. We used retroviral overexpression and siRNA knockdown of both C7 and LH3 in primary epidermal keratinocytes and fibroblasts. Western blot analysis and immunofluorescence staining revealed less LH3 in RDEB cells compared to normal cells (average fold-change 0.4). Overexpression of LH3 in normal and RDEB fibroblasts markedly elevated LH3 secretion (average fold-change 14.4 and 36.2) but only slightly increased the intracellular level of LH3 (average fold-change 2.2 and 1.3). Overexpression of C7 increased secretion of LH3 in both normal and RDEB fibroblasts (average fold-change 3.0 and 4.6). In addition, C7 knockdown resulted in a significant reduction in LH3 expression (fold-change 0.2). Interestingly LH3 overexpression increased C7 expression in normal fibroblasts (foldchange 20). Taken together these data demonstrate that changes in LH3 levels greatly impact LH3 secretion and that LH3 and C7 exhibit reciprocal regulation of expression and secretion. Given that reduction in LH3 secretion has a negative effect on extracellular matrix production, LH3 may represent a novel therapeutic target in RDEB.
Disease-targeted next generation sequencing identifies mutations in patients with epidermolysis bullosa A Saeidian1, L Youssefian1, H Vahidnezhad2, S Zeinali3, M Daneshpazhooh4, M Hamid5, A Ertel1, P Fortina1 and J Uitto1 1 TJU, Philadelphia, PA, 2 TJU, Pasteur Institute, Philadelphia, PA, 3 Pasteur Institute, Tehran, Iran, 4 TUMS, Tehran, Iran and 5 Pasteur Institute of Iran, Tehran, Iran Epidermolysis bullosa (EB) comprises a phenotypically and genotypically heterogeneous group of blistering disorders with the clinical hallmark of skin and mucosal fragility. The lack of definitive clinical findings suggestive of a candidate gene for molecular diagnosis of EB necessitated in the past a skin biopsy for electron microscopy and/or immune-epitope mapping as a screening method before formal mutational analysis. However, the antigen mapping is a complicated and demanding technique in terms of expertise and cost and is restricted to a few referral EB centers. To circumvent these problems, we developed an EBtargeted next generation sequencing panel consisting of 21 genes, including CD151, CDSN, CHST8, COL17A1, COL7A1, DSP, DST, EXPH5, FERMT1, ITGA3, ITGA6, ITGB4, JUP, KRT5, KRT14, LAMA3, LAMB3, LAMC2, PKP1, PLEC and TGM5 (18 genes reported to be causative for EB and 3 genes causing skin fragility disorders in the differential diagnosis). A total of 94 patients with clinical diagnosis of EB were sequenced. In 54 out of 94 families (detection rate of 57.5%) pathogenic variants were found in 10 different EB associated genes. Most common EB mutated genes in our Iranian cohort were COL7A1, COL17A1, LAMB3, KRT14 (17%, 14%, 7.5% and 6.5% of all EB cases, respectively) and collectively they comprise about 45% of mutated genes. A previously unreported splicing junction mutation, the second pathogenic variant in CD151, was disclosed in a patient with pretibial EB and nephropathy. The other 5 disclosed mutations were located in LAMA3, LAMC2, PLEC, FERMT1 and EXPH5. In conclusion, we developed a disease-targeted NGS approach which is rapid, minimally invasive, and cost effective in identifying mutations in EB patients.
www.jidonline.org S87
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Identification of a novel mevalonate kinase missense mutation in a case of disseminated superficial actinic porokeratosis R Stone1, G Glinos2, A Giubellino2, M Tomic-Canic1 and M Miteva2 1 University of Miami, Miami, FL and 2 Department of Dermatology and Cutaneous Surgery, University of MiamiMiller School of Medicine, Miami, FL Keratinocyte differentiation is a tightly controlled process that is essential for survival. The mevalonate/isoprenoid biosynthesis pathway has a central role in protein isoprenylation as well as in cholesterol synthesis by the epidermis, contributing to the processes of terminal differentiation and barrier formation in the skin. Disseminated superficial actinic porokeratosis (DSAP) is the most common form of porokeratosis, a group of inherited keratinization disorders characterized by lesions containing hyperkeratotic ridges that correlate histopathologically with the presence of a cornoid lamella. Several causal DSAP mutations have previously been identified in members of the mevalonate pathway, including mevalonate kinase (MVK). In this study, a 50 year old woman with a 15-year history of non-pruritic 3-5 mm pink papules with scaly rims on her arms and legs presented to the clinic. The diagnosis of DSAP was made based upon family history, characteristic clinical appearance of the lesions, and histologic findings on skin biopsy. Sanger sequencing of the MVK exons was performed on genomic DNA extracted from a paraffin-embedded biopsy specimen. A novel heterozygous missense mutation was identified in exon 5 (c.455:G>A), resulting in the substitution of tyrosine for cysteine (p.C152Y) in a predicted functional domain of the MVK enzyme. Impaired keratinocyte differentiation and apoptosis and defective sterol-mediated barrier formation resulting from mevalonate pathway mutations can account for the clinical DSAP phenotype.
Novel therapeutic approach using cell-mediated IL-12 gene transfer with adipose-derived mesenchymal stem cells for melanoma treatment T Arita1, T Kishida2, N Katoh1, O Mazda3 and J Asai1 1 Dermatology, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2 Immunology, Kyoto Prefectural University of Medicine, Kyoto, Japan and 3 Immunology, Kyoto Prefectural University of Medicine, Kyoto, Japan Recent studies have revealed that adipose-derived mesenchymal stem cells (AD-MSC) have the property of migrating toward tumor site, and therefore, could act as a vehicle for an antitumor gene therapy. IL-12 is one of the most potent cytokines having strong anti-tumor effects, however, systemic application is problematic due to its toxicity. Efficient local administration of IL-12 is thought to be promising and desirable approach to reduce its toxicity. The purpose of this study is to engineer IL-12 overexpressing AD-MSC and to evaluate therapeutic potential to melanoma. IL-12 overexpressing AD-MSC was generated with gene transfer using a retroviral vector. Successful transduction of the IL-12 in AD-MSC was confirmed by PCR and ELISA. Migration capacity and efficacy of treatment are under consideration by further experiments with melanoma mouse model. These data suggest that AD-MSC mediated gene delivery shows therapeutic potential for the treatment of melanoma.
A case of recessive dystrophic epidermolysis bullosa with a novel c.6885_6898del14 mutation S Shinkukma1, T Masunaga2, S Miyawaki2, S Takashima1, T Nomura1, Y Fujita1, H Nakamura1 and H Shimizu1 1 Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan and 2 Department of Dermatology, Ehime University, Ehime, Japan Recessive dystrophic epidermolysis bullosa (RDEB), an inherited blistering skin disorder, is caused by mutations in the COL7A1 gene encoding type VII collagen (COL7). Gene analysis in RDEB has so far disclosed certain genotype-phenotype correlations, and, therefore, it is important to understand the molecular mechanism of each mutation. A 2-day-old girl was referred to our department with extensive blistering and erosions on the whole body. Immunofluorescence mapping showed that COL7 at the dermal-epidermal junction was markedly reduced. Transmission electron microscopy showed clefts within the sublamina densa region and a few thin, poorly formed anchoring fibrils. These results led us to diagnose RDEB, generalized severe. Direct sequencing analysis revealed that the patient was compound heterozygous for the mutations, c.6573+1G>C and c.6885_6898del14. c.6573+1G>C is a well-known splice site mutation, resulting in aberrant splicing leading to a frameshift. On the other hand, pathogenic mechanism of the novel mutation (c.6885_6898del14) which is located at exon-intron boundary region is unclear. To identify the molecular mechanism of the mutation, we performed reverse transcriptase-PCR and sequencing of cDNA from fibroblasts that showed two splicing variants leading to a frameshift and a downstream premature termination codon.
Understanding mechanisms that govern premature termination codon read-through in human skin diseases V Atanasova1, Q Jiang1, J Uitto2 and A South2 1 Thomas Jefferson University, Philadelphia, PA and 2 TJU, Philadelphia, PA Up to 30% of mutant alleles contributing to genetic diseases harbor a premature termination codon (PTC). As result there is lack of functional protein synthesized and this often leads to severe clinical consequences, as is the case with recessive dystrophic epidermolysis bullosa (RDEB) where approximately 43% of patients harbor PTC mutations in COL7A1. RDEB is a rare genetic skin disease characterized by trauma induced skin blistering and extra-cutaneous complications. The aim of this study is to understand mechanisms determining endogenous and pharmacologically enhanced read-through of PTC mutations in RDEB patient derived skin cells. Unraveling the mechanisms which determine and allow for read-through induced protein synthesis would be a valuable tool in designing therapies for genetic diseases caused by PTC mutations. We evaluated two compounds which were shown to induce read-through e amlexanox and gentamicin e in RDEB patient skin cells harboring PTCs. We show by Western blotting that increase in full length type VII collagen is only evident in cells which initially exhibit endogenous read-through, detected as trace amount of protein. These data suggest that endogenous read-through, even at low level, is a pre-requisite for amlexanox or gentamicin induced read-through of PTCs. Furthermore, the data show that efficiency of readthrough protein synthesis correlates with increased phosphorylation of up-frameshift protein 1 (UPF1). UPF1 is an ATP driven RNA helicase critical for initiation of non-sense mediated mRNA decay (NMD). Read-through efficiency also correlated with an increase in PTC containing transcripts after treatment. This study is the first to elucidate mechanisms that govern read-through of PTC in human skin cells and forms the basis for development of future therapeutic approaches for genetic diseases caused by PTC mutations.
508
509
Reciprocal regulation of lysyl hydroxylase 3 and type VII collagen secretion: Pathomechanistic link in recessive dystrophic epidermolysis bullosa M Farshchian1, J Salas Alanis2 and A South3 1 Thomas Jefferson University, Philadelphia, PA, 2 RDEB SCC Sequencing Consortium, Monterrey, Mexico and 3 TJU, Philadelphia, PA We previously showed that the collagen modifying enzyme, lysyl hydroxylase 3 (LH3) localized to the basement membrane and is reduced in recessive dystrophic epidermolysis bullosa (RDEB) patient skin. RDEB is caused by loss of function mutations in COL7A1, the gene encoding type VII collagen (C7), and is characterized by trauma induced skin and mucosal membrane blistering. LH3 is the only lysyl hydroxylase (LH) to be secreted and is the only LH capable of attaching carbohydrates to hydroxylysine residues of collagen. However, the exact role of extracellular LH3 remains unclear. The aim of this study was to understand the relationship between C7 and LH3 expression and to determine the impact of reduced LH3 in RDEB patient skin. We used retroviral overexpression and siRNA knockdown of both C7 and LH3 in primary epidermal keratinocytes and fibroblasts. Western blot analysis and immunofluorescence staining revealed less LH3 in RDEB cells compared to normal cells (average fold-change 0.4). Overexpression of LH3 in normal and RDEB fibroblasts markedly elevated LH3 secretion (average fold-change 14.4 and 36.2) but only slightly increased the intracellular level of LH3 (average fold-change 2.2 and 1.3). Overexpression of C7 increased secretion of LH3 in both normal and RDEB fibroblasts (average fold-change 3.0 and 4.6). In addition, C7 knockdown resulted in a significant reduction in LH3 expression (fold-change 0.2). Interestingly LH3 overexpression increased C7 expression in normal fibroblasts (foldchange 20). Taken together these data demonstrate that changes in LH3 levels greatly impact LH3 secretion and that LH3 and C7 exhibit reciprocal regulation of expression and secretion. Given that reduction in LH3 secretion has a negative effect on extracellular matrix production, LH3 may represent a novel therapeutic target in RDEB.
Disease-targeted next generation sequencing identifies mutations in patients with epidermolysis bullosa A Saeidian1, L Youssefian1, H Vahidnezhad2, S Zeinali3, M Daneshpazhooh4, M Hamid5, A Ertel1, P Fortina1 and J Uitto1 1 TJU, Philadelphia, PA, 2 TJU, Pasteur Institute, Philadelphia, PA, 3 Pasteur Institute, Tehran, Iran, 4 TUMS, Tehran, Iran and 5 Pasteur Institute of Iran, Tehran, Iran Epidermolysis bullosa (EB) comprises a phenotypically and genotypically heterogeneous group of blistering disorders with the clinical hallmark of skin and mucosal fragility. The lack of definitive clinical findings suggestive of a candidate gene for molecular diagnosis of EB necessitated in the past a skin biopsy for electron microscopy and/or immune-epitope mapping as a screening method before formal mutational analysis. However, the antigen mapping is a complicated and demanding technique in terms of expertise and cost and is restricted to a few referral EB centers. To circumvent these problems, we developed an EBtargeted next generation sequencing panel consisting of 21 genes, including CD151, CDSN, CHST8, COL17A1, COL7A1, DSP, DST, EXPH5, FERMT1, ITGA3, ITGA6, ITGB4, JUP, KRT5, KRT14, LAMA3, LAMB3, LAMC2, PKP1, PLEC and TGM5 (18 genes reported to be causative for EB and 3 genes causing skin fragility disorders in the differential diagnosis). A total of 94 patients with clinical diagnosis of EB were sequenced. In 54 out of 94 families (detection rate of 57.5%) pathogenic variants were found in 10 different EB associated genes. Most common EB mutated genes in our Iranian cohort were COL7A1, COL17A1, LAMB3, KRT14 (17%, 14%, 7.5% and 6.5% of all EB cases, respectively) and collectively they comprise about 45% of mutated genes. A previously unreported splicing junction mutation, the second pathogenic variant in CD151, was disclosed in a patient with pretibial EB and nephropathy. The other 5 disclosed mutations were located in LAMA3, LAMC2, PLEC, FERMT1 and EXPH5. In conclusion, we developed a disease-targeted NGS approach which is rapid, minimally invasive, and cost effective in identifying mutations in EB patients.
www.jidonline.org S87