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205 Altering the splice pattern of COL17A1 with antisense oligonucleotides
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A comparison of minicircle vectors expressing COL7A1 under the control of human promoters for the treatment of recessive dystrophic epidermolysis bullosa F Alshehri, L Cutlar, I Lara-Sa´ez, D Zhou and W Wang Charles Institute of Dermatology, University College Dublin, Dublin, Ireland Recessive dystrophic epidermolysis bullosa (RDEB) is a blistering disorder caused by mutations in COL7A1 that encodes for the anchoring fibrils of type VII collagen. Our lab has demonstrated that non-viral gene delivery by polymer is able to restore the expression of type VII collagen (C7) to human RDEB skin and could overcome the possibility of generating an immune response when compared to viral vectors. Silencing caused by viral promoters is a major problem for an episomal non-viral gene therapy. To increase the safety profile of COL7A1 therapy more focus needs to be in the DNA cassette itself as an important tool for resisting transgene silencing and extending C7 expression. The feasibility of this approach was demonstrated using a novel gene delivery polymer as a non-viral vector combined with minicircle (MC) DNA encoding full-length COL7A1 under the control of three different promoters, including human COL7A1 promoter (C7p) as a tissue-specific promoter, human elongation factor one alpha (EF1a) and cytomegalovirus (CMV). The three MC plasmid (MCC7p-C7, MC-EF1a -C7 and MC-CMV-C7) were used to transfect human RDEB Keratinocytes (K) and Fibroblasts (F). Type VII collagen expression was detected and compared among the three promoters and confirmed in in vitro at the gene level using RT-qPCR as well as at the protein level using immuno-blotting. All the three MC constructs are capable of achieving expression levels comparable to native protein from healthy human primary cells, there is an indication of a 20 fold C7 expression change detected in transfected RDEBF cells compared to un-transfected cells. A multifaceted analysis of the promoters expression is being performed. In conclusion, our results suggest that an optimal promoter selection is an important enhancement that can be added to the minicircle vector in our non-viral system and therefore will enhance transgene expression and help to reduce immuneimmunogenicity.
Targeting tropomyosin receptor kinase (TRK) in CYLD defective tumours: A placebo-controlled early phase trial with pegcantratinib M Danilenko1, E Stamp2, D Stocken2, A Cranston2, R Plummer4, G Veal4, J Langtry5, A Ashworth3, J Burn1 and N Rajan1 1 Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom, 2 Institute of Health and Society, Newcastle, United Kingdom, 3 Helen Diller Cancer Centre, San Francisco, CA, 4 Northern Institute of Cancer Research, Newcastle, United Kingdom and 5 Royal Victoria Infirmary, Newcastle, United Kingdom Selective targeting of kinases in inherited human cutaneous CYLD defective tumours is a novel strategy for the non-surgical treatment of patients with CYLD cutaneous syndrome (CCS). Here we report the outcome of the first human trial of TRK targeting in CCS using a TRK inhibitor designed for topical application, pegcantratinib. We performed a Phase 1b trial in 8 patients with CCS to determine safety of daily application of pegcantratinib for 4 weeks to a single skin tumour. None of the 8 patients experienced any inflammation or ulceration at treated sites, however some reported reduction in pain from treated tumours. The positive safety data allowed for progression to a Phase 2a within patient placebo-controlled, double blinded trial. 15 patients were recruited, each with 10 small skin tumours that met prespecified tumour specific inclusion criteria, such that 5 matched lesions were selected on each side of the patient’s body (total tumours recruited ¼150). Patients were randomised to receive active treatment to either left or right sides. Treatment consisted of application of pegcantratinib 0.5% w/w and placebo once daily to allocated sides for 12 weeks. The primary outcome measure was the number of tumours meeting the predefined criteria for response, compared against a critical number (12) of pegcantratinib treated tumours in excess to the placebo treated tumours. This was not met. Secondary clinical outcome measures included assessment for safety, pain in small tumours, and concordance with trial protocol. This trial demonstrates safe and feasible short-term topical TRK targeting in CCS, and offers novel tumour natural history data for this rare disease.
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Preliminary therapeutic target validation of the IL-36 receptor in psoriasis SK Mahil1, M Peakman1, R Trembath1, J Wright2, J Barker1 and F Capon1 1 King’s College London, London, United Kingdom and 2 Bradford Royal Infirmary, Bradford, United Kingdom The IL-1 family cytokines IL-36 a, -b and eg are important in maintaining mucosal and epidermal immune homeostasis. As such, mutations in genes regulating IL-36 signalling (IL36RN and AP1S3) have been associated with inflammatory phenotypes e.g. pustular psoriasis. IL-36 may amplify IL-17 driven inflammatory responses and over-expression has been detected in common plaque psoriasis. IL-36 may thus be a promising therapeutic target, and antagonists directed against its receptor (IL-36R) are being developed. However, since the function of IL-36 in broader immune responses remains undetermined, pharmacological blockade may have unexpected adverse effects. We investigated the consequences of IL-36 signalling inhibition through the deep phenotyping of human knockouts who lack a functional IL-36R. These individuals were identified by querying the genotype data generated by “Born in Bradford”, a community-based initiative to exome sequence 2,162 British-Pakistanis. In keeping with the increased parental relatedness in this cohort, we identified numerous participants (n¼28) with low-frequency homozygous changes in IL1RL2 (encoding IL-36R). We recalled 6 subjects harbouring bi-allelic missense changes predicted to be deleterious. IL36 stimulation of PBMCs from these individuals did not elicit an inflammatory response. However, clinical examinations and medical records did not indicate anomalies in immune function. Full blood counts and antibody responses to past immunizations were normal. PBMC treatment with varied inflammatory stimuli (PMA, poly(I:C), C. albicans and Infanrix vaccine) induced up-regulation of inflammatory markers comparable to that observed in ethnically matched controls. Thus, these data suggest that IL-36 blockade may be compatible with normal immune function and pave the way for pre-clinical safety studies of IL-36 inhibitors. Our study also highlights the utility of phenotyping human knockouts in the preclinical assessment of novel drug targets.
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Epidermodysplasia verruciformis: Clinical, viral, and histopathological phenotype in patients with EVER3 mutation E Imahorn1, SJ de Jong2, I Spoerri1, W Kempf3, C Imhof4, P Ha¨usermann5, E Jouanguy2, J Casanova2, B Burger1 and P Itin1,5 1 Department of Biomedicine, University Hospital of Basel and University of Basel, Basel, Switzerland, 2 St. Giles Laboratory, The Rockefeller University, New York City, NY, 3 Kempf and Pfaltz Histological Diagnostics, Zurich, Switzerland, 4 Stadtpraxis Brig, Brig, Switzerland and 5 Department of Dermatology, University Hospital Basel, Basel, Switzerland Patients with the rare genodermatosis epidermodysplasia verruciformis (EV) are susceptible to infections with cutaneous HPV and have a high risk for development of non-melanoma skin cancer (NMSC). Biallelic mutations in TMC6/EVER1 and TMC8/EVER2 have been identified in many patients. Recently, a third gene EVER3 has been identified to be mutated in EV patients without TMC6/8 mutation. In the presented study we evaluated whether the phenotype of EVER3 deficient patients differs from the phenotype of TMC6/8 deficient patients. Therefore, we examined two unrelated Swiss EV patients. TMC6, TMC8, and EVER3 were sequenced on gDNA isolated from whole blood. HPV detection was performed by sequencing the L1 capsid gene after nested PCR on DNA isolated from skin biopsies. No loss of function mutation could be detected in TMC6 and TMC8 in these patients. Sequencing of EVER3 revealed a homozygous frameshift mutation leading to a premature stop codon. In both patients typical EV lesions were present on back of hands, neck, arms, legs, and cheeks comparable to TMC6 and TMC8 patients. First lesions appeared during early childhood. Histopathological examinations revealed EV characteristic features. Both patients developed various precanceroses, squamous cell carcinomas, and basal cell carcinomas. Typical HPV types could be detected in both patients. The results are discussed in context of a systematic literature review of all EV patients described to date. In sum, the two EVER3 deficient patients showed the typical clinical, histopathological, and viral phenotype of EV. No obvious phenotypic variability to TMC6/8 deficient patients could be observed.
Altering the splice pattern of COL17A1 with antisense oligonucleotides H Potocki, T Lettner, M Ablinger, M Wimmer, R Zauner, N Friedl, J Reichelt, JW Bauer and V Wally EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Austria, Laboratory for Molecular Therapy, EB House Austria, Salzburg, Austria Junctional epidermolysis bullosa (JEB) is a devastating disease of the skin and mucous membranes, characterized by blistering and erosions upon minor mechanical friction. Genotypically, mutations within the type XVII collagen gene underlie this phenotype. In this project, we targeted a cryptic splice site arising due to a mutation (c. 380-1 G>A) at the COL17A1 intron 6 / exon 7 junction. Utilization of this splice site leads to the loss of 16 nucleotides at the mRNA level. Subsequently, the reading frame is shifted so that a premature stop codon is generated within exon 9 and the mRNA is degraded by nonsense-mediated decay. Using three distinct antisense oligonucleotides (ASOs), we aimed to either restore the wild type splice pattern of COL17A1 or achieve skipping of exon 7, which would lead to a slightly truncated, but potentially functional type XVII collagen, since the reading frame remains intact. We transfected patient keratinocytes with either of the ASOs and analyzed overall COL17A1 expression, as well as the generation of alternative splicing products. Amplification and sequencing of the COL17A1 mRNA from exon 5 to exon 9 showed skipping of exon 7 as the most prominent result of ASO treatments in JEB keratinocytes. Additionally, alleles with a restored wild type splice pattern, as well as such showing concomitant skipping of exons 6 and 7, were identified. SqRT-PCR confirmed an increase in overall COL17A1 expression. Furthermore, full length (180 kDa) type XVII collagen was detected upon ASO treatment using Western blot analysis. Taken together, we conclude that targeting a cryptic splice site with ASOs may hold therapeutic potential for JEB and possibly other forms of EB with similar types of mutations.
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A comparison of minicircle vectors expressing COL7A1 under the control of human promoters for the treatment of recessive dystrophic epidermolysis bullosa F Alshehri, L Cutlar, I Lara-Sa´ez, D Zhou and W Wang Charles Institute of Dermatology, University College Dublin, Dublin, Ireland Recessive dystrophic epidermolysis bullosa (RDEB) is a blistering disorder caused by mutations in COL7A1 that encodes for the anchoring fibrils of type VII collagen. Our lab has demonstrated that non-viral gene delivery by polymer is able to restore the expression of type VII collagen (C7) to human RDEB skin and could overcome the possibility of generating an immune response when compared to viral vectors. Silencing caused by viral promoters is a major problem for an episomal non-viral gene therapy. To increase the safety profile of COL7A1 therapy more focus needs to be in the DNA cassette itself as an important tool for resisting transgene silencing and extending C7 expression. The feasibility of this approach was demonstrated using a novel gene delivery polymer as a non-viral vector combined with minicircle (MC) DNA encoding full-length COL7A1 under the control of three different promoters, including human COL7A1 promoter (C7p) as a tissue-specific promoter, human elongation factor one alpha (EF1a) and cytomegalovirus (CMV). The three MC plasmid (MCC7p-C7, MC-EF1a -C7 and MC-CMV-C7) were used to transfect human RDEB Keratinocytes (K) and Fibroblasts (F). Type VII collagen expression was detected and compared among the three promoters and confirmed in in vitro at the gene level using RT-qPCR as well as at the protein level using immuno-blotting. All the three MC constructs are capable of achieving expression levels comparable to native protein from healthy human primary cells, there is an indication of a 20 fold C7 expression change detected in transfected RDEBF cells compared to un-transfected cells. A multifaceted analysis of the promoters expression is being performed. In conclusion, our results suggest that an optimal promoter selection is an important enhancement that can be added to the minicircle vector in our non-viral system and therefore will enhance transgene expression and help to reduce immuneimmunogenicity.
Targeting tropomyosin receptor kinase (TRK) in CYLD defective tumours: A placebo-controlled early phase trial with pegcantratinib M Danilenko1, E Stamp2, D Stocken2, A Cranston2, R Plummer4, G Veal4, J Langtry5, A Ashworth3, J Burn1 and N Rajan1 1 Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom, 2 Institute of Health and Society, Newcastle, United Kingdom, 3 Helen Diller Cancer Centre, San Francisco, CA, 4 Northern Institute of Cancer Research, Newcastle, United Kingdom and 5 Royal Victoria Infirmary, Newcastle, United Kingdom Selective targeting of kinases in inherited human cutaneous CYLD defective tumours is a novel strategy for the non-surgical treatment of patients with CYLD cutaneous syndrome (CCS). Here we report the outcome of the first human trial of TRK targeting in CCS using a TRK inhibitor designed for topical application, pegcantratinib. We performed a Phase 1b trial in 8 patients with CCS to determine safety of daily application of pegcantratinib for 4 weeks to a single skin tumour. None of the 8 patients experienced any inflammation or ulceration at treated sites, however some reported reduction in pain from treated tumours. The positive safety data allowed for progression to a Phase 2a within patient placebo-controlled, double blinded trial. 15 patients were recruited, each with 10 small skin tumours that met prespecified tumour specific inclusion criteria, such that 5 matched lesions were selected on each side of the patient’s body (total tumours recruited ¼150). Patients were randomised to receive active treatment to either left or right sides. Treatment consisted of application of pegcantratinib 0.5% w/w and placebo once daily to allocated sides for 12 weeks. The primary outcome measure was the number of tumours meeting the predefined criteria for response, compared against a critical number (12) of pegcantratinib treated tumours in excess to the placebo treated tumours. This was not met. Secondary clinical outcome measures included assessment for safety, pain in small tumours, and concordance with trial protocol. This trial demonstrates safe and feasible short-term topical TRK targeting in CCS, and offers novel tumour natural history data for this rare disease.
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Preliminary therapeutic target validation of the IL-36 receptor in psoriasis SK Mahil1, M Peakman1, R Trembath1, J Wright2, J Barker1 and F Capon1 1 King’s College London, London, United Kingdom and 2 Bradford Royal Infirmary, Bradford, United Kingdom The IL-1 family cytokines IL-36 a, -b and eg are important in maintaining mucosal and epidermal immune homeostasis. As such, mutations in genes regulating IL-36 signalling (IL36RN and AP1S3) have been associated with inflammatory phenotypes e.g. pustular psoriasis. IL-36 may amplify IL-17 driven inflammatory responses and over-expression has been detected in common plaque psoriasis. IL-36 may thus be a promising therapeutic target, and antagonists directed against its receptor (IL-36R) are being developed. However, since the function of IL-36 in broader immune responses remains undetermined, pharmacological blockade may have unexpected adverse effects. We investigated the consequences of IL-36 signalling inhibition through the deep phenotyping of human knockouts who lack a functional IL-36R. These individuals were identified by querying the genotype data generated by “Born in Bradford”, a community-based initiative to exome sequence 2,162 British-Pakistanis. In keeping with the increased parental relatedness in this cohort, we identified numerous participants (n¼28) with low-frequency homozygous changes in IL1RL2 (encoding IL-36R). We recalled 6 subjects harbouring bi-allelic missense changes predicted to be deleterious. IL36 stimulation of PBMCs from these individuals did not elicit an inflammatory response. However, clinical examinations and medical records did not indicate anomalies in immune function. Full blood counts and antibody responses to past immunizations were normal. PBMC treatment with varied inflammatory stimuli (PMA, poly(I:C), C. albicans and Infanrix vaccine) induced up-regulation of inflammatory markers comparable to that observed in ethnically matched controls. Thus, these data suggest that IL-36 blockade may be compatible with normal immune function and pave the way for pre-clinical safety studies of IL-36 inhibitors. Our study also highlights the utility of phenotyping human knockouts in the preclinical assessment of novel drug targets.
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Epidermodysplasia verruciformis: Clinical, viral, and histopathological phenotype in patients with EVER3 mutation E Imahorn1, SJ de Jong2, I Spoerri1, W Kempf3, C Imhof4, P Ha¨usermann5, E Jouanguy2, J Casanova2, B Burger1 and P Itin1,5 1 Department of Biomedicine, University Hospital of Basel and University of Basel, Basel, Switzerland, 2 St. Giles Laboratory, The Rockefeller University, New York City, NY, 3 Kempf and Pfaltz Histological Diagnostics, Zurich, Switzerland, 4 Stadtpraxis Brig, Brig, Switzerland and 5 Department of Dermatology, University Hospital Basel, Basel, Switzerland Patients with the rare genodermatosis epidermodysplasia verruciformis (EV) are susceptible to infections with cutaneous HPV and have a high risk for development of non-melanoma skin cancer (NMSC). Biallelic mutations in TMC6/EVER1 and TMC8/EVER2 have been identified in many patients. Recently, a third gene EVER3 has been identified to be mutated in EV patients without TMC6/8 mutation. In the presented study we evaluated whether the phenotype of EVER3 deficient patients differs from the phenotype of TMC6/8 deficient patients. Therefore, we examined two unrelated Swiss EV patients. TMC6, TMC8, and EVER3 were sequenced on gDNA isolated from whole blood. HPV detection was performed by sequencing the L1 capsid gene after nested PCR on DNA isolated from skin biopsies. No loss of function mutation could be detected in TMC6 and TMC8 in these patients. Sequencing of EVER3 revealed a homozygous frameshift mutation leading to a premature stop codon. In both patients typical EV lesions were present on back of hands, neck, arms, legs, and cheeks comparable to TMC6 and TMC8 patients. First lesions appeared during early childhood. Histopathological examinations revealed EV characteristic features. Both patients developed various precanceroses, squamous cell carcinomas, and basal cell carcinomas. Typical HPV types could be detected in both patients. The results are discussed in context of a systematic literature review of all EV patients described to date. In sum, the two EVER3 deficient patients showed the typical clinical, histopathological, and viral phenotype of EV. No obvious phenotypic variability to TMC6/8 deficient patients could be observed.
Altering the splice pattern of COL17A1 with antisense oligonucleotides H Potocki, T Lettner, M Ablinger, M Wimmer, R Zauner, N Friedl, J Reichelt, JW Bauer and V Wally EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Austria, Laboratory for Molecular Therapy, EB House Austria, Salzburg, Austria Junctional epidermolysis bullosa (JEB) is a devastating disease of the skin and mucous membranes, characterized by blistering and erosions upon minor mechanical friction. Genotypically, mutations within the type XVII collagen gene underlie this phenotype. In this project, we targeted a cryptic splice site arising due to a mutation (c. 380-1 G>A) at the COL17A1 intron 6 / exon 7 junction. Utilization of this splice site leads to the loss of 16 nucleotides at the mRNA level. Subsequently, the reading frame is shifted so that a premature stop codon is generated within exon 9 and the mRNA is degraded by nonsense-mediated decay. Using three distinct antisense oligonucleotides (ASOs), we aimed to either restore the wild type splice pattern of COL17A1 or achieve skipping of exon 7, which would lead to a slightly truncated, but potentially functional type XVII collagen, since the reading frame remains intact. We transfected patient keratinocytes with either of the ASOs and analyzed overall COL17A1 expression, as well as the generation of alternative splicing products. Amplification and sequencing of the COL17A1 mRNA from exon 5 to exon 9 showed skipping of exon 7 as the most prominent result of ASO treatments in JEB keratinocytes. Additionally, alleles with a restored wild type splice pattern, as well as such showing concomitant skipping of exons 6 and 7, were identified. SqRT-PCR confirmed an increase in overall COL17A1 expression. Furthermore, full length (180 kDa) type XVII collagen was detected upon ASO treatment using Western blot analysis. Taken together, we conclude that targeting a cryptic splice site with ASOs may hold therapeutic potential for JEB and possibly other forms of EB with similar types of mutations.
www.jidonline.org S227