- Email: [email protected]
- mice: Potential treatment of PXE and GACI
Genetic Disease, Gene Regulation & Gene Therapy | ABSTRACTS 400
Amlexanox: Efficacy and mechanism for potential therapy for RDEB V Atanasova, Q Jiang, J Uitto and A South Thomas Jefferson University, Philadelphia, PA Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin blistering disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (ColVII). ColVII is the main, if not exclusive, component of anchoring fibrils that adhere the epidermis to the dermal layer of the skin. Mutations in COL7A1 lead to defective anchoring fibrils and subsequent perturbed adhesion between the two skin layers resulting in severe skin fragility and constant wounding without ability to heal. A subgroup of RDEB patients harbors nonsense mutations in COL7A1 which create premature termination codons (PTCs) of translation in the mRNA molecule. This leads to mRNA instability and lack of functional protein synthesized. A group of drugs have been reported for their ability to induce “readthrough” and allow for insertion of an amino acid at the site of PTC, thus producing fulllength protein. The mechanism by which these drugs act is currently not known. Here we focus on a PTC read-through drug, amlexanox, previously approved by the FDA for mouth ulcers. Our data show that amlexanox is able to induce full-length synthesis of ColVII in HpV immortalized RDEB patient derived keratinocytes and fibroblasts, as shown by western blotting. After 48 hours of treatment keratinocytes showed up to 14 fold increase in full-length ColVII protein synthesis as compared to non-treated controls. The same treatment in HpV fibroblasts led up to 3 fold increase in ColVII as compared to non-treated controls, or 25% of normal human fibroblasts. While the mechanism by which amlexanox is able to recover protein synthesis from the PTC containing COL7A1 gene is currently unclear, activation of UPF1 by its phosphorylation has been shown to be required for assembly of mRNA degradation machinery. Our data show that amlexanox treatment increases UPF1 phosphorylation, which may be part of the PTC read-through mechanism. In conclusion, we present data to support amlexanox as a read-through agent for the treatment of RDEB in cases with PTC in COL7A1.
402
401
Dermal fibroblasts with loss of TSC2 express increased galectin-3, a potential biomarker for tuberous sclerosis complex P Klover1, R Thangapazham1, J Kato2, J Wang1, S Li1, C Dalgard3, M Wilkerson3, E Mccart1, J Moss2 and T Darling1 1 Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD, 2 Cardiovascular and Pulmonary Branch, NIH/NHLBI, Bethesda, MD and 3 Anatomy, Physiology, and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD Tuberous sclerosis complex (TSC) is an autosomal genetic disorder in which tumors form in multiple organs due to biallelic inactivating mutations in TSC1 or TSC2 and increased mTORC1 signaling. In order to discover biomarkers for TSC, we studied our mouse model of TSC with conditional deletion of Tsc2 in dermal mesenchyme (Tsc2cKO), testing the potential clinical relevance of findings using human TSC skin tumors. RNA-sequencing analysis of cultured Tsc2-null and wild-type mouse dermal fibroblasts identified galectin-3 as overexpressed by the mutant cells. Levels of both intracellular and secreted galectin-3 protein were approximately 3-fold greater in Tsc2-null fibroblasts than wild-type fibroblasts. Tsc2-null cells incubated in the presence of 20 nM sirolimus, an mTORC1-inhibitor, showed decreased galectin-3 expression but not to the level of wild-type fibroblasts, suggesting that overexpression of galectin-3 is only partially mTORC1-dependent. Serum levels of galectin-3 were 68% greater (p¼0.0015) in Tsc2 cKO than wild-type mice and sirolimus treatment for 4 weeks decreased galectin-3 serum levels 25% (p¼0.036) in the Tsc2cKO. Fibroblast-like cells grown from human TSC skin tumors, including angiofibromas, fibrous cephalic plaque, and periungual fibromas, showed greater intracellular and secreted galectin-3 levels than patient normal fibroblasts. Immunohistochemical analysis showed abundant galectin-3 positive dermal cells in TSC skin tumors but nearly absent dermal staining in paired samples of patient normal-appearing skin. Our results demonstrate that loss of TSC2 in dermal fibroblasts is associated with increased galectin-3 and suggest that this molecule should be investigated as a biomarker for TSC and possibly other diseases associated with increased mTORC1 activation.
403
Precision medicine intervention in xeroderma pigmentosum R Ono2,1, SG Khan2, T Masaki1, C Kuschal2, C Nishigori1, M Chu2, D Tamura2, J DiGiovanna2 and KH Kraemer2 1 Derm, Kobe Univ, Kobe, Japan and 2 Derm, NCI, Bethesda, MD Xeroderma pigmentosum (XP) is a rare autosomal recessive, cancer-prone DNA repair disease with mutations in one of 8 nucleotide excision repair genes (XPA through XPG) or bypass polymerase eta (XPV)). Some XP-C patients have premature termination codon (PTC) mutations and decreased mRNA. We found that some PTC can be read-through by agents that disrupt ribosomal fidelity, including aminoglycoside antibiotics. A minimal increase in functional message may be of clinical benefit since cell lines from clinically normal obligate heterozygotes (parents of XP-C patients) have reduced levels of XPC mRNA. Also, patients with low levels (3-5 % of normal) XPC mRNA have a mild clinical phenotype. As part of the pre-clinical basis for a precision medicine directed XP treatment protocol, potential therapeutic compounds were tested on cell lines from an expanded cohort. Of 256 XP cell lines, 70 had XPC mutations and 13(18%) of these had primary PTC mutations in at least one allele. These included 3 new patients: XP518BE, a 5 y/o Korean-American boy with a heterozygous p.R579X mutation; XP115KO a 6 y/o Korean girl with a homozygous p.R579X mutation; and XP358BE, a 13 y/o Caucasian girl with a heterozygous p.K692X mutation. As is typical of XPC, none of these patients had acute burning on minimal sun exposure or neurologic abnormalities. XP358BE had skin cancer at age 2. Cells from the 3 new patients had very low mRNA levels typical of XPC. All had increases in XPC mRNA after treatment with geneticin, confirming PTC readthrough activity. In cells with PTC mutations, mRNAs with PTC can be removed by nonsense mediated decay (NMD). To increase XPC mRNA we identified amlexanox, an anti-inflammatory compound with activity as an inhibitor of NMD. Amlexanox (75-150 mM) increased XPC mRNA in the cells with primary PTC. We are studying combinations of these compounds to assess for improvement in XPC mRNA levels and DNA repair and plan to translate these findings into a pilot clinical study.
Etidronate prevents but does not reverse ectopic mineralization in Abcc6-/- mice: Potential treatment of PXE and GACI Q Li1, J Kingman1, D Wang1, JP Sundberg2, MA Levine3 and J Uitto1 1 Thomas Jefferson University, Philadelphia, PA, 2 The Jackson Laboratory, Bar Harbor, ME and 3 Children’s Hospital of Philadelphia, Philadelphia, PA Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are heritable ectopic mineralization disorders. Most cases of PXE and many cases of GACI harbor mutations in the ABCC6 gene. There is no effective treatment for these disorders. In this study we explored the potential efficacy of etidronate, a first generation bisphosphonate, on ectopic mineralization in the muzzle skin of Abcc6-/- mice, a model of PXE. Previous feeding studies demonstrated that etidronate prevents ectopic mineralization in Abcc6-/- mice at relatively high dose (12x of the human equivalent dose). Considering the low level of absorption of oral etidronate (<1%), in this study we tested the Abcc6-/- mice with subcutaneous injections of etidronate, 0.08 and 0.96 mg/kg/day (0.01x and 0.12x), twice a week, in both prevention and reversal experiments. In the prevention study, treatment of mice was initiated at 4 weeks of age before ectopic mineralization becomes evident, and the degree of mineralization was assessed at 12 weeks of age. In the reversal study, mice received treatment initiated at 12 weeks of age when ectopic mineralization is evident, and continued up to 24 weeks of age. Ectopic mineralization in the dermal sheath of vibrissae in muzzle skin, an early and progressive biomarker of the mineralization process, was determined by histopathologic analysis and by direct chemical assay for calcium content. Subcutaneous injection of etidronate prevented ectopic mineralization but did not reverse existing mineralization. The effect of etidronate was accompanied by alterations in the trabecular bone microarchitecture, determined by mCT. The results suggest that etidronate may offer a potential treatment modality for PXE and GACI caused by ABCC6 mutations. Etidronate therapy should be initiated in PXE patients as soon as diagnosis is made, with careful monitoring of potential side effects.
404
405
BET proteins as therapeutic targets for inflammatory skin disease: Differential recruitment of BET proteins to keratinocyte IL-6 and IL-8 promoters and suppression of cytokine production by a synthetic histone mimic (I-BET151) KC Wu1, NR Harker2, R Prinjha2, M Donaldson2, MA Morse2 and NJ Reynolds1 1 Newcastle University, Newcastle Upon Tyne, United Kingdom and 2 GlaxoSmithKline, Stevenage, United Kingdom Increasing evidence indicates epigenetic alterations, including histone acetylation, play an important role in regulating inflammatory responses. Bromodomain extraterminal (BET) proteins (Brd2/3/4/T) contain tandem bromodomains which bind acetyl-lysine residues on histones and orchestrate assembly of chromatin complexes including recruitment of transcription factors to regulate transcription. As BET inhibitors abrogate macrophage immune responses and Brd4 binds to p65/relA, we tested the hypothesis that BET proteins regulate keratinocyte inflammatory responses using the inhibitor I-BET151. In primary human keratinocytes (NHEKs), I-BET151 reduced secreted IL-6 protein (w90% inhibition, p<0.001) induced by TNFa (10ng/ml) + IL-17 (100ng/ml) and to a lesser extent IL-8 protein (w60% inhibition, p<0.001). I-BET151 inhibited IL-6/-8 mRNA expression (IC50 ¼ 134nM and 56nM, respectively), consistent with targeting transcription. In line with these results, chromatin immunoprecipitation (ChIP) studies showed TNFa/IL-17 induced dynamic, gene-specific alterations of the epigenome, including histone hyperacetylation, with co-ordinated recruitment of BET proteins and RNA pol II to IL-6/-8 promoters. Moreover ChIP studies showed w10-fold enrichment of Brd4/p65 at the IL-6 promoter compared to the IL-8 promoter, indicating differential regulation of IL-6 and IL-8 by BET proteins. Notably, I-BET151 reduced recruitment of Brd4 to the IL-6 promoter and Brd3/4 to the IL-8 promoter, with corresponding decreases in RNA pol II binding at both promoters, in TNFa/IL-17 stimulated NHEKs, consistent with the inhibitory effect of I-BET151 on IL-6/-8 mRNA expression. These data demonstrate TNFa/IL-17 induce chromatin remodelling, provide mechanistic insight into the role of BET proteins in regulating cutaneous inflammation and highlight BET proteins as potential therapeutic targets for inflammatory dermatoses.
Hydrophobically modified siRNAs (hsiRNAs) provide a platform to silence gene expression in inflammatory skin diseases M Rashighi1, M Nikan2,3, AH Coles2,3, A Khvorova2,3 and JE Harris1 1 Department of Medicine, Division of Dermatology, University of Massachusetts Medical School, Worcester, MA, 2 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA and 3 Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA RNA interference (RNAi) is a highly conserved biological process in which small RNA molecules inhibit gene expression. It has great potential to treat different human diseases, including inflammatory skin disorders. Despite promising results in vitro, therapeutic application of RNAi has been limited mainly due to difficulties in efficient delivery of the therapeutic oligonucleotides to target cells. Here we describe that direct conjugation of fully stabilized siRNA (hsiRNAs) to cholesterol (Chol) or biologically occurring lipids results in widespread distribution in the skin following local administration. Flow cytometry to detect fluorescently labeled Chol-hsiRNA in mouse skin indicated efficient delivery to multiple cell types, including T lymphocytes, Langerhans cells, keratinocytes, fibroblasts and endothelial cells, with preferential uptake in CD45+ cells (48.5% compared to 11% of CD45- cells in epidermis; and 69.7% compared to 39.7% of CD45- cells in dermis). To test silencing efficacy, an hsiRNA was designed to silence Flt1 mRNA, the gene that encodes vascular endothelial growth factor receptor found on vascular endothelial cells. A single intradermal injection of 0.3 nmoles of Flt1-Chol-hsiRNA resulted in target silencing in 49% of the dermal endothelial cells, which was statistically significant compared to the pretreatment levels and vehicle-treated controls (p-value¼0.0027). Taken together, hydrophobically modified siRNA demonstrates wide distribution, efficient delivery, and significant silencing capacity upon intradermal administration. Future studies will test whether this approach could be successfully used to effectively target key pathogenic proteins in the context of inflammatory skin diseases.
www.jidonline.org S71
Amlexanox: Efficacy and mechanism for potential therapy for RDEB V Atanasova, Q Jiang, J Uitto and A South Thomas Jefferson University, Philadelphia, PA Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin blistering disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (ColVII). ColVII is the main, if not exclusive, component of anchoring fibrils that adhere the epidermis to the dermal layer of the skin. Mutations in COL7A1 lead to defective anchoring fibrils and subsequent perturbed adhesion between the two skin layers resulting in severe skin fragility and constant wounding without ability to heal. A subgroup of RDEB patients harbors nonsense mutations in COL7A1 which create premature termination codons (PTCs) of translation in the mRNA molecule. This leads to mRNA instability and lack of functional protein synthesized. A group of drugs have been reported for their ability to induce “readthrough” and allow for insertion of an amino acid at the site of PTC, thus producing fulllength protein. The mechanism by which these drugs act is currently not known. Here we focus on a PTC read-through drug, amlexanox, previously approved by the FDA for mouth ulcers. Our data show that amlexanox is able to induce full-length synthesis of ColVII in HpV immortalized RDEB patient derived keratinocytes and fibroblasts, as shown by western blotting. After 48 hours of treatment keratinocytes showed up to 14 fold increase in full-length ColVII protein synthesis as compared to non-treated controls. The same treatment in HpV fibroblasts led up to 3 fold increase in ColVII as compared to non-treated controls, or 25% of normal human fibroblasts. While the mechanism by which amlexanox is able to recover protein synthesis from the PTC containing COL7A1 gene is currently unclear, activation of UPF1 by its phosphorylation has been shown to be required for assembly of mRNA degradation machinery. Our data show that amlexanox treatment increases UPF1 phosphorylation, which may be part of the PTC read-through mechanism. In conclusion, we present data to support amlexanox as a read-through agent for the treatment of RDEB in cases with PTC in COL7A1.
402
401
Dermal fibroblasts with loss of TSC2 express increased galectin-3, a potential biomarker for tuberous sclerosis complex P Klover1, R Thangapazham1, J Kato2, J Wang1, S Li1, C Dalgard3, M Wilkerson3, E Mccart1, J Moss2 and T Darling1 1 Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD, 2 Cardiovascular and Pulmonary Branch, NIH/NHLBI, Bethesda, MD and 3 Anatomy, Physiology, and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD Tuberous sclerosis complex (TSC) is an autosomal genetic disorder in which tumors form in multiple organs due to biallelic inactivating mutations in TSC1 or TSC2 and increased mTORC1 signaling. In order to discover biomarkers for TSC, we studied our mouse model of TSC with conditional deletion of Tsc2 in dermal mesenchyme (Tsc2cKO), testing the potential clinical relevance of findings using human TSC skin tumors. RNA-sequencing analysis of cultured Tsc2-null and wild-type mouse dermal fibroblasts identified galectin-3 as overexpressed by the mutant cells. Levels of both intracellular and secreted galectin-3 protein were approximately 3-fold greater in Tsc2-null fibroblasts than wild-type fibroblasts. Tsc2-null cells incubated in the presence of 20 nM sirolimus, an mTORC1-inhibitor, showed decreased galectin-3 expression but not to the level of wild-type fibroblasts, suggesting that overexpression of galectin-3 is only partially mTORC1-dependent. Serum levels of galectin-3 were 68% greater (p¼0.0015) in Tsc2 cKO than wild-type mice and sirolimus treatment for 4 weeks decreased galectin-3 serum levels 25% (p¼0.036) in the Tsc2cKO. Fibroblast-like cells grown from human TSC skin tumors, including angiofibromas, fibrous cephalic plaque, and periungual fibromas, showed greater intracellular and secreted galectin-3 levels than patient normal fibroblasts. Immunohistochemical analysis showed abundant galectin-3 positive dermal cells in TSC skin tumors but nearly absent dermal staining in paired samples of patient normal-appearing skin. Our results demonstrate that loss of TSC2 in dermal fibroblasts is associated with increased galectin-3 and suggest that this molecule should be investigated as a biomarker for TSC and possibly other diseases associated with increased mTORC1 activation.
403
Precision medicine intervention in xeroderma pigmentosum R Ono2,1, SG Khan2, T Masaki1, C Kuschal2, C Nishigori1, M Chu2, D Tamura2, J DiGiovanna2 and KH Kraemer2 1 Derm, Kobe Univ, Kobe, Japan and 2 Derm, NCI, Bethesda, MD Xeroderma pigmentosum (XP) is a rare autosomal recessive, cancer-prone DNA repair disease with mutations in one of 8 nucleotide excision repair genes (XPA through XPG) or bypass polymerase eta (XPV)). Some XP-C patients have premature termination codon (PTC) mutations and decreased mRNA. We found that some PTC can be read-through by agents that disrupt ribosomal fidelity, including aminoglycoside antibiotics. A minimal increase in functional message may be of clinical benefit since cell lines from clinically normal obligate heterozygotes (parents of XP-C patients) have reduced levels of XPC mRNA. Also, patients with low levels (3-5 % of normal) XPC mRNA have a mild clinical phenotype. As part of the pre-clinical basis for a precision medicine directed XP treatment protocol, potential therapeutic compounds were tested on cell lines from an expanded cohort. Of 256 XP cell lines, 70 had XPC mutations and 13(18%) of these had primary PTC mutations in at least one allele. These included 3 new patients: XP518BE, a 5 y/o Korean-American boy with a heterozygous p.R579X mutation; XP115KO a 6 y/o Korean girl with a homozygous p.R579X mutation; and XP358BE, a 13 y/o Caucasian girl with a heterozygous p.K692X mutation. As is typical of XPC, none of these patients had acute burning on minimal sun exposure or neurologic abnormalities. XP358BE had skin cancer at age 2. Cells from the 3 new patients had very low mRNA levels typical of XPC. All had increases in XPC mRNA after treatment with geneticin, confirming PTC readthrough activity. In cells with PTC mutations, mRNAs with PTC can be removed by nonsense mediated decay (NMD). To increase XPC mRNA we identified amlexanox, an anti-inflammatory compound with activity as an inhibitor of NMD. Amlexanox (75-150 mM) increased XPC mRNA in the cells with primary PTC. We are studying combinations of these compounds to assess for improvement in XPC mRNA levels and DNA repair and plan to translate these findings into a pilot clinical study.
Etidronate prevents but does not reverse ectopic mineralization in Abcc6-/- mice: Potential treatment of PXE and GACI Q Li1, J Kingman1, D Wang1, JP Sundberg2, MA Levine3 and J Uitto1 1 Thomas Jefferson University, Philadelphia, PA, 2 The Jackson Laboratory, Bar Harbor, ME and 3 Children’s Hospital of Philadelphia, Philadelphia, PA Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are heritable ectopic mineralization disorders. Most cases of PXE and many cases of GACI harbor mutations in the ABCC6 gene. There is no effective treatment for these disorders. In this study we explored the potential efficacy of etidronate, a first generation bisphosphonate, on ectopic mineralization in the muzzle skin of Abcc6-/- mice, a model of PXE. Previous feeding studies demonstrated that etidronate prevents ectopic mineralization in Abcc6-/- mice at relatively high dose (12x of the human equivalent dose). Considering the low level of absorption of oral etidronate (<1%), in this study we tested the Abcc6-/- mice with subcutaneous injections of etidronate, 0.08 and 0.96 mg/kg/day (0.01x and 0.12x), twice a week, in both prevention and reversal experiments. In the prevention study, treatment of mice was initiated at 4 weeks of age before ectopic mineralization becomes evident, and the degree of mineralization was assessed at 12 weeks of age. In the reversal study, mice received treatment initiated at 12 weeks of age when ectopic mineralization is evident, and continued up to 24 weeks of age. Ectopic mineralization in the dermal sheath of vibrissae in muzzle skin, an early and progressive biomarker of the mineralization process, was determined by histopathologic analysis and by direct chemical assay for calcium content. Subcutaneous injection of etidronate prevented ectopic mineralization but did not reverse existing mineralization. The effect of etidronate was accompanied by alterations in the trabecular bone microarchitecture, determined by mCT. The results suggest that etidronate may offer a potential treatment modality for PXE and GACI caused by ABCC6 mutations. Etidronate therapy should be initiated in PXE patients as soon as diagnosis is made, with careful monitoring of potential side effects.
404
405
BET proteins as therapeutic targets for inflammatory skin disease: Differential recruitment of BET proteins to keratinocyte IL-6 and IL-8 promoters and suppression of cytokine production by a synthetic histone mimic (I-BET151) KC Wu1, NR Harker2, R Prinjha2, M Donaldson2, MA Morse2 and NJ Reynolds1 1 Newcastle University, Newcastle Upon Tyne, United Kingdom and 2 GlaxoSmithKline, Stevenage, United Kingdom Increasing evidence indicates epigenetic alterations, including histone acetylation, play an important role in regulating inflammatory responses. Bromodomain extraterminal (BET) proteins (Brd2/3/4/T) contain tandem bromodomains which bind acetyl-lysine residues on histones and orchestrate assembly of chromatin complexes including recruitment of transcription factors to regulate transcription. As BET inhibitors abrogate macrophage immune responses and Brd4 binds to p65/relA, we tested the hypothesis that BET proteins regulate keratinocyte inflammatory responses using the inhibitor I-BET151. In primary human keratinocytes (NHEKs), I-BET151 reduced secreted IL-6 protein (w90% inhibition, p<0.001) induced by TNFa (10ng/ml) + IL-17 (100ng/ml) and to a lesser extent IL-8 protein (w60% inhibition, p<0.001). I-BET151 inhibited IL-6/-8 mRNA expression (IC50 ¼ 134nM and 56nM, respectively), consistent with targeting transcription. In line with these results, chromatin immunoprecipitation (ChIP) studies showed TNFa/IL-17 induced dynamic, gene-specific alterations of the epigenome, including histone hyperacetylation, with co-ordinated recruitment of BET proteins and RNA pol II to IL-6/-8 promoters. Moreover ChIP studies showed w10-fold enrichment of Brd4/p65 at the IL-6 promoter compared to the IL-8 promoter, indicating differential regulation of IL-6 and IL-8 by BET proteins. Notably, I-BET151 reduced recruitment of Brd4 to the IL-6 promoter and Brd3/4 to the IL-8 promoter, with corresponding decreases in RNA pol II binding at both promoters, in TNFa/IL-17 stimulated NHEKs, consistent with the inhibitory effect of I-BET151 on IL-6/-8 mRNA expression. These data demonstrate TNFa/IL-17 induce chromatin remodelling, provide mechanistic insight into the role of BET proteins in regulating cutaneous inflammation and highlight BET proteins as potential therapeutic targets for inflammatory dermatoses.
Hydrophobically modified siRNAs (hsiRNAs) provide a platform to silence gene expression in inflammatory skin diseases M Rashighi1, M Nikan2,3, AH Coles2,3, A Khvorova2,3 and JE Harris1 1 Department of Medicine, Division of Dermatology, University of Massachusetts Medical School, Worcester, MA, 2 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA and 3 Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA RNA interference (RNAi) is a highly conserved biological process in which small RNA molecules inhibit gene expression. It has great potential to treat different human diseases, including inflammatory skin disorders. Despite promising results in vitro, therapeutic application of RNAi has been limited mainly due to difficulties in efficient delivery of the therapeutic oligonucleotides to target cells. Here we describe that direct conjugation of fully stabilized siRNA (hsiRNAs) to cholesterol (Chol) or biologically occurring lipids results in widespread distribution in the skin following local administration. Flow cytometry to detect fluorescently labeled Chol-hsiRNA in mouse skin indicated efficient delivery to multiple cell types, including T lymphocytes, Langerhans cells, keratinocytes, fibroblasts and endothelial cells, with preferential uptake in CD45+ cells (48.5% compared to 11% of CD45- cells in epidermis; and 69.7% compared to 39.7% of CD45- cells in dermis). To test silencing efficacy, an hsiRNA was designed to silence Flt1 mRNA, the gene that encodes vascular endothelial growth factor receptor found on vascular endothelial cells. A single intradermal injection of 0.3 nmoles of Flt1-Chol-hsiRNA resulted in target silencing in 49% of the dermal endothelial cells, which was statistically significant compared to the pretreatment levels and vehicle-treated controls (p-value¼0.0027). Taken together, hydrophobically modified siRNA demonstrates wide distribution, efficient delivery, and significant silencing capacity upon intradermal administration. Future studies will test whether this approach could be successfully used to effectively target key pathogenic proteins in the context of inflammatory skin diseases.
www.jidonline.org S71