C7 Expression Research Articles

Splice modulation strategy applied to deep intronic variants in COL7A1 causing recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and most often severe genetic disease characterized by recurrent blistering and erosions of the skin and mucous membranes after minor trauma, leading to major local and systemic complications. The disease is caused by loss-of-function variants in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils, which form attachment structures stabilizing the cutaneous basement membrane zone. Alterations in C7 protein structure and/or expression lead to abnormal, rare or absent anchoring fibrils resulting in loss of dermal-epidermal adherence and skin blistering. To date, more than 1,200 distinct COL7A1 deleterious variants have been reported and 19% are splice variants. Here, we describe two RDEB patients for whom we identified two pathogenic deep intronic pathogenic variants in COL7A1. One of these variants (c.7795-97C > G) promotes the inclusion of a pseudoexon between exons 104 and 105 in the COL7A1 transcript, while the other causes partial or complete retention of intron 51. We used antisense oligonucleotide (ASO) mediated exon skipping to correct these aberrant splicing events in vitro. This led to increased normal mRNA splicing above 94% and restoration of C7 protein expression at a level (up to 56%) that should be sufficient to reverse the phenotype. This first report of exon skipping applied to counteract deep intronic variants in COL7A1 represents a promising therapeutic strategy for personalized medicine directed at patients with intronic variants at a distance of consensus splice sites.

Identification of novel small molecule-based strategies of COL7A1 upregulation and readthrough activity for the treatment of recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic disease caused by loss of function mutations in the gene coding for collagen VII (C7) due to deficient or absent C7 expression. This disrupts structural and functional skin architecture, leading to blistering, chronic wounds, inflammation, important systemic symptoms affecting the mouth, gastrointestinal tract, cornea, and kidney function, and an increased skin cancer risk. RDEB patients have an extremely poor quality of life and often die at an early age. A frequent class of mutations in RDEB is premature termination codons (PTC), which appear in homozygosity or compound heterozygosity with other mutations. RDEB has no cure and current therapies are mostly palliative. Using patient-derived keratinocytes and a library of 8273 small molecules and 20,160 microbial extracts evaluated in a phenotypic screening interrogating C7 levels, we identified three active chemical series. Two of these series had PTC readthrough activity, and one upregulated C7 mRNA, showing synergistic activity when combined with the reference readthrough molecule gentamicin. These compounds represent novel potential small molecule-based systemic strategies that could complement topical-based treatments for RDEB.

Disrupted Tuzzerella abundance and impaired l-glutamine levels induce Treg accumulation in ovarian endometriosis: a comprehensive multi-omics analysis

IntroductionThe microbial community plays a crucial role in the pathological microenvironment. However, the structure of the microbial community within endometriotic lesions and its impact on the microenvironment is still limited.MethodsAll 55 tissue samples, including ovarian ectopic (OEMs) and normal (NE) endometrium, were subjected to 16S rRNA sequencing, metabolomic and proteomic analysis.ResultsWe found the abundance of Tuzzerella is significantly lower in OEMs compared to NE tissue (p < 0.01). We selected samples from these two groups that exhibited the most pronounced difference in Tuzzerella abundance for further metabolomic and proteomic analysis. Our findings indicated that endometriotic lesions were associated with a decrease in l-Glutamine levels. However, proteomic analysis revealed a significant upregulation of proteins related to the complement pathway, including C3, C7, C1S, CLU, and A2M. Subsequent metabolic and protein correlation predictions demonstrated a negative regulation between l-Glutamine and C7. In vitro experiments further confirmed that high concentrations of Glutamine significantly inhibit C7 protein expression. Additionally, immune cell infiltration analysis, multiplex immunofluorescence, and multifactorial testing demonstrated a positive correlation between C7 expression and the infiltration of regulatory T cells (Tregs) in ectopic lesions, while l-Glutamine was found to negatively regulate the expression of chemotactic factors for Tregs.ConclusionIn this study, we found a clear multi-omics pathway alteration, “Tuzzerella (microbe)—l-Glutamine (metabolite)—C7 (protein),” which affects the infiltration of Tregs in endometriotic lesions. Our findings provide insights into endometriosis classification and personalized treatment strategies based on microbial structures.Graphical abstract

Splicing Modulation via Antisense Oligonucleotides in Recessive Dystrophic Epidermolysis Bullosa.

Antisense oligonucleotides (ASOs) represent an emerging therapeutic platform for targeting genetic diseases by influencing various aspects of (pre-)mRNA biology, such as splicing, stability, and translation. In this study, we investigated the potential of modulating the splicing pattern in recessive dystrophic epidermolysis bullosa (RDEB) patient cells carrying a frequent genomic variant (c.425A > G) that disrupts splicing in the COL7A1 gene by using short 2'-O-(2-Methoxyethyl) oligoribo-nucleotides (2'-MOE ASOs). COL7A1-encoded type VII collagen (C7) forms the anchoring fibrils within the skin that are essential for the attachment of the epidermis to the underlying dermis. As such, gene variants of COL7A1 leading to functionally impaired or absent C7 manifest in the form of extensive blistering and wounding. The severity of the disease pattern warrants the development of novel therapies for patients. The c.425A > G variant at the COL7A1 exon 3/intron 3 junction lowers the efficiency of splicing at this junction, resulting in non-functional C7 transcripts. However, we found that correct splicing still occurs, albeit at a very low level, highlighting an opportunity for intervention by modulating the splicing reaction. We therefore screened 2'-MOE ASOs that bind along the COL7A1 target region ranging from exon 3 to the intron 3/exon 4 junction for their ability to modulate splicing. We identified ASOs capable of increasing the relative levels of correctly spliced COL7A1 transcripts by RT-PCR, sqRT-PCR, and ddPCR. Furthermore, RDEB-derived skin equivalents treated with one of the most promising ASOs exhibited an increase in full-length C7 expression and its accurate deposition along the basement membrane zone (BMZ).

In vivo topical gene therapy for recessive dystrophic epidermolysis bullosa: a phase 1 and 2 trial

Recessive dystrophic epidermolysis bullosa (RDEB) is a lifelong genodermatosis associated with blistering, wounding, and scarring caused by mutations in COL7A1, the gene encoding the anchoring fibril component, collagen VII (C7). Here, we evaluated beremagene geperpavec (B-VEC), an engineered, non-replicating COL7A1 containing herpes simplex virus type 1 (HSV-1) vector, to treat RDEB skin. B-VEC restored C7 expression in RDEB keratinocytes, fibroblasts, RDEB mice and human RDEB xenografts. Subsequently, a randomized, placebo-controlled, phase 1 and 2 clinical trial (NCT03536143) evaluated matched wounds from nine RDEB patients receiving topical B-VEC or placebo repeatedly over 12 weeks. No grade 2 or above B-VEC-related adverse events or vector shedding or tissue-bound skin immunoreactants were noted. HSV-1 and C7 antibodies sometimes presented at baseline or increased after B-VEC treatment without an apparent impact on safety or efficacy. Primary and secondary objectives of C7 expression, anchoring fibril assembly, wound surface area reduction, duration of wound closure, and time to wound closure following B-VEC treatment were met. A patient-reported pain–severity secondary outcome was not assessed given the small proportion of wounds treated. A global assessment secondary endpoint was not pursued due to redundancy with regard to other endpoints. These studies show that B-VEC is an easily administered, safely tolerated, topical molecular corrective therapy promoting wound healing in patients with RDEB.

Genes and compounds that increase type VII collagen expression as potential treatments for dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is a skin-blistering disease caused by mutations in COL7A1, which encodes type VII collagen (C7). There is no cure for DEB, but previous work has shown potential therapeutic benefit of increased production of even partially functional C7. Genome-wide screens using CRISPR-Cas9 have enabled the identification of genes involved in cancer development, drug resistance and other genetic diseases, suggesting that they could be used to identify drivers of C7 production. A keratinocyte C7 reporter cell line was created and used in a genome-wide CRISPR activation (CRISPRa) screen to identify genes and pathways that increase C7 expression. The CRISPRa screen results were used to develop a targeted drug screen to identify compounds that upregulate C7 expression. The C7_tdTomato cell line was validated as an effective reporter for detection of C7 upregulation. The CRISPRa screen identified DENND4B and TYROBP as top gene hits plus pathways related to calcium uptake and immune signalling in C7 regulation. The targeted drug screen identified several compounds that increase C7 expression in keratinocytes, of which kaempferol, a plant flavonoid, also significantly increased C7mRNA and protein in DEB patient cells.

Development of Minicircle Vectors Encoding COL7A1 Gene with Human Promoters for Non-Viral Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin disorder caused by mutations in the COL7A1 gene that encodes type VII collagen (C7). The development of an efficient gene replacement strategy for RDEB is mainly hindered by the lack of vectors able to encapsulate and transfect the large cDNA size of this gene. To address this problem, our group has opted to use polymeric-based non-viral delivery systems and minicircle DNA. With this approach, safety is improved by avoiding the usage of viruses, the absence of bacterial backbone, and the replacement of the control viral cytomegalovirus (CMV) promoter of the gene with human promoters. All the promoters showed impressive C7 expression in RDEB skin cells, with eukaryotic translation elongation factor 1 α (EF1α) promoter producing higher C7 expression levels than CMV following minicircle induction, and COL7A1 tissue-specific promoter (C7P) generating C7 levels similar to normal human epidermal keratinocytes. The improved system developed here has a high potential for use as a non-viral topical treatment to restore C7 in RDEB patients efficiently and safely, and to be adapted to other genetic conditions.

High Expression of Complement Component C7 Indicates Poor Prognosis of Breast Cancer and Is Insensitive to Taxane-Anthracycline Chemotherapy.

BackgroundBreast cancer is the most commonly diagnosed cancer worldwide. However, the well-known biomarkers are not enough to meet the needs of precision medicine. Novel targets are desirable and highly valuable for improved patient survival. In this regard, we identified complement component C7 as one of the candidates based on data from the OCOMINE database.MethodsC7 expression was examined by immunohistochemistry in 331 cases of invasive ductal carcinoma (IDC), 45 cases of ductal carcinoma in situ (DCIS), and 52 cases of non-neoplastic tissues adjacent to tumor. Then, C7 expression was further confirmed by Western blot analysis based on IDC specimens and non-neoplastic breast specimens. The relationship between the C7 expression and prognosis of breast cancer patients was analyzed in order to investigate the function of C7 in breast cancer patients. Meanwhile, we also analyzed the relationship between the C7 expression and prognosis of 149 patients treated with conventional TE (taxane and anthracycline)-based chemotherapy. Then, a cohort of patients (22 cases) treated with TE neoadjuvant chemotherapy was used to further confirm the relationship between the C7 expression and TE-based chemosensitivity.ResultsIn our present study, we reported for the first time that C7 was an independent prognostic factor of breast cancer and C7 expression of IDC tissues was higher than non-neoplastic tissues adjacent to tumor and DCIS. In a cohort of 331 IDC patients, high expression of C7 indicated poor prognosis especially in the triple negative subtype and luminal B subtype. Furthermore, C7 was also a promoting factor for triple negative subtype patients to develop bone metastasis. Meanwhile, we provided the first evidence that patients with high C7 expression were insensitive to TE (taxane and anthracycline)-based chemotherapy by analyzing a cohort of 149 patients treated with TE-based chemotherapy and another cohort of 22 patients treated with TE neoadjuvant chemotherapy.ConclusionsIn summary, high expression of C7 may promote breast cancer development and might be insensitive to TE-based chemotherapy. Our present study laid a foundation to help clinicians improve the identification of patients for TE-based chemotherapy by C7 in the era of precision medicine.

Fludarabine modulates expression of type VII collagen during haematopoietic stem cell transplantation for recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe, complicated inherited blistering skin disease with few treatment options currently available. Recently, haematopoietic stem cell transplantation (HCT) has been used as an alternative therapy that can improve skin integrity, but it is not known if the preparative HCT regimen also contributes to the therapeutic response. To determine whether chemotherapy drugs used in the HCT preparative regimen influence type VII collagen (C7) expression, which is inherently reduced or absent in RDEB skin, and to explore the pathomechanisms of such responses, if present. Drugs from the HCT preparative regimen (busulfan, cyclophosphamide, ciclosporin A, fludarabine and mycophenolate) with inhibitors (PD98059, U0126, LY294002, SR11302, SIS3 and N-acetyl-l-cysteine) were added to normal human dermal and human RDEB fibroblasts. C7 expression was measured using reversetranscription polymerase chain reaction and immunoblotting. We uncovered a previously unknown consequence of fludarabine whereby dermal fibroblasts exposed to fludarabine upregulate C7. This effect is mediated, in part, through activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase, phosphoinositide 3-kinase/protein kinase B and transforming growth factor-β pathways. Activation of these pathways leads to activation of downstream transcription factors, including activator protein 1 (AP-1) and SMAD. Subsequently, both AP-1 and SMAD bind the COL7A1 promoter and increase COL7A1 expression. Fludarabine influences the production of type VII collagen in RDEB fibroblasts.

CC-type glutaredoxin, OsGrx_C7 plays a crucial role in enhancing protection against salt stress in rice

Soil salinity is one of the critical issue worldwide that adversely affect soil fertility. Salt stress significantly limits crop yield and grain quality; therefore, there is an urgent need to develop a strategy to improve salt stress tolerance. In present study, we reported that rice glutaredoxin (OsGrx_C7) plays a positive response in salt induced stress. Gene expression analysis, silencing, and overexpression of OsGrx_C7 gene were used to discover the role of OsGrx_C7 in response to salt stress. Gene expression analysis suggested that OsGrx_C7 expression was induced under salt stress and ubiquitously expressed in rice including root and shoot. The silencing of osgrx_c7 gene leads to increased sensitivity to salt stress, indicating its importance in salt stress tolerance. A gain-of-function approach showed that OsGrx_C7 may act as an important determinant in salt stress, compared with WT, and revealed higher biomass accumulation, improved root and plant growth under salt stress. Under salt stress condition, OsGrx_C7 overexpressing rice plants showed lower level of lipid peroxidation and Na+/K+ ratio, while proline accumulation, soluble sugar content and GSH/GSSG ratio was higher compared to WT. Furthermore, expression analysis suggested that OsGrx_C7 acted as positive regulator of salt tolerance by reinforcing the expression of transporters (OsHKT2;1, OsHKT1;5 and OsSOS1) engaged in Na+ homeostasis in overexpressing plants. Overall our study revealed that OsGrx_C7 emerged as a key mediator in response to salt stress in rice and could be used for engineering tolerance against salt stress in rice and other crops.

Complement C7 (C7), a Potential Tumor Suppressor, Is an Immune-Related Prognostic Biomarker in Prostate Cancer (PC).

Objectives: Prostate cancer (PC) is the second most frequent tumor in men, which has a high recurrence rate and poor prognosis. Therefore, this study aimed to identify novel prognostic biomarkers and therapeutic targets for immunotherapy and small molecule drugs for PC treatment.Materials and Methods: The Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) algorithm was applied to calculate immune scores and stromal scores of TCGA-PRAD data. Differentially expressed genes (DEGs) were identified using R package “limma.” GO, KEGG, and DO analyses were performed to analyze DEGs. Overall survival and disease-free survival analyses were conducted for hub gene identification. To validate the hub gene at the mRNA and protein expression levels, genetic alterations were measured, and CCLE and Cox regression analyses were performed. Connectivity map (CMap) analysis and GSEA were performed for drug exploration and function analysis, respectively.Results: Immune scores ranged from −1795.98 to 2339.39, and stomal scores ranged from −1877.60 to 1659.96. In total, 45 tumor microenvironment (TME)-related DEGs were identified, of which Complement C7 (C7) was selected and validated as a hub gene. CMap analysis identified six small molecule drugs as potential agents for PC treatment. Further analysis demonstrated that C7 expression was significantly correlated with clinical T, pathological N, and immune infiltration level.Conclusions: In conclusion, of the 45 TME-related DEGs, C7 was shown to correlate with PC prognosis in patients, indicating it as a novel prognostic biomarker and immunotherapy target in PC. Additionally, six small molecule drugs showed strong therapeutic potential for PC treatment.

304 Type VII collagen NC2 domain expression differentiates severe from milder recessive dystrophic epidermolysis bullosa subtypes

Recessive dystrophic epidermolysis bullosa (RDEB) results from type VII collagen (C7) deficiency due to COL7A1 gene mutations. C7 contains collagenous domains (COL) flanked by amino (NC1) and carboxyl (NC2) non-collagenous domains. The genotype spectrum of RDEB is well studied but does not entirely explain the highly variable clinical phenotype. In this retrospective study, we aimed to identify a disease severity and prognostic indicator, from an extensively characterized RDEB cohort. 41 RDEB patients were studied with mild, intermediate and severe clinical phenotypes in 7, 11 and 23 patients respectively. 22 of 23 severe RDEB patients showed negative NC2 expression, with variable NC1 staining and variable AF morphology. 7 of 7 mild RDEB patients showed at least one allele with missense mutations in COL domains and positive NC2 and NC1 expression. 5 of 11 intermediate patients showed positive NC2 expression with missense and/or splice site COL domain mutations. 6 intermediate patients with negative NC2 expression showed bi-allelic PTC mutations and very low NC1 staining. Given their paucity of C7 NC2 expression, its possible these latter 6 patients may evolve into a more severe clinical phenotype over time or other factors outside of C7 expression may be playing a role in dictating disease severity. This remains to be further studied. We conclude that genotype or anchoring fibril morphology do not entirely explain the phenotypic variability and C7 NC1 expression did not prove to be a good indicator of disease severity. However, of all the tests utilized, C7 NC2 expression proved the most useful in distinguishing mild from severe RDEB clinical phenotypes.

Efficient and Robust Highly Branched Poly(β-amino ester)/Minicircle COL7A1 Polymeric Nanoparticles for Gene Delivery to Recessive Dystrophic Epidermolysis Bullosa Keratinocytes.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe congenital skin fragility disease caused by COL7A1 mutations that result in type VII collagen (C7) deficiency. Herein, we report a synergistic polyplex system that can efficiently restore C7 expression in RDEB keratinocytes. A highly branched multifunctional poly(β-amino ester) (HPAE), termed as HC32-122, was optimized systematically as the high-performance gene delivery vector for keratinocytes, achieving much higher transfection capability than polyethylenimine, SuperFect, and Lipofectamine 2000 without inducing obvious cytotoxicity. Concurrently, a 12 kb length minicircle DNA encoding ∼9 kb full-length COL7A1 (MCC7) devoid of bacterial sequence was biosynthesized as the therapeutic gene. Combining the highly potent polymer and the miniaturized gene structure, HC32-122/MCC7 polyplexes achieve 96.4% cellular uptake efficiency, 4019-fold COL7A1 mRNA enhancement, and robust recombinant C7 expression. Structure-property investigations reveal that HC32-122 can effectively condense MCC7 to form small, uniform, compact, and positively charged spherical nanoparticles with high DNA release flexibility. Moreover, formulation study shows that sucrose is conductive to lyophilized HC32-122/DNA polyplexes for maintaining the transfection capability. Direct frozen polyplexes can maintain full gene transfection capability after one-year storage. High efficiency, biocompatibility, facile manipulation, and long-term stability make the HC32-122/MCC7 system a promising bench-to-bed candidate for treating the debilitating RDEB.

Human Mesenchymal Stromal Cells Engineered to Express Collagen VII Can Restore Anchoring Fibrils in Recessive Dystrophic Epidermolysis Bullosa Skin Graft Chimeras

Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating genodermatosis caused by loss-of-function mutations in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils at the dermal-epidermal junction. With no curative treatments presently available, retrovirally transduced autologous epidermal grafts and intradermal lentivirally engineered fibroblast injections are being investigated. Alternative approaches aim to infuse allogeneic mesenchymal stromal cells (MSCs) to provide a more generalized treatment for RDEB. We investigated whether healthy human MSCs could be engineered to overexpress C7 and correct RDEB in a human:murine chimeric model. Initially, engineered MSCs incorporated exvivo into RDEB grafts, their presence confirmed by fluorescence in situ hybridization, revealed recovery of function of the dermal-epidermal junction with no signs of blister formation. Importantly, the detection of anchoring fibrils by transmission electron microscopy corroborated structural recovery. Next, MSCs cotransduced to express C7 and luciferase were delivered intradermally into grafted RDEB skin, resulting in localized MSC persistence with deposition of de novo C7 at the site. Notably, C7 expression was sufficient to restore anchoring fibril density to normal levels. In contrast, intravenously injected engineered MSCs were undetectable within grafts and lacked anchoring fibril reconstitution. Our data suggest that although localized correction may be achievable using engineered MSCs, strategies for systemic administration require further modeling.

Effects of feeding time on complement component C7 expression in Pelteobagrus vachellii subject to bacterial challenge

Shifting the feeding time for fish from daytime to nighttime could alter their digestive behavior, disturb their metabolism, and may affect immune-related genes. This study aimed to clone complement component C7 and analyze the different expression of C7 mRNA in fish fed during either the day or at night and then challenged with Aeromonas hydrophila infection. The Pv-C7 cDNA of Pelteobagrus vachellii contained 2 647 bp with an open reading frame encoding a protein of 818 amino acids. Multiple sequences analysis indicated that Pv-C7 included eight domains, which was similar to results for other species. Quantitative PCR analysis showed that Pv-C7 was mainly expressed in the liver, spleen, intestine and head kidney tissues of healthy P. vachellii. Quantitative PCR analysis showed that C7 mRNA transcript in the liver, spleen and head kidney also increased significantly when the fish were fed at nighttime (20:00). In addition, the expression of Pv-C7 mRNA significantly increased with A. hydrophila challenge in the liver (48–96 h), spleen and head kidney (12–96 h) tissues of P. vachellii. Pv-C7 mRNA expression of the fish fed at nighttime showed significant higher than that in the fish fed at day time at 12–48 h in head kidney and 12–24 h in spleen. This study indicates that altering the feeding time from daytime to nighttime could increase Pv-C7 mRNA expression, and feeding time may affect the immune response involving C7.

Allogeneic fibroblast cell therapy in the treatment of recessive dystrophic epidermolysis bullosa

Abstract Background The non-healing wound is a characteristic clinical sign in recessive dystrophic epidermolysis bullosa (RDEB). Methods Six patients with recessive dystrophic epidermolysis bullosa were injected with suspension of allogeneic fibroblasts into erosion margins. Erosions (>1 month old) with surface area between 2 cm2 and 28 cm2 were selected for treatment. The patients were administered with 1 mL suspension of allogeneic fibroblasts with concentration 5 × 106 cells/ml, 10 × 106 cells/ml and 20 × 106 cells/ml. Paired erosions were injected with vehicle solution alone. All erosions were assessed for healing rate and biopsied at baseline and at two weeks after treatment. Immunofluorescence antigen mapping (IFM) was performed to detect C7 expression. Results An increase of healing rate was detected 14 days after fibroblasts and vehicle alone injections, in some cases with complete erosion closure. The results of the IFM in patients treated with allogeneic fibroblasts and vehicle solution demonstrated the increase of collagen VII expression in dermo-epidermal junction, more intensive in patients injected with 20 × 106 cells/ml. Conclusions Intradermal administration of allogeneic fibroblasts is a safe and effective method of treatment of non-healing wounds in recessive dystrophic epidermolysis bullosa.

Vaccinia virus host-range factor C7 has dual inhibitory functions in type I IFN production and signaling

Abstract Vaccinia virus C7 protein is an important host-range factor for vaccinia virus life cycle in mammalian cells. C7L homologs are present in almost all of the poxviruses that infect mammalian hosts. Type I IFN plays an important role in host defense of viral infection, and yet, the role of C7 in immune modulation of the IFN pathway is unclear. We have previously reported that the highly attenuated modified vaccinia virus Ankara (MVA) infection of conventional dendritic cells (cDCs) induces type I IFN via the cGAS/STING/TBK1/IRF3 pathway. In this study, we find that ectopic C7 expression blocks STING, TBK1, or IRF3-induced IFNB and ISRE (interferon stimulated response element) promoter activation. Murine or human macrophage cell lines that overexpress C7 have blunted innate immune responses to DNA or RNA stimuli, or the infection of DNA or RNA viruses. Overexpression of C7 also attenuates ISG gene expression induced by IFN-b treatment. MVA with deletion of C7L (MVAΔC7L) infection of cDCs induces higher levels of type I IFN than MVA. C7 blocks IFN-b-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway via preventing Stat2 phosphorylation. C7 directly interacts with stat2 as demonstrated by co-immunoprecipitation studies. Taken together, our results provide evidence that C7 has dual inhibitory roles in type I IFN production and signaling.

367 Phase I clinical trial for Recessive Dystrophic Epidermolysis Bullosa using genetically corrected autologous keratinocytes

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is an inherited genetic skin disorder caused by mutations in the COL7A1 gene encoding type VII collagen (C7). The Current therapy is limited to supportive palliation. We report the results from the first Phase I clinical trial of ex vivo gene therapy for the treatment of severe RDEB (NCT01263379). 4 adult, male RDEB subjects enrolled in this trial carried various heterozygous COL7A1 mutations resulting in expression of only truncated C7 protein undetectable by NC2 antibodies and displayed absent, or sparse rudimentary anchoring fibrils (AF) by EM. Autologous RDEB KC isolated from skin biopsies were transduced with GMP grade retrovirus carrying the full-length COL7A1. 6 ∼35cm2 autologous epidermal sheet grafts were manufactured and grafted onto prepared wound beds of each subject (N=24 grafts). The primary endpoints included expression of full-length C7 and restoration of AF at 3 and 6 months, as well as absence of RCR at 3, 6, 12 months post grafting. All 24 grafts were well tolerated by all subjects, and no serious adverse events were reported. At 3 months, 87% (21/24 grafts) and at 6 months, 67% (16/24 grafts) showed significant wound healing, as of 75% healing compared with baseline. Molecular analysis of the skin biopsies obtained from the grafted sites revealed robust C7 expression detected by both NC1 and NC2 C7 antibodies, with morphologically normal appearance and frequency of NC2 reactive AF identified at the basement membrane zone using IEM. These data indicate that COL7A1 ex-vivo gene transfer has a favorable safety profile and capable of cutaneous type VII collagen delivery highlighting the potential of durable cell based RDEB therapy in humans.

Complement proteins C7 and CFH control the stemness of liver cancer cells via LSF-1

Tumor-initiating cells are important for the formation and maintenance of tumor bulks in various tumors. To identify surface markers of liver tumor-initiating cells, we performed primary tumorsphere culture and analyzed the expression of cluster of differentiation (CD) antigen genes using NanoString. Interestingly, we found significant upregulation of the complement proteins (p = 1.60 × 10−18), including C7 and CFH. Further studies revealed that C7 and CFH are required to maintain stemness in liver cancer cells. Knockdown of C7 and CFH expression abrogated tumorsphere formation and induced differentiation, whereas overexpression stimulated stemness factor expression as well as in vivo cell growth. Mechanistically, by studying C7 and CFH-dependent LSF-1 expression and its direct role on stemness factor transcription, we found that LSF-1 is involved in this regulation. Taken together, our data demonstrate the unprecedented role of complement proteins on the maintenance of stemness in liver tumor-initiating cells.

Mucocutaneous Engraftment and Type VII Collagen (C7) Replacement after Allogeneic Hematopoietic Cell Transplantation (HCT) in Patients with Recessive Dystrophic Epidermolysis Bullosa (RDEB)

In RDEB-an incurable, often fatal, blistering genodermatosis-loss-of-function mutations in the COL7A1 gene result in diminished or absent C7, lack of attachment between the epidermal basement membrane and the dermal matrix, and a poor quality of life and early death as a result of chronic mucocutaneous blistering.In 2010, we reported the results of a 'first-in-human' study on the use of HCT in 6 patients with RDEB, suggesting that increased C7 expression and mucocutaneous wound healing paralleled integumentary donor cell engraftment (NEJM 363:629, 2010). However, myeloablative conditioning (MAC) resulted in high incidence of renal, pulmonary and hepatic complications, as well as opportunistic infections due to chemotherapy-induced mucocutaneous toxicity. Therefore, subsequent patients with RDEB received reduced intensity conditioning (RIC) and the addition of bone marrow (BM)-derived mesenchymal stem/stromal cells (MSC) in an attempt to reduce the conditioning-related toxicities and enhance tissue recovery.After 2010, 20 patients aged 0.4-20 years with generalized severe RDEB underwent allogeneic HCT at the University of Minnesota. Of these, 7 were treated with MAC (busulfan IV 0.8 mg/kg for >12 kg and 1.1 mg/kg for those <12 kg of body weight q6h on days -9 to -6, with pharmacokinetic targeting 1,000 mmol/min for the area under curve; fludarabine [FLU] IV 25 mg/m2/day on days -5 to -3, and cyclophosphamide [CY] IV 50 mg/kg/day on days -5 to -2). Subsequently, 13 were treated with RIC (CY IV 50 mg/kg/day on day -6, FLU IV 40 mg/m2/day on days -6 to -2, anti-thymocyte globulin IV 30 mg/kg/day on days -4 to -2, and total body irradiation [TBI] 200 cGy or 300 cGy on day -1). Uniform GvHD prophylaxis used was cyclosporine A and mycophenolate mofetil. The hematopoietic graft was HLA-matched related BM (N=10), unrelated BM (N=7) or umbilical cord blood (UCB, N=3). In addition, a single infusion of third-party HLA-mismatched MSC (dose: 2 x 106/kg) was administered on day 0 (7 with MAC; 13 with RIC).The probability of 2-year survival was 90% (95% CI, 47-99%) in recipients of RIC as compared to 69% (95% CI, 37-87%) in recipients of MAC (p=0.3). 3 individuals (2 MAC; 1 RIC) died within one year after treatment as a result of veno-occlusive disease (N=1) and fungal sepsis (N=2), and one patient treated with MAC died after one year from extensive chronic GvHD. Interestingly, acute GVHD was rare (overall grade 2, N=2). There were no statistically significant survival differences in comparison of related vs. unrelated grafts, BM vs. UCB grafts, and those who received additional MSC vs. those who did not. Complete hematopoietic chimerism was achieved in all patients after MAC; however, RIC was associated with partial engraftment-mean 31% (range, 0-93%) in the CD15 compartment and 55% (range, 25-100%) in the CD3 compartment by day 100 after HCT. Wild-type levels of C7 in the skin believed to be sufficient to protect from extensive blistering are 10-30%. The skin chimerism was a mean 37% (range, 0-97%) after MAC in contrast to 13% (range, 0-47%) after RIC with a trend to slightly higher proportion in recipients of TBI 300cGy relative to 200cGy TBI. The primary endpoint of the analysis was survival with objective evidence of reduced mucocutaneous blistering. When compared to MAC, RIC was better tolerated with reduced risks of end organ toxicities and infections: cumulative incidence was 63% (95% CI, 34-92%) in recipients of RIC as compared to 92% (95% CI, 65-100%) in recipients of MAC, (p=0.06). For the entire cohort of engrafted, surviving subjects with RDEB thus far (N=18 of 26), 14 individuals demonstrated partial to marked biochemical and clinical improvement in mucocutaneous disease as evidenced by >50% reduction in affected body surface area, and/or C7 expression; example from RIC-treated individual is shown in Figure 1 (nuclei are blue, epidermis [top] and dermis [bottom of each panel] with C7 expression [red] at the dermal-epidermal junction).These results suggest that [1] RIC is associated with less toxicity and potentially improved survival; [2] HCT can result in improved expression of C7 protein, with reduced mucocutaneous blistering characteristic of generalized severe RDEB; and [3] these observations set the stage for exploratory studies with hematopoietic cell therapy for tissue repair and regeneration in extramedullary sites, extending their use into treatment of a broad spectrum of non-hematologic disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.