Lentiviral Engineered Fibroblasts Expressing Codon-Optimized COL7A1 Restore Anchoring Fibrils in RDEB.
Bottom Line: Whereas allogeneic fibroblasts injected directly into the dermis can mediate transient disease modulation, autologous gene-modified fibroblasts should evade immunological rejection and support sustained delivery of type VII collagen at the dermal-epidermal junction.Expression and secretion of type VII collagen was confirmed with transduced cells exhibiting supranormal levels of protein expression, and ex vivo migration of fibroblasts was restored in functional assays.Gene-modified RDEB fibroblasts also deposited type VII collagen at the dermal-epidermal junction of human RDEB skin xenografts placed on NOD-scid IL2Rgamma() recipients, with reconstruction of human epidermal structure and regeneration of anchoring fibrils at the dermal-epidermal junction.
Affiliation: UCL Institute of Child Health, Molecular and Cellular Immunology Section & Great Ormond Street Hospital NHS Foundation Trust, London, United Kingdom.Show MeSH
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Mentions: Primary fibroblasts from patients with RDEB lacking C7 expression were transduced with a third-generation self-inactivating-LV vector encoding codon-optimized C7 (LV-COL7) under current good-manufacturing-practice compliant conditions using a single round of exposure at a multiplicity of infection 5 (Figure 1a). After 3 weeks of culture and expansion, flow cytometric analysis showed 9.3–12.8% of fibroblasts expressing C7 (Figure 1b–d), and this corresponded to an integrated proviral copy number of 0.12–0.14 copies/cell. In-cell Western blotting showed overexpression of C7 in transduced RDEB fibroblasts compared with untransduced and wild-type (WT) fibroblasts as measured by mean fluorescence intensity (Figure 1e and f). In situ cytostaining also detected C7 protein expression in transduced RDEB fibroblasts (Figure 2a), whereas there was no expression in untransduced RDEB fibroblasts. These results were further confirmed by Western blot analysis using a purified C7 antibody (a gift from Professor Mei Chen). Cell lysates from transduced RDEB fibroblasts revealed the expression of an approximately 290 kDa protein band corresponding to full-length C7 (Figure 2b) and expression was stable when reassessed after 8 weeks. Full-length protein was also detected in media harvested from cultured transduced RDEB fibroblasts (Figure 2c) indicating effective secretion of the recombinant protein. In view of previous reports that around a quarter of gamma retroviral vector integrants, particularly in keratinocytes, may encode truncated forms of the COL7A1 transgene, we screened cultures for aberrant protein forms, and found that only 3 of 49 single-cell clonal populations expressed abnormally sized protein. This greatly reduced frequency was attributed to our codon optimization of the transgene, with residual low-level recombination events during reverse transcription linked to a small number of persisting repeat sequences.
Affiliation: UCL Institute of Child Health, Molecular and Cellular Immunology Section & Great Ormond Street Hospital NHS Foundation Trust, London, United Kingdom.