Safety and early efficacy outcomes for lentiviral fibroblast gene therapy in recessive dystrophic epidermolysis bullosa
Su M. Lwin, et al.
Su M. Lwin, et al.
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Clinical Research and Public Health Dermatology Genetics

Safety and early efficacy outcomes for lentiviral fibroblast gene therapy in recessive dystrophic epidermolysis bullosa

Abstract

BACKGROUND Recessive dystrophic epidermolysis bullosa (RDEB) is a severe form of skin fragility disorder due to mutations in COL7A1 encoding basement membrane type VII collagen (C7), the main constituent of anchoring fibrils (AFs) in skin. We developed a self-inactivating lentiviral platform encoding a codon-optimized COL7A1 cDNA under the control of a human phosphoglycerate kinase promoter for phase I evaluation.METHODS In this single-center, open-label phase I trial, 4 adults with RDEB each received 3 intradermal injections (~1 × 106 cells/cm2 of intact skin) of COL7A1-modified autologous fibroblasts and were followed up for 12 months. The primary outcome was safety, including autoimmune reactions against recombinant C7. Secondary outcomes included C7 expression, AF morphology, and presence of transgene in the injected skin.RESULTS Gene-modified fibroblasts were well tolerated, without serious adverse reactions or autoimmune reactions against recombinant C7. Regarding efficacy, there was a significant (P < 0.05) 1.26-fold to 26.10-fold increase in C7 mean fluorescence intensity in the injected skin compared with noninjected skin in 3 of 4 subjects, with a sustained increase up to 12 months in 2 of 4 subjects. The presence of transgene (codon-optimized COL7A1 cDNA) was demonstrated in the injected skin at month 12 in 1 subject, but no new mature AFs were detected.CONCLUSION To our knowledge, this is the first human study demonstrating safety and potential efficacy of lentiviral fibroblast gene therapy with the presence of COL7A1 transgene and subsequent C7 restoration in vivo in treated skin at 1 year after gene therapy. These data provide a rationale for phase II studies for further clinical evaluation.TRIAL REGISTRATION ClincalTrials.gov NCT02493816.FUNDING Cure EB, Dystrophic Epidermolysis Bullosa Research Association (UK), UK NIHR Biomedical Research Centre at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London, and Fondation René Touraine Short-Exchange Award.

Authors

Su M. Lwin, Farhatullah Syed, Wei-Li Di, Tendai Kadiyirire, Lu Liu, Alyson Guy, Anastasia Petrova, Alya Abdul-Wahab, Fiona Reid, Rachel Phillips, Maria Elstad, Christos Georgiadis, Sophia Aristodemou, Patricia A. Lovell, James R. McMillan, John Mee, Snaigune Miskinyte, Matthias Titeux, Linda Ozoemena, Rashida Pramanik, Sonia Serrano, Racheal Rowles, Clarisse Maurin, Elizabeth Orrin, Magdalena Martinez-Queipo, Ellie Rashidghamat, Christos Tziotzios, Alexandros Onoufriadis, Mei Chen, Lucas Chan, Farzin Farzaneh, Marcela Del Rio, Jakub Tolar, Johann W. Bauer, Fernando Larcher, Michael N. Antoniou, Alain Hovnanian, Adrian J. Thrasher, Jemima E. Mellerio, Waseem Qasim, John A. McGrath

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Figure 6

PCR and semiquantitative PCR demonstrating the expression of COL7A1 transgene and endogenous mutant and WT cDNA respectively in the injected skin of subject 5.

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PCR and semiquantitative PCR demonstrating the expression of COL7A1 tran...
(A) PCR amplifications using 3 sets of primers targeting NC1, triple helix, and NC2 regions unique to codon-optimized COL7A1 transgene sequence (coCOL7A1 cDNA) (exon 12–13, exon 51–52, and exon 112–113, respectively) that are different from the endogenous WT COL7A1 cDNA sequence. This demonstrated clear bands (in boxes) at month 12 (lane 6) in the injected skin and transduced autologous fibroblasts before injection but not the noninjected skin or baseline skin; there was a band at 247 bp corresponding to the exon 12–13 fragment of coCOL7A1, at 231 bp corresponding to the exon 51–52 fragment, and 244 bp corresponding to the exon 112–113 fragment of coCOL7A1. Similar clear bands (in boxes) were also seen in the gene-modified fibroblasts (i.e., coCOL7A1-supplemented) before injections (lane 1), which represent the positive control. (B) Multiplex PCR amplifications of endogenous COL7A1 cDNA reversed transcribed from the total RNA extracted from the injected and noninjected skin using specific primers to detect mutant exon 87–skipped (without exon 87) sequence and WT (with exon 87) sequence of COL7A1 with GAPDH as internal control. Each band on the PCR gel image was used for quantification with GelQuant.NET. (C) Semiquantitative PCR demonstrated a relative increase in expression of both endogenous WT and mutant COL7A1 in the injected skin of subject 5 (WT at week 2, month 3, and month 12 after gene therapy, and mutant at month 12 after gene therapy), after normalization with the housekeeping gene GAPDH. The box-and-whisker plot represents triplicate measurements of respective COL7A1 expression. B, baseline; I, injected skin; M, month; NI, noninjected skin; W, week.