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

Abstract

BACKGROUNDRecessive 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.METHODSIn 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.RESULTSGene-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.CONCLUSIONTo 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 REGISTRATIONClincalTrials.gov NCT02493816.FUNDINGCure 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.

Keywords: Dermatology; Gene therapy; Genetic diseases; Genetics; Skin.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Illustration of Good Manufacturing Practice–compliant production of COL7A1-supplemented autologous fibroblasts for patients with recessive dystrophic epidermolysis bullosa in the present study (also referred to as the LENTICOL-F Trial).

(A) Configuration of a third-generation, 4-plasmid-system, self-inactivating lentiviral vector with deleted U3 region of the 3′ LTR, carrying full-length codon-optimized COL7A1 cDNA (SIN LV–coCOL7A1). An internal PGK promoter, mutated Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and HIV central polypurine tract (cPPT) are shown. (B) Steps involved in the GMP manufacture of ex vivo gene-modified autologous fibroblasts for intradermal injections in a subject with recessive dystrophic epidermolysis bullosa (RDEB). It took approximately 9–10 weeks from the time of obtaining the skin biopsy sample to the time of treatment.

Figure 2. CONSORT trial flowchart.

1Includes subject 1 twice, as he was withdrawn and reenrolled as subject 2 due to failure of delivery of the investigational medicinal product (IMP) — i.e., gene-modified autologous fibroblasts — as per consensus from the trial steering committee members and ethics approval. 2Trial completion includes all subjects followed up for 12 months after administration of gene-modified autologous fibroblasts.

Figure 3. Molecular characteristics of GMP-manufactured gene-modified autologous fibroblasts for subject 5.

Primary fibroblasts from 5 subjects. Four 4 patients in the trial were transduced with LV-PGK-coCOL7A1 at an MOI of 5, and the expression of C7 in transduced RDEB fibroblasts was assessed 4 weeks after transduction. (A) In subject 5, 9.0% of the transduced fibroblasts expressed C7 compared with untransduced RDEB fibroblasts as assessed by flow cytometry. (B) In situ cytoimmunofluorescence staining confirmed C7 (red: C7 and blue: nuclei) expression in both LV-PGK-coCOL7A1–transduced fibroblasts and normal human skin fibroblasts but not in untransduced RDEB fibroblasts. Scale bar: 50 μm. (C) C7 expression was further confirmed by immunoblotting, and results showed 290-kDa full-length C7 expression in transduced RDEB fibroblasts and normal human skin cells but not in untransduced fibroblasts. Vinculin represents internal loading control. NHS-FB, normal human skin fibroblasts; UT-RDEB-FB, untransduced RDEB fibroblasts; T-RDEB-FB, RDEB fibroblasts transduced with LV-PGK-coCOL7A1.

Figure 4. Type VII collagen (C7) expression in subjects 2–5 at baseline and in injected and noninjected skin at 2 weeks, 3 months, and 12 months.

(A) IF staining with a monoclonal LH7.2 antibody to C7 (targeting NC1 domain) on skin sections taken at specified time points from both injected (I) and noninjected (NI) sites alongside respective baseline samples. Trace C7 staining is visible in subjects 2 and 5, while brighter staining is seen in subjects 3 and 4. White arrows indicate the trace staining of C7 IF; asterisks indicate dermal-epidermal separation (B) The MFI of C7 NC1 expression along the DEJ in the injected and noninjected skin demonstrated in A for subjects 2–5 was further quantified for each sample in an assessor-blinded fashion using ImageJ, as adapted from a previous study (27). An average MFI value was calculated from 10 measurements taken at regular intervals using 8 × 8 pixels every 100 pixels along the DEJ. The C7 NCI MFI graphs in B correspond to the C7 IF images in A. (C) IF staining to demonstrate full-length C7 expression in subject 5 using a polyclonal antibody targeting the NC2 domain of C7 protein (ab198899; Abcam) after optimization. (D) The C7 NC2 MFI was then calculated using ImageJ as for the C7 NC1 MFI as described in B in an assessor-blinded fashion. The box-and-whisker plots in B and C represent the 10 repeat measurements of C7 MFI taken along the DEJ. *P < 0.05, **P < 0.005, ***P < 0.001. Error bars represent mean ± SEM. White arrows indicate C7 expression at the DEJ. B, baseline; C, control (normal skin); I, injected skin; M, month; NI, non-injected skin; W, week. Scale bars: 100 μm.

Figure 5. Transmission electron microscopy failed to demonstrate mature AFs along the dermal-epidermal junction of subject 5.

Thin sections of upper left arm control baseline (B), injected (I), and noninjected (NI) skin from subject 5 were obtained at 2 weeks, 3 months, and 12 months after treatment. No mature fan-shaped AFs were observed on any micrographs, but occasional thin wisp-like rudimentary AFs (black arrows) were visible at all time points. The epidermis was detached upon skin biopsy of the noninjected skin at month 12, and thus we were unable to obtain a meaningful intact dermal-epidermal junction transmission electron microscopy images. B, baseline; I, injected skin; M, month; NI, noninjected skin; W, week. Scale bar: 0.25 μm.

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.

(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.