SIN retroviral vectors expressing COL7A1 under human promoters for ex vivo gene therapy of recessive dystrophic epidermolysis bullosa

Abstract

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by loss-of-function mutations in COL7A1 encoding type VII collagen which forms key structures (anchoring fibrils) for dermal-epidermal adherence. Patients suffer since birth from skin blistering, and develop severe local and systemic complications resulting in poor prognosis. We lack a specific treatment for RDEB, but ex vivo gene transfer to epidermal stem cells shows a therapeutic potential. To minimize the risk of oncogenic events, we have developed new minimal self-inactivating (SIN) retroviral vectors in which the COL7A1 complementary DNA (cDNA) is under the control of the human elongation factor 1alpha (EF1alpha) or COL7A1 promoters. We show efficient ex vivo genetic correction of primary RDEB keratinocytes and fibroblasts without antibiotic selection, and use either of these genetically corrected cells to generate human skin equivalents (SEs) which were grafted onto immunodeficient mice. We achieved long-term expression of recombinant type VII collagen with restored dermal-epidermal adherence and anchoring fibril formation, demonstrating in vivo functional correction. In few cases, rearranged proviruses were detected, which were probably generated during the retrotranscription process. Despite this observation which should be taken under consideration for clinical application, this preclinical study paves the way for a therapy based on grafting the most severely affected skin areas of patients with fully autologous SEs genetically corrected using a SIN COL7A1 retroviral vector.

Figures

Figure 1

Ex vivo transduction of primary RDEB keratinocytes and fibroblasts. Immunodetection of type VII collagen in (a–c) RDEB keratinocytes and (d–f) RDEB fibroblasts before and after transduction. (a,d) RDEB keratinocytes and fibroblasts transduced with the pCMS-EF1α-COL7A1 retroviral vector produced with the stable producer clone. (b,e) RDEB keratinocytes and fibroblasts transduced with the pCMS-COL7A1-COL7A1 retroviral vector. (c,f) Nontransduced RDEB keratinocytes and fibroblasts. An average of 60% of the keratinocytes and 70% of fibroblasts were transduced in these experiments. EF1α, elongation factor 1α RDEB, recessive dystrophic epidermolysis bullosa.

Figure 2

Plating efficiency analysis. Transduced cells with both the pCMS-COL7A1-COL7A1 and the pCMS-EF1α-COL7A1 viral vectors showed similar plating efficiency when compared to untransduced cells of the same experiment. Cells arised from the same patient biopsy were transduced at passage 5 and maintained for 7 up to 13 consecutive passages depending on the experiment. *Cells transduced with pCMS-EF1α-COL7A1 supernatant produced using the producer clone. EF1α, elongation factor 1α NT, nontransduced.

Figure 3

Characterization of the integrated provirus. Southern blot experiments were carried out on genomic DNA of keratinocytes and fibroblasts transduced with either the pCMS-EF1α-COL7A1 or the pCMS-COL7A1-COL7A1 SIN retroviral vectors. HindIII + EcoRV fragments of genomic DNA were probed with a 9 kb 32P-labeled fragment corresponding to the full-length COL7A1 cDNA. Single bands of 9.6 kb (EF1α-COL7A1 construct) or 10 kb (COL7A1-COL7A1 construct) corresponding to the full-length provirus (P) were detected together with a 15 kb fragment corresponding to the endogenous COL7A1 gene (E). *Cells transduced with pCMS-EF1α-COL7A1 supernatant produced using the producer clone. cDNA, complementary DNA; EF1α, elongation factor 1α.

Figure 4

Characterization of biochemical properties of recombinant type VII collagen. SDS-PAGE and immunoblot analysis of type VII collagen was carried out with monoclonal antibody (a) LH7:2 or (b,c) a polyclonal antibody. (a) Protein extracts from NHK, Krdeb, Kcol7, and Kef1α culture supernatant run on a 4% acrylamide gel. A single band around 300 kd corresponding to type VII collagen is present in NK, Kcol7, and Kef1α while absent in Krdeb. (b) Type VII collagen FPLC-purified from transduced cells supernatant analyzed on a 4–12% gradient gel either directly on nonreducing conditions (NR) or after reduction with β-mercaptoethanol (R), showing the presence of both monomers (M, 300 kd) and homotrimers (H, ~900 kd) of type VII collagen. (c) Purified type VII collagen run on a 7% gel. Type VII collagen monomers (300 kd) were detected without protease treatment (NT), whereas digestion with pepsine (Pep) released the triple-helical domain (TH) and its two pepsin-resistant fragments, P1 and P2. Collagenase digestion (Col) left the NC1 domain intact. *Cells transduced with pCMS-EF1α-COL7A1 supernatant produced using the producer clone. FPLC, fast protein liquid chromatography; NHK, normal human keratinocytes; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Figure 5

Analysis of type VII collagen rearrangements. (a) Some western blot experiments (3/12) of cell protein extracts from transduced keratinocytes using the polyclonal antitype VII collagen antibody showed the presence of shorter immunoreactive bands which likely correspond to truncated proteins arising from a rearranged transgene. (b) Southern blot experiments confirmed in some cases (3/9) the presence of shorter provirus fragment (R) in addition to the endogenous fragment (E) and the normal provirus (P). These shorter proviruses, probably resulted from a rearrangement event during reverse transcription. In contrast, no shorter protein fragment was detected by western blot analysis of transduced fibroblast extracts (seven experiments), although a shorter provirus form could be seen on one Southern blot experiment (out of three experiments). *Cells transduced with pCMS-EF1α-COL7A1 supernatant produced using the producer clone. EF1α, elongation factor 1α.

Figure 6

In vivo restoration of type VII expression and epidermal adherence in RDEB reconstructed skin. Histological analysis, type VII collagen immunostaining, and transmission electron microscopy analyses at 5 months postgrafting of SEs made of (a–c) NHK and NHF, (d–f) Krdeb and Frdeb, (g–i) Kcol7 and Fcol7, and (j–l) Kef1α and Fef1α. Type VII collagen accumulates along the dermal–epidermal junction in normal SE and in both genetically corrected SEs, whereas it is completely absent in SE made of uncorrected RDEB cells. Note the dermo–epidermal cleft (*) in the SE made of (d,e) uncorrected RDEB cells, whereas no blistering is seen in (g,h,j,k) genetically corrected SE. *Cells transduced with pCMS-EF1α-COL7A1 supernatant produced using the producer clone. Anchoring fibrils (arrows) were present below the lamina densa in all SEs except for the SE made of uncorrected RDEB cells (the picture was taken in a nonblistering area). The hemidesmosomes (HD) appear normal. Original magnification ×50,000. EF1α, elongation factor 1α NHF, normal human fibroblast; NHK, normal human keratinocytes; RDEB, recessive dystrophic epidermolysis bullosa; SE, skin equivalent.

Figure 7

Differentiation pattern in reconstructed skins. At 5 months postgrafting, the expression of three major epidermal differentiation markers was compared between corrected SE, normal reconstructed skin and normal human foreskin. The immunostaining patterns of (a–d) loricrin, (e–h) keratin 10, and (i–l) keratin 14 are identical throughout. *Cells transduced with pCMS-EF1α-COL7A1 supernatant produced using the producer clone. EF1α, elongation factor 1α.