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Lentiviral Engineered Fibroblasts Expressing Codon-Optimized COL7A1 Restore Anchoring Fibrils in RDEB.

Georgiadis C, Syed F, Petrova A, Abdul-Wahab A, Lwin SM, Farzaneh F, Chan L, Ghani S, Fleck RA, Glover L, McMillan JR, Chen M, Thrasher AJ, McGrath JA, Di WL, Qasim W - J. Invest. Dermatol. (2016)

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.

View Article: PubMed Central - PubMed

Affiliation: UCL Institute of Child Health, Molecular and Cellular Immunology Section & Great Ormond Street Hospital NHS Foundation Trust, London, United Kingdom.

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Expression of C7 in gene-corrected RDEB fibroblasts using a SIN-LV-COL7A1 vector. (a) Configuration of pCCL-PGK-COL7A1 lentiviral transfer plasmid shows a third-generation, split-packaging SIN vector with the deleted U3 region of the 3′LTR, internal PGK promoter, mutated woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and central polypurine tract (cPPT). Transgene COL7A1 was codon-optimized (co-COL7A1) encoding the full-length COL7A1 sequence. (b, c) Average expression of C7 in LV-COL7-transduced and untransduced (UT) primary RDEB-1 and -2 fibroblasts by intracellular staining and flow cytometry with corresponding mean fluorescence intensity (MFI) (d). (e) In situ expression of C7 in RDEB-1 and -2 LV-COL7 fibroblasts using in-cell Western blotting (ICWB). Green lanes represent C7 expression; red lanes represent loading control (β-actin) expression with average immunoreactivity (f). LTR, long terminal repeat; LV, lentiviral; PGK, phosphoglycerate kinase; RDEB, recessive dystrophic epidermolysis bullosa; SD , standard deviation; SIN, self-inactivating. Error bars represent SD of four replicates.
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fig1: Expression of C7 in gene-corrected RDEB fibroblasts using a SIN-LV-COL7A1 vector. (a) Configuration of pCCL-PGK-COL7A1 lentiviral transfer plasmid shows a third-generation, split-packaging SIN vector with the deleted U3 region of the 3′LTR, internal PGK promoter, mutated woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and central polypurine tract (cPPT). Transgene COL7A1 was codon-optimized (co-COL7A1) encoding the full-length COL7A1 sequence. (b, c) Average expression of C7 in LV-COL7-transduced and untransduced (UT) primary RDEB-1 and -2 fibroblasts by intracellular staining and flow cytometry with corresponding mean fluorescence intensity (MFI) (d). (e) In situ expression of C7 in RDEB-1 and -2 LV-COL7 fibroblasts using in-cell Western blotting (ICWB). Green lanes represent C7 expression; red lanes represent loading control (β-actin) expression with average immunoreactivity (f). LTR, long terminal repeat; LV, lentiviral; PGK, phosphoglycerate kinase; RDEB, recessive dystrophic epidermolysis bullosa; SD , standard deviation; SIN, self-inactivating. Error bars represent SD of four replicates.

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.


Lentiviral Engineered Fibroblasts Expressing Codon-Optimized COL7A1 Restore Anchoring Fibrils in RDEB.

Georgiadis C, Syed F, Petrova A, Abdul-Wahab A, Lwin SM, Farzaneh F, Chan L, Ghani S, Fleck RA, Glover L, McMillan JR, Chen M, Thrasher AJ, McGrath JA, Di WL, Qasim W - J. Invest. Dermatol. (2016)

Expression of C7 in gene-corrected RDEB fibroblasts using a SIN-LV-COL7A1 vector. (a) Configuration of pCCL-PGK-COL7A1 lentiviral transfer plasmid shows a third-generation, split-packaging SIN vector with the deleted U3 region of the 3′LTR, internal PGK promoter, mutated woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and central polypurine tract (cPPT). Transgene COL7A1 was codon-optimized (co-COL7A1) encoding the full-length COL7A1 sequence. (b, c) Average expression of C7 in LV-COL7-transduced and untransduced (UT) primary RDEB-1 and -2 fibroblasts by intracellular staining and flow cytometry with corresponding mean fluorescence intensity (MFI) (d). (e) In situ expression of C7 in RDEB-1 and -2 LV-COL7 fibroblasts using in-cell Western blotting (ICWB). Green lanes represent C7 expression; red lanes represent loading control (β-actin) expression with average immunoreactivity (f). LTR, long terminal repeat; LV, lentiviral; PGK, phosphoglycerate kinase; RDEB, recessive dystrophic epidermolysis bullosa; SD , standard deviation; SIN, self-inactivating. Error bars represent SD of four replicates.
© Copyright Policy - CC BY
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getmorefigures.php?uid=PMC4759620&req=5

fig1: Expression of C7 in gene-corrected RDEB fibroblasts using a SIN-LV-COL7A1 vector. (a) Configuration of pCCL-PGK-COL7A1 lentiviral transfer plasmid shows a third-generation, split-packaging SIN vector with the deleted U3 region of the 3′LTR, internal PGK promoter, mutated woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and central polypurine tract (cPPT). Transgene COL7A1 was codon-optimized (co-COL7A1) encoding the full-length COL7A1 sequence. (b, c) Average expression of C7 in LV-COL7-transduced and untransduced (UT) primary RDEB-1 and -2 fibroblasts by intracellular staining and flow cytometry with corresponding mean fluorescence intensity (MFI) (d). (e) In situ expression of C7 in RDEB-1 and -2 LV-COL7 fibroblasts using in-cell Western blotting (ICWB). Green lanes represent C7 expression; red lanes represent loading control (β-actin) expression with average immunoreactivity (f). LTR, long terminal repeat; LV, lentiviral; PGK, phosphoglycerate kinase; RDEB, recessive dystrophic epidermolysis bullosa; SD , standard deviation; SIN, self-inactivating. Error bars represent SD of four replicates.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: UCL Institute of Child Health, Molecular and Cellular Immunology Section & Great Ormond Street Hospital NHS Foundation Trust, London, United Kingdom.

Show MeSH
Related in: MedlinePlus