Eva M. Murauer, Yannick Gache, Iris K

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Functional Correction of Type VII Collagen Expression in Dystrophic Epidermolysis Bullosa Eva M. Murauer, Yannick Gache, Iris K. Gratz, Alfred Klausegger, Wolfgang Muss, Christina Gruber, Guerrino Meneguzzi, Helmut Hintner, Johann W. Bauer Journal of Investigative Dermatology Volume 131, Issue 1, Pages 74-83 (January 2011) DOI: 10.1038/jid.2010.249 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Retroviral transduction of recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes with a 3′ pre-trans-splicing molecule (PTM). (a) Trans-splicing between the 5′-splice site (5′ss) of the endogenous COL7A1 pre-mRNA and the 3′-splice site (3′ss) of the exogenously transduced PTM-S6 results in a reprogrammed COL7A1 transcript, consisting of endogenous exons 1–64 (white) and delivered exons 65–118 (gray). (b) pLZRS-PTM-S6 vector comprising the Moloney mouse sarcoma virus long terminal repeat (LTR), the 3.6-kb PTM-S6, an internal ribosome entry (IRES), and a Zeocine-resistance gene. (c) Clonal analysis of keratinocyte cultures of clone no. 1 at passages 8 and 20 shows comparable clonogenic potential. (d) PCR analysis of PTM-S6 integration in the genome of RDEB- and PTM-S6-transduced keratinocytes. pLZRS, amplified product from plasmid pLZRS-PTM-S6. (e) Linear amplification-mediated-PCR analysis of genomic DNA of transduced RDEB cells showed one retroviral integration event. BP, branch point; CFE, colony-forming efficiency; PPT, polypyrimidine tract; wt, wild-type. Journal of Investigative Dermatology 2011 131, 74-83DOI: (10.1038/jid.2010.249) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Reexpression of type VII collagen in 3′pre-trans-splicing molecule (PTM)-S6-transduced recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes and at the dermal–epidermal junction of organotypic skin equivalents (SEs). (a) Immunofluorescence analysis of cultured keratinocytes showed no collagen VII staining in parental RDEB keratinocytes (RDEB), and positive staining in normal keratinocytes (NHK), PTM-S6-transduced cell pool (RDEB-PTM-S6), and clone no. 1 (RDEB-PTM-S6, clone no. 1). Bar=20μm. (b) Immunostaining for collagen VII is negative in SEs with RDEB keratinocytes (RDEB), and positive in SE with transduced RDEB keratinocytes at passages 4 and 20 (RDEB-PTM-S6, clone no. 1, p4/p20), comparable to the staining in normal SEs (NHK). Cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole dye. Bar=100μm. (c) Electron microscopy analysis shows no anchoring fibril (AF) in SEs of RDEB keratinocytes (RDEB), whereas rudimentary AFs are observed underneath the lamina densa in SEs of PTM-S6-transduced cells (RDEB-PTM-S6, clone no. 1). Bar=100nm. BM, basement membrane; HD, hemidesmosomes. Journal of Investigative Dermatology 2011 131, 74-83DOI: (10.1038/jid.2010.249) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Functional correction of type VII collagen expression by trans-splicing. (a) Light microscopy of cell cultures. Recessive dystrophic epidermolysis bullosa (RDEB) parental keratinocytes; arrow, unusual shape; arrowhead, unusual size; normal human keratinocytes (NHK); (RDEB-3′ pre-trans-splicing molecule (PTM)-S6) PTM-S6-transduced RDEB keratinocytes. Bar=10μm. (b) PTM-S6-transduced RDEB keratinocytes display impaired invasive potential compared with parental RDEB keratinocytes. Shown are mean±SD of triplicate values. (c) Immunoblot analysis indicates the absence of collagen VII in RDEB keratinocytes (1), whereas NHK (2) and PTM-S6-transduced RDEB keratinocytes (3) show secretion of full-length collagen VII. (d) Semiquantitative reverse transcription-PCR analysis of COL7A1 mRNA expression shows low levels in RDEB keratinocytes compared with NHK, and a twofold increase in PTM-S6-transduced RDEB cells. (e) Reverse transcriptase-PCR analysis of COL7A1 mRNA using trans-specific primers shows a band specific for trans-splicing in PTM-S6-transduced RDEB keratinocytes (1), undetectable in non-transduced RDEB keratinocytes (2). Journal of Investigative Dermatology 2011 131, 74-83DOI: (10.1038/jid.2010.249) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions