Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair.
RNA trans-splicing is a promising tool for mRNA modification in a diversity of genetic disorders. In particular, the substitution of internal exons of a gene by combining 3' and 5' RNA trans-splicing seems to be an elegant way to modify especially large pre-mRNAs. Here we discuss a robust method for designing double RNA trans-splicing molecules (dRTM). We demonstrate how the technique can be implemented in an endogenous setting, using COL7A1, the gene encoding type VII collagen, as a target. An RTM screening system was developed with the aim of testing the replacement of two internal COL7A1 exons, harbouring a homozygous mutation, with the wild-type version. The most efficient RTMs from a pool of randomly generated variants were selected via our fluorescence-based screening system and adapted for use in an in vitro disease model system. Transduction of type VII collagen-deficient keratinocytes with the selected dRTM led to accurate replacement of two internal COL7A1 exons resulting in a restored wild-type RNA sequence. This is the first study demonstrating specific exon replacement by double RNA trans-splicing within an endogenous transcript in cultured cells, corroborating the utility of this technology for mRNA repair in a variety of genetic disorders.
EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria. c.huettner@salk.at.
Clemens
C
Hüttner
EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria. e.murauer@salk.at.
Eva M
EM
Murauer
EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria. s.hainzl@salk.at.
Stefan
S
Hainzl
EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria. t.kocher@salk.at.
Thomas
T
Kocher
EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria. a.neumayer@salk.at.
Anna
A
Neumayer
EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria. j.reichelt@salk.at.
Julia
J
Reichelt
Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria. jo.bauer@salk.at.
Johann W
JW
Bauer
EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria. u.koller@salk.at.
Ulrich
U
Koller
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D001483:Base Sequence; D002478:Cells, Cultured; D024143:Collagen Type VII / Q000235:genetics*; D004820:Epidermolysis Bullosa / Q000378:metabolism / Q000473:pathology; D005091:Exons; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D020040:Trans-Splicing
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D024143:Collagen Type VII; D012330:RNA, Double-Stranded; D012333:RNA, Messenger
RNA therapy; double RNA trans-splicing; epidermolysis bullosa; genetic diseases; type VII collagen
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