title: Correction of DNA protein kinase deficiency by spliceosome-mediated RNA trans-splicing and sleeping beauty transposon delivery.
abstract: Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology for the repair of defective pre-messenger RNA (pre-mRNA) molecules. It is especially useful in the treatment of genetic disorders involving large genes. Although viral vectors have been used for achieving long-lasting expression of trans-splicing molecules, the immunogenicity and suboptimal safety profiles associated with viral-based components could limit the widespread application of SMaRT in the repair of genetic defects. Here, we tested whether the non-viral Sleeping Beauty (SB) transposon system could mediate stable delivery of trans-splicing molecules designed to correct the genetic defect responsible for severe combined immune deficiency (SCID). This immunological disorder is caused by a point mutation within the 12.4 kilobase (kb) gene encoding the DNA protein kinase catalytic subunit (DNA-PKcs) and is associated with aberrant DNA repair, defective T- and B-cell production, and hypersensitivity to radiation-induced injury. Using a novel SB-based trans-splicing vector, we demonstrate stable mRNA correction, proper DNA-PKcs protein production, and conference of a radiation-resistant phenotype in a T-cell thymoma cell line and SCID multipotent adult progenitor cells (MAPCs). These results suggest that SB-based trans-splicing vectors should prove useful in facilitating the correction of endogenous mutated mRNA transcripts, including the DNA-PKcs defect present in SCID cells.
authors: University of Minnesota Cancer Center, Department of Pediatrics, Division of Hematology-Oncology, Blood and Marrow Transplantation, University of Minnesota, Minneapolis, USA.
authors: Hatem
authors: H
authors: Zayed
authors: Lily
authors: L
authors: Xia
authors: Anton
authors: A
authors: Yerich
authors: Stephen R
authors: SR
authors: Yant
authors: Mark A
authors: MA
authors: Kay
authors: M
authors: M
authors: Puttaraju
authors: Gerard J
authors: GJ
authors: McGarrity
authors: David L
authors: DL
authors: Wiest
authors: R Scott
authors: RS
authors: McIvor
authors: Jakub
authors: J
authors: Tolar
authors: Bruce R
authors: BR
authors: Blazar
mesh_terms: D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D002460:Cell Line; D006801:Humans; D051379:Mice; D009154:Mutation / Q000235:genetics; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000172:deficiency* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D017381:Spliceosomes / Q000235:genetics*; D020040:Trans-Splicing / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics; D019895:Transposases / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D019895:Transposases; C494471:sleeping beauty transposase, human