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.
Studies have shown that KIR-ligand mismatching to predict NK cell alloreactivity may result in less relapse and better survival in patients with AML. KIR-ligands are distinguished by single nucleotide polymorphisms (SNPs) from HLA-B and HLA-C sequences. We hypothesized that pyrosequencing to determine KIR-ligand status by direct sequencing of the ligand epitope can be done as an alternative to high resolution HLA-typing. Pyrosequencing is rapid and would be particularly useful in analysis of retrospective cohorts where high resolution HLA-typing is unavailable or too expensive. To validate this assay, RNA and DNA from 70 clinical samples were tested for KIR-ligand by pyrosequencing. Primer binding to invariant regions without known SNPs was critical for KIR-ligand assignment by pyrosequencing to be in full concordance with high resolution HLA-typing. Pyrosequencing is sensitive, specific, high-throughput, inexpensive, and can rapidly screen KIRligand status to evaluate potential alloreactive NK cell or transplant donors.
Gene correction is an attractive strategy for gene therapy since it allows the corrected gene to remain regulated within its native genome location. We have explored gene correction of murine severe combined immunodeficiency (SCID) with single-stranded DNA oligonucleotides (SSO). Murine SCID is characterized by severe T- and B-cell lymphopenia and is caused by a point mutation in the DNA protein kinase subunit (DNA-PK). To correct the mutant missense sequence (T to A substitution), a silent mutation was introduced by synthesizing the SSO non-transcribed sequence (45 bp) surrounding the site of the SCID mutation and replacing the T nucleotide with a C nucleotide to permit production of wild-type (wt) DNA-PK protein. Since the fetus is potentially an ideal permissive environment for gene correction due to the high proliferative rate of its tissues, SSO were injected in utero either directly into the liver of the fetus or transplacentally (via hydrodynamic infusion to the pregnant dam). E15/16 BALB/c-SCID recipients (N = 78) were injected with SSO (20 mcg/fetus). Twenty nine mice survived to term and, when evaluated by peripheral blood (PB) FACS at 15–30 weeks of life, 11 had significant phenotypic evidence of immune restoration defined as ≥ 2% CD4+ or CD8+ T cells: 6 had both CD4+ and CD8+ T cells, 2 had CD4+ cells only and 3 had CD8+ T cells only. The highest level of CD4+ cells seen was 9%, the highest level of CD8+ cells was 2% and both had TCR rearrangement and 27% and 15% genotypic correction of the mutated bp by quantitative pyrosequencing (PSQ) of DNA isolated from whole blood. Since placental membranes are permeable to some molecules, SSO were hydrodynamically delivered to pregnant BALB/c-SCID dams (100 mcg). Two of 8 evaluable mice injected on day E5/6 had significant numbers of T cells, one of which had 20% CD8+ with 3% CD4+ cells at 13 weeks of life, and PSQ showed a 13% correction rate. Sixteen offspring injected at E13/14 were analyzed: 2 had 4% and 5% CD4+ cells and the latter also had 6% of CD8+ cells with PSQ correction rates of 22% and 11%, respectively. Of 40 mice evaluated after transplacental injections at age E15/16, 9 had >2% CD4+ or CD8+cells. The four with the highest T cell count had a genotypic correction of 12–25% of wt levels. Notably, littermates with no phenotypic correction had no evidence of gene correction at the DNA-PK mutation site. However, in all immune-restored animals that were analyzed for gene correction, (2/78 after in utero; 7/64 after transplacental delivery) an A to T rather than the anticipated A to C correction occurred. This is consistent with the hypothesis that SSO stimulated homologous recombination with a preferred utilization of the endogenous T rather than the exogenous C due to preferential pairing of two pyrimidines (A with T) than pyrimidine with purine (A with C). In summary, we show that SSO therapy for correction of DNA-PK mutation is possible when SSO are injected in utero at late gestation or are hydrodynamically delivered to the pregnant dam. These findings also suggest that while DNA homology around the mutation site is necessary for correction, the wt nucleotide is favored by the endogenous DNA repair pathway.
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