RESUMO
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 KIR-ligand status to evaluate potential alloreactive NK cell or transplant donors.
Assuntos
Antígenos HLA-B/genética , Antígenos HLA-C/genética , Células Matadoras Naturais/imunologia , Receptores Imunológicos/imunologia , Análise de Sequência de DNA/métodos , Alelos , Sequência de Bases , Bases de Dados de Ácidos Nucleicos , Antígenos HLA-B/imunologia , Antígenos HLA-C/imunologia , Teste de Histocompatibilidade , Humanos , Internet , Isoantígenos/genética , Isoantígenos/imunologia , Ligantes , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Receptores KIR , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Imunologia de Transplantes/genética , Imunologia de Transplantes/imunologia , Interface Usuário-ComputadorRESUMO
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.
