RAG1 reversion mosaicism in a patient with Omenn syndrome.

PURPOSE: To identify mechanisms of disease in a child born to consanguineous parents, who presented with Omenn syndrome (OS) and was found to carry a heterozygous RAG1 mutation in peripheral blood DNA. METHODS: Mutation analysis was performed on whole blood and buccal swab DNA. Recombination activity of the mutant RAG1 protein and diversity of T cell repertoire were tested. RESULTS: Apparent heterozygosity for a novel, functionally null RAG1 mutation in peripheral blood DNA from a patient with OS was shown to be secondary to true somatic reversion. Analysis of T cell repertoire demonstrated expression of various TCRBV families, but an overall restricted pattern. CONCLUSIONS: This is the first case of true somatic reversion of a RAG1 mutation in a patient with OS. The reversion event likely occurred at a stage where only a limited pool of T cell progenitors capable of performing V(D)J recombination could be generated. This work emphasizes the importance of performing functional studies to investigate the significance of novel genetic variants, and to consider somatic reversion as a possible disease modifier in SCID.

Erythroid-specific expression of beta-globin by the sleeping beauty transposon for Sickle cell disease.

Sickle cell disease (SCD) results predominately from a single monogenic mutation that affects thousands of individuals worldwide. Gene therapy approaches have focused on using viral vectors to transfer wild-type beta- or gamma-globin transgenes into hematopoietic stem cells for long-term expression of the recombinant globins. In this study, we investigated the use of a novel nonviral vector system, the Sleeping Beauty (SB) transposon (Tn) to insert a wild-type beta-globin expression cassette into the human genome for sustained expression of beta-globin. We initially constructed a beta-globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer chicken beta-actin promoter (CAGGS) and full-length beta-globin cDNA, as well as truncated forms lacking either the 3' or 3' and 5' untranslated regions (UTRs), to optimize expression of beta-globin. Beta-globin with its 5' UTR was efficiently expressed from its cDNA in K-562 cells induced with hemin. However, expression was constitutive and not erythroid-specific. We then constructed cis SB-Tn-beta-globin plasmids using a minimal beta-globin gene driven by hybrid promoter IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, ankyrin-1 promoter), IHbetap (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, beta-globin promoter), or HS3betap (HS3 core element from human betaLCR, beta-globin promoter) to establish erythroid-specific expression of beta-globin. Stable genomic insertion of the minimal gene and expression of the beta-globin transgene for >5 months at a level comparable to that of the endogenous gamma-globin gene were achieved using a SB-Tn beta-globin cis construct. Interestingly, erythroid-specific expression of beta-globin driven by IHK was regulated primarily at the translational level, in contrast to post-transcriptional regulation in non-erythroid cells. The SB-Tn system is a promising nonviral vector for efficient genomic insertion conferring stable, persistent erythroid-specific expression of beta-globin.