Towards RNA Repair of Diamond-Blackfan Anemia Hematopoietic Stem Cells.

Diamond Blackfan anemia (DBA) is a well known inherited bone marrow failure syndrome mostly caused by mutations in ribosomal protein (RP) genes but also rarely in the hematopoietic transcription factor gene, <i>GATA1</i>, or <i>TSR2</i>, a ribosomal protein (Rps26) chaperone gene. About 25% of patients have heterozygous mutations in the <i>RPS19</i> gene, which leads to haploinsufficiency of Rps19 protein in most cases. However, some <i>RPS19</i> missense mutations appear to act in a dominant negative fashion. DBA typically leads to a hypoplastic anemia that becomes apparent during the first year of life, and standard treatment includes steroids or red blood cell transfusions, each modality having attendant side effects. The only curative therapy is allogeneic stem cell transplantation, but this option is limited to patients with a histocompatible donor. DBA-mutant embryonic, induced pluripotent, and hematopoietic stem cells exhibit growth abnormalities that can be corrected by DNA gene transfer, suggesting the possibility of ex vivo autologous gene therapy. We have been interested in the application of Spliceosome Mediated mRNA trans-splicing (SMaRT) technology to RNA repair of DBA stem cells. Compared with gene replacement or other RNA re-programming approaches, the RNA trans-splicing technology offers several advantages: 1) it obviates the need to deliver the entire cDNA of a normal gene, which significantly reduces the transgene size for packaging and delivery; 2) expression of the corrected gene is completely dependent on expression of the endogenous mutant gene, and genes with large and complex transcriptional elements can be repaired in situ while keeping gene expression under the control of endogenous regulatory elements; 3) RNA trans-splicing utilizes the endogenous spliceosome that exists and functions in all types of cells; 4) RNA trans-splicing converts mutant transcripts into therapeutically useful mRNA, and thus, may be capable of treating disorders caused by dominant negative mutations such as in DBA.