SPLICEOSOME MEDIATED RNA TRANS-SPLICING IN STEM CELLS

摘要

The present invention provides methods and compositions for generating novel nucleic acid molecules through targeted spliceosomal mediated trans-splicing in stem cells. The compositions of the invention include stem cells engineer ed to express pre-trans-splicing molecules (PTMs) designed to interact with a target precursor messenger RNA molecule (target pre-mRNA) and mediate a tran s~- splicing reaction resulting in the generation of novel chimeric RNA molecule s (chimeric RNA). In particular, the stem cells of the present invention are genetically engineered to express a PTM that will interact with a specific target pre-mRNA expressed within a stem cell as it differentiates so as to result in correction of a genetic defect responsible for a genetic disorder. The methods of the invention encompass transferring a nucleic acid molecule capable of encoding a PTM of interest into a stem cell followed by transplantation of the PTM modified stem cell into a host. As the stem cell differentiates the target pre-mRNA is expressed thereby providing the substrate for a trans-splicing reaction. The present invention is based on t he successful transfer and expression of a nucleic acid molecule encoding a PTM capable of interacting with a cystic fibrosis transmembrane conductance regulator (CFTR) pre-mRNAinto primary human surface airway progenitor cells. The methods and compositions of the present invention can be used to correct genetic defects associated with a variety of different disorders such as cystic fibrosis, hemophilia, sickle cell anemia, Tay-Sachs disease, thalassemias, polycystic kidney disease and muscular dystrophy, to name a fe w.