An ideal therapy for genetic blood diseases would correct defective genes in the patient's own hematopoietic stem cells (HSC). Homologous recombination (HR) can, in principle, achieve this;however, its practicality is restricted by the scarcity of HSC and the low frequency of HR. Nonetheless, we have previously demonstrated HR in myeloid colony-forming cells (CFCs). We now propose to attempt gene correction in multipotent hematopoietic progenitor cells (MPC), which can be taken from an affected individual, corrected in vitro by HR and then used for intermediate-term therapy. To explore this possibility, we will expand MPC from mice carrying a 2-globin gene mutation (C654T) causing thalassemia, and then use HR to correct the mutation in the expanded MPC (eMPC). We will use our recently developed procedure for isolating gene-corrected cells by fluorescence-activated cell sorter (FACS), which allows efficient isolation of correctly targeted cells, achieving >30% """"""""purity"""""""", >2000 fold enrichment, with recovery H20%. The """"""""corrected"""""""" MPC will then be tested for therapeutic potential by engraftment into thalassemic mice.
Specific aim (i) will test methods for optimizing gene correction in eMPC.
Specific Aim (ii) will test whether FACS can be used to purify eMPC in which the C654T mutation has been corrected by HR. If the frequency of targeting proves to be too low, we will collaborate with Dr. Mathew Porteus to determine if zinc-finger nucleases (ZFNs) specific for the human 2-globin gene can increase the frequency of HR in eMPC.
Specific Aim (iii) will test whether gene-corrected C654T eMPC can provide short term, and possibly long term, improvement in the anemia of C654T thalassemic mice. Our goal is to isolate multipotent progenitors having a faulty gene corrected by HR and establish their usefulness for therapy. If successful, we will have made a significant step toward using autologous gene-corrected cells for at least short-term therapy.
Our aim is to correct an abnormal human gene that causes an inherited form of anemia. We will isolate bone marrow cells from mice with the same abnormal human gene, and will try correcting the gene in the isolated cells. We will then transplant the corrected cells back to the anemic mice to determine whether we have helped their anemia.
| Hatada, Seigo; Walton, William; Hatada, Tomoko et al. (2011) Therapeutic benefits in thalassemic mice transplanted with long-term-cultured bone marrow cells. Exp Hematol 39:375-83, 383.e1-4 |
