There are an estimated 10,000 human single-gene disorders, which impose a significant burden on human health worldwide. The 5-year goals of this NDC are to develop a clinically applicable gene correction technology to treat single-gene disorders, and to demonstrate the efficacy of this approach in treating sickle cell disease (SCD) using a mouse model. SCD is caused by a single (A-T) mutation in the beta-globin gene; it is a painful and life shortening disease and afflicts primarily persons of African origin.
To develop the gene correction approach for treating SCD, we will engineer and optimize nucleases including zinc finger nuclease (ZFN) and TAL effector nuclease (TALEN) proteins that bind specifically to the beta-globin gene, deliver them as well as wild-type donor templates into the nuclei of hematopoietic stem and progenitor cells (HSPCs) to induce a DNA double strand break (DSB) or a nick at a preselected site near the beta-globin locus, shepherding the broken DNA ends into the homologous recombination (HR) pathway for gene correction. The autologous gene-corrected HSPCs will be re-engrafted in a mouse model of SCD to produce healthy red blood cells and replace sickle cells. HSPCs are the normal precursors of all blood cells, including the oxygen-carrying erythrocytes rendered dysfunctional in sickle cell patients. These cells are relatively rare in the body, but possess potent regenerative potential in that transplantation of a small amount of HSPCs is sufficient to rebuild the entire blood system of an organism. Thus, by isolating HSPCs that carry the sickle mutation, correcting this mutation ex vivo, and then transplanting the gene-corrected HSPCs back into affected recipients, we would be able to provide enduring replacement of the blood-producing cells of SCD patients with unaffected precursors, thereby supplying healthy red blood cells and effectively curing the disease.
There are many practical and technological challenges in achieving our goals, including increasing the spontaneous rate of gene correction by many orders of magnitude, achieving high specificity of nuclease activity, highly efficient delivery of nucleases and donor templates, and avoiding or reducing off-target effects and gene rearrangements. We propose to overcome these challenges using nanotechnology and nanomedicine approaches, which will allow us to observe, control, and systematically optimize each step in the gene correction process. The team will also explore scale-up and IND/IDE issues such as safety, high throughput delivery and quality control, with the goal of being ready to begin clinical trials at the end of the 5-year project period.