An estimated 10,000 human single-gene disorders impose a significant burden on human health worldwide. The 5-year goals of this Nanomedicine Development Center are to develop a clinically applicable gene correction technology for single-gene disorders and to demonstrate its efficacy for treating sickle cell disease (SCD) in a mouse model. SCD is caused by a single (A to T) mutation in the beta-globin gene. It is painful and life shortening, and there is no general cure. In the US, SCD afflicts primarily persons of African origin and thus contributes to minority health disparities.

To develop the gene correction approach for treating SCD, Center investigators will engineer and optimize zinc finger nuclease (ZFN) proteins that cleave specifically within the beta-globin gene. They will deliver these engineered proteins, together with wild-type donor (correction) DNA templates, into the nuclei of the patient’s own hematopoietic stem cells (HSCs). The ZFNs will induce a DNA double strand break (DSB) or a nick at a preselected site near the SCD mutation (see figure), thus activating the gene for correction. The broken DNA ends will enter the homologous recombination repair pathway, which will use the genetic information provided by the donor template to correct the SCD mutation. The novelty of the approach lies in three factors: the use of modified ZFNs to provide precise temporal control, manipulation of the DNA damage response to shepherd the broken ends into the HR pathway, and the development of efficient delivery systems to introduce ZFN proteins and donor template directly to the HSC nuclei. The Center’s proposed approach represents a significant paradigm shift in current gene correction/gene therapy technology in that no virus or foreign DNA is used.

The Center’s five-year goal is to demonstrate the efficacy of their technology in a mouse model of sickle cell disease. Center investigators will isolate HSCs from mice, perform gene correction, and re-engraft gene-corrected HSCs in new SCD mice to achieve enduring replacement of sickle cells with healthy red blood cells. HSCs 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 even a single HSC is sufficient to rebuild the entire blood system of an organism.

The Center has chosen to work with patient-derived HSCs, rather than other types of stem cells, because this approach is more clinically practical. Human HSC transplantation has been available for more than 50 years and is used to treat more than 50,000 patients each year. Approaches based on induced pluripotent stem cells, or on embryonic stem cells, still face substantial barriers to translation. Notably, it remains difficult to induce these other types of stem cells to form precursors of red blood cells.

The Center’s proposed approach rests on established scientific principles and there are no conceptual barriers to its implementation. There are, however many practical and technological challenges in translating the proposed gene correction approach to clinical practice. These include shifting repair pathway choice from non-homologous end joining) toward HR, increasing the spontaneous rate of gene correction by many orders of magnitude, achieving spatial and temporal control of ZFN activity, avoiding or rejecting unwanted mutations and gene rearrangements, and establishing a high-throughput delivery capability. The Center proposes to overcome these challenges by developing laser light-activated ZFNs and microneedle arrays, optimizing overall design of the gene correction device, and applying novel imaging probes and methods developed observe a each step in the gene correction process.