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2013 Progress Report- Executive Summary

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. We will carry out the proposed studies with the following aims:

Aim 1. Design, validate and optimize nucleases and donor templates for efficient gene correction
We will systematically optimize nucleases (ZFNs and TALENs) design and production, establish the efficacy of β-globin-directed nucleases for gene correction in living cells, and identify nuclease pairs that have the optimal performance. We will also optimize the design of donor templates and evaluate their functions in cells.

Aim 2. Control and alter DSB repair pathway choice
We will develop methods to quantify DSB repair pathway choice in living cells and identify approaches to shift pathway choice toward HR, the pathway that leads to productive gene correction, by suppressing competing pathways of NHEJ and alternative NHEJ. We will evaluate the potential effect of shifting pathway choice on the gene rearrangements.

Aim 3. Optimize nucleases and donor delivery
We will develop and optimize different methods for delivering nucleases and donor templates into HSPCs, including      those     based     on     receptor      mediated      endocytosis,      single     glass-needle      injection, microfluidic/microstamping,  and microneedle  arrays. The goal is to have a robust delivery system with high efficiency and throughput. We will also systematically evaluate the effect of deliver on the potency of HSPCs.

Aim 4. Quantify off-target effect and minimize genomic risk
The off-target cleavage of nucleases may be a major safety concern, since DNA breaks at off-target sites can lead to mutations, gene knock-outs or translocations. We will develop methods to identify the off-target sites of nucleases and investigate the incidence and biological effects of undesired nuclease-induced mutations and gene rearrangements.

Aim 5. Achieve high gene correction rate in HSPCs and optimize their engraftment
We will perform ex vivo gene correction in HSPCs, determine the gene correction efficiency in these cells and evaluate their function, demonstrate that gene-corrected HSPCs can reconstitute the mouse hematopoietic system, and measure reversal of the SCD phenotype in a mouse model of SCD.


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