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Nanomedicine Center for Nucleoprotein Machines

2011 Progress Report – Executive Summary

There are over 6,000 human single-gene disorders, among them is sickle cell disease (SCD). SCD, or sickle-cell anaemia is caused by a single A-T point mutation in the beta-globin chain of haemoglobin. The association of two wild-type alpha-globin subunits with two mutant beta-globin subunits forms haemoglobin S (HbS), which leads to the non-covalent polymerisation (aggregation) of haemoglobin under low oxygen conditions, distorting red blood cells into a sickle shape and decreasing their elasticity. The sickle cells are unable to deform as they pass through narrow capillaries, leading to vessel occlusion and ischaemia, as well as destruction of the sickle red blood cells. SCD is a painful and life shortening disease, reasonably common, and afflicts primarily persons of African origin. Bone marrow transplantation, the only available curative therapy, is practical in only a small subset of patients (~15%). 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 using a knockout-transgenic sickle cell mouse model. The long­term goal is to conduct clinical trials to determine the efficacy of the gene correction approach for treating SCD patients.

To develop the gene correction approach for treating SCD, we have been engineering and optimizing 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 cells (HSCs) 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 HSCs will be re-engrafted in a mouse model of SCD to produce healthy red blood cells and replace sickle 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. Thus, by isolating HSCs that carry the sickle mutation, correcting this mutation ex vivo, and then transplanting the gene-corrected HSCs back into affected recipients, we should be able to provide enduring replacement of the blood-producing cells of SCD patients with unaffected precursors, thereby supplying healthy red blood cells (RBCs) and effectively curing the disease. Research in our NDC represents a significant paradigm shift in current therapeutic strategies in treating SCD.

There are many practical and technological challenges in translating the gene correction approach to clinical practice. These include shifting repair pathway choice from non-homologous end joining (NHEJ) toward HR, increasing the spontaneous rate of gene correction by many orders of magnitude, achieving spatio- te m po ra l control of ZFN/TALEN activity, reducing or avoiding off-target effects (including unwanted mutations and gene rearrangements), and establishing a high-throughput delivery capability. We have been working to overcome these challenges by developing photoswitchable ZFNs and photoactivatable donors, a microneedle array based high throughput delivery device, optimizing ZFN/TALEN and donor template designs, as well as applying novel imaging probes and methods developed in our NDC, which will allow us to observe and systematically optimize each step in the gene correction process. We will explore scale-up and IND/IDE issues such as safety and quality control, with the goal of being ready to begin clinical trials at the end of the 5-year project period.

Current and future work of the NDC has been carried out with the following three specific aims:

Aim 1. Quantify and control DSB repair pathway choice in cells undergoing gene correction
We have been develop ing methods to shift DSB repair pathway choice toward HR, the pathway that leads to productive gene correction, by suppressing competing pathways of NHEJ and alternative NHEJ. To guide and direct these studies, we aim to identify robust early markers to distinguish HR, NHEJ, and alt-NHEJ, and we will use these to perform temporal studies of repair complex assembly in cells undergoing gene correction. We will determine whether interventions to shift pathway choice result in improved outcome, defined as more efficient gene correction with a reduction in nonspecific mutations and gene rearrangements.

Aim 2. Design and optimize zinc finger nucleases and donor templates for controllable gene correction
We have been refining and optimizing ZFN design and production, developing photoactivatable ZFNs for better temporal control of ZFN activity. We will investigate the fate and dynamics of ZFNs and donor template in living cells, and investigate the incidence and biological effects of undesired ZFN-induced mutations and gene rearrangements. We will also design and optimize TALENs for gene correction in HSCs.

Aim 3. Demonstrate efficient gene correction of the HbS allele in preclinical mouse models of SCD
We will optimize methods for delivery of ZFNs/TALENs and donor template in HSC-containing cell populations, perform ex vivo gene correction in these cells with extensive controls for stem cell function, demonstrate that gene-corrected cells can reconstitute the mouse hematopoietic system, and measure reversal of the SCD phenotype.

During the last 12 months, we have made significant progress towards achieving our translational goals. The major advances include: (1) demonstrated the feasibility of achieving gene correction by direct delivery of an engineered ZFN nuclease, rather than by delivery of a plasmid or viral vector to the target cell; (2) established protocols for optimizing ZFNs and designing TALENs for better DNA cutting efficiency and reduced off-target effects; (3) developed methods to predict and measure, in a genome-wide fashion, off target effects induced by introduction of ZFNs into living cells; (4 ) developed molecular imaging and cell biology approaches to understand pathway choice by examining the cell cycle dependence of pathway choice (NHEJ vs HR) and observing the assembly/disassembly of the DSB repair machinery at a single or two DSB reporter locus in living cells; (5) developed endocytosis-based ZFN delivery for gene correction. These and other advances have demonstrated the feasibility of the gene correction approach and provided a basis for further development of the gene correction technology for treating SCD.

Modern medicine – allopathic medicine – focuses on treating symptoms, commonly through small-molecule enzyme inhibitors and receptor agonists/antagonists. It does not address the underlying genetic causes of disease. Consistent with the vision of the Nanomedicine Initiative, the Center investigators envision that the allopathic model will someday be replaced by therapies that directly modify information contained in a patient’s DNA.

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