Disorders affecting the blood, including Sickle Cell Disease (SCD), are the most common genetic disorders in the world. SCD causes significant suffering and early death, despite major improvements in medical management and advances in understanding the complex disease-related biology. A bone marrow transplant (BMT) can greatly benefit patients with SCD, by providing a life-long source of normal red blood cells. However, BMT is limited by the availability of suitable donors and immune complications, especially for the more than 80% of patients who lack a matched sibling donor. An alternative treatment approach for SCD is to isolate some of the patient’s own bone marrow and then use gene therapy methods to correct the sickle gene defect in the blood stem cells before transplanting them back into the patient. The gene-corrected stem cells could make normal blood cells for the life of the patient, essentially eliminating the SCD. Such an approach would avoid the complications typically associated with transplants from non-matched donors. We will define the optimal techniques to correct the sickle gene mutation in the bone marrow stem cells to develop as a therapy for patients with SCD.
Development of methods for regenerative medicine using stem cells will have widespread applications to improve the health and to provide novel, effective therapies for millions of Californians and tens of millions of people worldwide. Many severe medical conditions can be cured or improved by transplantation of blood-forming hematopoietic stem cells (HSC), including genetic diseases of blood cells, such as sickle cell disease and inborn errors of metabolism, cancer and leukemia, and HIV/AIDS. Precise genetic engineering of stem cells to repair inherited mutation may be the best way to correct genetic defects affecting the mature cells they produce. This project will advance methods to precisely repair the genetic defect that underlies sickle cell disease in hematopoietic stem cells, which can then be transplanted to ameliorate the disease. These advances will have direct and immediate applications to enhance current medical therapies of sickle cell disease and will more broadly help to advance the capacities for regenerative medicine. All scientific findings and biomedical materials produced from our studies will be publicly available to non-profit and academic organizations in California, and any intellectual property developed by this Project will be developed under the guidelines of CIRM to benefit the people of the State of California.
Sickle-cell disease (SCD) is characterized by a single point mutation in the seventh codon of the beta-globin gene. Site-specific correction of the sickle mutation in adult bone marrow hematopoietic stem cells (HSCs) would allow for permanent production of normal red blood cells. Site-specific correction can be achieved using proteins called zinc-finger nucleases (ZFNs) which recognize and bind the region of the genome surrounding the sickle mutation. The ZFNs are able to create a break in the DNA which the cells repair using existing repair machinery. If, at the time of repair, a homologous donor template containing the corrective base is present, the cells' repair machinery can use this template and the resulting cell genome will contain the wild-type base instead of the sickle mutation. By doing this in hematopoietic setm cells, the cell is permanently corrected and each red blood cell (RBC) derived from this corrected stem cell will produce normal, non-sickle RBCs. In this report, we show efficient targeted cleavage by the ZFNs at the beta-globin locus with minimal off-target modification. In addition, we compare two different homologous donor templates (an integrase-defective lentiviral vector [IDLV] and a single-stranded DNA oligonucleotide [oligo]) to determine the optimal donor template. In both wild-type as well as sickle cell disease patient CD34+ HSCs, we are able to deliver the ZFN and donor templates and specifically correct the genome at rates of up to 30%. When these cells are differentiated into RBCs in vitro, we demonstrate that they are not altered in their differentiation capacity and are able to produce wild-type hemoglobin at high levels (35% of all hemoglobins) by HPLC. These results provide a strong basis for moving forward with this work as we begin our efforts to increase the number of treated cells to achieve clinical levels of corrected cells as well as characterize the ability of these cells to engraft a murine model in vivo. The progress made in this year is an exciting step towards a clinical therapy and potential treatment for sickle cell disease.