The most common genetic diseases worldwide affect the production of normal hemoglobins, the red blood proteins that carry oxygen to our organs. These disorders result in the diseases known as sickle cell disease (SCD) and thalassemia, common in patients of African, Mediterranean, Middle Eastern, and Asian origins. In California, due to the large African American and Asian American populations these diseases have resulted health problems that have significant social and economic ramifications.
Most patients with SCD require lifelong care that includes treatment of painful crises due to blockage of blood vessels, strokes at an early age, frequent infections throughout life, and kidney damage. Treatment involves management of the crises and infections as well as transfusions for strokes and severe anemia. Hydroxyurea can alleviate some symptoms and anemia in selected patients; however, the long-term effectiveness of this treatment has yet to be determined.
Thalassemia comes in two major forms; alpha thalassemia, where the affected fetus usually dies in the third trimester of pregnancy or soon after birth, and beta thalassemia (beta-thal), where children are born healthy, but develop severe anemia within weeks or months, due to the lack of adult type of hemoglobin. Patients require regular monthly blood transfusions that carry with them large, toxic amounts of iron that will cause organ damage over time and therefore need to take medications throughout life to remove iron from their bodies.
Both diseases impose heavy burdens on affected families and on healthcare costs. At present, the only effective cure is bone marrow or cord blood transplantation where there is a compatible donor. However, affected families usually have few children, and compatible donors are rare.
This development proposal offers an alternative to conventional bone marrow transplantation. Its aims are to institute a treatment approach that converts a patients cells into induced pluripotent (iPS) cells, corrects the disease causing beta-globin mutation, differentiates the corrected iPS cells into cells that reconstitute the hematopoietic system, and returns these cells to the patient for a cure.
While carrier screening or prenatal diagnosis and subsequent selective abortion of the affected fetus can control these diseases, this proposal offers cure through transplantation as an alternative to abortion. We have successfully reprogrammed cells from chorionic villus sampling (CVS) used for prenatal diagnosis between weeks 9-12 of pregnancy, into iPS cells. If SCD or beta-thal is detected by CVS or amniocentesis, cells cultured from these procedures will be converted into iPS cells, the mutation will be corrected, corrected cells will be differentiated into hematopoietic stem cells, and then transfused into the baby after birth. Since prenatal diagnosis is early in pregnancy, adequate time is available for corrective measures to be instituted.
Statement of Benefit to California:
Sickle cell disease (SCD) and thalassemia (thal) are among the most common genetic diseases in the world. Of the thalassemias, patients with alpha-thal generally die in the 3rd trimester or shortly after birth, while beta-thal patients are symptomless at birth, but develop severe anemia with weeks to months due to the lack of adult hemoglobin. In California, these diseases have become important and significant health problem because of the large African and Asian American populations. Of the approximately, 500,000 new births in California every year, the rate of SCD incidence is about 1 in 6,600 births every year, while beta-thal occurs less frequently at 1 in 55,000 births. Most patients with these diseases require lifelong care that involves expensive therapies to treat the many complications that result in organ damage and severe pain. At present, the only cure is by bone marrow or cord blood transplantation. However, these families usually have a small number of children and compatible donors are difficult to find. Aside from the human cost and suffering caused by SCD and beta-thal, the economic impact to California is substantial. Treatment of SCD is about between $12,000 - $45,000/yr/patient, while the cost of treatment for a beta-thal patient is about $100,000/yr/patient. Given that SCD patients have predicted life-span of 50 yr and beta-thal patients now live to 40 yr, the economic impact of treating this cohort of patients for their lifetime by conventional means would be significant. One approach to prevent the disease has been through early detection and prenatal diagnosis. At present, the usual outcome of prenatal diagnosis is selective abortion of the affected fetus. This development project offers an alternative possibility of curing both newborn and adult patients with their own cells that have been reprogrammed, corrected and converted to cells that will regenerate their hematopoietic system. The benefit to California would be to eliminate emotional and physical suffering in patients and mitigate the economic impact that the life-long treatment has on the medical system.
The project proposes to develop stem cell-based therapy for patients with sickle cell disease and beta-thalassemia. To this end, the applicant will reprogram somatic cells from each patient to induce pluripotent stem cells (iPSC) that will eventually be converted to hematopoietic stem cells and used to reconstitute the patient's hematopoietic system. Induced pluripotent stem cells will be derived using either non-integrating adenovirus vectors encoding key reprogramming genes or through the use of activating double-stranded RNA to transiently enhance expression of these genes. Once iPSCs have been created, the disease-causing mutations will be corrected by gene targeting, either through classical homologous recombination or oligonucleotide-based homologous replacement. Investigators will test conditions to direct the corrected cells along the hematopoietic lineage to generate multipotent hematopoietic stem cells (HSC). As proof of concept, these corrected, iPSC derived HSC will be tested for their capacity to both engraft and reconstitute the hematopoietic system in myeloablated, immuno-compromised mice.
Sickle cell anemia and beta thalassemia are the most commonly inherited genetic diseases worldwide. Current treatment options, including HSC transplantation from HLA identical siblings, unrelated donors, and cord blood transplants, have been curative. However, transplants are associated with morbidity and occasional mortality. Development of autologous therapies for these diseases would avoid the problem of rejection. The PI has a solid publication record in homologous gene transfer technology (SFHR), and has assembled a team that includes a pioneer in hemoglobinopathies as well as experts in homologous recombination and RNAa techniques. This application has achievable goals, but reservations regarding the experimental plan, in particular the animal model, tempered enthusiasm. The applicant proposed adenovirus as a non-integrating tool for the generation of iPSC; however, low rates of integration can occur with these vectors, necessitating extensive testing of the resultant lines. Mutant globin genes will be corrected in the patient cell lines. The difficulty of achieving homologous recombination in human cells was a key concern, and if successful, this process might alter iPSC genetic stability and phenotype. An alternative proposed gene correction strategy, SFHR, avoids this process, and submitted preliminary data has demonstrated SFHR successfully introduced mutations in the beta globin gene of human HSC. The final phase of the study, the conversion of corrected iPSC into reconstituting hematopoietic stem cells, remains a formidable hurdle. The applicant is aware that introducing HoxB4 cDNA, while effective in this conversion, has been complicated by the development of HoxB4-associated leukemia; therefore a transient, endogenous HoxB4 gene activation strategy was proposed. In order for transplants to be curative, long-term reconstitution must be achieved. Transplantation into myeloablated, immunodeficient mice is the gold standard test for reconstitution. Unfortunately, the short mouse lifetime limits the demand on transplanted HSC and therefore limits the ability to predict long-term results. Reviewers felt that a more relevant clinical model may be required for clinical translation.
In summary, this is a focused application addressing an unmet medical need with a good chance of success. The group has extensive expertise and they are using the current state-of-the-art techniques. However, this approach may not be adequate to overcome some substantial technical obstacles.