Gene Therapy for Increasing Fetal Hemoglobin Production in Sickle Cell Disease

Robin Meadows

BIOL 770

Dr. Bert Ely

November 6, 2009

Sickle cell disease is an inherited autosomal recessive disorder in which a valine molecule is substituted for glutamic acid in the sixth position of the beta-globin (β-globin) chain of hemoglobin creating hemoglobin S (HgbS). This abnormal hemoglobin becomes polymerized into rigid rod-like polymers when deoxygenated, resulting in deformation and damage to the red blood cell (Pestina et al., 2009). These abnormally shaped sickle cells lead to vaso-occlusion by becoming lodged in small blood vessels which causes repeated painful vaso-occlusive crises, chronic hemolysis with resulting anemia, chronic inflammation, cumulative organ damage (Perumbeti et al., 2009), and increased risk of infection, especially by encapsulated organisms due to autoinfarction of the spleen. National statistics from 2007 reveal about 72,000 people in the United States have sickle cell disease (Lopez-De Fede et al., 2008) resulting in an estimated annual expenditure of $475 million dollars (Perumbeti et al., 2009).

Sickle cell patients who have a persistent expression of fetal hemoglobin (α2γ2)have a less severe disease expression than those who do not express increased levels of fetal hemoglobin. Since fetal hemoglobin does not contain β-globin chains, it inhibits sickling by interfering with the polymerization of the abnormal HgbS (Charache et al., 1995). Hydroxyurea, a ribonucleotide reductase inhibitor (Ma et al., 2007), was found to increase fetal hemoglobin in anemic monkeys. Platt et al. (1984) treated two sickle cell patients with five-day bursts of the drug,and found that the hydroxyurea dramatically increased the percentage of red blood cells that contained fetal hemoglobin. They also discovered that the drug reduced the methylation of M2 and/or M4 Hpa II sites 5’to the gamma globin genes, which is critical for gamma globin expression. Although hydroxyurea is a cytotoxic drug, it did not result in clinically significant bone marrow suppression (Platt et al., 1984).

Due to the results of the above study and others demonstrating the efficacy of hydroxyurea, Charache et al. (1995) conducted a double-blind clinical Multicenter Study of Hydroxyurea (MSH)use in sickle cell patients to reduce painful vaso-occlusive crises. They enrolled 299 patients from 21 centers around the United States and planned to follow them for 24 months. The study showed an increase in fetal hemoglobin in those patients receiving hydroxyurea as well as a decrease in the number and frequency of pain crises in these patients. The study ended early due to the consistent and remarkable results seen (Charache et al., 1995).

Although the above studies demonstrate the usefulness of hydroxyurea in treating sickle cell, they do not address the variability in patient response to the drug. This led to a study by Ma et al. (2007)which examined 320 single nucleotide polymorphisms (SNPs) in 137 patients from the MSH study discussed above. They found that seventeen SNPs were significantly associated with an increase in fetal hemoglobin. Two SNPs were particularly associated with an increase in fetal hemoglobin. The first, was SNP rs2182008 in the FLT1 (Fms-related tyrosine kinase 1) region of chromosome 13. It codes for a vascular endothelial growth factor which is involved in cell proliferation and differentiation. The presence of SNP rs2182008 in FLT1 was associated with a strong response to hydroxyurea, and patients with the A allele of this SNP showed a marked increase in fetal hemoglobin over those patients with the GG genotype. The second SNP to be associated with an increase in fetal hemoglobin was SNP rs10483801in ARG2 (arginase type II) in chromosome 14. This coding region is important in hydroxyurea metabolism. It codes for proteins that lead to an increase in nitric oxide, which in turn increases cGMP, causing an increase in the expression of the γ-globin genes by binding to transcription factors (Ma et al., 2007).

Hydroxyurea has improved the lives of some sickle cell patients, but it requires a lifelong commitment to daily drug use and frequent monitoring of blood counts; therefore, medical noncompliance is an issue. This limitation has led other researchers to investigate gene therapy to provide a cure for this chronic and disabling disease. Perumbeti et al. (2009) investigated the use of a lentiviral vector to insert a γ-globin gene into hematopoietic stem cells (HSCs) of Berkeley “humanized” sickle mice. These mice exclusively express α and sickle β-globin chains of hemoglobin just as patients with sickle cell disease, and the mice also exhibit the same signs and symptoms of the disease as humans. A β-globin regulatory gene was attached to the γ-globin gene to ensure its expression in the mice. The mice received a myeloablative dose of radiation to destroy any endogenous erythroid bone marrow cells before transplantation with the vector treated HSCs. The investigators found an amelioration of the disease after transplantation when there was an increase of greater than 10% fetal hemoglobin of the total hemoglobin with at least two-thirds of the circulating red blood cells containing fetal hemoglobin, and 20% of the HSCs in the bone marrow contained the vector copy. Not only did the increase in fetal hemoglobin result in preventing the complications of sickle cell disease, but it also reversed organ damage that had already occurred (Perumbeti et al., 2009).

Researchers at St. Jude’s Children’s ResearchHospital, Pestina et al. (2009), also conducted a study of lentiviral vector treated HSCs of Berkeley “humanized” mice. They studied two different γ-globin vectors. One vector contained the γ-globin gene as well as β-globin regulatory sequences. The other vector was the same, except that the γ-globin 3’-untranslated region (3’-UTR) was replaced with the β-globin 3’-UTR. HSCs were treated with these vectors and transplanted into Berkeley mice that had received myeloablative radiation therapy as in the previous study. These investigators also found an increase in fetal hemoglobin after transplantation with a total hemoglobin level resulting from HSCs treated with the first vector similar to those found by Perumbeti et al. (2009). Pestina et al. (2009) found that the mice transplanted with HSCs treated with the vector containing the β-globin 3’-UTR resulted in higher total hemoglobin levels and a slightly higher percentage of fetal hemoglobin, although statistically not significant, and fewer sickle cells seen on blood smear compared to the mice transplanted with the HSCs treated with the vector containing the γ-globin 3’-UTR. They theorize the β-globin 3’-UTR confers stability to the γ-globin mRNA due to the increase in this mRNA seen in the cells treated with the β-globin 3’-UTR vector. They also suggest that a 30% level of vector-transduced HSCs has a major effect on sickle cell disease (Pestina et al., 2009).

Sickle cell disease is a common genetic disorder that can produce devastating consequences of repeated painful vaso-occlusive crises, a shortened lifespan and results in a large impact on health care because of the chronic nature of the disease. Therapy for these patients has improved, but no treatment for a cure has been found. The use of gene therapy to alter the expression of fetal hemoglobin is a promising development. Research in humans may be conducted in the near future, and a cure for this devastating disease may be found. I look forward to the day when I no longer see a child suffer from the consequences of this genetic disorder.

References

Charache, S., Terrin, M., Moore, R., Dover, G., Barton, F., Eckert, S., McMahon, R., Bonds, D., & the Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia (1995). Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia [Electronic version]. The New England Journal of Medicine, 332(20), 1317-1322.

Lopez-De Fede, A., Mayfield-Smith, K., Payne, T., Stewart, J., Suddeth, D., et al. Sickle Cell and SC Medicaid Recipients: SFY 2007 Factsheet. (2008). Columbia, SC: Institute for Families in Society, University of South Carolina.

Ma, Q., Wyszynski,D., Farrell, J., Kutlar, A., Farrer, L., Baldwin, C., & Steinberg, M. (2007). Fetal hemoglobin in sickle cell anemia: genetic determinants of response to hydroxyurea [Electronic version]. The Pharmacogenomics Journal,7, 386-394.

Perumbeti, A., Higashimoto, T., Urbinate, F., Franco, R., Meiselman, H., Witte, D., & Malik,P. (2009). A novel human gamma-globin gene vector for genetic correction of sickle cell anemia in a humanized sickle mouse model: critical determinants for successful correction [Electronic version]. Blood, 114(6), 1174-1185.

Pestina, T., Hargrove, P., Jay, D., Gray, J., Boyd, K., & Persons, D. (2009). Correction of murine sickle cell disease using γ-globin lentiviral vectors to mediate high-level expression of fetal hemoglobin [Electronic version]. Molecular Therapy, 17(2), 245-252.

Platt, O., Orkin, S., Dover, G., Beardsley, G., Miller, B., & Nathan, D. (1984). Hydroxyurea enhances fetal hemoglobin production in sickle cell anemia [Electronic version]. Journal of Clinical Investigation, 74, 652-656.

’