Gene Therapy for Increasing Fetal Hemoglobin Production in Sickle Cell Disease
Gene Therapy for Increasing Fetal Hemoglobin Production in Sickle Cell Disease
by
Robin Meadows
BIOL 770
Dr. Bert Ely
November 6, 2009
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Gene Therapy for Increasing Fetal Hemoglobin Production in Sickle Cell Disease
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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 (βglobin) 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
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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 rs10483801 in ARG2
(arginase type II) in chromosome 14. This coding region is important in hydroxyurea
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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 Research Hospital, Pestina et al. (2009), also
conducted a study of lentiviral vector treated HSCs of Berkeley “humanized” mice. They
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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
Gene Therapy for Increasing Fetal Hemoglobin Production in Sickle Cell 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.
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Gene Therapy for Increasing Fetal Hemoglobin Production in Sickle Cell Disease
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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.
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