Sickle cell anaemia (SCA) is the most common blood related disorder in India with a high prevalence among ethnic groups that have a socio-economic disadvantage, such as tribal populations. Every year approximately 5,00,000 children are born with SCA worldwide with India accounting for nearly 50% of the cases. SCA is a genetic disease caused by a point mutation in the sixth codon of the β-globin expressing gene resulting in the replacement of glutamic acid by valine, which under deoxygenation state oligomerizes with α-globin and gives rise to a type of haemoglobin named as Haemoglobin sickle (HbS). The mutated valine favours the hydrophobic interactions between the β subunits of the haemoglobin tetramers leading to the HbS polymerization and formation of long haemoglobin fibers. These fibers deform the disc shaped RBCs to sickle shaped cells. The sickle shaped cells lose flexibility with reduced oxygen carrying capacity and induce dehydration in the cells. Due to irregular shape of these cells, they are prone to physical stress leading to hemolysis and capillary occlusion. Individuals suffering from sickle cell disease show symptoms such as body pain, clotting, dyspnea, anaemia, jaundice, pneumonia, repeated infection etc. Their lifespan is usually reduced to 42-48 years with 50% of children with SCA dying before the age of 5. A major cause of premature mortality in sickle disease patients is infection. The clinical course is also known to be variable and several factors including genetic constitution of an individual is said to be important. For example, although SCA does not confer resistance to infection, it protects from severe malaria but mortality in such patients is higher once malaria develops. The disease management is usually symptomatic and hydroxyurea is the major therapeutic agent that is available for its treatment. Hence early and affordable detection, treatment as well as preventive measures are important in managing this disease. With this consideration we propose a project with holistic approach for management of SCA which will include (1) population screening, genetic testing and counselling for SCA in selected districts of Chhattisgarh having high prevalence of this disease or trait for management of genetic burden of SCA as well as understanding the genetic basis of clinical course/differential response to hydroxyurea (HU) therapy. (2) Discovery and development of new lead molecules for management of SCA to improve the quality of life with better life expectancy. This will involve screening pure natural/ synthetic/ semisynthetic compounds and botanicals against various validated sickle cell anaemia targets such as sickle shape modulation, induction of fetal γ-globin expression, Nitric Oxide inducers, inhibitors of Histone methyl/acetyl transferases, Phosphodiesterases, Cell-adhesion, HbS polymerization, NLRP3 inflammasome, inflammatory cytokines and pain management. This project will also facilitate the process of getting the DCGI approval for hydroxyurea therapy for SCA in India and conduct phase I/II clinical trials of a herbal formulation to be developed based on the plants (or their substitute plants in India) used in the preparation of Niprisan. It is also proposed to explore the co-crystallization of drugs currently being prescribed for the management of SCA. (3) Genome editing and stem cell research approach for the treatment of SCA. This approach will involve dedifferentiating the peripheral blood mononuclear cells of SCA patients to human induced pluripotent stem cells (hiPSCs), which will further undergo gene editing by using CRISPR-Cas9 to correct the SCA mutation. The corrected hiPSCs will be reprogrammed to the blood lineage and can be an unlimited source for cell replacement therapy and it is proposed to validate functional efficacy of gene-corrected HSCs using ex vivo erythroid differentiation and engraftment into NSG mice. (4) Development and on-ground implementation of an affordable, accurate and accelerated diagnostic kit which will provide simultaneous quantitative estimation of: (a) sickle/aggregated vs. normal Hb, (b) white blood count, and (c) plasma gelsolin. Patent search at all stages of project will be an integral part of the project. Finally, it is planned to establish competent International collaborations for successfully achieving the objectives of the project and to develop competence and human resources in the area of SCA research.
India is a vast, ethnically diverse country and the people inhabiting it are as diverse as the land itself. As many as over 4635 different ethnic groups form the panoramic cultural mosaic of the country. Though the exact reason is not known, the sickle cell anaemia (SCA) is mainly concentrated in the scheduled tribal, scheduled caste and other backward caste populations of Madhya Pradesh, Orissa, Chhattisgarh, Jharkhand, Gujarat, Andhra Pradesh, where carrier frequencies range between 5-40% or more. Consanguineous marriages and resistance to malarial infection, especially falciparum malaria are considered important factors that have contributed to maintenance of gene frequency of sickle cell anaemia. The disease follows an autosomal recessive pattern and hence parents of an affected child are usually obligate carriers which aids in offering genetic testing, prenatal diagnosis and genetic counselling. Hydroxyurea, the only US FDA approved drug for SCA has several side effects including increased risk for developing infections and tumor formation. Further this drug does not show any efficacy in 1/3rdof the patients. Therefore, new therapies need to be developed for effective management of SCA with fewer side effects. In this direction, we plan to discover new chemical compounds which can modulate the known or validated drug targets related to the management of the SCA. Niprisan is an approved herbal drug formulation used for the treatment of sickle cell anemia in Nigeria. It is an extract mixture of four plants, Piper guineenses, Pterocarpus osun, Eugenia caryophyllum, and Sorghum bicolor.
SCA is caused by a mutation in the Haemoglobin β (HBB) gene found on Chromosome 11. The Haemoglobin protein is necessary for maintaining proper oxygen transport to tissues and deformities in the structure of the protein impairs this process. Red blood cells (RBCs) carrying HB-A (the normal variant of Haemoglobin) have a smooth texture and round morphology allowing them to glide through blood vessels. When the gene is mutated, such as in SCA patients, abnormal Haemoglobin molecules (HB-S) stick to each other and become long, rod-like structures that cause RBCs to become stiff and develop a sickle shape. Due to these gross morphological changes in RBCs, they pile up and their oxygen carrying capacity is severely compromised. Hemolysis and adherence of leucocytes with the endothelium leads to release of free haemoglobin and heme. Much of free haemoglobin and heme are scavenged by plasma proteins hemopexin (Hx) and haptoglobin (Hp), however, due to excessive hemolysis, SCA patients have large amount of free haemoglobin and heme in their blood. Extracellular heme is a potent inflammatory agonist and oxidant, and a classic damage associated molecular pattern (DAMP). Heme can induce the canonical pathway leading to NF-κB activation through TLR4 in SCA, followed by release of proinflammatory cytokines and chemokines. Additionally, heme has been reported to be an activator of NLRP3 inflammasome, therefore, can lead to subsequent release of inflammatory cytokine IL-1β. Heme in the circulation induces another pathological effect by binding and depleting the NO levels in the endothelial cells resulting in vasoconstriction, pulmonary hypertension, leg ulcers, priapism, and cerebrovascular disease. NO is known to regulate the expression levels of cell adhesive molecules and hence prevent the platelet aggregation. α4β1 integrin expressed on the erythrocytes surface is known to interact with the endothelium through fibronectin, vascular cell adhesion protein 1 (VCAM-1), intracellular adhesion molecule 1 (ICAM-1), and E-selectin. NO also regulates the activity of guanylyl cyclase (sGC) which further stimulates the expression of fetal haemoglobin (HbF) in erythroleukemic cells and primary erythroblasts. Proinflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-8 contribute to chronic inflammation and vaso-occlusive crises in SCA. The occlusion of blood capillaries due to increased interaction of sickle erythrocytes with other blood cells and endothelium is the main reason for the morbidity. The pathological progression of SCA is very complex in nature; however, it provides scope for pharmacological intervention at several steps, this proposal explores the possibility of using drug intervention for the treatment/management of SCA. We also propose to provide a proof of concept for utilizing the gene editing tools to correct the genetic mutation from the SCA patient cells. Finally, we propose to develop an accurate, accelerated and affordable kit for the screening of SCA patients. On account of being a genetic disease, SCA has no effective cure available. The sustained efforts including genetic counseling, medical intervention for symptom management and bone marrow transplant therapies have significantly decreased the morbidity and improved the quality of life and life expectancy of SCA patients.
The average life expectancy for individuals with SCA is estimated to be between 42 and 48 years of age 13, among which ~85% survives for at least 20 years of age. The clinical outcome of sickle cell disease (SCD) varies widely from mild to severe with acute to chronic clinical manifestations, including vaso-occlusive episodes and multi-organ damage, painful crisis, etc14, 15. Previous studies have demonstrated that the SCD course is milder in Indians because of the Arab-Indian haplotype16-18. Clinical variability of SCD in India is not uniform in tribal and non-tribal populations despite high fetal haemoglobin levels19. Region–specific genetic heterogeneity of HBB haplotypes suggests that the mutant beta globin gene arose separately in these locations20. However, there is no systematic study that has investigated potential modifiers of the SCD severity in Indians. In addition, the clinical presentation and onset of various complications ranges from mild to severe in different patients. Furthermore, response to hydroxyurea therapy, the only FDA approved treatment for SCD is highly variable and depends on many factors including fetal haemoglobin levels in the patient. Hence, we will compare the genomic profile of patients with different clinical course and response to hydroxyurea therapy and attempt to identify and validate genetic markers that can be used for deciding management strategies in the SCA patients. The genetic data might also help us in identifying novel targets that can be investigated further for any role as possible drug target for haemoglobinopathies. Overall these strategies will help in reduce the sickle cell anaemia cases and will help in identifying the genetic markers which will help the patients.
Hydroxyurea treatment in itself has several side effects including increased risk for developing infections and tumour formation. Further this drug does not show any efficacy in 1/3rd of the patients. Therefore, new therapies need to be developed for effective management of SCA with fewer side effects. In this direction we plan to discover new chemical compounds which can modulate the known or validated drug targets related to the management of the SCA. Some of these targets include sickled shape modulation, induction of fetal γ-globin and nitric oxide (NO), inhibition of histone methyl/acetyl transferases, phosphodiesterases (PDEs), cell-adhesion, HbS polymerization, NLRP3 inflammasome, inflammatory cytokines and pain management. Currently the only cure for the SCA is the stem cell transplant. This approach, however, is not fully successful due to non availability of allogenic donors and high rejection rates. Thus, new genome editing technologies like CRISPR-Cas9 complex may be used to make precise changes in the genome of SCA patients and generate a corrected version of the gene. It is also proposed to generate and characterize expansion of gene-corrected autologous CD34+ hematopoietic stem / progenitor cells derived from a cohort of Indian SCA patients and validate the functional efficacy of gene-corrected human stem cells (HSCs) in NOD scid gamma (NSG) mice.