Sickle Cell Disease
Sickle cell anemia (SCA) is an inherited disorder of blood wherein a single mutation in HbB gene [encoding for the beta subunit of hemoglobin protein] results in a faulty protein that eventually gives the red blood cells a sickled shape. These cells tend to adhere to each other within the patient’s blood vessels, restricting blood flow to various organs, thereby leading to organ failure, severe pain, in some cases: vision loss, stroke, or even death. Adjoining symptoms such as anemia, breathlessness, body pain, jaundice, repeated infection result in a stark reduction in lifespan. Annually, 5 lakh children are born bearing SCA with only one nation- India- accounting for about 50 % of the cases.
Sickle Cell Disease burden in India has not been systematically recorded, but a high incidence of SCD patients and carriers exist in tribal regions of Kerala, Tamil Nadu, Madhya Pradesh, Maharashtra, Gujarat, Chhattisgarh and Odisha. This high prevalence of sickle cell disease in our country is estimated to make India the second most affected country in the world. Another factor that adds onto the gravity of this situation is the much higher prevalence of the disease in low socio-economic status communities.
A typical SCD care model includes screening, inpatient and outpatient standardized protocols being applied, translational research, genetic counselling, and monitoring and follow-up of patients. Genetic counseling and screening by various government* and non governmental organizations** have been recently initiated in Maharashtra, Gujarat, Odisha, and Chattisgarh. Sympyomatic disease management is usually followed up after screening as no permanent treatment exists. The current treatment approaches are restricted to use of hydroxyurea, blood transfusions and eventually stem cell transplant.
CRISPR-Cas based clinical trials have been initiated around the world to find a permanent cure and some of the initial results are promising. We at RNA Biology Lab are working on a CRSIPR-Cas based ex vivo therapeutics for SCD with the support of Ministry of Tribal Affairs and Department of Science and Technology.
A crucial way to tackle this growing number of patients is the identification of carriers and counseling of patients besides finding an effective, robust, and cheap gene editing method. With the support of the Ministry of Tribal Affairs (MoTA) and Department of Science & Technology (DST), CSIR-IGIB (Dr. Debojyoti Chakraborty, Dr. Souvik Maiti, and Dr. Sivaprakash Ramalingam) in collaboration with clinical (AIIMS New Delhi, SCIC Raipur) and non-clinical (CSIR-IICT, ICMR-NIIH Mumbai, NNF Bangalore, University of Washington Seattle) partners has undertaken the challenge of Sickle Cell Disease gene therapy using CRISPR-Cas.
Prevalence of SCD in India ranges from 1-40%. Madhya Pradesh is dominated by the tribal groups of Gonds and Bhils and bears the highest load with more than half of its districts under the SCD belt.
The prevalence in Maharashtra varies from 0-35%, with a majority in the eastern tribes of Pardhans, Otkars, Madhias, and Bhils. There isn’t much epidemiological data available for many Indian states particularly, Uttar Pradesh, Uttarakhand, Bihar and the complete north-eastern region.
Most Indian SCD screening programmes employ the Sickling or the Rapid Solubility Test that is followed by hemoglobin electrophoresis for confirmation. Developed in India itself, the dithionate qualitative solubility test is easy and cost-effective, works at room temperature, and offers very high sensitivity and specificity making it a credible first-line screen for SCD. The presence of turbidity due to a difference in the lysis reaction of a soluble HbA and an insoluble HbS aids in the point-of-care assessment.
More recently, the High-performance Liquid Chromatography (HPLC) is being widely used for analyzing samples for potential sickled RBCs and in identifying carriers of SCD. Even capillary electrophoresis is being done at some centers.
Hydroxyurea is a medication that is usually consumed at 10-15 mg/kg/day. It acts by increasing fetal hemoglobin levels. It can also reduce WBCs production that is considered therapeutic as it works against vaso-occlusion. It has proven to decrease the chances of vaso-occlusive crisis and need for blood transfusion.
Blood transfusion therapy is based on the principle that normal hemoglobin would compensate for sickled hemoglobin related acute complications. An acute transfusion can reverse severe anemia while a chronic transfusion therapy can prevent primary stroke or its recurrence and may reduce chances of an acute chest syndrome in cases where hydroxyurea proves ineffective.
Other preventive measures include:
Regular oral prophylactic penicillin from birth up to 5 years
Opioid therapies to manage acute pain
Non-opioid pain-killers for managing chronic pain
Patients older than 2 years are also prescribed pneumococcal disease vaccine and folic acid tablets.
An SCD associated 3 episodes of pain crisis and hospitalization or 3 administrations of blood transfusion within a year act as selective criteria for severe SCD patients.
There is no existing gene therapy in Indian clinical settings that can permanently cure SCD. There are a few symptom-management strategies that are proven to help many patients lead a healthier life.
CSIR has developed a Mission Mode Project on Sickle Cell Anemia, to understand and manage this disease with the following aims at its core:
Managing Genetic Burden of Sickle Cell Anemia and Understanding Genetic Basis of Differential Response to Hydroxyurea Therapy
Development and on-ground implementation of an affordable, accurate and accelerated diagnostic kit
Drug discovery and development for the management of SCA
Genome editing and stem cell research approach for the treatment of SCA
Our Lab's Contribution:
Reprogramming patient-derived peripheral blood mononuclear cells (PBMCs) into induced pluripotent cells (iPSCs).
Performing gene correction of HbS gene with the help of CRISPR-Cas9 system (FnCas9).
Establishment of a rapid, field-deployable nucleobase detection and identification assay using FnCas9 (FELUDA).
Screening SCD using CRISPR-Cas
The search for an affordable, rapid, robust, and sensitive point-of-care diagnostic test for SCD led us to work with CRISPR-Cas based diagnostics. FELUDA that utilizes FnCas9 (Cas9 derived from a bacterium Francisella novicida) has an extremely high sensitivity and specificity to rapidly identify and distinguish between carrier (heterozygous) and sickle patient (homozygous).
It uses a RNA-Cas9 complex that is guided by the RNA to bind and then cleave the specific DNA sequence that harbors the SCD point mutation. The assay can be adapted in a way that detection can be done at either the binding or the cleavage step.
The fragments generated by cleavage can be detected by gel or capillary electrophoresis. Alternatively, the binding can be detected by using a dipstick or by measuring fluorescence.
Treating SCD with CRISPR-Cas9
SCD patients who need to undergo bone marrow transplant face complications like graft versus host disease that has a high mortality. Using an individual's own stem cells reduces such complications. With gene editing techniques like CRSIPR Cas the hematopoietic stem cells can be corrected in the lab ex vivo and administered back into the patient.
We are developing FnCas9 based gene editing technique, which uses a RNA to guide the Cas9 protein to the target DNA sequence, to correct the mutation or to reactivate the expression of fetal hemoglobin in the stem cells. These stem cells will give rise to RBCs in the patient's body that will no longer express the sticky beta globin chain that causes sickling of RBCs.
Know more about SCD
Important Research Articles
Screening and Counseling Organizations