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RESOURCES

CRISPR-Cas-Based Diagnostics and Therapeutics

We at RNA Biology Lab have developed CRISPR-Cas based diagnostics -FELUDA for multiple pathogenic conditions. In addition to diagnostics, we are also in the process of developing CRISPR therapeutics for Sickle Cell Disease and other ocular and neuropathological disorders. For more details, please write to us.

SICKLE CELL DISEASE

Sickle cell anemia (SCA) is an inherited hematological disorder caused by a single point mutation in the HbB gene, which encodes the beta subunit of hemoglobin. This mutation results in the production of an aberrant hemoglobin variant that induces erythrocytes to adopt a characteristic sickled morphology. These deformed cells exhibit increased adhesiveness within the vasculature, impeding blood flow and leading to ischemic organ damage, severe pain episodes, and potentially severe complications such as vision loss, stroke, or mortality. Additionally, SCA is associated with anemia, dyspnea, chronic pain, jaundice, and recurrent infections, cumulatively leading to reduced life expectancy. It is estimated that approximately 500,000 children are born with SCA annually, with India contributing to about 50% of these cases.

The burden of Sickle Cell Disease (SCD) in India is notably high, particularly among tribal populations in Kerala, Tamil Nadu, Madhya Pradesh, Maharashtra, Gujarat, Chhattisgarh, and Odisha, rendering India the second most affected country globally. The prevalence is exacerbated within communities of lower socioeconomic status, highlighting a significant public health challenge. 

Current SCD management primarily involves symptomatic treatment, including hydroxyurea administration, blood transfusions, and hematopoietic stem cell transplantation, as no definitive cure is presently available. Recent initiatives by governmental and non-governmental organizations have focused on genetic counseling, systematic screening, and patient monitoring in high-prevalence regions.

Advancements in CRISPR-Cas-based gene editing therapies offer promising avenues for a permanent cure. The RNA Biology Lab at CSIR-IGIB is actively developing CRISPR-Cas9-based ex vivo therapeutics for SCD, supported by the Ministry of Tribal Affairs (MoTA) and the Department of Science and Technology (DST). In collaboration with clinical and research partners, including AIIMS New Delhi, SCIC Raipur, CSIR-IICT, ICMR-NIIH Mumbai, NNF Bangalore, and the University of Washington Seattle, the lab is pioneering robust, cost-effective gene editing strategies to address the growing SCD burden in India.

PRELEVANCE

The prevalence of Sickle Cell Disease (SCD) in India varies significantly, ranging from 1% to 40% across different regions. Madhya Pradesh, home to major tribal groups such as the Gonds and Bhils, bears the highest disease burden, with more than half of its districts classified as high-prevalence zones. In Maharashtra, SCD prevalence ranges from 0% to 35%, predominantly among the eastern tribal communities, including the Pardhans, Otkars, Madhias, and Bhils. However, comprehensive epidemiological data remains scarce for several states, particularly Uttar Pradesh, Uttarakhand, Bihar, and the entire northeastern region.

SCREENING APPROACHES


Most SCD screening programs in India utilize the Sickling Test or the Rapid Solubility Test, followed by confirmatory hemoglobin electrophoresis. The dithionate qualitative solubility test, developed indigenously, is widely employed due to its cost-effectiveness, simplicity, and high sensitivity and specificity at room temperature, making it a reliable first-line screening tool. The test relies on the turbidity resulting from differential lysis of soluble HbA versus insoluble HbS, enabling efficient point-of-care assessment.

In recent years, High-Performance Liquid Chromatography (HPLC) has gained popularity for the accurate identification of sickle cell carriers and affected individuals. Additionally, some centers have adopted capillary electrophoresis for enhanced diagnostic precision.

CURRENT TREATMENT MODALITIES


Although no definitive cure for SCD is currently available in Indian clinical settings, several therapeutic strategies help manage symptoms and improve patient quality of life:

Hydroxyurea Therapy: Administered at 10-15 mg/kg/day, hydroxyurea promotes the production of fetal hemoglobin, thereby reducing the proportion of sickled cells. It also decreases leukocyte counts, mitigating vaso-occlusion and reducing the frequency of vaso-occlusive crises and blood transfusion requirements.

Blood Transfusion Therapy: Acute transfusions are employed to correct severe anemia, while chronic transfusion regimens are used to prevent primary strokes or recurrent episodes. Chronic transfusions also reduce the risk of acute chest syndrome, particularly when hydroxyurea is ineffective.

SUPPORTIVE AND PREVENTIVE MEASURES

  • Prophylactic Penicillin: Administered from birth until five years of age to prevent severe infections.

  • Pain Management: Acute pain crises are managed with opioid analgesics, while chronic pain is controlled using non-opioid painkillers.

  • Vaccinations and Supplements: Patients older than two years are recommended to receive pneumococcal vaccines and folic acid supplementation to support erythropoiesis.


Severe SCD is typically characterized by three or more pain crises requiring hospitalization or three or more blood transfusions within a year, warranting more aggressive management strategies.
Despite significant advancements in gene therapy worldwide, no permanent curative gene therapy for SCD is currently available in Indian clinical settings. Ongoing research and clinical trials in CRISPR-Cas9 gene editing are showing promise for potential curative interventions. Meanwhile, comprehensive symptom management and preventive strategies continue to play a crucial role in improving patient outcomes and quality of life.
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CSIR MISSION MODE PROJECT ON SICKLE CELL ANEMIA

CORE OBJECTIVES

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Managing the Genetic Burden of Sickle Cell Anemia

 Investigating the genetic basis of SCA and understanding the differential response to hydroxyurea therapy among patients.

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Drug Discovery and Development

Identifying novel therapeutic compounds for the effective management of SCA symptoms.

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Development of Affordable and Accurate Diagnostic Kits

Creating rapid, sensitive, and cost-effective diagnostic tools suitable for on-ground implementation.

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Genome Editing and Stem Cell Research

Exploring gene editing and stem cell-based therapies to achieve a permanent cure for SCA.

OUR LABORATORY'S CONTRIBUTION 
Our lab is actively involved in advancing gene therapy for SCA, focusing on the following innovative approaches:  

Reprogramming of Patient-Derived Cells

​Reprogramming peripheral blood mononuclear cells (PBMCs) from SCA patients into induced pluripotent stem cells (iPSCs). These iPSCs serve as a model system for gene correction studies and facilitate personalized therapeutic strategies.

 

CRISPR-Cas9 Mediated Gene Correction

Employing the FnCas9 variant (derived from Francisella novicida) to precisely correct the sickle cell mutation (HbS) in patient-derived iPSCs. The corrected stem cells are subsequently differentiated into hematopoietic stem cells for autologous transplantation, thereby reducing complications such as graft-versus-host disease.

Field-Deployable Diagnostic Development (FELUDA)

Establishing a rapid, affordable, and accurate nucleobase detection assay known as FELUDA (FnCas9 Editor Linked Uniform Detection Assay) using the FnCas9 system. FELUDA enables point-of-care testing to distinguish between SCA carriers (heterozygous) and affected individuals (homozygous) with high sensitivity and specificity.

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CRISPR-Cas9 Based Gene Therapy for SCA


Current treatment options for SCA, such as bone marrow transplants, are associated with high risks, including graft-versus-host disease. To address this, we are developing an ex vivo CRISPR-Cas9 gene editing therapy that uses a patient’s own hematopoietic stem cells, minimizing the risk of immune rejection.

FnCas9 Based Gene Editing Approach:

The FnCas9 protein is guided by RNA to target and correct the HbS mutation in hematopoietic stem cells. Alternatively, it is used to reactivate the expression of fetal hemoglobin, which can compensate for the defective beta-globin chain, thereby preventing sickling of red blood cells.


Therapeutic Advantage:

Edited stem cells are administered back to the patient, where they differentiate into healthy red blood cells devoid of the sticky beta-globin chains responsible for sickling. This approach offers the potential for a one-time curative treatment.

CRISPR-Cas Based Diagnostics for SCA


The quest for a rapid, robust, and affordable point-of-care diagnostic tool for SCA has led to the development of FELUDA, which leverages the high specificity of FnCas9. 

Mechanism of Action:

FELUDA utilizes an RNA-guided FnCas9 protein complex to bind to and cleave the DNA sequence harboring the sickle cell mutation. The system can be configured to detect the mutation either at the binding or cleavage step, enhancing its adaptability for various diagnostic platforms.


Detection Techniques:

Cleavage fragments are identified using gel or capillary electrophoresis.
Alternatively, binding events are detected using dipstick assays or fluorescence measurements, offering versatile detection options suitable for resource-limited settings.
This CRISPR-Cas-based diagnostic approach ensures high specificity and sensitivity, making it a powerful tool for mass screening and early diagnosis of SCA.

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PARTNERS

01

All India Institute of Medical Sciences (AIIMS), New Delhi

02

Sickle Cell Institute Chhattisgarh (SCIC), Raipur

03

CSIR-Indian Institute of Chemical Technology (IICT)

04

ICMR-National Institute of Immunohematology (NIIH), Mumbai

05

Narayana Nethralaya Foundation (NNF), Bangalore

06

University of Washington, Seattle

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