Executive Summary : | Objective: a) Extending our developed comprehensive model for screening of sickle cell anaemia (SCA) in other districts and states and understanding genetic basis of differential response to hydroxyurea therapy (CSIR-CCMB).
b) Development and on-ground implementation of affordable, accurate and accelerated diagnostic kit for sickle cell anemia (CSIR-CCMB, CSIR-IGIB, CSIR-URDIP).
c) Develop blood-based biomarkers using multi-pronged strategies to aid in management of SCA (CSIR-CCMB, CSIR-IGIB, CSIR-URDIP).
d) Genome editing and stem cell research approach for the treatment of sickle cell anaemia (CSIR-IGIB, CSIR-IICB).
e) Drug development for management of SCA (CSIR-NCL, CSIR-IIIM, CSIR-URDIP). Summary: Sickle Cell Disease (SCD) is a neglected chronic single-gene disorder of increasing global importance, with India estimated to have the second highest burden of the disease after Sub-Saharan Africa. However, some of the highest ßS allele frequencies have been reported in Indian populations with 42,016 (interquartile range: 35,347–50,919) babies estimated to be born with SCD in 2010. Further, as many as 6-7 states in India present with a very high prevalence of the disease making the disease as one of serious nature. In India, SCD is also said to be more prevalent in communities with low-socio economic status making the treatment burden unmanageable for them. Although the molecular lesion is a single-point mutation, the sickle gene is pleiotropic in nature causing multiple phenotypic expressions that constitute various complications of SCD in general and sickle cell anemia in particular. Individuals suffering from sickle cell disease show symptoms such as body pain, dyspnoea, anemia, jaundice, pneumonia, repeated infections etc. Given the complexity of pathophysiology, many different definitions of severity are possible complicating a full understanding of its genetic foundation. Thus, clinical course is variable and several factors including genetic constitution of an individual is said to be important. After 100 years of identification of the SCA genetic locus, there is no well-established consensus among health-care providers on the management of the complications of SCD due in part to lack of evidence and in part to differences in the experience of providers.
In Phase I project, a comprehensive model was developed for population screening to unravel the hidden burden of SCA by discovering carriers and patients, managing existing patients with better approaches, educating patients and family members through counselling and prevention of SCA by preventing birth of diseased children through prenatal diagnosis. A central Database has been created for all patients’ demographic, laboratory and clinical information which will be available for access to all partners. Point of care procedures and devices were developed for patients and tested successfully and can be taken forward for on-field testing and reporting. With NHLBI recommendation that HU treatment be offered to SCA children starting at nine months after birth regardless of clinical symptoms. In these regards, HU was provided to all patients attending camps and drug response was captured through pre- and post -HU blood collection and clinical examination. Patients were categorized on the basis of severity scores to address the phenotypic heterogeneity as well as drug response. Whole genome sequencing (WGS) was done on 350 well characterized SCA patients who were given HU treatment towards development of genetic markers to predict the clinical course and the drug response. Apart from HU, other Plant-based extracts from Sorghum and Piper were found to have anti-sickling properties. Other molecules like NO-conjugates, NLRP3a inhibitors, Gardos Channel Inhibitors, Gamma-Globin Inducers and GBT-440 analogues were identified that showed improvement in specific activity in patients’ cells. Co-crystals related to HU augmented the HU activity which will helpful in further managing the disease through these modified drugs. However, considering the translational potential and with time dimensions, only few activities especially, polyherbal nutraceutical product and the GBT440 analogues will be taken forward. Others may continue as institutional activity and based on results may be brought back in the programme at a later stage. In terms of genome editing, there is no substantial evidence available till date in literature of treating SCA patients in India or abroad using this approach except a report on single patient from US. In Phase I, a CRISPR-Cas9 setup has been established for genome editing and proof of principle has been provided. Techniques and protocols related to SCA gene correction in iPSCs and HPSCs from patients have also been done successfully; additionally a new and novel subtype of Cas9, called FnCas9 with better specificity and no off-target effects has been discovered. Now, with positive outcomes from the Phase I, we propose for the Phase II of CSIR Sickle Cell Anaemia Mission.
The Phase II of CSIR Sickle Cell Anaemia Mission is proposed now with a holistic approach for management of SCA which will include (1) Extension of established comprehensive model for population screening, genetic testing and counselling for SCA in other districts of Chhattisgarh and Maharashtra as well as in two other states (Jharkhand and Madhya Pradesh), ultimately extendable to the whole country for provision of genetic diagnosis cards and management of genetic burden of SCA as well as understanding the natural history of the disease and genetic basis of clinical course/differential response to hydroxyurea (HU) therapy. (2) Rapid Diagnostic protocols/POCs (Blood PCR, CrisprDx (FELUDA) and Paper- based test) will be established in the respected screening protocol to provide fast and reliable screening results. (3) Multi-pronged strategies to develop blood-based biomarkers from plasma and RBCs of SCA patients to aid management of SCA. (4) Established Genome editing and stem cell research approach for the treatment of SCA will be investigated for functional efficacy of gene-corrected iPSCs/HSCs in Humanized mice and further validated in a greater number of animals. After successful characterization, clinical trials on human subjects will be conducted. Patent search at all stages of project will be an integral part of the project, and, (5) Top hits identified from screening of thousands of extracts of plants in phase I will be taken forward towards their evolution into management and treatment of SCA patients. Especially, priority will be given for compounds from plants like Sorghum and Piper to be used as nutraceuticals. Further, the GBT440 analogues will be synthesised with an eye on freedom to operate (FTO) and evaluated for functional efficiency with GBT440 as the standard. This will help reduce the cost of treatment and help a large number of SCA patients. Finally, it is planned to further put serious efforts towards addition of POCs and genetic marker panel to upgrade this comprehensive model for overall SCA management, beginning from early detection to prevention to treatment and to develop competence and human resources in the area of SCA research. |