A major thrust of the center has been on developing gene therapy towards clinical trials. Two areas are being developed in collaboration with different groups - the first is the gene therapy program for hemophilia using AAV vectors and the second gene therapy program is towards the major hemoglobin disorders. A clinical trial for gene therapy of hemophilia B is proposed and will be initiated shortly.
The emphasis of this theme is on regeneration of musculoskeletal tissues such as cartilage (physeal and articular cartilage), bone and muscle using stem cells and differentiated cells. Major areas of research are cartilage tissue engineering for treatment of physeal and articular cartilage defect. A clinical trial in currently ongoing with the aim is to effectively translate musculoskeletal stem cells and differentiated cells for commonly encountered clinical problems which have less than satisfactory available treatment.
Cellular Reprogramming and its Applications – Haplobanking and Disease Modeling
CSCR is also engaged with the creation of an induced pluripotent stem cell “haplobank”. iPSCs derived from individuals with homozygous HLA haplotype will be banked for future research and potential therapeutic applications. iPSC models for some rare diseases are created to study the disease biology and explore novel therapeutic approaches.
Overview of gene therapy research at CSCR
The gene therapy program is coordinated by Alok Srivastava with RV Shaji, Saravanabhavan Thangavel, Mohankumar Murugesan and Srujan Marepally and involves two major areas at present – The first is directed towards a clinical trial for AAV vector-based gene therapy for haemophilia B in collaboration with Emory University, Atlanta, USA and the Powell Gene Therapy Centre as well as scientist at the University of Florida, Gainesville, USA. Given the success of AAV-based gene therapy reported in the last 4 years, the plan here is to apply a similar yet innovative approach to initiate a clinical trial in India with a novel AAV. Towards this end, apart from these scientific elements, regulatory processes are being established through ICMR, CDSCO, and DBT in India. The possibility of vector production at an industrial level is also being explored through a pharmaceutical partner in India. The second part of the gene therapy program involves pre-clinical models for lentiviral vector-based gene therapy through hematopoietic stem cell for the major hemoglobin disorders. This is in collaboration with the Emory University, USA. Lentiviral vectors carrying the beta globin gene are tested in human ex-vivo erythropoietic systems developed at CSCR. Work towards using genome editing technologies towards therapeutic gene corrections in stem cells has also been initiated. Other non-vector mediated gene transfer technologies are also being explored.
A major thrust in the last year has been on developing gene therapy towards clinical trials. Two areas are being developed in collaboration with different groups: A. The first is the gene therapy program for hemophilia using AAV vectors. Based on the successful clinical trial with the scAAV8 vector for gene therapy of FIX deficiency, several more clinical trials have been initiated in the last 2 years. There is also more recent data from Dr. Arun Srivastava’s laboratory that compared to all other AAV serotypes, it is AAV3 that has the highest tropism for the human liver. This then becomes one of the best options for gene transfer to the human hepatocyte. In collaboration with Dr. Trent Spencer and his group at Emory University, Dr. Arun Srivastava and his group along with Dr. Barry Byrne, Director of the Powell Gene Therapy Center both at the University of Florida, Gainesville, USA we are now developing a complete package for a clinical trial for gene therapy for hemophilia B in India. Preclinical data has shown the advantage of this serotype over the others in terms of transduction efficiency. We are now working rapidly to identify the best transgene construct in different models which will then be finally tested in both humanized mice as well the non-human primate models to decide on efficacy and safety parameters before non-human primate models to decide on efficacy and safety parameters before taking it to a clinical trial in India. Discussion with regulatory agencies (ICMR & DBT) in India is also being done in parallel to ensure that those aspects are also resolved in the next 6-9 months as the preclinical work reaches completion. The recent grant obtained from the DBT Towards Novel Applications in Hematological Diseases (NAHD), the efforts for this trial has got a very major boost. We are now in a position to put together a timeline.
The second gene therapy program is towards the major hemoglobin disorders. There has been much progress in this area of work also in the last 1-2 years. The initial results of the lentiviral vector-based gene therapy sponsored by Bluebird Bio are encouraging with 4-6g/dl increase in transgene Hb over 3-6 months after autologous gene-modified hematopoietic stem cell (HSC) transplantation. Several new studies have been initiated using a similar approach with novel transgene and vector constructs both for beta thalassemia major and sickle cell disease. Continuing our collaboration with Dr. Trent Spencer of Emory University, USA and his group this work will now be developed further in pre-clinical models using a marked transgene in human HSC systems. This work will be done in CSCR in collaboration with Dr. R V Shaji. In other related work being developed at CSCR, two new scientists (Dr. Saravanabhavan Thangavel and Dr. Mohankumar Murugesan) who have joined the gene therapy team will work also work on hemoglobin disorders using the genome-editing techniques. This is also part of the recently sanctioned NAHD project.
The main goal is to setup genome editing as a therapeutic option for patients with genetic disorders. The research involves two broad areas:
1. Genome editing mediated correction of Hemoglobin disorders
Existing treatment options for haemoglobin disorders are supportive and do not correct the fundamental genetic defect. Recent progress in genome editing technologies, particularly with the CRISPR/Cas9 systems has opened the possibility of precise gene corrections for treating Hemoglobin disorders
Dr. Saravanabhavan Thangavel and Dr. Mohankumar Murugesan are exploring the possibilities of therapeutic genome editing for correcting Hemoglobin disorders. Genome corrections are performed in patient Hematopoietic stem cells which allows for autologous transplantation of genetically manipulated HSCs
2. Genome editing mediated Gene therapy for primary immunodeficiency disorders
Primary immunodeficiency disorders (PID) are a diverse group of more than 300 rare, incurable diseases that occur as a result of genetic mutations in genes involved in the immune response cascade. The affected individuals are susceptible to infections that can be fatal. Allogeneic hematopoietic stem cell (HSC) transplantation is the existing treatment for the most severe forms of PID. Ex vivo genetic correction of autologous HSCs by gene therapy is an emerging option for those who do not have an available HLA- matched donor. Recent gene therapy clinical trials using viral vectors for delivering the functional gene into HSCs treats the immune deficiency, but also poses associated oncogenic risks.
As an alternative to virus-mediated gene therapy for PID, we propose a safer, non-viral gene therapy by combining the new generation clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) based gene-editing tools with a target specific expression of a functional gene. We aim to test this gene editing approach using Wiskott- Aldrich syndrome (WAS) as a candidate for PID. Wiskott-Aldrich syndrome is a life-threatening X-linked recessive disorder characterized by thrombocytopenia, eczema and immunodeficiency. WAS is caused by mutations in the WAS gene, which leads to compromised expression of the Wiskott-Aldrich Syndrome Protein (WASP). WASP plays a key role in hematopoietic actin cytoskeleton reorganization, and the deregulation of this process is responsible for the pathophysiology of WAS. We hypothesize that genome editing mediated targeted expression of WASP would potentially reverse WAS phenotype. This work is carried out by Dr. Saravanabhavan Thangavel.
CRISPR/Cas9 - system holds a great promise in treating genetic diseases, owing to its safe and precise genome editing. The technology can be used to remove and correct genes or mutations, and to introduce site-specific therapeutic genes in human cells. However, major challenge lies in achieving the therapeutically relevant frequencies with high fidelity for translating the technology into clinics. To this end, we are developing library of lipids varying the structural parameters including hydrophilic head group, hydrophobic tail and linker functionality. We are fabricating the nanoparticle system with these lipids and also evaluating the efficacies in delivering genome editing tools. We aim to apply this this technology in HSCs for knocking out BCL11A gene to improve fetal globin production, which has a great significance in stem cell gene therapy. We anticipate that developing the efficient toolset for CRISPR-Cas9 application will make a paradigm shift in the treatment of several genetic diseases such as hemophilia, beta thalassemia and sickle cell anemia.
This program is coordinated by Vrisha Madhuri with her team. The major focus is on clinical translations related to physis, articular cartilage and bone regeneration. For articular cartilage regeneration, ongoing small and large animal studies have articular defect reconstruction with differentiated MSCs on indigenous scaffolds. The continued follow up for human physeal regeneration with culture expanded autologous chondrocytes has shown success at 2.5 years follow-up and work is ongoing for similar physeal regeneration using 3D scaffolds in large animals. In a first of its kind study, reconstructions of bone defects in children with MSCs differentiated to osteoblasts on ceramic scaffolds have shown good outcome in the first 5 cases. A larger trial is being planned.
Cellular reprogramming and its applications - Disease modeling and Haplobanking
The area of cellular reprogramming technology is coordinated by R. V. Shaji at CSCR. This is now being applied to two areas of disease modeling and haplobanking. Towards understanding the mechanisms of reprogramming, a shRNA library is being used to investigate the role of epigenetic factors in different stages of reprogramming. Results so far have identified specific histone methylases and protein arginine methylases involved in the late stages of reprogramming. The reprogramming technology is also being applied to the development of disease models of various bone marrow failure syndromes – Fanconi anemia, Diamond Blackfan anemia and congenital dyserythropoietic anemia. A major translational effort has also been initiated towards establishing a “haplobank”, where the field and clinical aspects are being coordinated by Dolly Daniel and Alok Srivastava. This involves obtaining blood mononuclear cells from HLA haplotype homozygous normal individuals and creating a bank of these cells from which iPSCs are generated in a GMP compliant manner. This is part of an international consortium called the Global Alliance for iPSC Therapies (GAiT) for potential use in regenerative medicine in the future.