doi:10.1038/nindia.2018.100 Published online 7 August 2018
Geneticist Debojyoti Chakraborty has been tinkering with samples collected from patients of sickle cell anaemia, a genetically-inherited disease that affects more than 4.4 million people around the world every year1, mostly in sub-Saharan Africa, India and the Arabian peninsula. The disease, estimated to have the highest number of carriers in India2, turns round red blood cells the shape of sickles that stack against one another, robbing the body’s capacity to ferry oxygen around. If both parents carry the ‘sickle cell trait’, they pass it on to the child, who then suffers from the incurable, often fatal, disease.
The usual practice to prevent the disease until now has been either through genetic screening before childbirth to rule out that the baby is affected or by counselling carriers of the trait not to conceive together. But the gene editing tool CRISPR-Cas9 may now make it possible to nip the gene responsible for the mutation and correct the anomaly even if the child is born with sickle cell anaemia.
At Delhi’s Institute of Genomics and Integrated Biology (IGIB), Chakraborty and his lab mates alongside chemist Souvik Maiti's lab are trying to correct such mutations using CRISPR Cas9 on sickle cell anaemia patient samples. They receive these samples from repositories such as the Sickle Cell Institute in Chattisgarh, New Delhi-based All India Institute of Medical Sciences or the Government Medical College in Nagpur, Maharashtra. The blood disorder is widely prevalent among tribal populations in the Indian states of Chhattisgarh, Maharashtra, Gujarat, and Odisha. The tool cleaves a target DNA molecule – in this case the ‘haemoglobin B’ locus – where mutation in a single base pair causes the disease. The break in the DNA can be fixed by the cells’ own repair machinery based on a new DNA template with the mutation-free gene sequence.
The researchers choose cells in which they have corrected the mutation and grow more of them in the lab for potential transfusion back into the patient. The repair is either done in the blood-generating (or haematopoietic) stem cells from the red bone marrow or in reprogrammed stem cells generated from adult cells (called induced pluripotent stem cells). “Since the cells are the patient’s own, he or she will generally not reject a graft when the gene-corrected cells are put back into the body,” Chakraborty explains.
His team, working on the tool for over a year, is now trying to show a proof of this concept in human induced pluripotent stem cells. The next step would be to use the concept in mouse models. “The idea is to eventually follow it up in human trials, and develop gene therapy for sickle cell anaemia.”
Monogenic blood disorders – those controlled by a single gene – are easiest to approach since a single gene locus needs to be corrected. Practically, a large number of monogenic disorders in the Indian population can be tackled if gene therapy using CRISPR is made to work in a precise fashion, Chakraborty points out.
Beta-thalassemia is another such genetic anomaly – the other most prevalent inherited haemoglobin disorder in India. “These genetic mutations were identified several decades back but there is no cure available till now,” says Sivaprakash Ramalingam, a senior scientist at IGIB. Although some patients have been treated with bone marrow transplants, the scope of such therapies remains limited due to the difficulty in finding compatible donors and transplant-associated clinical complications.
Ramalingam has been researching the possibility of a gene therapy for beta-thalassemia for over 18 months. He says his lab has seen some exciting first results in reactivating foetal haemoglobin (HbF) by editing human erythrocytes, the most common type of blood cells. This could form the basis of a viable therapy – a one-stop solution, as he calls it – for both sickle cell disease and beta-thalassemia. The team is also using this tool for genetic correction of other monogenic haematological diseases such as haemophilia-A and haemophilia-B.
“Gene or cell therapy is not science fiction any longer, given the success in autologous cell therapies, particularly for haematological disorders,” says Amitava Sengupta, who heads a research group at the Indian Institute of Chemical Biology (CSIR-IICB) trying to harness the potential of epigenetics and genome editing for gene or cellular therapy in blood disorders.
Sengupta, who started dabbling in the possibility of a gene therapy for haemophilia a decade back at the Saha Institute of Nuclear Physics in Kolkata, feels geneticists will be able to help customise these therapies for personalised transfusion medicine, especially for monogenic blood disorders. His team is also trying to better understand through epigenetic studies how the deadly blood cancer myeloid leukaemia develops in the body.
For developing countries, such “one patient, one product” cell replacement therapies may be extremely difficult to scale due to large populations and massive needs. “Off-the-shelf universal stem cell-based products developed by tinkering genes that are essential for immune rejection will be less expensive," Ramalingam says. Such products, he says, can aid in cell therapies for diseases like sickle cell anaemia or beta-thalassemia.
In 2017, Chakraborty and his colleagues at IGIB realised that the country’s labs have not yet warmed up to using the technology, mostly due to a lack of exposure. This prompted them to start a peer discussion that sought to tickle the interest of the community and assuage apprehensions around using CRISPR.
Earlier in March 2018, they brought in international experts to a workshop that taught young scientists how to use the cutting edge tool. The workshop was attended by 26 scientists, graduate students and physicians from across India with interests ranging from human cells to bacterial genome manipulation. It saw significant interest among the young turks seeking to customise CRISPR for individual research needs.
“In recent times, some labs in India have started using the tool for basic research questions, such as constructing knockouts (rendering a gene inoperative in an organism), knock-ins (inserting a DNA sequence) or tagging (labelling a gene),” Chakraborty says. IGIB also sells CRISPR products such as Cas9 proteins and its variants to educational institutes at a fraction of the commercial price so that the technology can be widely used. The institute is currently involved in developing better Cas9-like proteins that have high specificity and therapeutic relevance.
There’s also great interest in producing superior producer cell lines, says Vaibhav Jadhav from the Austrian Centre of Industrial Biotechnology. Jadhav works on Chinese hamster ovary (CHO) cells, the workhorse in production of recombinant therapeutics due to their capacity to express complex proteins with human-like post-translational modifications. The rapid surge in the CRISPR-Cas9 genome editing tool has accelerated biochemical pathway engineering in industrially important CHO cell lines, he says.
Since CRISPR has the power to change genetic make-ups, a large number of ethical issues, such as whether it should be used to produce designer babies or super humans, are being debated across the world. “The technology needs to be regulated,” Chakraborty says.
Recently, the controversial CRISPR-backed technology ‘gene drive’ capable of altering the genomes of entire species and in the process wiping them off, was applied for first time on mammals – lab mice. The gene drive technology has gained popularity particularly due to its promise in controlling malaria. “It will still take some time to see how far this will be accepted by nature since it will cause a balance shift,” Chakraborty notes.
DIY CRISPR kits sell online for as low as $159 making one believe that anyone can hack a genome. “It is not as easy as portrayed, scientists and student who newly adapt these techniques are perplexed with general and specific technical challenges,” he adds.
India banned the use of stem cell therapy for commercial use last year following concerns over 'rampant malpractice'. However, the national guidelines do exempt the use of such therapy to treat certain forms of blood cancers, malignant lymphomas and tumours. The non-malignant diseases exempt from the ban include sickle cell disease and thalassemia major.
Another major concern around taking CRISPR to the clinic is the unintended editing of genome by the CRISPR complex, called 'off-targeting'3. This might result in harmful mutations that can jeopardize the outcome of gene therapy. "We are finding ways to tackle this issue by designing and validating better Cas9 proteins that maintain high specificity for its intended target so that only the sickle cell anaemia mutation can be reverted," Chakraborty says. His lab is looking at other naturally occuring Cas9 proteins and rationally engineering them for use.
Sengupta says their experiments use genetically engineered mice models and once a proof of the concept is achieved, a national committee will look at how to translate it clinically.
1. GBD 2015 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990–2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet 388, 1545–1602 (2016)
2. Arjunan, A. Prevalence of the βeta-S gene and sickle cell disease in India. Univ. Pittsburg (2013) Link
3. Keep off-target effects in focus. Nat. Med. (2018) doi: 10.1038/s41591-018-0150-3