Sickle cell disease (SCD) is the most common fatal genetic disease, affecting more than 300,000 newborns worldwide each year. It causes chronic pain, organ failure and premature death in patients. A team led by researchers at the Broad Institute of MIT and Harvard and St. Jude Children’s Research Hospital has now demonstrated a basic editing approach that effectively corrects the mutation underlying SCD in patients’ blood stem cells and in mice. This gene editing treatment has saved symptoms of the disease in animal models, allowing for the sustainable production of healthy blood cells.
The root of SCD is made up of two mutated copies of the hemoglobin gene, HBB, which causes red blood cells to transform from a circular disc into a sickle shape, triggering a chain of events leading to organ damage, recurrent pain and early mortality. In this study, the researchers used a molecular technology called basic editing to directly convert a single letter of pathogenic DNA into a harmless genetic variant of HBB in human blood-producing cells and in a mouse model of SCD.
“We were able to correct the disease-causing variant in cell and animal models using a custom base editor, without requiring double-stranded DNA breaks or inserting new DNA segments into the genome. Says co-lead author David Liu, Richard Merkin Professor and Director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad Institute, Professor at Harvard University and Researcher at Howard Hughes Medical Institute. “It was a major team effort, and our hope is that the Core Edition will provide a promising basis for an upcoming treatment strategy for sickle cell disease.”
“Our study illustrates the power and enthusiasm of multidisciplinary collaborations to create novel mechanism-based cures for genetic diseases,” says co-lead author Mitchell Weiss, chair of the Department of Hematology at St. Jude. “In particular, we have combined our expertise in protein engineering, base editing and red blood cell biology to create a new approach to the treatment and possibly cure of sickle cell disease. “
The work appeared in Nature, edited by co-first authors Gregory Newby at the Broad Institute and Jonathan Yen, Kaitly Woodard and Thiyagaraj Mayuranathan at St. Jude Children’s Research Hospital.
An improved approach
Currently, the only established method of curing SCD is a bone marrow transplant – but it is difficult to find a suitable bone marrow donor for a patient, and patients who have a transplant can experience dangerous side effects. While there are a number of gene editing treatments in development that avoid these risks by directly modifying a patient’s bone marrow, these experimental therapies rely on the introduction of new DNA or cleavage. of genomic DNA in cells, which can also cause side effects.
For this work, the research team used what is called an “adenine base editor”, a molecular tool developed in Liu’s lab that can target a specific genetic sequence and convert the base pair to A * T DNA to G * C, modifying a gene at a single pair of nucleotides. The core editor used in this study consists of a lab-grown Cas9 variant – a CRISPR-associated protein that positions the core editor at the mutated HBB site in the genome – and a lab-grown enzyme that converts target A to a base that pairs like G. The base editor also guides the cell to repair the complementary DNA strand, completing the conversion of the target base pair A * T to G * C.
The only DNA mutation underlying sickle cell anemia is an A in the healthy hemoglobin gene that has been changed to T. Although a basic adenine editor cannot reverse this change, it can. converting this T to C. This change turns the dangerous form of hemoglobin into a natural, non-pathogenic variant called “Makassar hemoglobin”.
Editing in models
The team first introduced the basic adenine editor in blood stem cells isolated from sickle cell patients. In these experiments, up to 80 percent of the pathogenic hemoglobin variants were successfully modified into the benign variant of Makassar, with minimal instances of the editor causing undesirable changes in hemoglobin.
The researchers transferred these modified blood stem cells into a mouse model to observe how they worked in living animals. After 16 weeks, the modified cells were still producing healthy blood cells.
“Sixteen weeks after transplantation, the total frequency of changes maintained in stem cells – which could contain changes in both copies of their hemoglobin gene, in a single copy or in no copy – was 68%. And we were particularly excited. to see that almost 90 percent of the cells contained at least one modified copy of hemoglobin, ”says Newby. “Even those cells with a single edited copy appeared to be protected against sickling.”
In another set of experiments, the researchers took blood stem cells from mice carrying the human sickle cell variant, modified them, and transplanted the modified cells into another group of recipient mice. Control mice transplanted with unmodified cells exhibited typical symptoms: sickle red blood cells, the consequences of a short lifespan of red blood cells, and an enlarged spleen. In contrast, mice transplanted with modified cells were improved over controls by each disease metric tested, with all measured blood parameters observed at levels almost indistinguishable from healthy animals.
Finally, to confirm the lasting editing of the target blood stem cells, the researchers performed a secondary transplant, removing bone marrow from mice that had received modified cells 16 weeks previously and transferring the blood stem cells to a new set. mouse. In the new animal cohort, the modified cells continued to function similarly to healthy blood stem cells, confirming that the effects of the core edit were long-lasting. The team determined that changing at least 20 percent of pathogenic hemoglobin genes was enough to keep blood measurements in mice at healthy levels.
“In these final experimental phases, we have demonstrated an editing threshold of about 20% which is necessary to alleviate this disease in mice. This basic editing strategy is effective enough to far exceed this benchmark,” explains Liu. “The approach shows promise as the basis of a potential one-time treatment, or perhaps even a one-time cure, for sickle cell anemia.”
Researchers and other partners are working to move this concept safely and effectively into further preclinical studies, with the ultimate goal of reaching patients.