Baseline editing shows potential superiority for treating sickle cell disease

St. Jude Hematology

image: (Left to right) Co-corresponding author Mitchell Weiss, MD, Ph.D., St. Jude Department of Hematology chair and co-corresponding author Jonathan Yen, Ph.D., St. Jude Therapeutic Genome engineering director at a microscope in the laboratory.
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Credit: St. Jude Children’s Research Hospital

Gene therapy that alters hemoglobin genes may be a response to treatment for sickle cell anemia (SCD) and beta thalassemia. These two common life-threatening anemias afflict millions of people around the world. Scientists a St. Jude Children’s Research Hospital and the Broad Institute of MIT and Harvard have used a next-generation genome editing technology, adenosine base editing, to restart the expression of fetal hemoglobin in cells from SCD patients. The approach elevated fetal hemoglobin expression to higher, more stable and more uniform levels than other genome editing technologies using the CRISPR/Cas9 nuclease in human hematopoietic stem cells. The results were published today in Genetics of nature.

SCD and beta thalassemia are blood disorders affecting millions of people; mutations in the gene that codes for an adult version of the oxygen-carrying molecule, hemoglobin, cause these disorders. Restoring gene expression of an alternative subunit of active hemoglobin in a developing fetus has previously shown therapeutic benefits in patients with SCD and beta thalassemia. The researchers wanted to find and optimize genomic technology to modify the fetal hemoglobin gene. An alteration installed by adenosine base editing was particularly potent for restoring fetal hemoglobin expression in postnatal red blood cells.

“We have shown that base redacters significantly increase fetal hemoglobin levels,” said the corresponding lead author. Jonathan Yen, Ph.D., director of the St. Jude Therapeutic Genome Engineering group. “Now, my therapeutic genome engineering team is already hard at work, starting to optimize baseline editing to bring this technology to the clinic.”

Hemoglobin holds the key

Adult hemoglobin, expressed mainly after birth, contains four protein subunits: two beta-globins and two alpha-globins. Mutations in the beta-globin gene cause sickle cell anemia and beta-thalassemia. But humans have another hemoglobin subunit gene (gamma-globin), which is expressed during fetal development instead of beta-globin. Gamma-globin combines with alpha-globin to form fetal hemoglobin. Normally around birth, expression of gamma-globin is switched off and beta-globin is switched on, switching from fetal to adult hemoglobin. Genome-editing technologies can introduce mutations that reactivate the gamma-globin gene, thereby increasing the production of fetal hemoglobin, which can effectively replace the defective production of adult hemoglobin.

“We used a based editor to create a novel TAL1 transcription factor binding site that causes a particularly strong induction of fetal hemoglobin,” said Yen. “Creating a new transcription factor binding site requires a precise base pair change, something that cannot be done using CRISPR-Cas9 without generating unwanted byproducts and other potential consequences from double-strand breaks.”

“Gamma globin [fetal hemoglobin] gene is a good target for baseline editing because there are very precise mutations that can reactivate its expression to induce expression after birth, which can provide a powerful “one size fits all” treatment for all mutations that cause SCD and beta-thalassemia,” said corresponding author Mitchell Weiss, MD, Ph.D., chair of the St. Jude Department of Hematology.

Therefore, scientists want to restore fetal hemoglobin expression because it is a more universal treatment for major hemoglobin disorders than correcting the SCD mutation or hundreds of mutations that cause beta thalassemia. Increased fetal hemoglobin expression has the potential to therapeutically benefit the majority of patients with SCD or beta thalassemia, regardless of their causative mutations. Researchers have already demonstrated proof of principle with multiple genome editing approaches, but this study is the first to systematically compare the effectiveness of these different strategies.

“We have carefully examined the individual DNA sequence results of the nucleases and base editors used to make therapeutic modifications to fetal hemoglobin genes. Because nucleases often generate complex and uncontrolled mixtures of many different DNA sequence outcomes, we characterized how each nuclease-modified sequence affects fetal hemoglobin expression. So we did the same for the basic editing results, which were much smoother,” said the correspondent authorDavid LiuPh.D., Richard Merkin, Professor atBroad Institute of MIT and Harvardwhose lab invented basic editing in 2016.

The study found that using baseline editing at the most potent site in the gamma-globin promoter achieved 2- to 4-fold higher HbF levels than Cas9 editing. They further demonstrated that these basic modifications could be maintained in blood stem cell engraftment from healthy donors and SCD patients by inserting them into immunocompromised mice.

Address security issues

“In the end, we’ve shown that not all genetic approaches are created equal,” Yen said. “Basic editors may be able to create more powerful and precise edits than other technologies. But we need to do more security and optimization testing.”

Compared with safety, baseline editing caused fewer genotoxic events, such as p53 activation and large deletions. Base editing was much more consistent in edits and products, a highly desirable safety property for a clinical therapy. In contrast to conventional Cas9, which generates uncontrolled mixtures of insertion and deletion mutations called “indels”, base editing generates precise nucleotide changes with few unwanted byproducts.

“In our comparison, we encountered unexpected problems with conventional Cas9 nucleases,” Weiss said. “We were somewhat surprised that not all Cas9 insertions or deletions increased fetal hemoglobin to the same extent, indicating the potential for heterogeneous biological outcomes with that technology.” The team found that individual RBCs derived from hematopoietic stem cells treated with the same Cas9 produce a more variable amount of fetal hemoglobin than cells treated with the baseline editing. Therefore, base editing has produced more powerful, reliable and consistent results, which are desirable therapeutic properties.

While the basic editing worked well, the researchers have yet to determine its safety in patients. In particular, basic editing may have some risks not presented by Cas9; for example, some first base editors can cause unwanted changes in genomic DNA or RNA at off-target sites. The team showed that these changes are relatively small and not expected to be harmful, but more extensive studies are needed to fully assess these risks.

The future of gene editing therapies

In the course of the study, the scientists directly compared the performance of Cas9 nucleases at two different target sites that induce fetal hemoglobin production in different ways and base modification. Base editing uses a distinct editing mechanism that directly converts one DNA base pair into another, rather than cutting the DNA double helix in two.

Cas9 nuclease approaches create mixtures of deletions and insertions that impair the expression or activity of BCL11A, a known repressor of the gamma globin gene. In contrast, base editing creates a new transcription factor binding motif within the gamma-globin promoter. Cas9 nuclease approaches and a different base editing approach are being tested through clinical trials. St. Jude is participating in some of these studies.

“It’s very important to test and compare different genome-editing approaches for treating SCD and beta-thalassemia because the best ones are not known,” Weiss said.

Authors and funding

The first authors of the study are Thiyagaraj Mayuranathan, St. Jude and Gregory Newby, Broad Institute. Other authors are Ruopeng Feng, Yu Yao, Kalin Mayberry, Cicera Lazzarotto, Yichao Li, Rachel Levine, Nikitha Nimmagadda, Erin Dempsey, Guolian Kang, Shaina Porter, Phillip Doerfler, Jingjing Zhang, Yoonjeong Jang, Jingjing Chen, Senthil Velan Bhoopalan, Akshay Sharma, Shondra Pruett-Miller, Yong Cheng and Shengdar Tsai, all of St. Jude; Henry Bell and Merlin Crossley, University of New South Wales and John Tisdale, National Heart, Lung, and Blood Institute and National Institute of Diabetes and Digestive and Kidney Diseases.

The study was supported by grants from the National Institutes of Health (U01 AI142756, RM1 HG009490, R01 EB022376, R35 GM118062, R01 HL156647, R01 HL136135, P01 HL053749, U01 AI157189, R35 GM133614 , HL163805, K01 DK132453 and P30 CA21765); the Bill and Melinda Gates Foundation; the Howard Hughes Medical Institute including a Helen Hay Whitney Postdoctoral Fellow; the St. Jude Collaborative Research Consortium for SCD; the Doris Duke Foundation; the Assisi Foundation of Menfi; Cooley’s Anemia Foundation Postdoctoral Research Award; the American Society of Hematology (RTAF) and ALSAC, St. Jude’s fundraising and awareness organization.

St. Jude Children’s Research Hospital

St. Jude Children’s Research Hospital is at the forefront of how the world understands, treats and cures childhood cancer, sickle cell disease and other life-threatening ailments. It is the only National Cancer Institute-designated Comprehensive Cancer Center dedicated exclusively to children. Judehave has helped push the overall childhood cancer survival rate from 20% to 80% since the hospital opened more than 60 years ago.St. Jude shares the discoveries she makes to help doctors and researchers at local hospitals and cancer centers around the world improve the quality of care and care for even more children. To find out more, visitwww.stjude.org,LightSt. Jude progressblogs,and follow St. Jude on social media at@stjuderesearch.



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