The fact that more than 30 different genetic mutations can cause Diamond-Blackfan anemia (DBA), which is a life-threatening disorder in which bone marrow cannot make mature, functioning red blood cells has hampered efforts to develop gene therapy for the disorder. Now, a team led by researchers at Harvard Medical School has overcome this difficulty, creating a universal gene therapy for DBA that targets the bone marrow defect regardless of a patient’s specific mutation. This experimental therapy is ready to be tested in clinical trials.
“We can develop a gene therapy that targets dozens of mutations with a single vector, and this is one of the first,” said Vijay G Sankaran, Paediatrician, Boston Children’s hospital, USA. “This work gives proof of concept that gene therapies for rare or complex blood diseases can overcome a problem caused by individual mutations, rather than correct specific mutations,” said the work’s team.
Over the past 15 years, Sankaran and Hanna Gazda, a scientist at Boston Children’s, discovered most of the DBA gene mutations known. These mutations mostly affect ribosomes. Further on down the line, Sankaran found that the ribosomal mutations make fewer functional ribosomes in cells, which in turn eliminates the capacity to manufacture GATA Binding Protein 1 (GATA1). When Sankaran added the GATA1 protein back to blood stem cells collected from patients with DBA, the cells also improved at differentiating into red cells.
They observed that if GATA1 levels were increased, DBA could be treated in patients with mutations within the GATA1 gene itself as well as patients with ribosome-related mutations. The team then went on to create a vector, or engineered, non-infectious lentivirus that could deliver the GATA1 gene to patients. In the new work, Richard Voit, then in Sankaran’s lab and now at UT Southwestern, and his colleagues devised a way to regulate GATA1 expression to allow the gene to be set to turn on only in the progenitors of red blood cells that reside in the bone marrow, and which travel that bone marrow but don’t turn on until they arrive.
Detailed analysis of the stem cells themselves showed that they were still stem cells, while lab experiments showed an increase in the production of mature red blood cells. The gene therapy vector lands GATA1 only at the intended spot in the genome also allayed concerns that the insert might happen elsewhere in the genome, near any cancer-causing genes, the researchers found. However, only a clinical trial will show if this indeed holds up in patients.
The team then examined human GATA1 accessible chromatin upstream of the GATA1 transcriptional start site in human hematopoietic stem cells (HSCs) and cells in erythroid differentiation to identify human regulatory enhancers supporting GATA1 lineage-restricted expression. Expression from parental hG1E cassette was confirmed to provide the most faithful restriction in HSCs and robust expression in developing erythroid cells. The next assessment considers the consequences of regulated GATA1 expression during erythropoiesis. Treatment with hG1E-GATA1 led to increased hG1E-GATA1 expression and increased levels of GATA1 per cell.
Incubation with exogenous GATA1 led to a modest acceleration of early erythroid differentiation with increased percentages of cluster of differentiation 71 (CD71+) and cluster of differentiation 235a (CD235a+) cells. In addition, they investigated clonal dynamics living in vivo using ISA on human CD45 selected cells sorted from the bone marrow of mice transplanted with hG1E-GATA1 treated DBA patients’ cells. The team detected integration events in 2,749 cells, from limited sample material. Together these data show that the clinical grade hG1E-GATA1 lentiviral vector supports clinical translation of regulated GATA1 as a universal gene therapy for DBA.Â
A successful gene therapy would help narrow the racial and ethnic disparity in DBA patients who lack bone marrow transplant donor matches. The implications for other gene therapies are also exciting to Sankaran. “We show for the first time that the reach of any hematopoietic gene therapy can be broadened if you can target the downstream mechanism instead of each of the individual components,” Sankaran said. That could lead to new avenues for other blood diseases.
Reference: Voit RA, Liao X, Caulier A, et al. Regulated GATA1 expression as a universal gene therapy for Diamond-Blackfan anemia. Cell Stem Cell. 2024;32:1-15. doi:10.1016/j.stem.2024.10.012‌
