In a case study published in the New England Journal of Medicine, researchers successfully used a CRISPR-based gene-editing approach in a patient with HIV and acute lymphocytic leukemia (ALL). Although the engineered cells persisted for 19 months and the patient’s leukemia was put into remission, the percentage of engineered cells appeared to be too low to lower the patient’s HIV viral load.
The report from Lei Xu, MD, PhD, from the Department of Hematopoietic Stem Cell Transplantation at the 307 Hospital of the People’s Liberation Army in Beijing, details the first attempt to use CRISPR-edited cells in a patient infected with HIV. Using CRISPR, the investigators engineered CCR5-ablated hematopoietic stem and progenitor cells; these were designed to mimic a natural immunity wherein CCR5-null blood cells are largely resistant to HIV infection.
The patient in this report is a 27-year-old man who was diagnosed with HIV/AIDS and ALL in May 2016. Antiretroviral therapy resulted in control of the HIV infection and the virus was undetectable in serum RNA after one year. For his ALL, the patient underwent six courses of standard chemotherapy. Following the sixth course of chemotherapy, measurable residual disease became undetectable.
After determining that the HIV was CCR5-tropic, the researchers identified a 33-year-old, fully human leukocyte antigen–matched male donor with the unmutated CCR5 gene. The patient then underwent allogeneic hematopoietic cell transplantation (alloHCT) with myeloablative conditioning, from which clinicians sorted CD34-positive hematopoietic stem and progenitor cells using magnetic beads from donor-derived mobilized peripheral-blood mononuclear cells.
They next performed CRISPR editing of the CCR5 locus. CRISPR editing resulted in a CCR5 insertion or deletion efficiency of 17.8%, and the proportion of CCR5 ablation in the genome of bone marrow karyocytes ranged between 5% and 8% during engraftment.
Newly edited CCR5 CD34-positive cells, as well as unedited CD34-depleted cells, were infused into the patient with HIV.
“In our study, Cas9 ribonucleoprotein was introduced by means of nonviral transfection to avoid the introduction of exogenous DNA and avert the long-term existence of Cas9 in targeted cells, which was a potential causative factor for unexpected off-target indels,” the study’s co-author Hongkui Deng, PhD, from the Peking University in Beijing, explained.
Neutrophil and platelet engraftment occurred at 13 days and 27 days following alloHCT, respectively. The investigators also observed increases in lymphocyte counts and T-lymphocyte subsets following transplant, and CD4-positive cell counts stabilized to a normal range at six months. The researchers also observed morphologic complete remission of ALL at week 4, which persisted over 19 months of follow-up.
The patient had “predictable” side effects after preconditioning, the authors reported, including anemia, neutropenia, and thrombocytopenia. There was no observed acute immune response following infusion of donor cells, but at two months, the patient developed acute graft-versus-host disease, urinary frequency and urgency, cytomegalovirus viremia, and herpes simplex reactivation.
Based on previous work showing that autologous CCR5-edited T cells may decrease viral load in patients during a four-week interruption of antiretroviral therapy, the investigators temporarily stopped the patient’s antiretroviral therapy at seven months post-alloHCT, when his CD4-positive cell count increased to within the normal range and HIV RNA copies remained undetectable.
At week 4 of the interruption, the patient’s serum viral load increased to 3×107 copies/mL, at which point antiretroviral therapy was resumed. In the following months, the viral load gradually reduced to an undetectable level. During therapy interruption, the level of CCR5 disruption in CD4-positive cells peaked at 4.39%, up from 2.96% before the interruption and at a level that was 1.6 times the mean level.
Despite achieving long-term engraftment, Dr. Deng and co-authors detected only a 5% CCR5 disruption in lymphocytes, indicating a need for further research into improving this approach. “The low efficiency of gene editing in the patient may be due to the competitive engraftment of the CD34-depleted cells, which contained 28.8% of total CD34-positive cells,” Dr. Deng explained.
However, using a high-throughput whole-genome assay to analyze samples before and after alloHCT, the investigators found no evidence of any single-nucleotide variants, large deletions, or chromosomal rearrangements related to CRISPR modification.
While the absence of adverse events associated with gene editing supported the safety of this CRISPR-based approach, Dr. Deng noted that “the current low efficiency of CCR5 targeting limited the depth of the off-target gene-editing analysis, so it will be necessary to analyze the safety of CRISPR–Cas9–mediated CCR5 ablation in hematopoietic stem and progenitor cells further under a higher gene-targeting efficiency.”
This report included data from just one patient and one center, limiting the generalizability of its findings.
The authors report no relevant conflicts of interest.
Xu L, Wang J, Liu Y, et al. CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia. N Engl J Med. 2019;381:1240-7.