Dr Dever identified a laundry list of problems with the study methodology. First off, research-grade material was used in the manufacture of the investigational therapies, which is generally not considered high-enough quality for a clinical trial. In fact, the CRISPR-Cas9 itself was from New England BioLabs, a company that supplies research-grade reagents. Secondly, the stem cells were edited — separately — across several batches and then blended together before reinfusion into the patient.
“That is something that you would never do in a clinical trial,” Dr Dever observed.
As Carl June, MD, described in a corresponding editorial, at the University of Pennsylvania it took about 5 years from the time of acquiring proof-of-concept animal data to treating patients with gene-edited CD4 T cells in a clinical trial. It took only about 2 years between when CRISPR was first tried in animals and when this case report was reported.5 According to Dr Dever, the additional years of research could have been spent fine-tuning and improving the methods used to edit the target cells and developing a proper manufacturing protocol that can be applied in the clinic.
Beyond the cited methodology concerns, the gene-editing efficiency achieved with this CRISPR system both in previous animal data and the current patient was low. Previously, the CCR5 gene was successfully ablated in human hematopoietic stem cells that were transplanted into mice at 27%, a rate which Dr Dever described as “incredibly low” for a study conducted in 2017.6 In the current study, the editing efficiency was even lower — 17.8% for edited stem cells before infusion and between 5% and 8% for bone marrow karyocytes during engraftment.3
“When you’re doing these trials, there’s a balance of safety and efficacy, and everyone always leans toward it being safe, but you have to make sure that you’re convinced that it would actually have a chance to work,” Dr Dever remarked.
At present, laboratories routinely achieve an editing efficiency of 80% or 90%, according to Dr Dever. However, achieving such a high editing efficiency does not come automatically. It’s the product of tinkering with the CRISPR-Cas9 system until maximal efficiency is achieved. This can be done by swapping out one guide for another, trying a new target, or even just titrating the guide and Cas9 concentrations.
Although the study authors detected no off-target effects with whole-genome sequencing (WGS), because the efficiency was so low, it’s unclear whether WGS provides a true picture of the safety of the alterations. The possibility remains that if the editing efficiency were higher, off-target effects may have been seen.
“It was almost impossible to be able to detect any off-target mutation,” said Dr Dever. He explained that with only 17.8% of transplanted stem cells having an on-target event, a deeper sequencing approach would probably be needed to even find an off-target event.
Despite his various concerns about the study, Dr Dever did acknowledge a positive outcome of the study: the edited stem cells remained engrafted in the patient in the long term — that is, 19 months — and were able to produce lineages that had the ablated CCR5 gene.
- Uldrick TS, Gonçalves PH, Abdul-Hay M, et al. Assessment of the safety of pembrolizumab in patients with HIV and advanced cancer — a phase 1 study. JAMA Oncol. 2019;5(9):1332-1339.
- Abramson JS, Irwin KE, Frigault MJ, et al. Successful anti-CD19 CAR T-cell therapy in HIV-infected patients with refractory high-grade B-cell lymphoma. Cancer. 2019;125(21):3692-3698.
- 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(13):1240-1247.
- Maier R, Akbari A, Wei X, Patterson N, Nielsen R, Reich D. No statistical evidence for an effect of CCR5-Δ32 on lifespan in the UK Biobank cohort. BioRxiv. doi: 10.1101/787986
- June CH. Emerging use of CRISPR technology — Chasing the elusive HIV cure. N Engl J Med. 2019;381(13):1281-1283.
- Xu L, Yang H, Gao Y, et al. CRISPR/Cas9-mediated CCR5 ablation in human hematopoietic stem/progenitor cells confers HIV-1 resistance in vivo. Mol Ther. 2017;25:1782-1789.