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NIH Panel Approves CRISPR-Edited T Cell Safety Trial, First In-Human Use

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NEW YORK (GenomeWeb) – The National Institutes of Health's Recombinant DNA Advisory Committee (RAC) has reviewed and approved a protocol for a Phase I trial of a cancer immunotherapy that would be the first use of CRISPR/Cas9 gene editing in human patients.

In April, University of Pennsylvania researchers led by Edward Stadtmauer proposed a trial to test the safety and feasibility of autologous T cell therapy using CRISPR/Cas9 to knock out three genes contributing to two proteins: endogenous T cell receptor (TCR) genes alpha and beta, as well as PD-1, a gene coding for an immune checkpoint protein implicated in cancer. The cells would also be edited using lentiviruses to include transgenic TCR NY-ESO-1, a receptor found in several types of cancer cells.

At a meeting held yesterday in Bethesda, Maryland, the UPenn investigators presented preliminary data, including data on off-target editing rates, and protocol considerations to the RAC. After some discussion of the genome-editing approach that would be used in the trial, follow-up protocols, patient enrollment, conflicts of interest, and other topics, the RAC voted unanimously to approve the protocol, with one member abstaining. The trial still requires several more approvals, including institutional review board approval from UPenn and approval of an investigational new drug application by the US Food and Drug Administration, but the RAC decision sets the stage for development of ex vivo therapies using CRISPR/Cas9 genome editing.

"The past year has seen major advances in the efficiency of CRISPR," Alexander Marson, a professor at the University of California, San Francisco, told GenomeWeb. Marson is an expert on T cell editing and is not involved with the trial, although UCSF is listed as a proposed trial enrollment site. "There's work ahead to figure out the efficiency and the safety, but I think [CRISPR] is going to be a powerful addition to the set of tools available. There will be a lot of science to be done as part of these clinical trials to make sure this is safe and effective."

According to documents obtained by MIT Technology Review, the RAC notified principal investigator Edward Stadtmauer in May that the proposal to test the safety and feasibility of editing autologous T cells for cancer immunotherapy using CRISPR/Cas9 would require in-depth review. The committee is authorized by federal statutes to provide advice to the NIH Director on activities related to recombinant DNA.

The trial builds on previous work pioneered by UPenn researcher Carl June, who presented to the RAC and is a scientific advisor for the study, to edit the genomes of patient-derived T cells using lentiviruses, so the cells would recognize particular tumor antigens, such as NY-ESO-1, which is highly expressed in melanomas, sarcomas, and myelomas. Transgenic NY-ESO-1 T cell therapies are currently being developed by Adaptimmune.

The rationale for additionally knocking out PD-1 comes from years of research in immune system regulation. PD-1 is an immune checkpoint and prevents T cell activation. Anti-PD-1 drugs such as Bristol Myers Squibb's Opdivo (nivolumab) and Merck's Keytruda (pembrolizumab) have been hyped as next-generation cancer therapies and have spurred development of companion diagnostics to help predict patient response to the drugs.

In addition, NanoString Technologies is currently developing a companion diagnostic for Keytruda, while Myriad Genetics has a similar deal to use its myChoice HRD as a companion diagnostic to predict response to a combination therapy of Keytruda and a PARP inhibitor.

The UPenn proposal follows a growing body of literature suggesting that CRISPR/Cas9 and other genome-editing technologies could be used to edit PD-1 in T cells. In December 2015, scientists led by Marson and UC-Berkeley professor Jennifer Doudna demonstrated the ability to edit genes in primary T cells using Cas9-guide RNA ribonucleoproteins (PD-1 happened to be one of the genes they successfully edited). In January, scientists from Nanjing University led by Xingxu Huang and Baorui Liu published a study in Scientific Reports demonstrating the ability to edit T cells derived from cancer patients.

In the proposed trial, patients diagnosed with multiple myeloma, sarcoma, and melanoma will receive a single infusion of their own edited T cells. UPenn's Clinical Cell and Vaccine Production Facility will manufacture the cells.

Three different guide RNAs will target each gene, with at least 13 base pair mismatches from other sites to minimize off-target editing. The review has revealed some preliminary data on what CRISPR/Cas9 editing looks like in human cells.

The researchers said that approximately 95 of the on‐target indels are small deletions, with deletions of 1 nucleotide being the most common, accounting for approximately 15 percent of all indels. The longest indel was a 53bp deletion, they said.

Investigator Joseph Melenhorst of UPenn also presented preclinical data on off-target effects of editing in healthy donors. Based on the gRNAs used to target the genes, there were potential off-target sites in 148 genes; however, in two experiments in donor cells, only one gene, ANK1, was found to have been edited and the edit was in an intron.

The RAC committee raised several questions about how the investigators planned to look for both on-target and off-target editing. The investigators presented several methods that could be used for one or the other.

For on-target edits, they proposed using flow cytometry, T7 surveyor assays, and digital PCR assays for gene disruption. For off-target edits, they proposed targeted deep sequencing, gene panel sequencing, and even whole-genome or whole-exome sequencing, as well as long-term culture to detect clonal advantage or transformation. And the researchers plan to follow patients for up to 15 years to gather data on the effects of off-target edits, should they occur.

The researchers acknowledged that even better tools were needed to characterize genome editing in the cells. All the assessments so far had been done in bulk cell populations and they said they were working on developing single-cell assays to better understand the cells.

Because there are four genomic alterations — the addition of NY-ESO-1, and deletion of TCR alpha, TCR beta, and PD-1 — the researchers said that they expect the transfusion to contain 16 different "species" of T cells, since not all genomic edits are 100 percent efficient. Preclinical data suggested that CRISPR edited PD-1 at efficiencies between 20 percent to 55 percent and the TCR genes at approximately 60 percent to 90 percent efficiency.

One RAC reviewer asked if there were any estimates on how many cells carried all three CRISPR edits.

"Without a single-cell assay, we can't say," June said, but added that previous trials with other edited T cells showed efficacy when as few as 3 percent of all transfused cells were genetically modified.

But efficacy studies will require different considerations than safety trials. While the RAC approved the study, it also offered some suggestions, including modifications to the consent form, declarations of conflict of interest, and patient support budget.

Additionally, they suggested that the investigators highlight the trial would be a historic first.

"This is the first in-human use of CRISPR," said Laurie Zoloth, a bioethicist at Northwestern University and RAC member. "It should say that. Because this is the first one, we want this to be abundantly clear to the participants and to the watching public."

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