By Kevin Davies, PhD
A key FDA Advisory Committee met on Tuesday to discuss Vertex Pharmaceuticals’ groundbreaking IND application of exa-cel, a CRISPR-based therapy for sickle cell disease (SCD).
Originally sponsored by CRISPR Therapeutics, the Vertex exa-cel trial was initiated in 2018. The strategy uses CRISPR-Cas9 to target the binding site of a key inhibitor of the gamma-globin gene, BCL11a. Releasing the brake on fetal globin expression mitigates the effects of the SCD point mutation in the beta globin gene.
Investigators have published and presented exciting clinical results in dozens of treated patients, including Victoria Gray, who has been profiled in a series of interviews on National Public Radio. The FDA has set a Prescription Drug User Fee Act (PDUFA) date of December 8, 2023, for a final decision on approval.
The FDA’s Cellular, Tissue, and Gene Therapies Advisory Committee, chaired by Tabassum Ahsan, PhD (City of Hope), convened a panel of 14 experts in hematology, gene editing, virology and bioethics, as well as patient advocates. Members included Alexis Komor, PhD (UC San Diego), the co-developer of base editing; John Tisdale, MD, a leading hematologist at NIH who has treated SCD patients with bone marrow transplantation and gene therapy; and Scot Wolfe, PhD (UMass Chan Medical School), an expert in genome editing.
The panel was charged with assessing specific questions regarding the potential clinical significance of so-called off-target genome edits and to consider recommendations for additional studies that the sponsor might be asked to perform to assess the risk of off-target editing.
Before a series of presentations from Vertex executives, consultants and trial clinicians, FDA invited a pair of genome-editing experts to provide context and analysis on the subject of off-target edits: Fyodor Urnov, PhD (Innovative Genomics Institute) and Daniel Bauer, MD, PhD (Harvard Medical School).
Urnov reviewed three decades of progress in genome editing, with the CRISPR community over the past decade delivering a total of more than 27,000 citations to Cas9 in PubMed. During that time, there has been great progress in characterizing guide RNA (gRNA) behavior to direct clinical genome-editing studies.
“Knowing a given patient’s genome has a partial match is a first step to understanding whether the off target will be cut by that enzyme in cells from that patient,” he said. “Some guide RNAs just don’t work… Each cell imposes its own rules on what Cas9 can do.”
Urnov said the art of genome editing design requires designing appropriate gRNAs and screening in the relevant cell type. Overall, the success of genome editing depends on a host of variables besides off-target editing. They include the type of Cas9 nuclease, choice of gRNA, nature of the target sequence, mode of delivery, handling of transfected cells, and more.
The therapeutic goal, Urnov concluded, is to “find the Goldilocks conditions of maximum potency.” The only way to find them is to transplant cells into trial volunteers and see what happens.
Dan Bauer, MD, PhD, a hematologist at Boston Children’s Hospital who led some of the foundational studies that supported the BCL11a targeting strategy, highlighted one particular off-target site relevant to the Vertex trial, that dominated discussion throughout the hearing.
In a paper published in Nature Genetics earlier this year, Bauer and colleagues used a software tool called CRISPRme to screen for potential off-target sites. One such site (rs114518452) is a rare single nucleotide polymorphism (SNP) on the long arm of chromosome 2, which creates a PAM sequence that enables binding of Cas9 in a sequence that resembles the BCL11A target site, with three nucleotide mismatches. This SNP occurs in a gene called CSP1 at a frequency of 4.5% in populations of African descent.
Furthermore, Bauer said that the co-location of this off-target site and the target site (on the short arm of the same chromosome) raised the possibility that dual Cas9 cleavage could result in a large DNA flip—a 150-megabase pericentric inversion. Such rearrangements were indeed found, albeit rarely, with a frequency of 1:600.
“The biological significance of these inversions is uncertain—and may be negligible,” Bauer stressed. He added that tracking gene editing distribution over time would be analogous to a viral vector integration site analysis in gene therapy studies.
In a lively Q&A round, Bauer was asked how he would explain the risk of off-targets to a SCD patient and his/her family members? “There’s often uncertainty about the functional significance of off-target edits,” Bauer said. “Most of the [human] genome is non-coding… It’s likely that many places can tolerate an off-target edit with no functional consequence… The only way to know is careful follow-up.”
He added: “It’s a relatively small risk. It’s new, it’s unknown, but it’s easily measurable… We have to be humble and open to learning from these brave [SCD] patients who are participating.”
An advantage of working with a blood disease, Bauer continued, is that “it’s easy to follow blood samples over time and follow inversions or rearrangements. If [a rearrangement] is non-functional, we can supply reassuring data on this point.” But there was no preclinical analysis that was so convincing to say the approach is certainly safe.
Urnov concurred: although we have 30 years’ experience in gene editing, “there is always some uncertainty.” Today, he said, the field is in a different place. “The technology is ready for prime time. I don’t know what else to do at this point.”
A series of short presentations hosted by Vertex reviewed the exa-cel trial data and made a strong clinical case for approval. Physician Alexis Thompson MD (Children’s Hospital, Philadelphia) said SCD patients “greatly need a curative treatment,” with only a subset eligible for bone marrow transplants.
William Hobbs MD, Vertex vice president of clinical development, said the exa-cel therapy resulted in a “transformational clinical benefit” for patients.
Moreover, Hobbs added, the results are consistent in both adults and adolescents, and have been shown to be durable in the first treated patient (Gray) for four years. Only two of the patients in the original cohort experienced any VOCs: one had a single pain crisis, more than 12 months post treatment, that occurred during a parvovirus infection, which is known to be life-threatening in untreated patients. “This patient recovered quickly, without any needed transfusions,” Hobbs said. Another patient has also experienced VOCs but has not required hospitalization.
Vertex chief science officer, David Altshuler MD, reviewed the non-clinical safety data, and handled most of the panel’s questions. The strategy was to limit exposure to CRISPR; select a unique target site with no other match in the human genome; and optimize the gRNA for efficacy and specificity by screening hundreds of potential gRNAs. Candidate off-target sites were screened using both an in silico approach as well as the GUIDE-seq assay.
The closest matches for potential off-target sequences have three nucleotide mismatches in the gRNA sequence, Altshuler said, with the likelihood of editing just 1.6%. In all experiments, including deep sequencing of 14 individuals including six SCD patients, no detectable off-target edits were observed. Altshuler’s team also investigated 50 candidate off-target sites from a deep analysis of the 1000 Genomes database (more than 660 individuals of African ancestry). In short, he said, “This is the most comprehensive evaluation of off-target editing performed to date.”
Vertex picked up the CPS1 variant during its initial homology search, which configured an alternative PAM sequence. But experiments showed no off-target editing at the site and none of the 14 donors tested carried the risk allele. Altshuler said the SNP occurs in a non-coding intron of the gene, with no relevant tissue expression or any putative role in malignancy.
The Vertex presentations concluded with Haydar Frangoul, MD, the clinician who led the exa-cel trial and was the senior author on the first major publication from the trial in 2021. That report dazzled researchers by showing continued positive results for Gray a year after treatment for SCD in the CLIMB-121 (NCT03745287) study, including elimination of vaso-occlusive episodes.
Three months after treatment, two additional SCD patients showed similarly positive results. Soon after, Vertex agreed to pay CRISPR Therapeutics $900 million upfront to lead the global development, manufacturing, and commercialization of exa-cel, then known as CTX001
“I’ve seen first-hand the impact exa-cel has on my patients. It’s been such a rewarding experience to be part of this program,” he told the committee.
He highlighted the progress of his patient Gray, spending time with her family and working full-time, as well as another patient, a 13-year-old girl. Following exa-cel therapy, she had experienced no pain crises or hospitalizations, and was “enjoying her teenage years.”
“I see the same effect on the other patients,” Frangoul said. “Many adult patients wish they could’ve been treated younger, to enjoy the benefits longer.” And he added: “I’d be happy to have this therapy available for my adolescent patients.”
Doubted, dismissed, and judged
Later in the agenda, a one-hour session for public statements was dominated by SCD patients speaking passionately about their struggles with pain before treatment, and the hope they’ve received from the exa-cel trial or other transformative therapies.
Opening the proceedings was Gray herself. One patient, who signed off by calling herself “surviving sickle cell warrior”, said she had been “doubted, dismissed and judged” despite being hospitalized 100 times in 2022. Another said she was so glad to see “hope on the horizon,” and that patients might be spared the choice between “dignity and death.”
In the final analysis, it was hard not to place that question in the broader context of the massive unmet medical need servicing an estimated 100,000 SCD patients in the United States.
Wolfe said Vertex had done about as much as could be expected in testing for off-target editing. The GUIDE-seq assay was the “gold standard” in the field, he said. As for the CPS1 variant, Wolfe thought it would be “interesting to study in patient samples… It should be possible to look at the persistence of edits at that off-target site,” as well as the presence of the inversion. “We could learn quite a bit about the outcomes of genome editing,” he said.
Komor, the other true genome editing expert on the panel, agreed. “Given the benefits of this treatment/cure, and what these patients are dealing with [without treatment], the benefits far outweigh the risks here,” she said. What would the perfect off-target analysis look like, Komor asked? Should we “sequence [each] patient and use that as ‘the reference’ and analyze every off target?”
Wolfe said he would like to see a molecular follow-up to study the treated patient’s hematopoietic cells to monitor whether the spectrum of indels at the edited target site change over time—which might be a surrogate to something going wrong. “It would be good to follow up patients that have been treated so far.” With 45 SCD patients treated so far, that would suggest there are 4–5 putative CPS1 off-target variants to study. Wolfe even suggested pre-screening patients for the CPS1 variant.
Tisdale agreed. He said he wasn’t sure that further experiments were needed, but he said, “it would be interesting and [Vertex] could do it.”
Nicole Verdun, MD, super office director, CBER, FDA, reminded the panel that their task was not to discuss the well-established benefits of exa-cel. “We want to focus on any additional [off-target] studies. Those could be in the post-market setting,” she said.
But Wolfe seemed to speak for most if not all members of the committee when he concluded: “We don’t want to let perfect be the enemy of the good. At some point we have to try things out in patients. There is a huge unmet need for sickle cell disease. It’s important to advance therapies. I certainly think that this is one of them.”
The FDA’s decision will be announced within the next six weeks.