Interview on genome-editing and xenotransplantation

I was pleased to be invited to reflect on the future of xenotransplantation and human genome editing recently in an interview on ABC’s The World with Beverley O’Connor. The interview was broadcast following news of the unfortunate passing of Richard “Rick” Slayman — the first human to have received a genome-edited pig kidney. Mr Slayman passed less than two months following the xenotransplantation, although the General Massachusetts Hospital has stated that there is no indication that the transplant was the cause of death.

As I note in the interview, it is likely that the pig from which the kidney was sourced was a cloned pig. While this has not been confirmed (to my knowledge) by eGenesis, there are reports that the firm uses cloned pigs.

Moreover, the protocol by which pigs are prepared for this process is confirmed in the academic literature as involving somatic cell nuclear transfer (SCNT). SCNT is a kind of cloning that makes use of cultured fibroblasts — skin cells that excrete proteins like collagen — that are transferred into the enucleated nucleus of endogenous oocytes (eggs) . Willard Eyestone and colleagues describes the process in this way:

A major step forward in the generation of pigs as organ donors was the advent of somatic cell nuclear transfer (SCNT). In the pig, cultured fibroblasts were used as nuclear donors to replace the endogenous nuclei of porcine oocytes. Upon fusion with an enucleated oocyte, fibroblast nuclei were reprogrammed to totipotency by factors in oocyte cytoplasm. The newly reconstructed oocyte then developed into a new individual with the genetic constitution of the donor nucleus. SCNT technology opened the door for genetic modification of cultured somatic cells, which could be used to generate pigs bearing those modifications.

In essence, pig oocytes or eggs are enucleated — their nucleuses removed — and then they are fused with the somatic (adult) nuclei of the fibroblast cells. The fused or engineered egg undergoes ‘reprogramming’ as a result of this process. This means that the genes within the engineered egg cells are expressed differently once the fusion occurs; indeed, the so-called ‘fate’ of the cell is ‘switched.’ This means that ‘potency’ (or differentiation pathway) of the egg cell is changed. By ‘differentiation pathway,’ I mean the cells’ ability to differentiate into other cell types.

Cell biologists speak of several different cell potencies. Stem cells can express different degrees of potency, and may be classified as totipotent, pluripotent, multipotent, oligopotent, and unipotent cells:

  • totipotent stem cells can differentiate into all adult somatic cell types, as well as tissues of the placental and fetal membranes; eg, the zygote (until the 16-cell stage)
  • pluripotent stem cells are capable of differentiation into all adult somatic cells in all three germ layers: the endoderm, mesoderm and ectoderm. They have two defining features: the ability to form teratomas when injected in immune-deficient mice and the ability to form chimera or contribute to the germline of a mouse if injected into the blastocyst. An example is an embryonic stem cell or a somatic cell reprogrammed into the pluripotent state using somatic cell nuclear transfer (SCNT).
  • multipotent stem cells are capable of differentiating into multiple but limited cell types, usually within one germ layer: eg, hematopoietic stem cells can differentiate into lymphoid, myeloid, erythroid and megakaryocyte precursors; mesenchymal stem cells, which are often used for attempt to regenerate tissue and other cells, can differentiate into osteogenic, chondrogenic, and adipogenic cells.

As the pig oocytes undergo reprogramming through the fusion process, they become pluripotent cells. This means they can give rise to another new form of life (eg, a pig may be ‘cloned’ from this engineered donor cell). This is how the pig was likely to have been created in respect of this process; and the pig kidney would have been harvested from the pig that was made through this process.

I suspect the 69 gene edits that were made to the pig, which included pig gene knockouts, human gene knock-ins, and pig endogenous retrovirus (PERV) gene knockouts, was done at the pre-fertilisation stage — that is, that were applied to the engineered pig oocyte.

In any event, it will be important to understand how well these treatments last, especially given that they will continue to be offered. A second recipient of a xenotransplanted pig kidney, a woman from New Jersey, was given her xenotransplanted organ at the New York University Langone Health around 24 April 2023. The organ, however, also included the pig’s thymus glad, according to reports.

Around the same time as the organ xenotransplant, the NYU surgical team also transplanted a mechanical heart pump into this patient. It is noted in reports that the patient was given these treatments under an FDA emergency authorisation; that would mean that it was likely authorised by an Investigational New Drug licence under pt 312 of Title 21 of the Code of Federal Regulations.

Salient details about this pig kidney from the media release include the following points:

  • The genome-edited pig kidney was sourced from biotech firm United Therapeutics Corporation
  • It was an investigational xenokidney that ‘matched’ the donee (presumably this is something like a HLA match?)
  • Although chronic kidney failure ordinarily rules out patients from receiving a mechanical heart pump, the potential for this patient to live without a need for kidney dialysis (provided the xenotransplant succeeds) meant that the heart pump could be given to this patient
  • The pig kidney was engineered to “knock out” the gene responsible for producing the sugar known as alpha-gal
  • NYU Langone studies (although it is not clear what kinds of studies — presumably non-human primate studies?) demonstrated that removing alpha-gal was sufficient to prevent an antibody reaction that causes hyperacute rejection
  • The donor pig’s thymus gland, which is said to “educate” the immune system, was included: it was surgically placed under the covering of the kidney to reduce the likelihood of rejection
  • The xenokidney and the thymus tissue combined are called a UThymoKidney
  • The gene edits, pig breeding, and production of the investigational UThymoKidney used in this procedure were performed by United Therapeutics Corporation. No other unapproved devices or medications were used in the procedure.

CRISPR’d pig kidneys for xenotransplantation

Last week, the biotech firm eGenesis supplied a donor kidney sourced from a genome-edited pig to a surgical team at Massachusetts General Hospital (MGH) in the US. The donated kidney was transplanted into a patient with end-stage renal disease whose existing kidney transplant (‘allotransplanted’ from a human) was wearing out. The FDA had granted an expanded access authorisation for the procedure and, as of today’s date, the patient is reported to be doing well.

It is not clear to me from any of the materials online how exactly the pig was edited, other than that it was broadly subject to CRISPR/Cas9 editing of three kinds. First, there was so-called knock out editing of the glycan antigens (which lead to hyperacute rejection); second, there was knock in editing of seven human transgenes, which are thought to help with ‘acceptance’ (or which mitigate immunogenic rejection); and, finally, there was editing directed towards inactivating the genes known to cause porcine endogenous retroviruses (PERVs).

In total, eGenesis and MGH report that sixty-nine (69) discrete gene edits were made. That appears to be a record for a xenotransplantation, because the previous two pig-derived heart xenotransplants from 2022 and 2023 in the US are reported to have only been subject to about 10 edits.

I am assuming that the edits were made to the germline cells of a pig embryo fertilised in the laboratory, as opposed to being made on primordial germ cells or embryonic stem cells, but I am not able to find any specific protocols for this firm’s method. I suspect that somatic cell nuclear transfer (SCNT; ie, cloning) was not used, even though SCNT is technically legal in the US. In any event, I wrote a piece for the Conversation on some of the issues arising in relation to this recent news item here.

Case note: Fidge v Pfizer Australia Pty Ltd [2024] FCA 161

On 1 March 2024, Rofe J of the Federal Court of Australia (FCA) delivered a decision concerning an application brought by a medical practitioner, Dr Julian Fidge, in July 2023, against the pharmaceutical companies Pfizer Australia Pty Limited and Moderna Australia Pty Limited (the respondents).

The applicant sought an injunction under s 147(1) of the Gene Technology Act 2000 (Cth) (GT Act), which permitted an ‘aggrieved person’ to obtain an injunction from the FCA as against any conduct that was or would be an offence under the GT Act or the Gene Technology Regulations 2000 (Cth) (GT Regulations). An injunction so ordered would restrain the offending party from engaging in the conduct.

It is important to note that the applicant had formed the view that the vaccines sponsored by the respondents, which are generally known as mRNA vaccines (mRNA vaccines), were, or had contained, genetically modified organisms (GMOs) as defined under s 10 of the Gene Technology Act 2000 (Cth) (GT Act). While the meanings of these words might well be ‘at large’ in the scientific literature, the application in this case was framed in terms of the legal meaning of this expression, which was provided under s 10 the GT Act, as follows:

genetically modified organism means:

(a) an organism that has been modified by gene technology; or

(b) an organism that has inherited particular traits from an organism (the initial organism), being traits that occurred in the initial organism because of gene technology; or

(c) anything declared by the regulations to be a genetically modified organism, or that belongs to a class of things declared by the regulations to be genetically modified organisms;

but does not include:

(d) a human being, if the human being is covered by paragraph (a) only because the human being has undergone somatic cell gene therapy; or

(e) an organism declared by the regulations not to be a genetically modified organism, or that belongs to a class of organisms declared by the regulations not to be genetically modified organisms.

As I have argued elsewhere, the applicant’s submission appeared to completely omit any reference the GT Regulations. This was unusual, since the GT Regulations declare a broad range of organisms to to be not GMOs (notwithstanding s 10 of the GT Act) pursuant to the subsection (e), above. For instance, ‘Schedule 1: Organisms that are not genetically modified organisms’ contains 10 items; and Schedule 1A: Techniques that are not gene technology’ contains 11 items, which appear to affect many GMOs. Indeed, it might be argued, as I have argued, that the applicant’s failure to consider the applicability of these exemptions rendered the submissions inadequate.

In any event, the applicant had formed the view that the mRNA vaccines were GMOs under the GT Act as a result of reading various sources. These included some refereed articles, some preprint publications concerning the genetic profile of the mRNA vaccines, various online documents, and other sources (many of which were presented on a webpage published by the Australian Medical Professional Society in respect of the proceedings). However, none of these sources appeared to consider the legal meaning of the expression GMO under the GT Act.

Nevertheless, on the basis of the above view, the applicant asserted that the respondents were required to obtain licenses under the GT Act from the Office of the Gene Technology Regulator (OGTR) to ‘deal with’ those mRNA vaccines pursuant to s 40 of the GT Act. The requirement to obtain licences in respect of the mRNA vaccines followed from the asserted conclusion that they were GMOs under the GT Act. Since the respondents had not obtained those licences, the applicant submitted, the importation, distribution and transportation of the vaccines (among other acts) constituted offences under ss 32 and 33 of the GT Act. In committing the offences, the conduct of the respondents could now be restrained by the FCA under s 147(1) of the GT Act — although it was not clear whether the relief sought (the injunction) was intended to apply to the impugned activities in general or only insofar as they related to the applicant.

While the evidence relating to the applicant’s belief that the mRNA vaccines were, or contained, GMOs was submitted to the FCA, Rofe J disregarded this evidence because the question of law underlying the applicant’s action — were the mRNA vaccines GMOs? — was unneccessary to determine in these summary judgment proceedings. That was because the proceedings really concerned the anterior procedural question whether the applicant had standing to make the application.

The reason a summary judgment was delivered was because summary judgment had been sought by the respondents. They averred that the applicant lacked standing in reliance of s 147(1) of the GT Act, which effectively defined what an ‘aggrieved person’ was in respect of the GT Act:

If a person has engaged, is engaging, or is about to engage in any conduct that is or would be an offence against this Act or the regulations, the Federal Court of Australia (the Court) may, on the application of the Regulator or any other aggrieved person, grant an injunction restraining the person from engaging in the conduct.

The respondents submitted that the purpose of s 147(1) was to confine standing to persons aggrieved by actions done contrary to the GT Act [42]. Pfizer submitted, in effect, that not any person affected by (or even especially affected by) any product regulated or partially regulated by the GT Act would attain standing by virtue of their status as an affected person; instead, s 147(1) is drafted so as to narrow the scope of standing to those persons who are aggrieved directly by regulated dealings under the GT Act and where the regulation of those dealings are virtually the sole province of the GT Act. In other words, the provision, as Rofe J summarised the argument (at [42]),

is not intended to afford a broad remedy for any person with either general or specific grievances with GMOs arising from activities, uses or conduct not regulated by the GTA. This includes health and safety risks to consumers that may only be indirectly related to dealings under the Act, especially where such risks are specifically regulated by other statutory regimes or bodies.

As against this submission, the applicant contended that the GT Act has a far broader purpose, which includes the protection of the health and safety of the public. It thus followed that the applicant would have standing because, if the mRNA vaccines were or contained GMOs as the applicant submitted, and if this meant the mRNA vaccines had the potential to harm patients, then the applicant, as a person who had consumed the mRNA vaccines and had administered the mRNA vaccines, was a person protected by the GT Act, and in particular its object to regulate ‘risks to the end user or recipient of a GMO product, in particular the safety and efficacy of a therapeutic GMO product’ [43].

Given the above submissions, Rofe J understood the task of the proceedings as resolving the question of law whether the applicant was an aggrieved person based on the characterisation of the GT Act averred by the applicant. In this way, the case revolved not so much on whether the mRNA vaccines were, or contained GMOs, but on whether the applicant was protected as a consumer of a therapeutic good by the GT Act, whose purpose, as the applicant contended, was protective of such a class of persons.

On top of that, the applicant had to show that, even if this was accepted, that he, above others, had a ‘special interest’ in the alleged protective purpose of the Act, as was required by the general (common law) of standing [43]. This was always going to be a difficult thing to prove, because, after all, the applicant was one of millions of consumers of the mRNA vaccines, as well as one of thousands of health practitioner to have administered them to patients.

In the end, Rofe J found that the applicant did not have standing. The authority most analogous and relevant to the applicant’s submissions was Ogle v Strickland (1987) 13 FCR 306 (Ogle). That case involved a Christian ministers of religion identifying themselves as persons aggrieved by a decision by a censorship agency to permit an allegedly blasphemous film into Australia (see [129]). Although the authority of Ogle has been questioned several times since — including as observed by Jagot J in Australian National Imams Council Ltd v Australian Communications and Media Authority (2022) 404 ALR 323 at [91] — Rofe J found that her Honour was bound to follow the decision because it had not been overturned and the respondents did not challenge it.

Again, Ogle was a case in which certain Christian ministers argued that they were, and were found to be, persons aggrieved in respect of a decision of a censorship agency to permit an allegedly blasphemous film to be imported into Australia. The relevant statutory provision regarding aggrieved persons in that case appeared in s 5(1) of the Administrative Decisions (Judicial Review) Act 1977 (ADJR Act). That provision simply stated that certain aggrieved persons would be able to apply to the (then) federal court in various circumstances. Lockhart J found that the clergymen were persons aggrieved (at 318) notwithstanding that they did not have any legalistic special interest in the subject matter of the proceeding. As Lockart J wrote,

It is true that the appellants have no special interests in the subject matter of the decision in the sense of legal or equitable rights or proprietary or pecuniary interests; but they are persons aggrieved because to repel blasphemy is a necessary incident of their vocation. To deny them standing would deny an important class in the community an effective means and procedure for challenging decisions of the kind involved in this case.

When applied to the facts in this matter, Ogle enlivened an important test, which might be called the ‘necessary incident of the profession’ test. Applied to the medical profession, that question became ‘whether the matters agitated by Dr Fidge are a necessary incident of being a medical practitioner’ [131]. Rofe J’s consideration of this question is instructive for cases involving medical practitioners who seek remedies in circumstances where they allege a general legal breach and take action to protect patients generally. Rofe J found, in this case, that challenging decisions or actions of regulators did not form an incident of the medical profession in the same way as challenging a censorship decision did form an incident of the clergy in Ogle.

As Rofe J writes,

it is not a necessary incident of the medical profession that doctors oppose any or every breach of legislation that may (in their view) pose a risk to the health and safety of people. The sources of obligation referred to by Dr Fidge — the AHPRA Code of Conduct, Hippocratic Oath and AMA Code of Ethics — do not require doctors to challenge alleged breaches of legislation in order to “do no harm” or properly inform their patients. It is certainly not a necessary incident of being a doctor to challenge alleged breaches of the GTA. Nor do doctors have a professional obligation to “speak for all members of the Australian community” about potential health risks.

In my capacity as a legal scholar, I agree with Rofe J that neither the various ethical codes (Codes) regulating health practitioners in Australia, nor the Hippocratic Oath, go so far as to require, compel, or even permit health practitioners to challenge alleged offences under law to protect their patients. While the Codes do not explicitly prevent practitioners from challenging laws, they do not explicitly support such actions either. And consideration of the disciplinary actions taken by regulators responsible for enforcing the Codes soon indicates that, with respect to vaccines, health practitioners have been expected to have been bound to comply with the law in respect of protecting patients: see, eg, Health Care Complaints Commission v Parmenter [2023] NSWCATOD 136. In general, it might be said that compliance with the law to protect patients is an incident of the profession whereas challenging laws to allegedly protect patients is not.

In divining the legal character of Dr Fidge’s action, Rofe J concluded that it derived from an ethical position — one of ‘strong personal beliefs’ [135] — rather than a professional or legal duty:

Dr Fidge’s concern ultimately arises from a sense of moral obligation, not professional obligation or legal duty imposed on him as a medical practitioner. Although the ministers in Ogle may have felt that they had a moral obligation to challenge the decision of the Censorship Board, the majority in Ogle did not hold that a moral obligation is sufficient to establish standing.

Given that the applicant failed to demonstrate that he was an aggrieved person, the question of whether the mRNA vaccines were, or contained, GMOs did not arise. However, as I plan to explore in much more detail in future writing on this case, the decision does indicate that the purpose of the GT Act is to regulate GMOs on a ‘gap-filling’ basis, the result of which is that the GT Act does not regulate every therapeutic good that may or does contain a GMO, or something akin to a GMO. Rofe J ascertained this conclusion by means of a detailed purposive analysis of the GT Act as a whole, which included an independent construction of the text and (historical and legislative) context of the GT Act, as well as its interaction with other statutes, such as the Therapeutic Goods Act 1989 (Cth).

Indeed, the idea that the GT Act would be a ‘super regulator’ whose purpose was to become a ‘one stop shop’ for the regulation of GMOs and GM products in Australia, ‘regardless of whether the GMOs or GM products were also therapeutic goods, foods, agricultural and veterinary chemicals or industrial chemicals,’ was explicitly rejected by the Commonwealth Parliament when the GT Act was enacted (see [66]).

Instead, the GT Act was enacted explicitly as a gap filler directed at filling gaps related to ‘dealings with live viable organisms,’ such as ‘laboratory research involving the genetic modification of animals, plants, bacteria and viruses and the growing of crops, animals and fish.’ Where the Therapeutic Goods Administration (TGA) had approved a therapeutic product that contained certain gene-related products, the GT Act was not intended to, and did not, per force of its objects or legislative purpose, also apply as a second layer of regulation. Indeed, as I have argued in respect of this proceedings and more generally, the risk-based regulation (RBR) approach adopted to product regulation in Australia and abroad has always stood against duplication of this kind.

Thus, although Rofe J’s decision did not answer the question as to whether Dr Fidge, if found to have been aggrieved, would have been correct in identifying the respondents’ failure to obtain licences as breaches of the GT Act under ss 32 and 33, it is certainly apparent from her Honour’s analysis of the GT Act that such alleged breaches are unlikely to be made out by any future litigant.

Casgevy: bioethical concerns should not be written off just yet — even though the treatment has been approved

On BioEdge, Dr Patrick Foong and I quickly run through the bioethical perturbations that have come with the relatively rapid approval of Casgevy, the first-ever approved genome-editing therapy that utilises CRISPR/Cas 9. In some ways, it could be said that Casgevy is the first-ever approved genome therapy ‘full-stop’, because other categories of gene therapy have not been understood to create ‘edits’ to the human genome, as such.

For instance, Luxturna (and other AAV therapies) are used to ‘swap out’ mutated genes with ‘healthy’ or unmutated versions of the same. While this may seem like an ‘edit,’ it is also arguably a form of “gene replacement therapy.”

Similarly, forms of therapy using CAR T cells have been characterised as therapies that use cell reprogramming or engineering, rather than cell ‘editing,’ to create their therapeutic effects. This is because the immune cells that are extracted from a patient in a CAR T therapy process, and then reprogrammed to recognise and target cancer cells, is not as much a process by which genes or cells are ‘edited’ as much as one in which they are adapted, ex vivo.

As I have noted previously on Cells and Statutes, one of the most concerning aspects of the process in which Casgevy was approved was that the admitted lack of acceptable data about so-called off-target effects was not deal-breaker: neither for the FDA in the United States nor the MHRA in the United Kingdom.

Closer to home, it will be interesting to see what happens when an evidence dossier lands with the Australian regulator: the TGA. Given the ongoing pivot towards making cell and gene therapies faster to approve, the broader governmental investment in cell and gene therapies (see the 2021 senate inquiry on the future of cell and gene therapy here), and the historical tendency of the TGA to follow the FDA’s decisions, both for scientific and regulatory reasons, I suspect it will be approved without too much agony.

Exa-cel on review at the FDA

Just last week, on 31 October, the FDA’s Cellular, Tissue, and Gene Therapies Advisory Committee met to discuss Vertex Pharma’s Biologics License Application for Exagamglogene autotemcel (or exa-cel, and formerly known as CTX-001) — a cell-based gene therapy designed to treat sickle-cell diseases.

I have written about exa-cel many times before, both on this blog (here) and in published academic writing too. I have also spoken about it in this podcast. Exa-cel is a therapeutic product that is composed of the patient’s own (autologous) hematopoietic stem cells; however, those cells — specifically differentiation 34+ (CD34+) cells, have been edited using CRISPR/Cas 9 editing machinery (CRISPR).

In short, a CRISPR-Cas endonuclease system (CRISPRs) is a naturally occurring adaptive immune system that exists in most bacteria. These systems prevent bacteria from being infected by foreign genetic elements, such as viruses and phages. Where an infection exists, the Cas9 protein in the CRISPR will cleave or cut one strand each of double-stranded DNA to cause a double-strand break and thus decrease production of progeny viruses. Following that DSB, the genome will be repaired naturally, usually through a naturally occurring process called non-homologous end joining or NHEJ.

But this CRISPR/Cas 9 system can be ‘hijacked’ by science for therapeutic purposes. Using a guided template, the DSBs made by the Cas9 protein can be repaired in a precise and controllable manner, allowing the editing machinery of cell repair to be redirected toward doing repairs or edits that it would otherwise not do unguided.

This is how exa-cel works (in a nutshell). After the patient’s cells have been extracted, they are subject to guided disruption and repair by CRISPR. The CRISPR system make precise DSBs at the erythroid lineage specific enhancer region of B-cell lymphoma/leukemia 11A (BCL11A) gene on chromosome 2. In turn, this process disrupts GATA1 binding and abrogates BCL11A expression. Having turned off the expression of BCL11A, another gene, γ-globin (HBG1/HGB2) is expressed, creating fetal hemoglobin (HbF) production. It is quite complicated; but, in essence, the production of fetal hemoglobin allows people with sickle-cell diseases (red blood cells shaped like sickles because the cells are starved of oxygen) to be restored to health.

There are many issues and risks with this therapy, including the fact that sickle-cell disorder could be a protective disorder against malaria. But one especially concerning prospect, which is really at the core of the BLA on review currently, is the chance that the CRISPR may create cuts or DSBs at a site on the genome locus that is not in the right place. These misplaced or unforeseen cuts are known as ‘off-target effects’ or, alternatively, as indels — which means (usually unintended) insertions or deletions. The BLA puts the risks well:

One of the main concerns related to genome editing technology is risk of cleavage of genomic DNA at unintended sites due to imperfect pairing between the gRNA and the target DNA sequence. A subset of these imperfectly paired sites can be cleaved by the Cas9 endonuclease resulting in unintended edits across the genome. These sites can tolerate up to 6-mismatches between the gRNA and the genomic DNA. Since unintended edits can disrupt gene expression if present in the coding or regulatory DNA sequences, it is critical that the specificity of the gRNA be thoroughly screened to ensure off-target genome editing is minimized.

I am still trying to get my head around the recent report of the results of the off-target analysis present in the BLA. The FDA’s BLA report states as follows:

For the cellular off-target analysis, the Applicant used three samples from healthy donors and three samples from subjects with SCD of African American ethnicity. Given the impact of the SCD on [hematopoietic stem cell] function, which can potentially change the chromatin landscape and can impact off-target editing, the merits of using healthy donor samples for such analysis is not clear.

Additionally, it is not clear if the small number of samples used in the cellular GUIDE-seq offtarget analysis is sufficient to adequately assess off-target editing in exa-cel.

The report then continues: In Silico Analysis Off-Target Analysis Data for Exa-cel

The Applicant used three publicly available in silico algorithms to nominate potential off-target sites for the sgRNA SPY101 (Figure 6) based on its homology to the reference sequence.

Notably, however, when you get to the next page on the analysis of these risks, there are a number of redactions, no doubt because these are commercially protected contents that the regulator must not disclose. On first view, it appears that these ‘in silico algorithms’ to nominate potential off-target sites is, as is said below, a ‘part of the tool.’ I am not quite sure whether that means that the tool — the CRISPR system used be Vertex — is also the same system that conducts the off-target search, or something else. Have a read:

In any case, it looks like the so-called ‘indel frequency’ is very low. As the report noted later, “In this analysis, there were no statistically significant off-target editing events observed at any of the off-targets nominated using in silico analysis.” Although it remains unclear to me precisely how the indel assessment takes place, it is worth noting that the report’s view of the findings of the sponsor are very ambiguous, and tend towards a finding that the results of the study are inadequate. As the report notes in its conclusion of the safety summary section (4.1.2):

These changes have the potential to impact the chromatin landscape of SCD donor derived CD34+ HSPCs. Since chromatin accessibility can influence off-target activity, it is not clear if GUIDE-seq analysis of healthy donor derived CD34+ HSPCs can adequately capture potential off-target editing occurring in patient cells. However, availability of SCD donor cells can be limited and should also be considered. The Applicant used a total of four samples that were from donors of African American ethnicity. Three of these samples were from SCD donors that were used in the GUIDE-seq experiment and hybrid capture sequencing experiment, and one sample was from healthy donor that was used in the hybrid capture sequencing experiment. Given the limited number of SCD samples that were used in the cellular off-target analysis, it is not clear if the GUIDE-seq analysis adequately assessed the potential off-target editing by exa-cel.

Given the ambiguity of the FDA’s assessment, which states that Vertex’s pharmacovigilance plan is still under review, it remains to be seen whether more studies will need to be provided before the FDA consider exa-cel ready for the clinic.