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.

https://link.springer.com/chapter/10.1007/978-3-030-49127-7_6

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.

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.

https://www.fda.gov/media/173414/download

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.

https://www.fda.gov/media/173414/download

The report then continues:

4.1.1.1 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.

https://www.fda.gov/media/173414/download

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.

New book chapter on law/bioethics of somatic cell genome editing (and sickle-cell diseases)

After a long journey that should have been no surprise whatsoever (not when one looks at the scale and size of this volume), a book chapter I authored with Emeritus Distinguished Prof Dianne Nicol from the University of Tasmania’s Centre for Law and Genetics, way back in 2021, has now been published.

The chapter deals with the bioethics of somatic cell genome editing, which, so it is said — and as the literature mostly suggests — does not raise many ethical issues at all. But that, of course, is not quite right. ‘SCGE’ — like all cellular therapies — presents an array of bioethical and biopolitical challenges to patients, including those related to safety and accessibility.

This is not least because those treatments that are now emerging, the frontrunner among which is exa-cel (previously CTX-001), are designed to treat hemoglobinopathies — that is, blood diseases — that disproportionately impact people in sub-Saharan Africa. And it appears they will be prohibitively expensive.

Additionally, the treatment of these disorders may have impacts beyond our current predictions. After all, the sickle-shaped red blood cells (RBCs) that this treatment will treat and resolve have also been identified as providing protection against malaria. That’s because haem — a component of haemoglobin — was found to confer advantage in experimental studies.

In essence, haem, which is present in a free form in the RBCs of mice with the sickle cell trait, but mostly absent from mice without the trait, seems to protect against malaria. When mice without sickle cells were injected with haem, and then later infected with malaria, it was found that the injected haem helped to guard against malaria in those previously normal — and malaria-unprotected — mice. It follows, as the authors note, that ‘Sickle human hemoglobin (Hb) confers a survival advantage to individuals living in endemic areas of malaria, the disease caused by Plasmodium infection.’ The question, then, is whether this conferred advantage will change at all among those who are treated for sickle-cell diseases, such as beta thalassemia, with exa-cel. I should note, however, this is not a question explicitly addressed in the chapter.

Instead, our chapter deals with the technical history of somatic cell genome editing, and the way in which this form of genome editing is different to heritable genome editing. SCGE is not heritable because it is performed on post-natal humans whose gametes (reproductive cells) have fully materialised and developed. Once the gametes have developed, they maintain their genomic content for life (as far as we know). By contrast, genome editing prior to fertilisation (ie, genome editing performed on IVF or ART embryonic cells prior to their being fertilised) is heritable, and will carry over to the future generations.

Although the science of SCGE might sound relatively boring when compared to heritable human genome editing, it really is not. For a start, SCGE is happening right now. It could be decades — perhaps centuries — before we start editing pre-fertilised human embryos using CRISPR Cas-9. But we are using the same technology — CRISPR Cas-9 endonucleases — on adult stem cells today. Speaking generally of SCGE (and not specifically about exa-cel), the process is essentially this:

  • cells are removed from the adult patient’s body;
  • the patient undergoes ablative therapy to kill all pathogenic/diseased cells (eg, the sickle RBCs) in their body (and clearly they are in a very serious condition at this time);
  • the patient’s removed cells are shipped off to a laboratory, where they are subjected to the action of the endonuclease (the CRISPR) through flow electroporation or a similar technique (which enables the CRISPR to work by manipulating the materials at a nuclear level);
  • the resulting ‘product’ of the ‘manufacturing’ process is the therapeutic good (ie, exa-cel)
  • the CRISPR-Cas is essentially a cutting and repair tool that cuts and then repairs double-strand breaks in human DNA (changing the nucleotides as it repairs them);
  • the repairs created by the CRISPR-Cas are ‘guided’ or played out on a ‘template’; and
  • the edited cells, which are not affected by disease (because they have been edited) are then reinfused into the body, and replace the cells that existed previously

There is much more to say about SCGE, but that is all in the chapter. I also wrote about it earlier this year, on this website, here.

But it’s great to have the book chapter finally out there. I attach a few images of the contents pages to illustrate the huge scope of the volume — which is one of two. The book’s full title is the Handbook of Bioethical Decisions, Volume I: Decisions at the Bench. It is edited by Erick Valdés and Juan Alberto Lecaros, whom I thank for including our chapter; and it is published by Springer.