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.

Chimeric monkey sheds light on what can be done with embryonic stem cells

I was quoted in this article today regarding a newly published study in Cell that demonstrates, in the author’s words, that ‘mammalian pluripotent stem cells possess preimplantation embryonic cell-like (naive) pluripotency.’ As the summary notes, this discovery about embryonic stem cells can now be said to have been demonstrated experimentally through the generation of a chimeric animal — a monkey whose embryonic development has been ‘complemented’ by homologous embryonic stem cells derived from another ‘donated’ line of cells. The monkey, in short, has developed from a blastocyst that is a compound of two embryonic stem cell lines.

Unsurprisingly, news stories have been focusing on one of the eye-grabbing aspects of this experiment: that the monkey in question has fluorescent green fingers and eyes. Unfortunately, the monkey died after only 10 days — which is still the longest period of time for which such a chimeric organism has lived before.

The reason that the monkey has these features is because the researchers used green fluorescent protein (GFP) to ‘label’ the embryonic stem cells (ESCs) that were incorporated into the host embryo at the blastocyst stage. And so what one is looking at when one sees the monkey with green fingers and eyes (visible even to the naked eye) is visual evidence that the embryonic stem cells have survived the process of being ‘complemented’ into the blastocyst of the host monkey and have spread throughout its body. In other words, the cells have been incorporated into the monkey’s cellular DNA; the monkey has both its ‘natural’ DNA and a ‘foreign’ line of DNA. Indeed, as the images indicate, there is a proliferation of these complemented ESCs throughout the monkey’s organs, including plenty in the brain and ileum (small intestine).

As the ‘Highlights’ section of the article points out, when these embryonic stem cells (ESCs) in the body of the monkey were ‘characterised’ (assessed), it was revealed that they remained in a so-called pluripotent state. In other words, the ESCs seem to have been able to differentiate into the different kinds of cellular categories: glial (brain) cells, heart cells (myocytes), lung cells (epithelial cells), and so on. Indeed, they continue to be in this pluripotent state, even as they maintain a ‘functional’ presence in the monkey’s body.

The news.com.au story quotes me as follows:

Sydney University lecturer in health law Dr Christopher Rudge told new.com.au the medical experiment had been on the cards for a long time.

“This is another step along the journey,” he said.

“The advancement here is that scientists have never been able to show such a prolific survival / proliferation of donated (or ‘complemented’) embryonic cells through a single organism.

“You’ve got more of these donated or secondary cells throughout the organism in a mammal.”

But he cautioned whether it would lead to anything substantive.

“Regenerative medicine has been hyped since the late 1990s,” he said. “Unfortunately it has not borne fruit.”

See https://www.news.com.au/technology/science/stunning-monkey-born-with-glowing-eyes-and-fingers/news-story/27d84700628476da1579968e76cbda5d

Obviously this scientific study demonstrates that certain new techniques can be adopted to expand the capacity of scientists to create chimeras. Scientists have long had the capacity to infuse mouse and rat blastocysts with pluripotent stem cells to generate live chimeric animals that feature this high proliferation of homologous cells. What is new here is that this capacity now extends to non-human primates — a species of animal much closer, in evolutionary terms, to humans.

It is arguably another step along the way in discovering how stem cells, including pluripotent embryonic stem cells, can be used as technologies of biological inquiry (for diagnosis, and to study developmental mechanisms) and, ultimately, to biological treatments. Of course, there is still so much more to learn.

Whether an experiment of this nature would be approved in Australia is an interesting question. If nothing else, this finding indicates that discoveries in stem cell medicine are continuing apace. Of course, given that this involved the effective fusing of two monkey embryos (or embryonic cell lines), the more serious bioethical questions regarding human-monkey chimeras, which have been posed before, do not arise in this instance.

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.

Updates from ‘Cellular Horizons’

Medical Research Future Fund project investigating how to to improve decisions about accessing cellular therapies

I recently presented updates from our MRFF project, which investigates how to improve decision-making, primarily among patients, about how and whether to access cellular therapies. The project has so far focused primarily on mesenchymal stem cell interventions: ie, ‘regenerative medicine’ treatments for osteoarthritis and conditions involving cell dysfunction. As a chief investigator in the ‘legal and regulatory affairs’ sub-team on the project, my focus has been on how these treatments are regulated.

New cellular therapies

However, as new cell-based interventions emerge as we speak, the project will likely also consider these novel interventions. In a very recent trial, immune cells (CAR-T cells) have been ‘programmed’ to attack cancerous T-cells by means of CRISPR ‘base editing.’ Specifically, this trial is aimed at treating a patient with T-cell acute lymphoblastic leukemia, or T-ALL.

There have been two issues for treating this disorder in the past.

The first relates to what is called T-cell aplasia. This is a process whereby the antigens on the T-cells are attacked, which destroys not only the cancerous T-cells but also destroys the normal T-cells. The means that T-cell numbers are decreased significantly.

Cell aplasia happens when patients undergo CAR T-cell therapy. Thus, when the cancer is a B-cell malignancy, the patient will generally experience B-cell aplasia. However, with B-cell aplasia, immunoglobulin replacement therapy can be administered to manage the problem. This is not so for T-cell aplasia. T-cell aplasia is not generally tolerated in humans, and persistent T-cell aplasia is life threatenting. Therefore, CAR T-cell therapy has generally not been possible for T-ALL.

The second issue is that CAR-T cells programmed to recognise and destroy T-cell antigens will inevitably attack healthy T-cells too in what is described as ‘T-cell fratricide.’ Sometimes, this problem is called ‘T v T’ fratiricide. This is described in the video below, which is a basic introduction to base editing published by Great Ormond Street Hospital:

While it is an extremely complicated molecular process, in CRISPR base editing, nucleotide bases in donor cells are edited at the atomic level so that the gene for CD7 (a genetic marker in blood cancers) is changed from cytosine to a thymine. In this process, the base editing produces a so-called ‘stop codon’ that terminates the production of CD7 (acting like a molecular ‘full-stop’). These edited cells are then transplanted into the patient to treat relapsed lymphoblastic leukaemia.

In an article published in Leukemia, authored by the team that are conducting the trial, the authors write that

Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells.

https://doi.org/10.1038/s41375-021-01282-6

While it is far too early to determine whether base editing might present an option to patients with T-ALL and whose options are exhausted, it is promising to see that base editing appears to be possible in humans. Using CRISPR base-edited cells in humans represents a new form of cellullar therapy — it could be called somatic cell genome editing; the only other trial for SCGE that I’m aware of is the trial for exagamglogene autotemcel  or exa-cel (formerly known as CTX-001); however, that is not a base-edited genome therapy. Rather, Exa-cel uses CRISPR to treat blood disorders (hemoglobinopathies) such as sickle-cell disorder.

Unlike T-ALL base editing treatment above, where base editing is applied to donor cells, exa-cel edits the patient’s own cells (ie, it is an autologous treatment), which are removed prior to the treatment. With the cells removed, the patient is given ablative therapy while the hematopoietic stem cells are edited using CRISPR-Cas9 to produce high level of fetal hemoglobin. This treatment promises to ensure that vaso-occlusive crises (blocked blood flow, depriving tissues of oxygen, and usually caused by the ‘sickle’ shape of the red blood cell), which is a symptom of the blood disorders, is avoided.

New Priority Review Pathway for Biologicals

In the context of such cellular therapies emerging in recent times, the Australian drug regulator, the Therapeutic Goods Administration, has recently introduced new expedited pathways for drug sponsors and manufacturers to fast-track novel biological therapies (another name for therapies involving cell and tissue products) through the approval process. In a recent article published in the Journal of Law and Medicine, our team analysed the new ‘priority pathway,’ which has since been approved. We also wrote a submission to the TGA, which can be found here.

In early December, I presented some of our work on these matters to another team of experts working on cellular therapies in Australia. Presentation slides from that meeting are available here.

Earlier in the year, in April, I presented a brief introduction on the same subject to our internal team. That presentation is also made available here.