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.

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.

Podcast interview: ‘Learn Me Right in Health Law and Bioethics’

Learn Me Right in Health Law and Bioethics is a podcast produced by QUT’s Australian Centre for Health Law Research. I was fortunate to be invited to join the podcast by its scholar producers, Sinead Prince and Ruthie Jeanneret, and to discuss my work on somatic cell genome editing.

One thing that I probably failed to do was to distinguish somatic cell genome editing (perhaps more helpfully described as non-heritable adult genome editing) from another type of therapy involving genes, which is known simply as ‘gene therapy.’ I failed to make this distinction, even though, on listening back, Sinead prompted me to do so when asking about whether gene treatments are a once-and-for-all cure. I should have understood this question to have probably been directed towards some of the gene therapies that are currently on the market, such as Luxturna, Yescarta, Zolgensma, Kymriah (tisagenlecleucel) or Hemgenix. Many of these therapies are a one-time treatment, and most of them are incredibly expensive. Hemgenix, for instance, which is a treatment for Hemophilia B, costs about 3.5 million US dollars. Isn’t that a remarkably expensive form of medicine?

The important distinction between gene therapies and adult genome editing is as follows. In a book chapter soon to be published authored by me and Dianne Nicol, we state the following:

Although there are various forms of genome editing, their key features are ‘programmable nucleases’ designed to create precise breaks in both strands of the DNA molecule (double strand breaks). Thus, somatic cell genome editing is usually distinguished from ‘traditional’ somatic cell gene therapy (SCGT) (World Health Organization 2021a)—or simply ‘gene therapy’—because the latter employs random insertion methods, including by using adenovirus-associated viral vectors (AVVs) or other delivery tools, to replace mutated genes or add new ones. The former, by contrast, seeks to alter existing nucleotides (Chang et al., 2018).

See Rudge and Nicol, “Bioethical decision-making about somatic cell genome editing: Sickle-cell disease as a case study” in Erick Valdés Meza et al, eds. Springer Handbook of Bioethical Decisions (Springer, forthcoming; contracted in 2022).

So, in short, gene therapies like those named above involve inserting new genes into the cellular DNA, including by replacing mutated genes with new genes (non-faulty genes) not associated with dysfunction. These new genes are ‘inserted’ through AAVs, which are versatile viral vectors that can be engineered for specific functions and are used as vectors through which new DNA can be delivered to target cells. It is important that these AAVs lack any viral genes but instead contain the ‘good’ replacement DNA sequences for the relevant therapeutic application.

In contrast, as can be heard in the podcast, non-heritable genome editing involves removing cells from a patient, usually hematopoietic stem cells, then editing those cells, and then reinfusing those edited cells back into the patient’s body. Those edited cells are usually edited with a CRISPR-Cas9 endonuclease, which may make targted breaks and then repair (using an RNA-guided nuclease) those double strand breaks (DSBs) in the human cellular DNA in a programmed way, so that, when the DSB is repaired, it is repaired in such a way that the cells are no longer pathogenic.

I should note that nuclease-mediated SCGE may occur either outside the body (ex vivo), as I discuss in the podcast, or inside the body itself (in vivo). I am unfamiliar with much of the latter actually going on. A third route, in which edited somatic cells may be administered to fetuses (in utero), is also under investigation.

In terms of the process that I am describing — which is an ex vivo application — when the extracted cells are taken from the patient’s body, they are separated from the so-called vascular fraction (heterogenous extraction of cells) so that enriched hematopoietic stem cells (CD34+ cells) can be isolated.

Following this ‘enrichment’ process, the cells are then edited by means on instrument called an electroporator. This instrument delivers a precise electrical pulse to the enriched cells in a protective medium that contains the CRISPR-Cas9–guide RNA complex. The electroporation increases the permeability of the cell membranes, allowing the RNA-guided nuclease to be introduced (or electrotransferred) into the cell. Another way in which cells are edited is through as process called nucleofection.

A description of both methods is given in Synthego’s guide to using CRISPR, in its chapter on transfection protocols, as follows:

Traditional electroporation and nucleofection are based on the same overall methodology. Cells are first mixed with an electroporation or nucleofection reagent, and then added to CRISPR components (shown here as RNPs). The cell-RNP mix is then put into a Nucleocuvette (for nucleofection) or Neon pipette tip (for electroporation) and inserted into a Nucleofector or electroporator. Once in the machine, an electric pulse is applied to the cells. The electric field temporarily causes pores to form in the plasma membrane, enabling RNPs to enter the cells. Nucleofection also enables nuclear entry of RNPs. Once the electric field is removed, the plasma membrane is repaired.

See How To Use CRISPR: Your Guide to Successful Genome Engineering, chapter 6.
nucleofection.svg
Source: Synthego, How To Use CRISPR: Your Guide to Successful Genome Engineering, chapter 6.

Following this process, the cells are left to recover, during which the editing, as it were, occurs. After some time, the cells are then cryopreserved to facilitate manufacturing quality analysis before being thawed. These edited cells are then reinfused in the patient.

Although I do not go into this kind of detail in the podcast, I do describe the manner in which one new somatic cell genome therapy for sickle-cell disease has been described in the provisional ‘proof of principle’ studies. That description is based on exa-cel (formerly CTX-001) — a genome editing therapy currently undergoing the regulatory submission provess in the US.

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.

Two conference papers to wrap up the year

In November I delivered two conference papers. I summarise them below.

From cell transplants to genome edits: Regulation and bioethics of existing and emerging interventions for sickle cell disease

This first paper was a ‘rapid-fire’ talk at the Australian Association of Bioethics and Health Law (AABHL) conference (‘Making Connections’) in Hobart, Tasmania, on 17 November. This talk summarised some of the work I have done on the bioethics of somatic cell genome editing with my colleague Prof Dianne Nicol. The most promising and translation-ready genome editing treatment around today seems to be CTX-001, manufactured by Vertex Pharmaceuticals. CTX-001 has been developed to treat sickle-cell disorders (SCDs).

SCDs comprise a group of genetic disorders of red blood cells (RBCs). Hemoglobin in RBCs usually carries oxygen from the lungs to the tissues and removes carbon dioxide. In the SCDs, a ‘sickle’ hemoglobin (‘HbS’) molecule is expressed within the RBCs. Generally speaking, if you have two HbS genes, you might have sickle-cell anaemia. If you have one HbS gene (heterozygous), you might have beta thalassemia.

The HbS molecule in the RBCs comes from the HbS gene. The HbS gene is a mutation thyat occurs when glutamic acid is replaced by valine. GAG becomes GTG on the HbS gene (at chromosome 11p15.5), which results in the HbS gene. Pathophysiologically, once the HbS gene mutation occurs, the HbS molecule within the RBCs results in a situation where, under reduced oxygen tension, you get polymerisation of the RBC, and this turns the cells (erythrocytes) into the characteristic sickle cell shape. When the RBCs are in this sickle shape, they obstruct blood flow, causing ischemias or vaso-occlusive crises. This then deprives the tissues of oxygen, creating respiratory issues that can be very serious.

CTX-001 is a new treatment to treat and potentially cure SCD. This paper examined SCD and its prevalence, identified its significant impacts on African American and African populations and analysed the bioethics of the best existing treatment (the allogeneic hematopoietic stem cell transplant, or a bone-marrow transplant). The paper then contrasted the bone-marrow transplant with the CTX-001, the yet-to-be-approved somatic cell genome therapy, and briefly noted the bioethical implications of administering CTX-001.

The paper was largely based on a book chapter contribution that I have written with Dianne Nicol titled Bioethical decision-making about somatic cell genome editing; Sickle-cell disease as a case study, which has been accepted by the editors of the Springer Handbook of Bioethical Decisions and will presumably be published in 2023. Slides from my talk (title page below) are viewable here.

Teaching constraints: Why we should (but don’t) teach the Commonwealth ‘spending’ power (among other things)

The second paper was presented at a symposium of law academics from around Australia held at the University of Sydney on 29 and 30 November and called Teaching Material: Symposium On The Pedagogy Of Political Economy In Australian Law Schools (program here).

This paper was one of my first serious attempts as a legal scholar to write about public finance law. In writing the paper, I learnt a lot, including from Will Bateman’s excellent book Public Finance and Parliamentary Constitutionalism (CUP, 2020). The essence of my talk was the ‘spending power,’ which is generally understood to be reposed in section 83 of the Australian Constitution. However, sections 81, 3 and 66 also deal with the Executive Government’s ability to spend by reference to Consolidated Revenue Fund, and so I made reference to those sections as well. But the main claims I was making were as follows.

There is no textbook dealing with public finance law in Australia, or in the UK, Canada or New Zealand; this represents an almost unbelievable lacuna in legal knowledge that shall continue to dog learning and epistemic understanding until we build a textual knowledge base.

There are three examples of how complicated public finance law can be; but, when we look at these examples, we can readily see (1) just how easily these complications can be resolved, and (2) why it is so important to resolve them.

The first complication is the notion of the spending power under s 81 of the Constitution; that power is not generally a parliamentary power but one exercised exclusively by the Executive Government of the Commonwealth; in other words, federal MPs not a part of the Executive are powerless to block spending or ‘block supply,’ with the effect being that there is really nothing anyone can do outside the Executive to control how much, or how little, the Commonwealth spend on its projects

The second complication is that the Consolidated Revenue Fund, which exists by dint of the ‘appropriations power’ (Constitution s 81; but also ss 83, 3, 66) is not actually a finite ‘kitty’ into which taxpayers’ taxes are deposited (eg, by the Australian Taxation Office), as appears to be largely assumed. Rather, the CRF is more complicated. It is a legal concept; and it is notionally self-executing and does not actually appear to be accessible, or its full quantum knowable, at any single point in time. This may be as confusing for students as it has been for apex courts!

The third complication is the seemingly unknown fact that special appropriations and standing appropriations can set aside (‘hypothecate’) or even ‘bake in’ an unspecified amount to be spent on a government program over an unspecified period of time. This means that spending can be effectively automated. For example, Medicare: will a person entitled to a rebate ever have their request declined because there are ‘insufficient funds’ in the CRF? No.

The point of bringing this into relief is to show that, as with legal knowledge about the legal ‘abstraction’ that is the CRF, knowledge about appropriations, and specifically ‘special appropriations’ (including knowledge about the common law and operations of the finance department), shows us: (1) precisely how government can lawfully spend money (for what purposes); (2) how it determined this (and it appears to be self-determined and open-ended (see Brown v West; Combet v Cth); (3) whether an appropriation that is an ‘Advance to the Finance Minister’ (AFM), for example, is lawful (it is: see Wilkie v Cth; Aus Marriage Equality Ltd v Minister for Finance); and (4) whether appropriations can include money that is not government money (seemingly they can, through ‘net appropriation’ agreements determined by the Finance Minister under the Financial Management and Accountability Act 1997 (Cth) s 31).

As the slides reveal, for each of these ‘complications’, there is a respective ‘teachable’ that shows us so much more about the reality of government spending. The paper then goes on to illustrate its claims through an example of a fictitious Act, the Bigger Medicare Act 2022 (Cth). I am hoping to write this up as a paper in the new year, once I finish some other work. The slides (title below) are viewable here.