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.

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.
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.

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.