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.
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.