13.2 Gene Editing

Genetic Engineering

Many types of genetic engineering have yielded clear benefits with few apparent risks. Few would question, for example, the value of our now abundant supply of human insulin produced by genetically engineered bacteria. However, many emerging applications of genetic engineering are much more controversial, often because their potential benefits are pitted against significant risks, real or perceived. This is certainly the case for gene therapy, a clinical application of genetic engineering that may one day provide a cure for many diseases but is still largely an experimental approach to treatment.

Historically, clinical trials have shown the clear hazards of attempting genetic manipulation in complex multicellular organisms like humans. In some patients, the use of an adenovirus vector can trigger an unanticipated inflammatory response from the immune system, which may lead to organ failure. Moreover, because viruses can often target multiple cell types, the virus vector may infect cells not targeted for the therapy, damaging these other cells and possibly leading to illnesses such as cancer. Another potential risk is that the modified virus could revert to being infectious and cause disease in the patient. Lastly, there is a risk that the inserted gene could unintentionally inactivate another important gene in the patient’s genome, disrupting normal cell cycling and possibly leading to tumor formation and cancer. Because gene therapy involves so many risks, candidates for gene therapy need to be fully informed of these risks before providing informed consent to undergo the therapy.

No discussion of gene editing would be complete without introducing CRISPR. Genome editing (also called gene editing) is a group of technologies that give scientists the ability to change an organism’s DNA. These technologies allow genetic material to be added, removed, or altered at particular locations in the genome. Several approaches to genome editing have been developed. A well-known one is called CRISPR-Cas9, which is short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9. The CRISPR-Cas9 system has generated a lot of excitement in the scientific community because it is faster, cheaper, more accurate, and more efficient than other genome editing methods.

CRISPR-Cas9 was adapted from a naturally occurring genome editing system that bacteria use as an immune defense. When infected with viruses, bacteria capture small pieces of the viruses’ DNA and insert them into their own DNA in a particular pattern to create segments known as CRISPR arrays. The CRISPR arrays allow the bacteria to “remember” the viruses (or closely related ones). If the viruses attack again, the bacteria produce RNA segments from the CRISPR arrays that recognize and attach to specific regions of the viruses’ DNA. The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which disables the virus.

Researchers adapted this immune defense system to edit DNA. They create a small piece of RNA with a short “guide” sequence that attaches (binds) to a specific target sequence in a cell’s DNA, much like the RNA segments bacteria produce from the CRISPR array. This guide RNA also attaches to the Cas9 enzyme. When introduced into cells, the guide RNA recognizes the intended DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location, mirroring the process in bacteria. Although Cas9 is the enzyme that is used most often, other enzymes (for example Cpf1) can also be used. Once the DNA is cut, researchers use the cell’s own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence.

Genome editing is of great interest in the prevention and treatment of human diseases. Currently, genome editing is used in cells and animal models in research labs to understand diseases. Scientists are still working to determine whether this approach is safe and effective for use in people.

CRISPR – Applications in Humans

Applications of CRISPR in humans include treatment for sickle cell disease, which causes severe pain and premature death in millions of people worldwide. Scientists use CRISPR to treat sickle cell disease by removing blood stem cells from a patient with sickle cell disease, editing the genome of those cells to remove the sickle cell mutation, and then re-insert the modified cells into the person’s bone marrow.

Another CRISPR application now entering human clinical trials aims to combat human immunodeficiency virus (or HIV) infection. HIV enters human white blood cells and then alters those cells’ genomes. Then, it makes copies of itself to infect the person’s immune system, making them vulnerable to other infections. CRISPR is now being investigated for use in either cutting out the HIV-derived DNA from the genome as well as engineering a person’s genome so that HIV cannot enter their cells. However, it is important to stress that these techniques are still relatively new and very much still in testing mode.

Oversight of Gene Therapy

Presently, there is significant oversight of gene therapy clinical trials. In the US, at the federal level, three agencies regulate gene therapy in parallel: the Food and Drug Administration (FDA), the Office of Human Research Protection (OHRP), and the Recombinant DNA Advisory Committee (RAC) at the National Institutes of Health (NIH). Along with several local agencies, these federal agencies interact with the institutional review board to ensure that protocols are in place to protect patient safety during clinical trials. Compliance with these protocols is enforced mostly on the local level in cooperation with the federal agencies. Gene therapies are currently under the most extensive federal and local review compared to other types of therapies, which are more typically only under the review of the FDA. Some researchers believe that these extensive regulations actually inhibit progress in gene therapy research. In 2013, the Institute of Medicine (now the National Academy of Medicine) called upon the NIH to relax its review of gene therapy trials in most cases (Grens, 2013). However, ensuring patient safety continues to be of utmost concern.

In Canada, gene therapy products are regulated in the same way as other pharmaceuticals, under the Food and Drugs Act and Food and Drug Regulations (Jorgensen et al., 2024). Gene therapy products are classified by Health Canada as biologic drugs, which are under the purview of the Health Canada (HC) Biologic and Radiopharmaceutical Drugs Directorate (Jorgensen et al., 2024). Gene therapy products qualify for expedited approval via HC Notice of Compliance with Conditions (NOC/c), approving drugs that have evidence of potential to treat serious life-threatening or life-limiting conditions, subject to further testing. As such, these drugs are usually not subject to phase 3 clinical trials (Jorgensen et al., 2024).

As of June 6, 2024, there were ten approved gene therapy products in Canada, including treatments targeting rare and severe conditions such as blood cancers and spinal muscular atrophy (Jorgensen et al., 2024). Six of these are CAR-T therapies for blood cancer treatment and the other four are AAV-based therapies that restore gene function (Jorgensen et al., 2024). As of November 21, 2024, there were 41 FDA-approved gene and cell therapies in the US, including treatment for several types of cancer, Duchenne muscular dystrophy, hemophilia A and B, type 1 diabetes, spinal muscular atrophy, and sickle cell disease (USFDA, 2024).

In 2024 Health Canada approved CASGEVY® (exagamglogene autotemcel), an autologous CRISPR-Cas9 genome-edited hematopoietic stem cell therapy to treat sickle cell disease (SCD). This is the first CRISPER-based gene editing therapy approved in Canada. For those who qualify, this will bring relief for this severely debilitating and progressive disease. However, the cost of these medications is exorbitant and it is unclear in the long term whether provincial healthcare plans or insurance companies will continue to pay. CASGEVY, for example, costs $2.2 million for a one-time treatment (Watt, 2024). According to Watt (2024), LENMELDY is one of the most expensive at $4.25 million per dose. However, the cost of managing SCD using other previously existing therapies, including hospital stays for crisis management, is comparable, if not more (Watt, 2024).

Ethical Concerns

Beyond the health risks of gene therapy, the ability to genetically modify humans poses a number of ethical issues related to the limits of such “therapy.” While current research is focused on gene therapy for genetic diseases, scientists might one day apply these methods to manipulate other genetic traits not perceived as desirable, which brings us back to the discussion on eugenics. This raises questions such as:

• Which genetic traits are worthy of being “corrected”?
• Should gene therapy be used for cosmetic reasons or to enhance human abilities?
• Should genetic manipulation be used to impart desirable traits to the unborn?
• Is everyone entitled to gene therapy, or could the cost of gene therapy create new forms of social inequality?
• Who should be responsible for regulating and policing inappropriate use of gene therapies?

What are off-target effects?

There are concerns that CRISPR might inadvertently alter regions of the genome other than the intended ones. These are called “off-target effects.” The worry is that CRISPR could change a beneficial gene, such as disabling a tumor-suppressing gene or activating one that causes cancer. Another concern is that because no two people’s genomes are identical, identifying off-target effects in individuals may be impossible. Researchers attempt to predict where in the genome off-target effects might occur using web-based algorithms, but there are concerns that this approach is not accurate enough.

In May 2017, an article published in the journal Nature Methods reported an alarming number of off-target mutations in mice whose genomes had been edited using CRISPR. However, experts voiced skepticism of the finding because only two mice were edited and unusual methods used. Scientists are attempting to address these concerns by developing more precise variants of the Cas9 enzyme used in the CRISPR system. Some of these enzymes have been shown to improve targeting in human tissue in the lab. Researchers have also focused on developing methods to more efficiently locate off-target mutations in the animals they study.

Somatic vs. Germline Editing

The ability to alter reproductive cells using gene therapy could also generate new ethical dilemmas. To date, the various types of gene therapies have been targeted to somatic cells, the non-reproductive cells within the body. Because somatic cell traits are not inherited, any genetic changes accomplished by somatic-cell gene therapy would not be passed on to offspring. However, should scientists successfully introduce new genes to germ cells, the resulting traits could be passed on to offspring. This approach, called germ-line gene therapy, could potentially be used to combat heritable diseases, but it could also lead to unintended consequences for future generations. Moreover, there is the question of informed consent, because those impacted by germ-line gene therapy are unborn and therefore unable to choose whether they receive the therapy. For these reasons, the U.S. government does not currently fund research projects investigating germ-line gene therapies in humans.

Following the announcement about the work of He Jiankui, in 2019, scientists called for a five-year global moratorium on all clinical uses of human germline editing (Ladner et al., 2019). The aim was to allow for a period of discussion about the potential medical, societal, and ethical issues germline editing might pose. This was to be followed by a period where nations would choose how to proceed and whether they would continue to impose a ban. The hope was for transparency and open communication amongst the scientific community. The ban did not apply to research using germline editing, provided there was no transfer of embryos to a human uterus (Ladner et al., 2019). In Canada, the TCPS2 guidelines, article 13.7, section G, addresses research involving gene transfer. It directs readers to the Assisted Human Reproduction Act which prohibits altering the human genome or in vitro embryo such that the alteration can be passed on to subsequent generations (Government of Canada, 2022). While there are no laws or regulations in many countries prohibiting germline editing, the moratorium is strictly voluntary. It has now been five years and, despite the potential ethical issues remaining, there is discussion that South Africa might be the first country to accept germline editing.

What are gene drives?

A gene drive is a natural phenomenon whereby the inheritance of a particular gene or set of genes is favorably biased, resulting in the increase in its frequency in the population. Gene drives can arise through a variety of mechanisms, and scientists have proposed using gene editing to engineer gene drives for specific purposes. These include preventing the spread of insects that carry pathogens, such as mosquitoes that transmit malaria, dengue, Zika and other diseases.

Here is how it works: This system uses genetically modified male mosquitos to deliver new genes along with a mechanism for copying the new sequences from one member of a chromosome pair to the other. In other words, a mosquito larva has a gene that came only from its father, yet has it as both a paternal and maternal copy. Thus, even a recessive gene will manifest its trait in all offspring. Furthermore, the offspring will spread the gene and trait to their own offspring. Since mosquitoes have a short life cycle, this means that in the course of just a summer, we could alter almost the entire population of a particular mosquito species in say the Brazilian rain forest, possibly wiping out the Zika disease. In August 2016 the U.S. Food And Drug Administration (FDA) issued a “Finding of No Significant Impact” to biotech company Oxitec’s plan to release genetically modified male Aedes aegypti mosquitoes into the Florida Keys.

Engineered gene drives have also been proposed to control invasive species, such as rodents that eat the eggs of endangered bird species in New Zealand, and for eliminating herbicide and pesticide resistance in crops.

Concerns about gene drives include the possibility that a mutation could happen during the engineered gene drive, which could spread unwanted traits with the drive. The spread of some other disease could be unexpectedly facilitated. Or the elimination of a link in the food chain could harm the local ecology. It’s also plausible that something could happen akin to the introduction of rabbits in 19th century Australia, where the population exploded, due to lack of predators, with major consequences for the ecosystem. There are also worries that an engineered gene drive could move beyond its target population, causing unintended impacts on other species and ecosystems.

Anti-biotechnology activists including Vandana Shiva, Jane Goodall and David Suzuki have advocated against the use of gene drives. In August 2017, they joined with other radical environmental groups to issue a well-publicized opposition statement [PDF] to gene drive technology, writing:

Given the obvious dangers of irretrievably releasing genocidal genes into the natural world, and the moral implications of taking such action, we call for a halt to all proposals for the use of gene drive technologies, but especially in conservation.

In 2016, the National Academy of Sciences issued its wide-ranging review of dozens of studies, Report on Gene Drives in Non-Human Organisms [PDF], which outlined a number of potential risks but urged more research and gave a cautious green light to “highly controlled field trials.” Some studies have come to different conclusions, among them: researchers at the University of California, San Diego and colleagues at Harvard created a mathematical model for CRISPRs likely success, concluding the a gene drive could be remarkably aggressive in the wild, spreading a new gene beyond its targeted population—possibly meaning that experiments in the real world are too risky on a case by case basis at this stage in the technology’s development.

What is “bio-hacking” and “DIY-bio?”

Do-it-yourself biology, also called “biohacking” or “DIY bio,” is a movement in which people are experimenting with biotechnology research and development methods outside of traditional research institutions. Some “biohackers” are trying to make these methods easier and more accessible, so that even non-scientists can use them. Because of its relative ease to deploy, CRISPR experiments can be performed even by high school students.

One of the most accessible forms of biohacking is through engineering microorganisms or plants. Experiments range from using plasmids to create fluorescent bacteria, controlling gene expression using light in bacteria and even using CRISPR to engineer the genomes of bacteria or yeast. Some biohackers have begun selling kits that allow you to use CRISPR at home. One kit, created as part of an Indiegogo crowd-funding project, was sold for $130 by biohacker Josiah Zayner.

The Future of CRISPR

Despite the serious ethical challenges, CRISPR/Cas 9 is a promising technology to treat a multitude of conditions.

Attribution & References

Except where otherwise noted, this page is adapted from 12.4 Gene Therapy In Microbiology by Nina Parker, Mark Schneegurt, Anh-Hue Thi Tu, Philip Lister and Brian M. Forster, CC BY 4.0. Access for free at Microbiology (OpenStax)

• CRISPR – Applications in Humans from Genome Editing, courtesy of National Human Genome Research Institute (NHGRI), Public Domain with attribution
• Paragraphs on gene therapy products in Canada and Ethical Issues spotlight written by Andrea Gretchev, CC BY-NC 4.0
• Genome Editing and Human Disease from What are genome editing and CRISPR-Cas9? In Help Me Understand Genetics Courtesy of MedlinePlus from the National Library of Medicine, Public Domain with attribution

References

Jorgensen, P.l., Heir, P., Daniele, D., & Wall, K. (2024, June 6). IP monitor: Gene therapy products in Canada: Regulation, IP and emerging issues. Norton Rose Fulbright.

Government of Canada Panel on Research Ethics. (2022). TCPS2 (2022) – Chapter 13: Human genetic research.

Grens, K. (2013, December 9). Ease Gene Therapy Reviews [Report]. The Scientist.

Lander, E. S., Baylis, F., Zhang, F., Charpentier, E., Berg, P., Bourgain, C., Friedrich, B., Joung, J. K., Li, J., Liu, D., Naldini, L., Nie, J. B., Qiu, R., Schoene-Seifert, B., Shao, F., Terry, S., Wei, W., & Winnacker, E. L. (2019). Adopt a moratorium on heritable genome editing. Nature, 567(7747), 165–168. https://doi.org/10.1038/d41586-019-00726-5

Sibbald, B.. (2001). Death but one unintended consequence of gene-therapy trial. Canadian Medical Association Journal, 164(11), 1612–1612. https://pmc.ncbi.nlm.nih.gov/articles/PMC81135/

US Food and Drug Administration (USFDA). (2024, November 21). Approved Cellular and Gene Therapy Products. https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellular-and-gene-therapy-products

Watt, A. (2024, August 2). Casgevy: Launch sequence and price analysis of the first marketed CRISPR therapy. Pharmaceutical Technology.