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CRISPR Genomics Precision Medicine

The State of Gene Therapies

Nima Karimi — McMaster University Health Sciences 2023

As the quest of mankind for optimal health continues,  different avenues for achieving this goal have emerged. One area, in particular, is looking at the role of genes in the pathophysiology of diseases, and consequently, investigating therapeutics that target those genes. Genes are the basic functional unit of heredity (1); in other words, they determine your height, the color of your eye and hair, and many other biological traits. Importantly, alterations of the genes and genome have been consistently linked to many pathological conditions. Cystic fibrosis (CF), sickle-cell anemia, and Huntington’s disease are some of the more prominent examples of genetic disorders (2). As these conditions have led to increased mortality and reduced quality of life, the scientific community has searched for potential therapeutics; one, in particular, being gene therapy.

The first human studies on gene therapy were done in the early 1990s. One such experiment involved the transfer of genes coding for a specific enzyme, to patients with severe combined immunodeficiency, which showed promising results (3). Since then, research into gene therapy has grown considerably. More recently, some studies have used gene therapy to treat CF, a progressive genetic condition that leads to the loss of lung function with no current treatment (4). Interestingly, the use of gene therapy in CF patients have shown to improve lung capacity (3). With CF being one of the most prevalent genetic disorders, the use of gene therapy shows a promising future for the treatment of this condition.

Now you may be wondering to yourself, how is genetic therapy actually done? Generally speaking, gene therapy involves the identification of cell types and DNA sequences that are defective, and then introducing a new DNA sequence containing the functional genes to offset the effects of the disease-causing genetic alterations (5). There are two different approaches to gene therapy (figure 1), and these involve alterations of different types of cells (6). Somatic gene therapy involves the transfer of DNA to different cells in our body that do not produce sperm or eggs. Given that these changes are not in the germline, any DNA alteration cannot be passed on from parents to their children (6). In contrast, germline gene therapy involves the transfer of DNA to cells that produce eggs or sperms, meaning these changes can be inherited (6).

Additionally, various techniques are being used in gene therapy. One such technique is gene augmentation therapy (6). This technique can be used to treat genetic abnormalities  that stem from mutation, where the gene in question does not produce its functional products (6, 7). As shown in figure 2, this therapy adds the DNA containing a functional gene back into the cell that is defective and ultimately, can reverse the abnormality (8). Another technique involves gene inhibition (6). This technique can be used in pathologies in which the overexpression of certain genes is causing the disease. In this approach, the aim is to introduce a new gene that either inhibits the expression of another faulty gene, or interferes with the activity of the product produced by the faulty gene (9), as shown in figure 3.

Overall, it is clear that gene therapy presents a very promising future for the treatment and management of diseases that were once deemed incurable. Today, there are more than 600 genes and cellular therapies that are being researched (10). In the coming years, one could expect the emergence of many genetic therapies for common and rare conditions. This emergence could both provide treatments for patients that lack therapeutics today, and also improve their overall quality of life.

References

1.         Kitcher P. Genes. The British Journal for the Philosophy of Science. 1982;33(4):337-59.

2.         Conrad Stöppler M. Genetic Diseases (Disorder Definition, Types, and Examples): MedicineNet;  [Available from: https://www.medicinenet.com/genetic_disease/article.htm.

3.         Steffin DHM, Hsieh EM, Rouce RH. Gene Therapy: Current Applications and Future Possibilities. Advances in Pediatrics. 2019;66:37-54.

4.         Davis PB. Cystic Fibrosis Since 1938. American Journal of Respiratory and Critical Care Medicine. 2006;173(5):475-82.

5.         Verma IM, Naldini L, Kafri T, Miyoshi H, Takahashi M, Blömer U, et al., editors. Gene Therapy: Promises, Problems and Prospects. Genes and Resistance to Disease; 2000 2000//; Berlin, Heidelberg: Springer Berlin Heidelberg.

6.         What is gene therapy? 2021 [Available from: https://www.yourgenome.org/facts/what-is-gene-therapy.

7.         Frazier S. Embryo Gene Editing: Changing Life As We Know It 2019 [Available from: https://the-gist.org/2019/05/embryo-gene-editing-changing-life-as-we-know-it/.

8.         Nóbrega C, Mendonça L, Matos CA. Gene Therapy Strategies: Gene Augmentation. In: Nóbrega C, Mendonça L, Matos CA, editors. A Handbook of Gene and Cell Therapy. Cham: Springer International Publishing; 2020. p. 117-26.

9.         James W. Towards Gene-Inhibition Therapy: A Review of Progress and Prospects in the Field of Antiviral Antisense Nucleic Acids and Ribozymes. Antiviral Chemistry and Chemotherapy. 1991;2(4):191-214.

10.       Dorholt M. We’re on the Verge of a Breakthrough for Gene Therapies 2021 [Available from: https://www.evernorth.com/articles/the-history-and-future-of-gene-therapy.

Categories
CRISPR

CRISPR and Ethics: a Tough yet Riveting Conversation

Jennifer Kraliz—McMaster University Honours Biology 2023

This past fall, biochemist Dr. Jennifer Doudna and microbiologist Dr. Emmanuelle Charpentier were awarded the Nobel Prize in Chemistry 2020 for their development of CRISPR/Cas9 gene editing technology (1). Charpentier and Doudna’s discovery has significantly progressed genome editing and has redefined the life sciences, unlocking new research avenues and an abundance of potential for further biotechnology and healthcare advancements  (1). Achieved through several different technologies, genome editing allows scientists to alter the DNA of organisms, which leads to phenotypic changes, such as in hair colour or disease susceptibility (2). The first of these technologies were invented in the late 1900s, essentially acting like scissors, cutting the organism’s DNA in certain places so that scientists can detach, add, or replace DNA segments (2). This procedure was drastically advanced in 2009 with the invention of the CRISPR method, making genome editing easier, cheaper, and faster than ever before (2).

Figure 1: CRISPR and other genome-editing technologies are often referred to as genetic scissors, or a “cut and paste” tool for DNA
SOURCE: Illustration by Johan Jarnestad, The Royal Swedish Academy of Sciences, for The Nobel Prize

Consisting of guide RNA and the DNA-cutting enzyme Cas9, the CRISPR (Clustered Regularly Interspaced Short Palindromic) tool was adapted from an immune defense against viruses observed in bacteria (3). With it, scientists can inactivate genes, add in new segments of DNA, and even edit single nucleotide bases (3)Due to the universal and foundational position DNA holds, genome editing tools have countless applications across many fields of the life sciences.

Gene therapy for humans, however, is arguably one of the most remarkable. CRISPR/Cas9 and other genome editing technologies have great potential to treat diseases with genomic bases, such as cystic fibrosis (2). Research on CRISPR-based cancer treatment exploded after the first U.S. trial tested it in 2019, and deemed it feasible (3). As full of potential CRISPR is, it is extremely important to remember its novelty, and therefore its imperfection. CRISPR is undoubtedly fascinating and influential, but it is far from being an errorless method of genome editing (2). In using the CRISPR method, there is risk of altering off-target DNA and mosaicism, which could have unpredictable, detrimental effects on the individual’s phenotype (4). The concerns surrounding the CRISPR/Cas9 technology and other technologies alike do not end there. The effects of germline therapies, which edit the genes of reproductive cells, are passed down from generation to generation. This raises concerns about interference with human evolution (2). Furthermore, it has also been proposed that germline editing could produce hierarchical classes or divisions among people, delineated by the quality of their engineered genome (4). Another potential aspect of genome editing that could cause further harm and alienation is its cost and accessibility. What if gene therapies are expensive and only accessible to the wealthy? This could worsen the health gap between the rich and the poor (2). Many believe that genome editing used to treat diseases could very easily lead to people using it for enhancement or non-health related purposes (4). Beliefs on what is considered a disease or an impairment to one’s health are not universal. How will genetic enhancement be managed by policy and regulation (4)? Who will make the decisions concerning this regulation? How will political agendas and religious beliefs affect the outcome of these decisions? 

In summary, genome editing technology is a powerful tool, and many fear it landing in the wrong hands. Current bioethical discourse on the issue often includes a comparison of gene editing technology to selective human reproduction, calling it a “renewal of eugenics” (5). Whether or not the potential benefits of genome editing technology outweighs its potential harm is an extremely difficult quandary to navigate. Many assert that restricting research and development in human genome editing is unethical because it completely eliminates any chance of a positive impact.Whatever one’s stance on the issue may be, it is undeniable that genome editing and CRISPR technology has opened a door for humankind that cannot be closed.

References

  1. The Nobel Prize in Chemistry 2020 [Internet]. NobelPrize.org. [cited 2021 Feb 19].Available from: https://www.nobelprize.org/prizes/chemistry/2020/popular-information/
  2. What is genome editing? [Internet]. Genome.gov. [cited 2021 Feb 19]. Available from:https://www.genome.gov/about-genomics/policy-issues/what-is-Genome-Editing
  3. How CRISPR Is Changing Cancer Research and Treatment – National Cancer Institute [Internet]. 2020 [cited 2021 Feb 19]. Available from: https://www.cancer.gov/news-events/cancer-currents-blog/2020/crispr-cancer-research-treat ment
  4. What are the Ethical Concerns of Genome Editing? [Internet]. Genome.gov. [cited 2021 Feb 19]. Available from: https://www.genome.gov/about-genomics/policy-issues/Genome-Editing/ethical-concerns
  5. Ranisch R. ‘Eugenics is Back’? Historic References in Current Discussions of Germline Gene Editing. Nanoethics. 2019 Dec 1;13(3):209–22.

Categories
CRISPR

CRISPR: the Future of Cancer Research

Paige Chandran Blair—McMaster University Biochemistry 2023

Introduction

Cancer has become one of the leading global public health issues, exemplified by its status as the second leading cause of death worldwide in 2015 (1). The magnitude of cancer’s impact is predicted to surge over the next two decades, with the instance rate expected to increase by 70%. Despite humanity’s long awareness of cancer, treatment design has proved to be incredibly difficult as cancer involves complex physiological alterations, several mutations, translocations and chromosomal insertions/deletions. Cancer involves changes in genetic material that either turn genes off (such as tumour suppressors), or turn genes on (such as oncogenes). This ultimately leads to unregulated cellular growth (2). Consequently, gene editing is rising to the forefront of developing treatments for cancer,with the CRISPR-Cas system leading the way. 

CRISPR Technologies

The CRISPR-Cas technique is based on a prokaryotic immune defence mechanism (1). Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems involve guide RNAs (gRNA) and an endonuclease known as the CRISPR-associated (Cas) protein (3). Guide RNAs are designed by researchers to identify and guide the Cas DNA cutting enzyme to the target DNA where Cas cuts the DNA to creates a double-stranded break, illustrated in the figure below.

How CRISPR Is Changing Cancer Research and Treatment - National Cancer  Institute
Figure 1: Mechanism of CRISPR-Cas9 technology showcasing the recognition of a target sequence, its cleavage, and the insertion of new genetic material. 
SOURCE: National Cancer Institute (2020)

Once the target gene is cut, the DNA can either be altered to inactivate the gene and create ‘knockouts’ or new segments can be added to create novel cellular functions. Researchers have taken advantage of this system because it presents the opportunity to accurately and easily make modifications at the genetic level such as adding and removing specific nucleotides without requiring separate cleaving enzymes (4). 

Current CRISPR-Based Cancer Treatments: 

CRISPR is a valuable asset to cancer treatments because cancer consists of several mutations making it difficult to target. Due to CRISPR’s versatility, accuracy, and precision, it is widely applicable to many branches of cancer research. CRISPR is currently driving research in cancer screening technologies, and providing drastic improvement on immunotherapies such as Chimeric Antigen Receptor (CAR) T-cell therapy (5). CRISPR-based applications to cancer screening are currently being conducted to identify genes related to drug resistance/sensitivity, genes pertaining to cellular vulnerability to environmental toxins, and genes with a role in the development of diseases. CAR T-cell therapy is a leading immunotherapy in which a patient’s T cells are removed, altered to express the chimeric antigen receptor (CAR) so they are better able to bind and kill cancer cells, then grown in large quantities, and reinserted into the patient as illustrated in the figure below (6).

PDF] Neurological updates: neurological complications of CAR-T therapy |  Semantic Scholar
Figure 2: General process of CAR T-Cell Therapy from synthesis of CAR T-cells to insertion back into patient. 
SOURCE: National Cancer Institute (n.d.)

CAR T-cell therapy has been previously limited due to a lack of proliferation and persistence of CAR T-cells in toxic tumour microenvironments common to patients with advanced stages of cancer (7). However, novel CRISPR-edited CAR T-cells have demonstrated increased potency against tumours. The CRISPR system is applied to enhance T-cell function by ‘knocking out’ a protein that limits CAR T-cell activation (8). 

The Future of CRISPR Research: 

CRISPR based technologies currently have several limitations which future studies must overcome. First, future technologies must improve the delivery of CRISPR treatments to cancerous cells (3). Currently, two major methods are being developed to address this delivery problem; viruses and nanocapsules. Viruses are exploited to deliver CRISPR components to a specific organ (e.g. liver) using a virus which naturally targets that organ, while nanocapsules are designed to bring CRISPR components directly to target cells. Human cancer research is solely ex vivo, meaning that cells are removed and edited outside of the body as described with CAR T-cell therapy. However, future treatments may move beyond this and begin in vivo treatment design. Finally, while treatments such as CAR T-cell therapy have had several positive results in treating hematologic malignancies (i.e. blood cancers) efficacy has yet to be demonstrated in treating solid tumours (7).With CRISPR based technologies rising to the forefront of personalized cancer treatments, new hope is growing in research to address cancer’s future health threat.

References

(1) Ratan ZA, Son Y-J, Haidere MF, Uddin BMM, Yusuf MA, Zaman S Bin, et al. CRISPR-Cas9: a promising genetic engineering approach in cancer research. Ther Adv Med Oncol [Internet]. 2018 Jan 1 [cited 2021 Feb 19];10:1758834018755089. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29434679 

(2) CRISPR Cancer Research [Internet]. Synthego. 2021 [cited 2021Feb19]. Available from: (3) NCI Staff. How CRISPR Is Changing Cancer Research and Treatment [Internet]. National Cancer Institute. 2021 [cited 2021Feb19]. Available from: https://www.cancer.gov/news-events/cancer-currents-blog/2020/crispr-cancer-research-treatment 

(4) Questions and Answers about CRISPR [Internet]. Broad Institute. 2018 [cited 2021Feb20]. Available from: https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/questions-and-answers-about-crispr

(5) Spencer NY. Overview: What is CRISPR Screening [Internet]. Integrated DNA Technologies. Integrated DNA Technologies; 2019 [cited 2021 Feb 19]. Available from: https://www.idtdna.com/pages/education/decoded/article/overview-what-is-crispr-screening

(6) NCI Staff. CAR T-Cell Therapy [Internet]. National Cancer Institute. [cited 2021 Feb 19]. Available from: https://www.cancer.gov/publications/dictionaries/cancer-terms/def/car-t-cell-therapy

(7) Salas-Mckee J, Kong W, Gladney WL, Jadlowsky JK, Plesa G, Davis MM, et al. CRISPR/Cas9-based genome editing in the era of CAR T cell immunotherapy. Hum Vaccin Immunother [Internet]. 2019 May 4 [cited 2021 Feb 19];15(5):1126–32. Available from: https://www.tandfonline.com/doi/full/10.1080/21645515.2019.1571893

(8) University of Pennsylvania School of Medicine. CRISPR-edited CAR T cells enhance fight against blood cancers [Internet]. ScienceDaily. ScienceDaily; 2020 [cited 2021Feb19]. Available from: https://www.sciencedaily.com/releases/2020/12/201207161535.htm

Categories
CRISPR

CRISPR and Genome Editing: a Split Path

Areeba Imran—McMaster Honours Life Sciences 2023

Should scientists genetically modify children the way plant biologists genetically modify corn? Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene-editing makes this a reality to take into consideration. This technology has the ability to edit human genomes to alter DNA sequences and consequently change gene function. (1) It does this by inserting cuts or breaks of DNA to disturb the natural sequence of the human genome. (1) The changed sequence can then be programmed to trick DNA repair mechanisms into deleting unwanted genes. (1)

SOURCE: Drug Target Review

CRISPR has the potential to remove unfavourable genetic mutations that have burdened the lives of many children and adults. Examples of common genetic diseases are down syndrome, cystic fibrosis and sickle cell anemia. (2) The root cause of genetic diseases, many of which have no cure, is in our DNA’s blueprint. As a result, people either live their entire lives suffering from their symptoms or die prematurely without the luxury of fulfilling their dreams.

Cómo se produce una mutación genética?
SOURCE: HealthWorld

CRISPR gene-editing has not reached its full potential in the medical field because of various ethical concerns and social implications. (3) One of the major concerns against CRISPR is that it is considered an unnatural intervention. However, the intention behind using this technology is no different from the reason we readily use prescription medications and allow other medical interventions like surgeries. The intention is to provide the best care to everyone, so they can wake up healthy every morning ready to turn their dreams into a reality. If used with the right intention, CRISPR has the ability to make genetic diseases a thing of the past. 

The most significant setback to making CRISPR a readily available technology is its impact on our society. (4) Would it lead to a society where everyone strives to be similar? Will differences no longer be celebrated? With a gene-editing technology available, people may look into it as a way to mitigate themselves from unfavourable genes and lead to a more uniform society. In doing this, diversity which is currently highly celebrated, especially in countries like Canada, with its multicultural idealism, will no longer hold such value.

Lorazepam pour dormir ce que c'est, les doses et les effets secondaires /  Drogues psychotropes | Psychologie, philosophie et réflexion sur la vie.
SOURCE: Pharmaceutical Pain Management

In addition to its various social implications, CRISPR can also be a promising solution used for vaccinations. (5) This may be very beneficial in today’s world, where the COVID-19 pandemic has changed our lives. It has been shown that CRISPR can engineer white blood cells to produce antibodies to respond to a disease like COVID-19 without exposing the body to the disease. (6) However, more clinical trials must be done before this technology can be used in this way. (5) With the COVID-19 variants on the rise and no foreseeable end to this pandemic, further research into CRISPR technologies may help vaccinate our population quicker and thus warrants our attention. 

The question remains: Are we willing to overlook the drawbacks of CRISPR gene-editing technology if it means limiting the occurrences of incurable genetic diseases and future pandemics? It is important to realize that every new technology comes with its disadvantages, and investing more time and research may lead to discoveries on ways to limit these drawbacks. Do we have the right to stop technology from taking over our world, or is it our duty to advocate for these changes?

Coronavirus & COVID-19 Overview: Symptoms, Risks, Prevention, Treatment &  More
SOURCE: WebMD

References 

1. Vidyasagar A. What Is CRISPR? [Internet]. LiveScience. Purch; 2018 [cited 2021Feb20]. Available from: https://www.livescience.com/58790-crispr-explained.html 

2. What You Need to Know About 5 Most Common Genetic Disorders [Internet]. Regis College Online. 2020 [cited 2021Mar29]. Available from: https://online.regiscollege.edu/blog/information-5-common-genetic-disorders/

3. Locke LG. The Promise of CRISPR for Human Germline Editing and the Perils of “Playing God.” The CRISPR Journal. 2020;3(1):27–31 

4. Doudna JA, Sternberg SH, Rosa WDL. Opinion: Should we use gene editing to produce disease-free babies? A scientist who helped discover CRISPR weighs in. [Internet]. ideas.ted.com. 2017 [cited 2021Feb20]. Available from: https://ideas.ted.com/opinion-should-we-use-gene-editing-to-produce-disease-free-babies -a-scientist-who-helped-discover-crispr-weighs-in/ 

5. Bussler F. 3 Ways CRISPR is Used to Fight the Coronavirus [Internet]. Medium. Data Driven Investor; 2020 [cited 2021Feb20]. Available from: https://medium.com/datadriveninvestor/3-ways-crispr-is-used-to-fight-the-coronavirus-33 19eddd6906 

6. Eatwell E, Maertens V, et al. Could CRISPR Create a COVID-19 Vaccine? [Internet]. BRINK. 2020 [cited 2021Feb20]. Available from: https://www.brinknews.com/crispr-and-the-fight-against-covid-19/