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Genetic Engineering

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When asked about gene therapy, what do you think of? A medication to treat faulty genes? a scientist with a really small knife going into your cells and cutting out the faulty genes? Perhaps the introduction of a new gene into your body to treat an illness you may have. 

 

Since secondary school, I've heard teachers and even some friends throwing around the phrase “gene therapy” , “crispr” and “genetic engineering” without ever fully understanding what it is and what it does. So today, I'm doing exactly that, so please sit back, grab some genetically modified popcorn and enjoy as I explore the miniature world of gene therapy and genetic engineering.

Prologue

First let us define gene therapy. Gene therapy involves the altering of genes inside your body in an effort to treat a disease, this can be done by replacing the faulty gene or adding a new gene to mask the faulty one.

Gene therapy may then be divided into 2 sub categories depending on the type of cells they impact:

  • Somatic cell gene therapy is when therapeutic genes are transferred into all cells EXCEPT stem cells and reproductive cells. Hence the gene alterations are not inherited by the subjects offspring.

  • Germline gene therapy, as its name suggests, impacts solely the reproductive cells, hence these genetic changes are inheritable by the subject's offspring.

Viral Vectors

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The next logical train of questioning is how then do we add the therapeutic genes to our all our cells and or remove the faulty gene. Surely we cant be expected to modify the genome of each of our cells one by one, that would quite literally take forever. Well, the answer is actually surprisingly obvious.Viruses! Yeap those little buggers that caused us and are still causing us a world of issues (especially right now with covid). 

 

Viruses or Viral Vectors are often used to administer the new gene into the patent. Their mechanism of administration would vary depending on the type of viruses in question. The main 3 types are:

  1. Retroviruses: inserts a copy of its RNA genome into the DNA of the host cells, changing the genome of the host cell. 

  2. Adenoviruses:  do not integrate into the genome and do not replicate during cell division. 

  3. Lentiviruses: they are a subtype of retrovirus but with the ability to integrate its genome into non-dividing cells whereas retroviruses only infect dividing cells.

 

Apart from viruses however, there are also non-viral ways (non-viral vectors) in which we are able to introduce the gene into the host patient. However, they produce a much lower level of transfection and gene expression of the therapeutic gene.

 

Now that we have a rough idea of what genetic therapy is and its mechanism of action, let's take a look at some of the uses of gene therapy and the diseases in which it has been treated.

Severe combined immunodeficiency (SCID)

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SCID is a rare genetic disorder that is characterized by the disturbed/imparied development of the functional T and B lymphocytes. Now to fully grasp how debilitating this condition is, let us first learn a little bit about the immune system.

 

The adaptive immune system can be divided into 2 mechanisms. The Humoral part consists of B lymphocytes which are involved in the “GAMED” (acronym) antibody production. The Cellular portion consists of the T lymphocytes: Thymus, Helper T & Killer T. Both of these immune mechanisms help to fend off invasive pathogens and work collaboratively to ensure our body is free of viruses and bacteria etc. Now,an impairment in one of these systems is bad, but not terrible, a person can still get away with his day to day activities with only one of the 2 systems functioning properly,but if BOTH of them aren't functioning, like in the case of SCID, then a whole slew of problems will arise. Most cases of SCID are due to mutations in the IL2RG gene which helps in the collaboration with proteins for the formation of lymphocytes. The gene codes for common gamma chains, a cytokine receptor, located on the surface of immature blood-forming cells.

 

SCID patients essentially have no immune system, so any infection, bacteria or virus like a cold is lethal to them. Hence they have to be kept inside an enclosed sanitary “bubble” their entire lives to ensure no pathogens reach them. It is also a X chromosome linked recessive disease, hence it has been unofficially dubbed the “bubble boy disease”

 

So given the debilitating nature of this illness, we have tried various forms of treatment methods and medication, but to no avail. But then came Gene therapy. Though not a full proof method, gene therapy has provided much more promising results during clinical trials compared to other treatment methods because it alters the very genome of cells, targeting the IL2RG gene. The first clinical trial of gene therapy was conducted on 12 child SCID patients in 2002. However, from this trial, 4 patients developed leukemia a few months later due to the accidental insertion of a gene-carrying retrovirus near an oncogene,setting back the progress of gene therapy a couple years. More recently though, gene therapy has been gaining more popularity again with a 2021 clinical trial on 50 children with SCID showed 48 exhibiting positive results through a new method using an altered version of the HIV virus as a lentivirus vector. 

 

One current shortfall to gene therapy is that it can only be used to treat (so far) single gene disorders such as hemophilia or muscular dystrophy. But why is that? This leads us very nicely to the next segment of this article, Crispr gene editing.

Crispr Gene Editing

Crispr is a genetic engineering technique in molecular biology by which genomes of living organisms may be modified. ( I mean what could possibly go wrong with people trying to play god right?)

Crispr is actually an acronym for: 

  • Clustered

  • Regularly

  • Interspaced

  • Short

  • Palindromic

  • Repeats

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So let's clarify what some of these terms shall we,cause there are some pretty big and intimidating words here. “Clustered'' simply means the genes are all in the same region. “Interspaced” means that between repeats of RNA in the crispr system will be “spacer DNA”. Palindromic means a sequence of letters AKA nucleotides that are read the same both forwards and  backwards, like EYE. From the above image, the short palindromic repeats are the DNA sections found between the Spacer DNA.

Spacer DNA is essentially a tool used by typical bacteria to identify invasive bacteriophages, essentially acting like memory T cells in our immune system, storing and recognizing the DNA of previous bacteriophages. 

At the end of this DNA sequence are “CAS genes” which make 2 proteins, DNA helicases and nucleases which are essential for the mechanism of CRISPR gene editing.

Crispr-CAS system (bacteria)

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From my poorly drawn representation of a bacteria, there are 2 distinct mechanisms of action for the bacteria when a virus injects its RNA into it, and this will depend on whether the RNA injected is recognized (ie found) in the Crispr-Cas system. 

In the first scenario (1), the bacteria injects an RNA that the bacteria recognizes since its found along Crispr as one of its spacer DNAs. In this case, the CAS genes will transcribe and translate proteins called the CAS complex. Concurrently, the crispr genes will transcribe the matching DNA of the virus's RNA and insert it into the CAS complex protein. Hence this creates what is referred to as crRNA that breaks apart the viral RNA that was injected by the virus.

In the second scenario (2), the injected RNA is not recognized among the crispr system, meaning it's a new type of virus that is trying to infect the bacterium. In such a case where the matching spacer DNA to the virus RNA is not found, the CAS genes create a class 1 CAS protein that takes in the viral RNA. The new RNA is then integrated as spacer DNA into the crispr system allowing the bacteria to now recognize the viral RNA and hence create the necessary CAS complex proteins and matching DNA for scenario 1 to occur.

It is actually scenario 2 in which scientists are most intrigued about, IE the cutting and integrating of a new DNA into the existing crispr genes.

CRISPR CAS 9 system

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The CRISPR CAS 9 system allows us now to pick and choose the specific gene which we want to integrate into a patient's gene. This diagram looks pretty confusing, I know it took be about 2 hours to understand it, so lets go through this system systematically shall we (pun intended)

Starting from the, the entire object shown is the CAS9 protein (nuclease) which is inserted into usually a viral vector where the therapeutic gene is allowed to propagate throughout the patients cells as the virus infects its host.  (gRNA) stands for guide RNA and it represents the “crisr” portion of this system as it contains the gene that needs to be cut, like a guide for the CAS9 nuclease. The CAS9 nuclease itself is the “cutter” that cleaves the DNA of cells at the specified gene locus as guided by the gRNA. dsDNA basically represents the host cell's DNA which is being fed through the CAS9 nuclease… because it's a nuclease. Hence as the cell's genome runs through the CAS9 nuclease, when a gene which corresponds to that of the gRNA is located, the CAS9 nuclease cuts away the specific gene. Upon this action, 2 different modifications may occur depending on the type of treatment provided:

  • Knock-in mutations is the scenario where the the faulty gene is cut out and replaced by a specific gene to provide a therapeutic effect (Homologous directed repair HDR)

  • Knock-out mutations result in the repair of the double stranded of the double stranded dna by non-homologous end joining NHEJ, which essentially means the faulty gene is deleted from the cells genome.


However it isn't enough to just understand the mechanism of CRISPR. We need to also consider some of the ethical concerns of using gene editing and perhaps some societal issues which may ensue if gene editing becomes more widespread and easily accessible.

Ethical issues of genetic engineering

Well let's first look at some of the upsides of genetic engineering and the potential it holds for the healthcare industry and our overall well being. Reusing the same example of  clinical trials on SCID patients mentioned earlier on in this article, it is evident that gene editing may become a viable solution to cure diseases which are otherwise deemed untreatable. Hence given the nature of such diseases and their debilitating nature and impact on affected people, a strong point can be made for the benefits of genetic engineering. Additionally, this isn't just limited to diseases in our current population, but also the unborn population. A Statistically significant amount of babies are borned with some genetic defects,due to no fault of their own, and would have to live with it for the rest of their lives. Given that its a right for everyone to be born free of diseases or ailments and deformities, then genetic engineering could provide a method for us to prevent such illnesses in our offspring. However it can also be argued that babies also have a right to remain genetically unmodified, with some people staunchly against genetic engineering on moral or religious grounds as they view it as “playing with fire”. 

 

In fact there is also an interesting story about this.

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In 2018,during the second international summit of human genome editing, Chinese researcher He Jian Kui announced to the world that he had genetically modified and brought to term “perfect babies' '.  From this announcement He received immense social pressure and outcrys from scientists who dubbed what he had done to be blatantly unethical and morally wrong.

 

Another area of concern would be gene doping where gene therapy is used for non-therapeutic purposes to enhance and improve the performance of athletes and competitors. While it remains true that many sports are already strewn with doping issues, gene doping encompasses a broader scope as it may spill over into intellectual competitions as well, with genes being introduced which may help with memory etc. Hence in my personal opinion as a rugby player and also semi-powerlifter, I think that advances in gene therapy such as CRISPR poses a threat to many sporting or competitive events, taking away the whole concept of rewarding someone for their hard work and “good genes” and compromising the spirit of competitions where people are driven to reach their win to the best of their own natural born abilities. 

 

So yea, there are certainly a lot of things to consider moving forward if more advances in gene editing are. I believe that comprehensive restrictions and regulations need to be put in place such as to prevent the misuse of such technology for selfish gain or unethical purposes.

References

  1. BBC. (2019, April 17). HIV used to cure 'bubble boy' disease. BBC News. Retrieved January 22, 2022, from https://www.bbc.com/news/world-us-canada-47969367

  2. Biolabs, N. E. (n.d.). CRISPR/Cas9 & targeted genome editing: New Era in Molecular Biology. NEB. Retrieved January 20, 2022, from https://www.neb.sg/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology

  3. bozemanbiology. (2016, February 18). What is CRISPR? YouTube. Retrieved January 20, 2022, from https://www.youtube.com/watch?v=MnYppmstxIs

  4. Cable News Network. (2018, November 29). Scientist defends gene-editing babies - CNN video. CNN. Retrieved January 22, 2022, from https://edition.cnn.com/videos/world/2018/11/29/gene-edited-babies-alexandra-field-lkl-vpx.cnn

  5. CRISPR clinical trials: A 2021 update. Innovative Genomics Institute (IGI). (2021, October 29). Retrieved January 20, 2022, from https://innovativegenomics.org/news/crispr-clinical-trials-2021/

  6. CRISPR injected into the blood treats a genetic disease for first time. Science. (n.d.). Retrieved January 20, 2022, from https://www.science.org/content/article/crispr-injected-blood-treats-genetic-disease-first-time

  7. Cyranoski, D. (2016, November 15). CRISPR gene-editing tested in a person for the first time. Nature News. Retrieved January 20, 2022, from https://www.nature.com/articles/nature.2016.20988

  8. Full stack genome engineering. Synthego. (n.d.). Retrieved January 22, 2022, from https://www.synthego.com/blog/crispr-scientists

  9. Huidu, A. (1970, January 1). Tailoring humans: The ethics of Genetic Engineering. IGI Global. Retrieved January 20, 2022, from https://www.igi-global.com/chapter/tailoring-humans/273067

  10. Key considerations for gene therapy commercialization. Cell Culture Dish. (2021, April 26). Retrieved January 22, 2022, from https://cellculturedish.com/key-considerations-for-gene-therapy-commercialization/

  11. Khan, L. (2019, April 7). Rewriting ourselves with CRISPR. Medium. Retrieved January 22, 2022, from https://medium.com/@ttlkhan/rewriting-ourselves-with-crispr-cdfae3dfd299

  12. Mayo Foundation for Medical Education and Research. (2017, December 29). Gene therapy. Mayo Clinic. Retrieved January 20, 2022, from https://www.mayoclinic.org/tests-procedures/gene-therapy/about/pac-20384619

  13. Molteni, M. (2018, April 27). Everything you need to know about CRISPR gene editing. Wired. Retrieved January 20, 2022, from https://www.wired.com/story/wired-guide-to-crispr/

  14. The Public Engagement team at the Wellcome Genome Campus. (2021, October 25). What is CRISPR-Cas9? Facts. Retrieved January 20, 2022, from https://www.yourgenome.org/facts/what-is-crispr-cas9

  15. TEDtalksDirector. (2015, November 12). HOW CRISPR lets us edit our DNA | Jennifer Doudna. YouTube. Retrieved January 20, 2022, from https://www.youtube.com/watch?v=TdBAHexVYzc

  16. U.S. National Library of Medicine. (2021, May 12). What is gene therapy?: Medlineplus Genetics. MedlinePlus. Retrieved January 20, 2022, from https://medlineplus.gov/genetics/understanding/therapy/genetherapy/

  17. Wikimedia Foundation. (2022, January 17). Gene therapy. Wikipedia. Retrieved January 20, 2022, from https://en.wikipedia.org/wiki/Gene_therapy#Gene_doping

  18. Wikimedia Foundation. (2022, January 9). CRISPR gene editing. Wikipedia. Retrieved January 20, 2022, from https://en.wikipedia.org/wiki/CRISPR_gene_editing#/media/File:CRISPR_transfection.png

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