Gene+Editing+in+Healthcare

=**Introduction **=

toc Genome editing is the practice that consists of making very specific changes in the DNA of any living organisms. It is done essentially by using enzymes or nucleases that have been specifically modified to affect a specific sequence of the DNA and change it [10]. There are different technologies that been developed around this practice, some more effective than others. The major goal of the practice is correct any genetic imperfection that natural mutation can create through reproduction. It is predicted to be very useful in healthcare but it still presents an array of issues that will be underlined below. The main difference between the different techniques in gene therapy is the kind of nuclease used to sever the DNA we are trying to edit.

**Background **
During the 1970s, gene therapy started to be developed but did not see its first trial until 1990. It was only seven years later in 1997, that the first clinical trial for infant HIV patient was conducted followed by a complete marrow ablation for an adult HIV patient in 1999. In 2002, a child in France was treated for Leukemia using gene therapy via intersectional mutagenesis [11]. =**Types of Nuclease **=

**ZFNs **
Zinc finger nucleases are artificial enzymes specifically designed to split a specific DNA sequence at two very precise points ins ide a complex genome. The particularity of this kind of nuclease is how they are obtained. This restriction enzyme is obtained by combining zinc finger DNA-binding domain and a nuclease which will do the splitting of the DNA inside the molecule. Consequently, a Zinc Finger Nuclease technology has been developed with the intent to cure HIV and genital herpes [9]. The main reason why Acquired Immune Deficiency Syndrome (AIDS) cannot be cured is because the HIV 1 provirus is actually inside the human genome and becomes part of it [8]. So in order to destroy the virus, the human genome has to destroy itself which can kill an organism. The Zinc Finger Nuclease technology helps to remedy to that dilemma. It essentially consists of targeting the [|proviral]HIV DNA and inducing a change of genetic information in the DNA which will results in a mutation [7]. The change is referred to as mutagenesis. This technology can effectively excise the virus from the human genome without any major damage of the gene.

However, the practice is not completely harmless. In order to administer the mutated genome, the patient has to go through a bone marrow transplant which represents more risks than the actual treatment. Moreover, by introducing more fingers into the genome, there is more possibility to sever the DNA and more spots than originally intended. There is also the cytotoxicity that tends to be really high with this practice and the cost to develop those enzymes is relatively high. These are the main challenges that prevent the technique to be available to everybody . For example, the case of Jesse Gelsinger, he was the first patient publicly identified as having passed away from the treatment of gene therapy. He was born with [|ornithine transcarbamylase deficiency] which is a genetic disease of the liver that prevents people from processing ammonia which is a byproduct of the decomposition of proteins. The clinical trial was conducted by the University of Pennsylvania and he got injected with an [|adenoviral vector] which was carrying the modified gene. Unfortunately, after only 4 days, he suffered a severe immune response as his body reacted aggressively to kill the viral agent that was carrying the gene which ultimately led to multiple organ failure and a brain death [12].

**TALENs **
Transcription activator-like effector nucleases are another kind of artificial enzymes engineered to cease a specific se quence of the DNA just like the Zinc finger nucleases. Unlike the ZFNs, their constitution is different. They are dimeric transcription nucleases that are constructed from 33 to 35 amino acides modules and each of them has the mission to target a single nucleotide [4]. When those arrays are assembled, they can be engineered to target any sequence in the DNA and cease it. TALENs are extremely precise but not very efficient when it comes to cutting the desired part of the DNA [5]. Both ZFNs and TALENs can be used any living organisms such as plants, humans and animals. Unlike what the scientific community initially thought. They thought that those two could only be used for mutagenesis in mouse embryonic stem cells [6].

**CRISPR **
<span style="font-family: Arial,sans-serif; font-size: 10pt;">Clustered Regularly Interspaced Short Palindromic Repeats is a group of DNA sequences in bacteria. Those sequences have fragment of DNA from viruses that have attacked the cell. The cell will subsequently use those fragments as information to recognize future attacks from any similar viruses and destroy them. The cell will identify the problematic sequence and destroy anything similar to that if it gets attacked again. A technology for gene editing was created based on this behavior. Jennifer Doudna and Emmanuelle Charpentier are the two scientists behind the breakthrough technology called CRISPR-Cas9. The technology was accidently developed while they were investigating how to fight viruses using bacteria. This technology could in theory manufacture specific properties for humans such as

<span style="font-family: Arial,sans-serif; font-size: 10pt;">high IQ, perfect vision or less susceptibility to have cancer [1].

<span style="font-family: Arial,sans-serif; font-size: 10pt;">The research was aimed at discovering how bacteria can destroy viruses. The reason why they were interested in bacteria is because of how they are structured. Many bacteria are constituted by an adaptive immune system that helps them survive by fixing any anomaly in their DNA. Using that unique properly, they found a way to introduce the bacteria into <span style="font-family: Arial,sans-serif; font-size: 10pt;">the virus DNA in order to make the bacteria think of that DNA as its own, which it would consequently fix by destroying. That would eventually destroy the virus. The part of the CRISPR system is this protein called Cas9. It is that protein that would act scissors to trim out the defective part of the DNA of the virus; hence, the name of the technology. The human DNA is a repository of information and trait that we primarily inherit from our parents whether they are good or bad. The technique unable to tinker with it would mean that we can not only correct any defective part of our genome but also potentially destroy the good parts. There is a risk of creating many unwanted mutations which could cause serious health complications [2]. While ground breaking, this discovery can also cause a lot of damage. For years now, it has been possible to do a test for disease predisposition. In fact, 7.9 million children around the globe are born with birth defect due to their inherited genes [3]. CRISPR-Cas9 while revolutionary, still need to be clinically tested on humans before being available to the public. It has the potential to prevent cancer or reduce the chances to have Alzheimer. The technology behind CRISPR-Cas9 is by far the most efficient, the simplest and the fastest of all methods so far. We can induce mutations on multiple genes at the same time by adding multiple gNRAs to the genes.

=**<span style="font-family: Arial,sans-serif; font-size: 10pt;">Conclusion **=

<span style="font-family: Arial,sans-serif; font-size: 10pt;">There is a lot more progress to do before having the technology ready because there are many issues that needs to be addressed. It is still extremely expensive to generate the necessary mutation and the medical procedure on humans so far is very dangerous. Furthermore, once the technology is fully developed and ready for clinical use, the ethical questions will need to be addressed as far as what scientist can and cannot do to any genome. It will need to be clearly defined like for any other technique or treatment to protect patients fearing to create more problems than solving. These revolutionary discoveries are milestones that will revolutionize healthcare.

=**<span style="color: #000000; font-family: Arial,sans-serif; font-size: 10pt;">References **=

<span style="font-family: Arial,sans-serif; font-size: 10pt;">[1] Doudna JA, Charpentier E (November 2014). "Genome editing. The new frontier of genome engineering with CRISPR-Cas9 <span style="font-family: Arial,sans-serif; font-size: 10pt;">[2] Mercola. 2017 Jun 13. The Very Real Dangers of New Gene-Editing Technology. <span style="font-family: Arial,sans-serif; font-size: 10pt;">[3] Darnovsky M. 2017 Oct 19. Pro and Con: Should Gene Editing Be Performed on Human Embryos? National Geographic. [accessed 2018 April 23]. https://www.nationalgeographic.com/magazine/2016/08/human-gene-editing-pro-con-opinions/ <span style="font-family: Arial,sans-serif; font-size: 10pt;">[4] Mohanraju P, Makarova KS, Zetsche B, Zhang F, Koonin EV, van der Oost J (2016). "Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems". <span style="font-family: Arial,sans-serif; font-size: 10pt;">[5] Barrangou R, van der Oost J (2013). CRISPR-Cas Systems : RNA-mediated Adaptive Immunity in Bacteria and Archaea. Heidelberg: Springer <span style="font-family: Arial,sans-serif; font-size: 10pt;">[6] Carroll, D (2011). "Genome engineering with zinc-finger nucleases". Genetics Society of America <span style="font-family: Arial,sans-serif; font-size: 10pt;">[7] Doyon, Y.; Vo, T. D.; Mendel, M. C.; Greenberg, S. G.; Wang, J.; Xia, D. F.; Miller, J. C.; Urnov, F. D.; Gregory, P. D.; Holmes, M. C. (2010). "Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures". Nature Methods <span style="font-family: Arial,sans-serif; font-size: 10pt;">[8] Shukla VK, Doyon Y, Miller JC, et al. (May 2009). "Precise genome modification in the crop species Zea mays using zinc-finger nucleases" <span style="font-family: Arial,sans-serif; font-size: 10pt;">[9] Morbitzer R, Elsaesser J, Hausner J, Lahaye T (July 2011). "Assembly of custom TALE-type DNA binding domains by modular cloning" <span style="font-family: Arial,sans-serif; font-size: 10pt;">[10] Valton J, Guyot V, Marechal A, Filhol JM, Juillerat A, Duclert A, Duchateau P, Poirot L (September 2015). "A Multidrug-resistant Engineered CAR T Cell for Allogeneic Combination Immunotherapy". <span style="font-family: Arial,sans-serif; font-size: 10pt;">[11] Friedmann T (2001) Stanfield Rogers: insights into virus vectors and failure of an early gene therapy model. Mol Ther 4(4):285–288 <span style="font-family: Arial,sans-serif; font-size: 10pt;">[12] Institute for Human Gene Therapy Responds to FDA – Almanac Between Issues". Upenn.edu. 2000-02-14. Retrieved 2010-11-16