CRISPR And The Effectiveness Of Gene Therapy

The Foundation of CRISPR

Clustered regularly interspaced short palindromic repeats or CRISPR as it is more commonly known would not be possible without the many scientific discoveries that enabled scientists to understand DNA. Many discoveries enabled this, such as Watson and Crick and Rosalind Franklin, however the most important was the use of recombinant DNA.The first use of recombinant DNA for humans occurred in 1986 when Pablo D. T. Valenzuela created the first recombinant vaccine(Broad Institute 2018). This vaccine was so successful that it immediately replaced the blood-derived vaccine that was currently in place. Following his success came Francisco Mojica’s discovery of the principles of CRISPR where he found the DNA in bacteria repeated and that it matched the viruses that attacked it. A mere three years later, in 1989 the first approved human gene therapy trial of Rosenberg was conducted. Then, in 1999 the entire human genome began to be sequenced, which was finished in 2003 (Broad Institute 2018). During this time, the first ever gene targeted drug therapy was approved to treat chronic myelogenous leukaemia (CML), a type of cancer. About ten years later, however, came the most significant achievement to date: the discovery of the CRISPR-Cas9 process. A few short years later, CRISPR was being used on humans. In 2015, a human embryo was edited with CRISPR for the first time by Junjiu Huang at the Sun Yat-Sen University in Guangzhou. (Pinello et. al 2019) However, his experiment was considered unethical by western medicine and thus was rejected until 2018. In 2018, Vertex Pharmaceuticals and CRISPR Therapeutics, created a joint initiative of experimental treatment for the blood disorder B-thalassemia which was approved to start clinical trials (Broad Institute 2018). This has paved the way for future uses of CRISPR, beyond the traditional methods of merely editing corn, cloning sheep, and other GMO products, to perhaps curing diseases in humans. CRISPR is now the most efficient way to edit genes which is what makes gene therapy possible.

Defining Gene Therapy

According to the United States National Library of Medicine, “Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient’s cells instead of using drugs or surgery” (United States National Library of Medicine 2019). Gene therapy is being tested via multiple approaches such as replacing disease causing genes with copies of the healthy gene, inactivating or damaging a mutated gene so that it ceases function, and introducing new genes into the body to fight diseases. Gene therapy is an incredible, new, experimental treatment option which is extremely promising as it can be used to fight many types of diseases, including hereditary disorders, some cancer types, and a set few viral infections. However, it is still risky and is being studied to make sure that it will be safe and effective, though currently it is only being tested for diseases with no other cures.

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Importance of CRISPR in Gene Therapy

CRISPR is essential for modern gene therapy. It is significantly more advantageous than traditional methods as it offers the ability to multiplex, editing multiple genes at the same time and is also significantly easier to use thanks to an easy to use design. This has led to continued development which in return has enabled it to become cost effective. CRISPR is particularly useful in correcting minor mutations in single gene disorders, preventing the disease phenotypes. This is the most promising field for gene therapy at the moment. In addition CRISPR shows great potential in gene therapy against cancer through deactivating oncogenic viruses and inducing oncosuppressor expressions. CRISPR is also essential in the process of inducing the DNA through one of two methods, viral and “naked” DNA. CRISPR is used for editing the viruses to make them non harmful to humans and incapable of reproduction, in addition to adding the actual gene. As for naked DNA, the other method, DNA is injected directly into the tissue, as a safer but less effective method that eliminates the risk of viral transformation (Ginn et. al 2018).

Types of Gene Therapy

Cancer

The majority of gene therapy trials (65% of all gene therapy trials) to date have been used for the treatment of cancer (Ginn et. al 2018). These trials have been used to target many different types of cancer, such as lung, gynaecological, skin, urological, neurological and gastrointestinal tumours, as well as haematological malignancies and paediatric tumours. Many different approaches have been taken in these therapies, such gene directed enzyme pro‐drug therapy, inserting tumour suppressor genes, oncolytic virotherapy, and immunotherapy. Primarily, the therapies involve the transmission of the p53 gene, a tumor suppressing gene, though it is not the only as BRCA‐1, Fus‐1 and endostatin have also been used. Oncolytic virotherapy uses viruses that are usually naturally oncolytic or are engineered to be. These viruses are then modified so that they encode suicide genes into the cancer cells, and convert pro‐drugs into cytotoxic drugs within the tumour and its immediate environment. (Ginn et. al 2018)

Cardiovascular

Cardiovascular gene therapy is the fourth most popular form of gene therapy at 6.9% (Ginn et. al 2018). Gene therapy in a cardiovascular application is hoped to be used for therapeutic angiogenesis, myocardial protection, regeneration and repair, prevention of restenosis following angioplasty, prevention of bypass graft failure and risk‐factor management. (Ginn et. al 2018) Primarily, cardiovascular gene therapy has been used for therapeutic angiogenesis to increase blood flow to ischaemic regions. Efforts to treat myocardial ischaemia, a result of coronary artery disease and lower limb ischaemia, a result of peripheral artery disease, have been test. Three types of growth factors have been used, fibroblast growth factor and vascular endothelial growth factor families, as well as a small number of trials using platelet‐derived growth factor to treat foot ulcers. A limited number of trials have also used the induction of hypoxia inducible factor to stimulate angiogenesis, though this has only occurred in 11 trials. (Ginn et. al 2018)

Inherited Monogenic Diseases

Monogenic diseases are treated with the aim of the treatment to transfer the functional gene into stem cells in order to ensure the permanence of the transfer. Monogenic diseases make up 11.1% of gene therapy clinical trials. More than thirty-two inherited monogenic diseases have been targeted, including Tay-Sachs, Sickle cell disease, and cystic fibrosis (Ginn et. al 2014). Cystic fibrosis is the most commonly targeted disease with 22.4% of monogenic disease trials aiming to cure it, as it is also the most commonly inherited genetic disease in both Europe and the US. Second most common are the combined immunodeficiency syndromes which are just over 20% of trials for monogenic disease. Chronic granulomatous disease is among these and has been treated with fairly good results.

Other Diseases

A total of 182 of trials, 7% of the overall amount performed were used to treat infectious disease, the third most popular use of gene therapy. The primary target of these trials was HIV, however adenovirus, cytomegalovirus, and tetanus were also attempted to be treated. In addition, some neurological diseases have been targeted. There have been 36 clinical trials which aim to treat many different diseases including common diseases such as Alzheimer’s and Parkinson’s disease as well as neurological complications of diabetes, amyotrophic lateral sclerosis, multiple sclerosis, and myasthenia gravis.

Success of Gene Therapy

As of a study conducted in November 2017, nearly 2600 gene therapy clinical trials have been approved and are ongoing or completed, worldwide, from 1989 to 2017. Since 2014 the number of trials conducted each year has continued to increase. These clinical trials have been conducted in 38 countries, with the United States making up the vast majority at 63.3%. 65% of diseases addressed by gene therapy clinical trials are cancer disease, followed my by monogenic at 11.1%, and a change from 2014 to 2017 has brought infectious disease up to 7% where it replaced Cardiovascular diseases (6.9%) for the third most common use of gene therapy (Ginn et. al 2018). Many monogenic diseases and cancer patients have experienced great success with gene therapy, however there is no definitive answer as to how successful gene therapy is overall. It is still a developing technology, with limited successes in small trials. Many clinical trials have succeeded in improving the quality of life for patients, such as a single dose gene replacement therapy for spinal muscular atrophy, in which “12 patients who had received the high dose, 11 sat unassisted, 9 rolled over, 11 fed orally and could speak, and 2 walked independently.” (single dose gene replacement). For some of these patients, this was an enormous success, but even the smallest improvement led to greatly increased quality of life for affected individuals. (Mendell et. al 2017)

Failures of Gene Therapy

Jesse Gelsinger was an 18 year old American male was the first person to die as the result of gene therapy. Gelsinger had a metabolic disorder called ornithine transcarbamoylase (OTC) deficiency, which is a somewhat uncommon disorder, affecting 1 in 40000 newborns. Typically, this disease is fatal, killing half of newborns within a month of birth and the rest before age 5. Gelsinger survived because he only had a partial OTC deficiency. Typically, OTC impedes the elimination of ammonia, but because of Gelsinger only had partial deficiency, he was able to keep it in check with a low protein diet and drugs. Gelsinger did not die as a result of his disease. Gelsinger was killed by a violent immune response to the delivery vector of the new gene, which was encased a recombinant adenoviral vector injected into his hepatic artery (Sibbald 2001). Jesse’s death created a huge setback for gene therapy. As Jennifer Doudna, one of the researchers who founded the CRISPR-Cas9 system described it, “That made the whole field of gene therapy go away, mostly, for at least a decade. Even the term gene therapy became kind of a black label. You didn’t want that in your grants. You didn’t want to say, ‘I’m a gene therapist’ or ‘I’m working on gene therapy.’ It sounded terrible.” (Rinde 2019) However, the industry has reacted very strongly to this. The lead researcher on the trial that killed Gelsinger has since dedicated himself to numerous cautionary and safety studies which have shaped the field as a whole with a safer approach.

Unfortunately, Gelsinger is not the only person to die during a gene therapy clinical trial. However, according to the resources used for the research for this paper, no other patients were found to have been killed as a direct result of the gene therapy, as Gelsinger was. This is not to say there have not been any more deaths. Multiple people have died during gene therapy clinical trials, however their deaths were the result of the diseases that the trial was attempting to treat or unrelated factors, such as the recent death of a baby in Novartis’s gene therapy trial, where it was determined after extensive investigation that the baby died of a respiratory infection (Novartis 2019).

Conclusion

In all, gene therapy is an extremely promising advancement in medical technology. It is so successful today as a result of many great researchers and advancements such as the CRISPR Cas9 mechanism (Khan 2019). While it is still in its early stages of development and has proven to be very costly, dangerous, and difficult, it is worth pursuing. Gene therapy holds so much promise that it could one day save or significantly improve the lives of millions of people worldwide. Gene therapy is not yet completely effective due to its early stages of development but shows significant promise that it will be one day and is worth pursuing, despite the costs.

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