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Tony Yang
Mrs. Greene
Capstone- 7th Period
18 November 2016

Final Research Paper Rough Draft

I.

Introduction

With the modern advances in biotechnology and increased capability of modern

machinery, scientists have developed ways to solve problems previously seen as unsolvable. One
such innovation, gene therapy, comes in the form of editing the very genetic information that
defines who we are. Although the technology is still in its earliest form today, gene therapy will
function as a futuristic technology to help cure illnesses that were previously incurable. Gene
therapy comes in many forms and works through the editing of genes inside of cells.
Implications of the research show promising support for the possibility of curing diseases like
cancers and genetically related diseases. Further research hinges on the ability to garner grants
and support from government and private companies. Thus, more money should be invested to
fund future research.
II.

Gene Therapy Overview

Gene therapy is a broad topic and comes in many differing forms. It allows humans to

artificially edit the DNA within an organism. At its core, gene therapy is a technique in which
genes are used to treat a disease or disorder. This stands in contrast to traditional methodology as
medicine for the last few centuries has revolved around the use of drugs and antibiotics. Today,
modern gene therapy can be seen in efforts like CAR T-cell immunotherapy as well as the
recently discovered gene-editing tool called CRISPR, which allows for scientists to scan the

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genome and effectively replace genes with new ones. As it stands today, the processes remain
risky and unreliable, however, with more money and research, there is great potential in the
possibility for gene therapy to become an essential part of 21st century medicine. One of the
challenges that currently stands in the way of gene therapy is finding a reliable way to transplant
a gene into an organisms cells. As of today, the main methods include using viruses that are
bioengineered to become harmless, nanoparticles guided by magnets, and direct injection into the
bloodstream. Viruses, however, stand the possibility of mutating and becoming harmful and
dangerous to the body, while nanoparticles and direct injection are not nearly as effective. Today,
direct injection is still the simplest way for administration of DNA (Pardoll). With that said, more
research is being done and new creative techniques are being developed while existing ones are
being made more effective and less harmful to the human body.
III.

CAR T-Cell Immunotherapy Overview

Gene therapy has the capability to solve many medical problems that were once

considered unsolvable. CAR T-cell immunotherapy, for example, has already advanced from an
idea to human trials for cancer treatments to certain types of lymphomas (Rosenburg). As current
treatments revolve around bone marrow transplants and such, gene therapy is easily
administered to outpatients and generally does not cause significant side effects (Pardoll). The
effectiveness of the process is also extremely efficient as it involves the use of the bodys own
immune system to fight against the disease. Developments include the FDA approval of antiCTLA4 therapy...encouraging preliminary clinical data... of the potential antitumour activity of a
patient's own endogenous immune system (Pardoll). What is essentially happening here is that
gene therapy infects your own immune systems white blood cells to active them and
essentially train them to be better equipped to identify the foreign cancer cells and eliminate

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them quickly and effectively. T cell-mediated immunity includes multiple sequential steps
involving the clonal selection of antigen-specific cells, their activation and proliferation in
secondary lymphoid tissues (Rosenberg). In many cancers, the problem with our bodys
immune system is that it lacks the ability to identify the cancerous cells as cancer. However,
CAR T-Cell immunotherapy will allow the T-cells to fight and eliminate the cancer.
IV.

CRISPR Overview
CRISPR, short for clustered regularly interspaced palindromic repeats, was initially

discovered as a bacterial immune system that together with associated cas genes, provided
resistance against phages (Barrangou). CRISPR essentially is a segment of prokaryotic DNA
that codes for the CAS molecule (Barrangou). The CAS molecule is able to go through DNA and
very accurately scan for foreign DNA. In the case that it does discoverer a virus, it can then
eliminate the virus from the DNA, purging it and preventing it from harming the bacteria. This
can be eventually useful when applied to humans to solve some of the most daunting genetic
diseases like hemophilia, sickle cell, Parkinsons, or even cancer. CRISPR essentially allows for
different type II CRISPR/Cas systems...Cas9 nucleases to be directed by short RNAs to induce
precise cleavage at endogenous genomic loci in human and mouse cells (Cong). All genetic
diseases have one thing in common, they originate from bad genes inside of the human genome.
If these genes could be taken out and replaced with functional ones, then the problem will
subside and go away. The Cas9 endonuclease permits a means for accomplishing this task
accurately and effectively. The possible benefits of gene therapy are endless and could allow for
human life expectancies to increase exponentially into the future. Concerns remain in terms of
the ethicality of CRISPR research. The ability to find undesirable genes and replace them with
desirable ones could be easily abused in the future. Things like designer babies who are

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changed to be stronger, taller, and more attractive could become the norm. This is a scary idea
that could manifest itself into standards for how humans should be born. For this reason, future
research of the implementation of CRISPR into humans needs to be monitored in order to
prevent abuse of the technology.
V.

CRISPRs Abilities
Gene therapy using CRISPR can be used to cure many diseases previously thought to be

incurable and help with the process of determining gene function. The human genome has
already been sequenced largely thanks to the help of The Human Genome Project, however
many genes still have undetermined functions and CRISPR has shown that AAV-mediated
SpCas9 genome editing can enable reverse genetic studies of gene function in the brain
(Sweich). While this particular application of CRISPR is not life-saving, it can help to give
researchers a better understanding about bodily functions. This, in turn, can lead to new
medicines and new innovative ways to cure genetic diseases. The more dramatic application that
CRISPR brings comes in the form of being able to change the DNA of cells. For example, in an
experiment with mice that have a mutated liver gene, CRISPR was able to replace the gene with
a normal one, leading to the prevention of death (Cheng). This also goes along with the ability to
enact a phenotypical change, which is essentially allowing for the DNA change to make physical
change. This works not only in vitro but in vivo as well, meaning that CRISPR can work in living
human organisms. In the same rat experiment it was demonstrated that the system could reach
the level of tissue-specific gene knockout, resulting in phenotypic changes (Cheng). For many
people currently afflicted with such ailments around the world, the development of CRISPR to
its potential not only means they can be cured, it could also be the difference between life and
death. Conditions like Huntingtons, Sickle Cell, and Cystic Fibrosis could eventually exist in

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nothing more than the pages of a textbook. With that said, its only logical that CRISPR
continues to be researched and that more funding is given so that it can one day become an
instrumental part in saving human lives.
VI.

Barriers to CRISPR Implementation

However, at the same time, many barriers prevent the implementation of CRISPR into

cells to serve its purpose. The principal problem is the presence of a cell membrane that prevents
the free flow of things in and out of cells. While this is a problem for CRISPR, the cell
membrane system exists for the logical reason of keeping good things in and bad things out. The
current methods that exist for breaking into a cell membrane include lentivirus delivery, which is
based off of HIV cells. AAV delivery, which is a nonenveloped parvovirus that has the ability to
trigger a small immune response and enter the cell, and non-viral delivery through the use of
electroporation, which is essentially delivering a charge to a cell to temporarily break down the
cell membrane, allowing things to pass through (Gori). While these methods of delivery are
viable to various degrees, it is without a doubt that better ones can be discovered or existing ones
improved with more research. On the other hand, another major problem with CRISPR is the
possibility of unintentionally changing DNA segments. Due to CRISPRs specificity in targeting
a particular sequence, another gene could contain the same sequence as the targeted gene,
resulting in an unwanted replacement, possibly leading to a number of unwanted effects. For this
reason, One major concern about this system is its specificity; although CRISPRCas9
mediated off-target mutation has been broadly studied, more efforts are required to further
improve the specificity of CRISPRCas9 (Till). CRISPRs specificity still needs to be improved
so that such mistakes do not happen before moving on to human trials. This is due to the fact that
an accidental replacement of a functional gene could lead to disastrous results like the

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inactivation of that gene or even the possibility of cancer (Till). Although these obstacles may
appear to be challenging, they are within reach of being solved through the power of human
innovation and creativity. Overcoming these obstacles means the saving of a countless number of
lives, something invaluable that no amount of money could equal
Lastly, more development of CRISPR is still needed to help its transition to humans.
Although the functionality of CRISPR can be applied to human cells, there are many
fundamental differences that exist between humans and other mammals. Because of this,
measuring and understanding the relationship between efficacy and specificity will be an
important aspect of ongoing CRISPR/Cas9 clinical development (Gori). The only way to solve
obstacles that exist and achieve the true potential of CRISPR is through the influx of more
money and increased funding, otherwise its nothing short of impossible for any progress to be
made. An increase in research will speed up the rate in which such problems can be solved and
result in new ideas that havent been thought of. Continuing on previously mentioned points,
improvements to CRISPR delivery methods and repair efficiency will be required for its broad
therapeutic application in humans (Till). While CRISPR can be tested on animals like mice,
humans hold distinct ethical laws put into place by the government that prevent researchers from
freely experimenting on humans. For this reason, delivering CRISPR for human trials will
require much more stricter guidelines and more effective ways of getting CRISPR into cells.
CRISPR serves as a landmark breakthrough that could signal a dramatic change in the
field of bioengineering as we know it. The powerful capability of creating genetic changes in
embryos and living organisms could mean that we as humans would soon have the capabilities to
cure diseases, give ourselves superhuman intelligence, and even change our outward appearance.
The question of ethics arises out of this discussion, however, the capability in curing diseases

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that up to the present day were thought of as incurable means that CRISPR should continue to be
researched. Overcoming the barriers that prevent widespread CRISPR implementation can soon
be solved, giving manking the opportunity to save lives and change the landscape of human
biology.
VII. CAR T-Cell Functionality
T-cell therapy is a process that utilizes many 21st century biotech advancements to
function as way to cure cancer. Through the late 1900s into the present era, developments like
electron microscopes and better research methods have given scientists a better understanding of
both the human immune system and cancer cells. A large portion of the immune system function
is based off of a special group of cells known as T-cells. T cells mature in the thymus... express
TCR (T cell receptor), and can express either CD8 glycoprotein on their surface and are called
CD8+ T cells (cytotoxic) or CD4 glycoprotein and are then called CD4 cells (helper T cells)
(Golubovskaya). These T-cells use molecules called antigens to differentiate between different
things inside of the body. In CAR T-cell therapy, artificial T-cell receptors that can bind to
specific antigens are engineered into T-cells. These CARs are derived from the antigen-binding
domain of a monoclonal antibody coupled with the intracellular signaling portion of the T cell
receptor (Kohn). The artificially made CARs can then be used to target specific antigens on
cancer cells as cancer cells hold distinct differences in their expressed antigens. While cancer
cells are normally able to escape detection through various methods like disguising themselves
as normal body cells, CARs will essentially allow for differentiation between normal and
cancerous cells, subsequently leading to an immune response.
In actual experiments on humans, CAR T-Cell Therapy has shown great promise in
studies done so far as more focus is being put into testing its viability. Very generally speaking, it

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has already been shown that T cell therapy with tumor-targeted chimeric antigen receptor
(CAR)modified T cells has... yielded outcomes that support the tremendous potential of this
approach to cancer therapy (Davila). However, despite the fact that success has been shown,
because of the fact that every type of cancer is different and unique in its own way, CAR T-cell
immunotherapy has shown greater efficacy in certain tumors. Particularly convincing results
have been seen with neuroblastoma as a potential alternative to conventional treatment
options, and B-cell lymphomas. The results of CAR targeted treatment have shown to result in
partial or complete remissions in... patients with advanced disease, with no persistent toxicities
(Onea). The advantage of using this treatment is it equips the body to fight cancer like a virus or
bacteria, rather than killing everything in a targeted area as with chemo and radiation therapy.
While these two conventional cancer treatments also work, their ability to kill cancer cells also
means that they will more than likely destroy normal healthy body cells as well. CAR T-cell
therapy on the other hand has the ability to target cancer cells specifically, which subsequently
leaves normal cells alone. This is revolutionary in that patients can leave treatment without the
feeling of pain or extreme sickness, and for that reason should have more research and funding to
allow for rapid development of the treatment.
VIII. CAR T-Cell Therapy Drawbacks
At the same time however, many prevalent drawbacks still exist in preventing CAR Tcell therapy from being utilized today. Firstly, much of the success in using CAR t-cell therapy
has come from use on liquid tumors like leukemia. For an unknown reason that scientists have
not been able to pinpoint, CARs are still not able to effectively work on solid tumors, however,
many hypothesis have yet to be tested (Golubovskaya). There is also a problem commonly called
antigen escape. Due to how CAR T-cell therapy exerts selective pressure on tumor cells, it will

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result in the development of new genetic variants that evade treatment (Golubovskaya).
Essentially what is happening is the natural selection of cancer cells. Due to the fact that CARs
target specific antigens and that each tumor is constantly mutating and thus may express different
antigens, it is possible for the engineered t-cells to be unable to kill all of the tumor mass.
Finally, due to an increased amount of T-cells being present, CAR therapy can cause prolonged
toxicities because the engineered T-cells can persist in the body even after treatment is no longer
required (Golubovskaya). This is a problem in that prolonged exposure to cytokines can
potentially result in a cytokine storm, a potentially fatal chain reaction due to a positive feedback
loop in the immune system. With the presence of all of these drawbacks, the only way to solve
them is through the use of increased research. With the great potential that exists for CAR T-cell
therapy, it is only logical that we allocate more resources so that development can be accelerated.
With the advent of modern technology and research methods, scientists are able to think
creatively and come up with new innovations. As a result, new methods of solving problems
have been created with only more to come. Using CARs to target cancer cells is an intuitive way
to utilize ones own immune system to fight cancer in a painless and effective way. Despite the
many drawbacks that still exist with this treatment method, through the use of more research and
funding, CARs can eventually be developed into an effective treatment to treat and prevent
cancer.
IX. Conclusion
While gene therapy is an exciting new technology that has the potential to really change
the world, many things still exist that hamper its ability to be successful today. Barriers like the
delivery of the genes or the need for more research in general make it so that gene therapy is still
an afterthought today. However, with more research and funding it could dramatically change the

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future landscape of medicine and biotechnology. Incurable diseases could be solved with a one
time cure, cancer could become an afterthought of the past, and age could even be reversed.
Although all of these things sound exciting and present us with a future to look forward to, none
of it will happen without funding and continued research. For this reason it is important that we
devote more efforts and research into gene therapy and its future as a life saving technology.

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