AN UNDERGRADUATE SEMINAR

ON

THE FUTURE OF GENE THERAPY: OPPORTUNITIES AND

CHALLENGES

BY
EFOSA PRINCE UYI
BMS1902123

PRESENTED TO

THE DEPARTMENT OF MEDICAL BIOCHEMISTRY, SCHOOL, OF

BASIC MEDICAL SCIENCES, UNIVERSITY OF BENIN

NOVEMBER, 2023

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 CERTIFICATION

We the undersigned hereby certify that EFOSA PRINCE UYI (BMS1902123) presented this
seminar paper to the department of Medical Biochemistry, School of Basic Medical Sciences,
University of Benin, in partial fulfillment of a B.Sc. degree from the above named
department.

Signed:

Prof. A. Omonkhua Date

(Seminar Supervisor)

Dr. F. E. Olumese Date

(Head of Department)

ii
 DEDICATION

iii
 ACKNOWLEDGMENTS

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 TABLE OF CONTENT

TITLE PAGE..…………………………………….………………………………………….i
CERTIFICATION............……………………………………………….………………......ii

DEDICATION.........................................................................................................................iii
ACKNOWLEDGMENT.........................................................................................................iv
TABLE OF CONTENT……….............................................................................................v
SUMMARY.............................................................................................................................vi
CHAPTER ONE: INTRODUCTION

1.1 Overview of Gene therapy............................................................................................1

CHAPTER TWO: LITERATURE REVIEW:

2.1 Background on Gene Expression, Gene Regulation and Gene editing……..……….3

2.2 Historical Perspective on Gene Therapy…………………....…………………….....6

2.3 Types of Gene Therapy..............................................................................................9

2.4 Opportunities in Gene Therapy…………...………………………...………….......12

2.5 Emerging Technologies in Gene Therapy.................................................................16

2.6 Challenges in Gene Therapy………………………………………...………..........22

2.7 Ethical Considerations in Gene therapy....................................................................26

CONCLUSION AND RECOMMENDATIONS….………………………………………30

REFERENCES…………………………………………………….…..……………………31

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 SUMMARY

The future of gene therapy is a riveting voyage into the domain of biology, with unlimited
prospects and daunting hurdles. Gene therapy, which includes the introduction, modification,
or silence of genes to cure or prevent illnesses, has gone a long way since its start. It is set to
change healthcare by treating genetic problems, opening the door for customized therapy, and
solving some of the most difficult illnesses known to mankind. While the possibilities are
alluring, the route to harnessing the full potential of gene therapy is defined by significant
challenges. Safety problems, ethical and regulatory difficulties, high prices, and access
discrepancies need to be solved to guarantee a future where gene therapy may be a reality for
patients in need. This detailed examination digs into the historical history, kinds of gene
therapy, prospects, problems, clinical triumphs, new technologies, regulatory environment,
and future uses. It addresses the ethics and patient advocacy that lead the way, stresses the
need of international cooperation, and presents a vision for the future of gene therapy.
Ultimately, the future of gene therapy involves a delicate balance between the wonderful
opportunities it presents and the crucial difficulties that must be managed to properly achieve
its potential influence on healthcare and society.

Gene therapy has made considerable progress from its early inception. The trip starts with the
idea that genes, the basic components of heredity, hold the key to a multiplicity of illnesses.
In the early phases of gene therapy, researchers envisioned replacing faulty genes with
healthy ones, laying the ground for a revolution in medicine. Historical achievements in gene
therapy include the successful treatment of a severe combined immunodeficiency (SCID)
patient in the 1990s and subsequent developments. These early wins established the road for
the future of gene therapy, changing it from a distant fantasy into a hopeful reality.

There are numerous forms of gene therapy, each with its particular technique and uses.
Germline gene therapy includes changing the genes in reproductive cells, possibly
influencing future generations. Somatic gene therapy, on the other hand, targets non-
reproductive cells and is meant to cure or reduce the symptoms of hereditary illnesses in
particular people. Ex vivo and in vivo gene treatments provide distinct approaches to insert
or change genes. Ex vivo techniques entail the removal of cells or tissues from the patient,
genetic change outside the body, and then reintroduction into the patient. In vivo treatments
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try to change genes inside the patient’s body directly. Each of these techniques provides great
options for treating certain medical issues. The prospects of gene therapy are immense and
transformational. Gene therapy presents the prospect of addressing genetic illnesses at their
source, perhaps giving cures rather than merely symptom treatment. The advent of
customized medicine is on the horizon, when treatment strategies are tailored to an
individual’s genetic composition. Complex disorders, previously believed untreatable, may
become topics of gene therapy research. Advancements in gene-editing technologies, notably
CRISPR-Cas9, have opened new possibilities for precise genetic changes. These prospects
are making gene therapy a beacon of hope for patients and medical practitioners alike.

Despite the great promise, gene therapy grapples with some daunting hurdles. Safety
considerations are a primary focus, since gene therapy may occasionally lead to
unanticipated results. Ethical concerns surround germline gene editing and debates regarding
the border between treatment and augmentation. Regulatory barriers and discrepancies make
it a hard sector to traverse, with various nations having differing methods. The high cost of
gene therapy restricts accessibility and affordability for many individuals. These obstacles
must be addressed to guarantee that the advantages of gene therapy are achieved without
sacrificing safety and ethics. A large portion of gene therapy’s future resides in the
accomplishments and breakthroughs that have previously been accomplished. Clinical
studies have revealed astonishing benefits, with examples of patients experiencing life-
changing changes. These accomplishments serve as beacons of hope and underline the
potential influence of gene therapy on patients’ lives. For instance, people with hereditary
retinal abnormalities have acquired eyesight, while others with specific genetic blood
problems have received relief via gene therapy.

Emerging technologies are taking gene therapy to new heights. The groundbreaking
CRISPR-Cas9 gene-editing technique has received worldwide interest for its accuracy and
efficiency. Other gene-editing technologies, such as TALENs and zinc-finger nucleases,
supplement the toolset for genetic changes. The development of viral and non-viral vectors
for delivering genes into target cells is progressing the science. RNA-based therapeutics,
particularly mRNA vaccinations like those utilized in the battle against COVID-19, provide
new prospects for gene therapy. These technologies are opening doors to novel solutions and
pushing the limits of what can be done. Genetic engineering and biotechnology businesses

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play a crucial role in advancing gene therapy research and development. These organizations
are at the forefront of innovative discoveries, clinical studies, and the manufacture of gene
treatments. They are important in pushing gene therapy from the laboratory to the patient’s
bedside. Leading firms in the sector are actively involved in research, development, and
marketing of gene treatments.

The regulatory landscape of gene therapy is a significant factor that impacts its development.
Regulatory bodies, including the U.S. Food and Drug Administration (FDA) and their foreign
equivalents, play a vital role in establishing the safety and effectiveness of gene treatments.
The process of receiving regulatory clearance is extensive, comprising rigorous testing and
examinations to assure patient safety. Harmonizing gene therapy rules across nations is an
ongoing topic that has to be addressed to develop a common worldwide framework.

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 CHAPTER ONE

1.0 INTRODUCTION
1.1 OVERVIEW OF GENE THERAPY
Gene therapy, sometimes termed gene transfer therapy, insertion of a normal gene into an
individual’s genome in order to fix a mutation that causes a genetic illness. When a normal
gene is injected into the nucleus of a mutant cell, the gene most likely to integrate into a
chromosomal position distinct from the faulty allele; while that may repair the mutation, a
new mutation may emerge if the normal gene integrates into another functioning gene. If the
normal gene replaces the mutant allele, there is a possibility that the transformed cells will
proliferate and create enough normal gene product for the whole body to be restored to the
undiseased phenotype (Roth and Marson, 2021).

Gene therapy is the outcome of man’s ambition to remove illnesses. Gene therapy has three
facets namely, gene silencing using siRNA, shRNA and miRNA, gene replacement where the
desired gene in the form of plasmids and viral vectors, are directly administered and finally
gene editing based therapy where mutations are modified using specific nucleases such as
zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and
clustered regulatory interspaced short tandem repeats (CRISPR)/CRISPR-associated protein
(Cas)-associated nucleases (Jensen et al., 2021). Transfer of gene is either through
transformation where under specific conditions the gene is directly taken up by the bacterial
cells, transduction where a bacteriophage is used to transfer the genetic material and lastly
transfection that involves forceful delivery of gene using either viral or non-viral vectors. The
non-viral transfection techniques are split into physical, chemical and biological. The
physical techniques include electroporation, biolistic, microinjection, laser, increased
temperature, ultrasound and hydrodynamic gene transfer. The chemical approaches employ
calcium- phosphate, DAE-dextran, liposomes and nanoparticles for transfection. The
biological methods are increasingly using viruses for gene transfer, these viruses could either
integrate within the genome of the host cell conferring a stable gene expression, whereas few
other non-integrating viruses are episomal and their expression is diluted proportional to the
cell division. So far, gene therapy has been utilized in a variety of disorders. However,
coherent and harmless delivery of genes is among the biggest barriers in the utilization of this
potential medicine (Sayed et al., 2022).
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Some parts of gene therapy, including genetic modification and selection, study on fetal
tissue, and testing on human beings, have sparked ethical debate and safety concerns. Some
objections to gene therapy are founded on the belief that people should not “play God” and
intervene in the natural order. On the other hand, some have claimed that genetic engineering
may be permissible when it is congruent with the goals of God as creator. Some skeptics are
especially worried about the safety of germline gene therapy, since any damage induced by
such treatment may be transferred to succeeding generations. Benefits, however, would also
be passed on permanently. There also has been worry that the use of somatic gene therapy
may harm germ cells (Joseph et al., 2022).

The purpose of this study article was to fully evaluate the growing landscape of gene therapy
and its role in determining the future of healthcare. This research intends to give insights into
the prospects and problems related with gene therapy, presenting a comprehensive
assessment of its potential influence on medical treatments, human well-being, and society.

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 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Background on Gene Expression, Gene Regulation and Gene editing

 Gene Expression

Gene expression is the process by which information from a gene is employed in the
synthesis of a functional gene product that permits it to create end products, proteins or non-
coding RNA, and eventually alter a phenotype. These products are frequently proteins, but in
non-protein-coding genes such as transfer RNA (tRNA) and small nuclear RNA (snRNA),
the output is a functional non-coding RNA (Statello et al., 2021). Gene expression is
described in the core dogma of molecular biology initially established by Francis Crick in
1958, later elaborated in his 1970 essay, and enlarged by the subsequent discoveries of
reverse transcription and RNA replication. The expanded core dogma of molecular biology
comprises all the cellular processes involved in the transfer of genetic information (Cobb,
2017). The process of gene expression is employed by all known living eukaryotes (including
multicellular organisms), prokaryotes (bacteria and archaea), and used by viruses—to build
the macromolecular machinery for life (Shapiro, 2016).

In genetics, gene expression is the most basic level at which the genotype gives birth to the
phenotype, i.e. observable characteristic. The genetic information encoded in DNA
constitutes the genotype, while the phenotype emerges from the “interpretation” of that
information. Such phenotypes are typically expressed by the creation of proteins that govern
the organism’s structure and growth, or that operate as enzymes catalysing certain metabolic
pathways. All stages in the gene expression process may be controlled (regulated), including
the transcription, RNA splicing, translation, and post-translational modification of a protein.
Regulation of gene expression allows control over the time, location, and quantity of a
specific gene product (protein or ncRNA) present in a cell and may have a dramatic influence
on the cellular structure and function. Regulation of gene expression is the foundation for
cellular differentiation, development, morphogenesis and the diversity and adaptability of
every organism. Gene control may thus act as a substrate for evolutionary change (Nussinov
et al., 2019).

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  Gene Regulation

Regulation of gene expression, or gene regulation, encompasses a broad variety of methods

that are utilized by cells to promote or reduce the creation of certain gene products (protein or
RNA). Sophisticated plans of gene expression are often seen in biology, for example to
activate developmental pathways, react to environmental stimuli, or adapt to new food
sources. Virtually each stage of gene expression may be regulated, from transcriptional start,
to RNA processing, and to the post-translational modification of a protein. Often, one gene
regulator regulates another, and so on, in a gene regulatory network (Steven, 2021).

Gene regulation is vital for viruses, prokaryotes and eukaryotes since it promotes the variety
and adaptability of an organism by enabling the cell to produce protein when required.
Although as early as 1951, Barbara McClintock showed interaction between two genetic loci,
Activator (Ac) and Dis-sociator (Ds), in the color formation of maize seeds, the first
discovery of a gene regulation system is widely considered to be the identification in 1961 of
the lac operon, discovered by François Jacob and Jacques Monod, in which some enzymes
involved in lactose metabolism are expressed by E. coli only in the presence of lactose and
absence of glucose (Shapiro, 2016). In multicellular animals, gene regulation causes cellular
differentiation and morphogenesis in the embryo, resulting to the development of various cell
types that feature varied gene expression patterns from the same genomic sequence. Although
this does not explain how gene regulation arose, evolutionary scientists include it as a partial
explanation of how evolution works at a molecular level, and it is essential to the discipline
of evolutionary developmental biology (“evo-devo") (Mora Van Cauwelaert et al., 2015).

 Gene Editing

Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in

which DNA is inserted, deleted, modified or replaced in the genome of a living organism.
Unlike early genetic engineering techniques that randomly inserts genetic material into a host
genome, genome editing targets the insertions to site-specific locations. The basic mechanism
involved in genetic manipulations through programmable nucleases is the recognition of
target genomic loci and binding of effector DNA-binding domain (DBD), double-strand
breaks (DSBs) in target DNA by the restriction endonucleases (FokI and Cas), and the repair
of DSBs through homology-directed recombination (HDR) or non-homologous end joining

4
(NHEJ) (Li et al., 2020). Genetic engineering as method of introducing new genetic elements
into organisms has been around since the 1970s. One drawback of this technology has been
the random nature with which the DNA is inserted into the hosts genome, which can impair
or alter other genes within the organism. Although, several methods have been discovered
which target the inserted genes to specific sites within an organism genome. It has also
enabled the editing of specific sequences within a genome as well as reduced off target
effects. This could be used for research purposes, by targeting mutations to specific genes,
and in gene therapy. By inserting a functional gene into an organism and targeting it to
replace the defective one it could be possible to cure certain genetic diseases (Yalew et al.,
2020).

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2.2 Historical Perspective on Gene Therapy

Gene therapy was initially envisioned in the 1960s, when the viability of adding new genetic
functions to mammalian cells started to be investigated. Several techniques to accomplish so
were attempted, including injecting genes with a micropipette straight into a live mammalian
cell, and exposing cells to a precipitate of DNA that included the necessary genes. Scientists
believed that a virus may potentially be employed as a vehicle, or vector, to transport new
genes into cells (Malech et al., 2022).

One of the first scientists to disclose the effective direct insertion of functioning DNA into a
mammalian cell was biochemist Dr. Lorraine Marquardt Kraus of the University of
Tennessee in Tennessee, United States. In 1961, she succeeded to genetically change the
hemoglobin of cells from bone marrow obtained from a patient with sickle cell anaemia. She
achieved this by culturing the patient’s cells in tissue culture with DNA recovered from a
donor with normal hemoglobin (Lorraine Kraus Obituary, 2023). In 1968, researchers
Theodore Friedmann, Jay Seegmiller, and John Subak-Sharpe at the National Institutes of
Health (NIH), Bethesda, in the United States successfully corrected genetic defects
associated with Lesch-Nyhan syndrome, a debilitating neurological disease, by adding
foreign DNA to cultured cells collected from patients suffering from the disease. The first
effort, an unsuccessful one, at gene therapy (as well as the first instance of medical transfer of
foreign genes into humans not including organ transplantation) was undertaken by geneticist
Martin Cline of the University of California, Los Angeles in California, United States on 10
July 1980. Cline stated that one of the genes in his patients remained active six months later,
however he never published this data or had it validated. After significant research on
animals during the 1980s and a 1989 bacterial gene tagging study on humans, the first gene
therapy generally acknowledged as a success was proven in a trial that began on 14
September 1990, when Ashanthi DeSilva was treated for ADA-SCID (Wirth et al., 2013).

The first somatic therapy that created a lasting genetic alteration was undertaken in 1993. The
objective was to treat malignant brain tumors by utilizing recombinant DNA to transfer a
gene making the cancer cells sensitive to a medicine that in turn would cause the tumour cells
to die. The polymers are either translated into proteins, interfere with target gene expression,

6
or maybe fix genetic mutations. The most frequent approach utilizes DNA that encodes a
functioning, therapeutic gene to replace a defective gene. The polymer molecule is wrapped
into a "vector", which conveys the molecule within cells (Khan et al., 2016).

Early clinical failures led to dismissals of gene therapy. Clinical accomplishments since 2006
rekindled researchers’ interest, however as of 2014, it was still mostly an experimental
procedure. These include therapy of retinal illnesses Leber’s congenital amaurosis and
choroideremia, X-linked SCID, ADA-SCID, adrenoleukodystrophy, chronic lymphocytic
leukemia (CLL), acute lymphocytic leukemia (ALL), multiple myeloma, haemophilia, and
Parkinson’s disease. Between 2013 and April 2014, US firms spent over $600 million in the
sector (Khan et al., 2016). The first commercial gene therapy, Gendicine, was authorized in
China in 2003, for the treatment of specific malignancies. In 2011, Neovasculgen was
registered in Russia as the first-in-class gene-therapy medication for treatment of peripheral
artery disease, including critical limb ischemia. In 2012, Glybera, a medication for a rare
hereditary condition, lipoprotein lipase deficiency, became the first medicine to be licensed
for clinical use in either Europe or the United States following its support by the European
Commission (Zhang et al., 2018).

Following early discoveries in genetic engineering of bacteria, cells, and tiny animals,
scientists began researching how to apply it to medicine. Two primary techniques were
investigated replacing or interrupting faulty genes. Scientists concentrated on disorders
caused by single-gene abnormalities, such as cystic fibrosis, haemophilia, muscular
dystrophy, thalassemia, and sickle cell anaemia. Glybera cures one such condition, caused by
a deficiency in lipoprotein lipase. DNA must be delivered, reach the injured cells, enter the
cell and either express or disrupt a protein. Multiple delivery modalities have been
considered. The original strategy combined DNA with an engineered virus to transfer the
DNA into a chromosome. Naked DNA techniques have also been examined, notably in the
context of vaccine development (Gonçalves and Paiva, 2017).

Generally, efforts concentrated on delivering a gene that causes a required protein to be

expressed. More recently, greater knowledge of nuclease function has led to more direct

7
DNA editing, employing tools like as zinc finger nucleases and CRISPR. The vector
integrates genes into chromosomes. The expressed nucleases then knock out and substitute
genes in the chromosome. As of 2014 these procedures include extracting cells from patients,
altering a chromosome and returning the changed cells to patients. Gene editing is a possible
way to modify the human genome to cure hereditary illnesses, viral diseases, and cancer. As
of 2020 these techniques are being investigated in clinical studies (Li et al., 2020).

8
2.3 Types of Gene Therapy

There are two fundamental forms of gene therapy that include germline treatment and
somatic gene therapy. Somatic cell gene therapy includes collecting blood cells from a person
with a genetic condition and then transferring a normal gene into the faulty cell. This sort of
gene therapy does not prevent the illness from arising in the future generation since it does
not influence the sperm and egg cells. Somatic cell gene therapy only influences the other
bodily cells. A working gene is frequently inserted in the new DNA to remedy the
consequences of a disease causing mutation. Somatic cell gene therapy needs to be done
multiple times during the course of the patient's life since the results do not endure very long
(Razi Soofiyani et al., 2013).

A somatic cell Is any cell in the body other than sperm and egg cells. Because somatic cells
are diploid, they contain two sets of chromosomes, one for each parent. Somatic cell
mutations may impact people, but they are not handed on to offspring. Somatic cells are body
cells that are not part of the germ line, which are the sexual organ cells that create sperm and
eggs. It is required for all living creatures to exist, yet it adds nothing to genetic transmission
or inheritance to the next generation. As a consequence, it serves just the live thing and has
no impact on what happens to the organism’s next generation (Saha et al., 2021).

Somatic gene therapy is a method that includes putting a normal gene into the proper cells of
a person with a genetic illness, therefore permanently curing the issue. The most fundamental
mechanisms of delivering genes into a person’s cells are viruses and liposomes. In the
nucleus of particular cells, the gene or genes are inserted into a chromosome. The target cells
might be bone marrow cells, which are readily isolated and replanted. Because bone marrow
cells grow throughout a person’s life in order to make blood cells, this approach is only
successful if the gene to be transferred has a biological purpose in the blood. A gene having a
biological role in the lungs, muscle, or liver, would need to be administered into those organs.
Accessing the right tissue, or ensuring that a gene is delivered where it is needed if it is
necessary in many tissues, is a substantial barrier in many circumstances (Razi Soofiyani et
al., 2013).

This treatment includes implanting a human gene into the somatic cells of a live individual,
which do not create the eggs and sperm that make the next generation. Somatic cell gene
therapy tries to treat an illness in the patient alone, not in their progeny. Some dangers are
also related with somatic gene therapy. It is likely that once viruses are introduced into the

9
body, they will recover their potential to cause sickness. If the additional genes are put in the
incorrect position in DNA, they may induce cancers (Zakrzewski et al., 2019).

Currently, this treatment is concentrating on illnesses that impact just one tissue, such as
cystic fibrosis and adenosine deaminase. It was first suggested as a way of implanting a
completely functioning copy of a gene into a person who had a genetic ailment as a
consequence of obtaining only partly functional copies. Since then, numerous forms of
somatic cell gene therapy have been researched for the treatment of illnesses such as AIDS
and cancer that are not largely caused by inherited genes. The genetic properties of somatic
cell gene therapy have been mainly uncontroversial. Gene therapy is really simply another
pharmaceutical delivery mechanism, a unique means of delivering a normal human protein to
the proper spot in the body (Razi Soofiyani et al., 2013).

The antithesis of somatic cell gene therapy is germline treatment. Germline treatment takes
place in the reproductive cells. It entails the genetic alteration of germ cells that will pass the
change on to the following generation. This form of gene therapy just needs to be done one
time to be permanent. One sort of germline treatment is to cure a pre-embryo that bears a
major genetic abnormality before it is implanted back in the mother via in vitro fertilization.
Another germline treatment aims to treat adult sperm and egg cells so the genetic abnormality
is not passed on to offspring. If a genetic modification happens, it will influence the
following generation. There have been no trials of human germ line gene therapy; fact, there
is an informal moratorium in the scientific community against performing such studies in
people. Both its feasibility and its worth are unknown (Rubeis and Steger, 2018).

New genes have been successfully inserted into the germ lines of other animals, although
with extremely low efficiency. At the same time, pre-implantation genetic diagnosis enables
parents to pick embryos based on their genetic differences, as long as the parents themselves
developed the desired variants. If not, donated eggs or sperm would be a more safer and
simpler approach to deliver the desired genes than somatic cell gene therapy. Germ line gene
therapy may turn out to be most essential as a barrier to somatic cell gene therapy. If germ
line gene therapy were forbidden, researchers utilizing somatic gene therapy could need to
make the challenging demonstrating that the transplanted genes could not ‘infect’ the

10
patient’s germ cells and therefore constitute unintended germ line gene therapy (Ranisch,
2020).

This creates several ethical problems. The next issue in gene therapy is how is the genetic
material given to the proper cells in the patient, and also how do you make the delivery
precise and safe. The first step in gene therapy is an accurate diagnosis of the genetic
abnormality. This is done by utilizing a DNA probe. The DNA probe is specific to a
complementary fragment of DNA. This technology employing DNA probes is more precise
that other standard ways of identifying genetic abnormalities in people. After the right
diagnosis is obtained, healthy DNA may be introduced into a virus, which has had infectious
genes stripped out of it. The virus is then combined with cells taken out of a patient and then
injected back into that patient. Viruses are utilized because they are like genes, but coated in a
unique protein shell. On the surface of this protein coat are specific proteins that bind to the
surface of cells. Once these viruses are in the body they lock in place on certain cells. The
cells then suck the viruses in or the viruses push their way into the cells (PBS) (Li et al.,
2020).

A retrovirus Is a kind of virus that is utilized in gene therapy. This is a virus that inserts its
genetic information directly into the chromosomes of the host cell (PBS). Other viruses are
utilized for various sorts of genetic disorders. One of them is the adenovirus which is utilized
for cystic fibrosis patients. To utilize a virus in gene therapy, the patient’s immune system
needs to be weakened so it will not fight against the virus. If weakening the patient’s immune
system is not an option then there are various vectors that may be employed instead of
viruses to undertake gene therapy. An additional gene therapy option that doesn’t require
viruses is to encapsulate the DNA into fatty liposomes. This procedure works because the
cell’s membranes are made up of fatty components so the liposomes may travel across the
membranes toward the center of the cell. Then the liposomes will release their DNA material
into the cell. Also the new DNA might be inserted directly into the skin cells or tumor cells.
Neither liposomes or direct DNA injections are as successful as viruses employed in gene
therapy (PBS) (Lundstrom, 2018).

There are various ethical and legal considerations involved with gene therapy. The
Recombination DNA Advisory Committee (RAC) is a review body that was designed to
govern research initiatives using recombinant DNA, manage gene marking studies, and

11
assess all gene therapy techniques. The Food and Drug Administration (FDA) collaborates
with the RAC on recommendations engaged in human trials (Coutts, 1998). Some of the
ethical difficulties in gene therapy originate from germline treatment procedures. This is
because germline gene therapy entails too much scientific uncertainty and hazards. The long-
term implications of this form of treatment remain unclear (Piccoli et al., 2019).

2.4 Opportunities in Gene Therapy

Gene therapy has great promise and potential in the realm of medicine. It is a cutting-edge
strategy that includes the insertion, deletion, or change of genes inside an individual’s cells to
cure or prevent illness. This revolutionary sector has the potential to change healthcare and
provides numerous enticing opportunities (Gonçalves and Paiva, 2017).

 Treatment of Genetic illnesses

One of the principal uses of gene therapy is the treatment of genetic illnesses. It holds the
potential to treat disorders caused by a single defective gene, such as cystic fibrosis, muscular
dystrophy, and sickle cell anaemia. By inserting a functioning copy of the faulty gene, gene
therapy may cure the underlying genetic cause of various illnesses. The first gene treatments
for monogenic illnesses are currently in use in clinical practice, and it is to be predicted that
the number of authorized gene therapies will expand further over the next years (Kirschner
and Cathomen, 2020).

 Cancer Therapy

Gene therapy provides new paths for cancer treatment. Researchers are developing gene
treatments that target cancer cells, either by transforming the patient’s immune cells to better
identify and eliminate cancer cells (CAR-T cell therapy) or by directly altering the genes
responsible for cancer development and proliferation. Recent progress in developing
programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like
effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat
(CRISPR)–Cas-associated nucleases, has greatly expedited the progress of gene editing from
concept to clinical practice (Belete, 2021).

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  Rare illnesses

Many uncommon illnesses lack effective medicines owing to their restricted patient
populations. Gene therapy may give a customized approach to treat uncommon illnesses,
giving it a viable option for tackling problems that typically go untreated or under-researched
(Jensen et al., 2021).

 Targeted Therapies

Gene therapy enables for precision targeting of certain genes or cells, reducing collateral
harm to healthy areas. This focused strategy is a crucial benefit in decreasing side effects and
enhancing the overall safety of therapies. The most frequent gene therapy vectors are viruses
because they can detect select cells and transmit genetic information into the cells’ genes
(Gonçalves and Paiva, 2017).

 Regenerative Medicine

Gene therapy may encourage tissue regeneration and repair. This offers tremendous promise
in treating degenerative disorders, such as Parkinson’s disease or age-related macular
degeneration, by stimulating the creation of healthy cells and tissues.Although non-viral
vectors-based gene therapy has yet to be licensed as therapies for neurodegenerative
illnesses, recent advancements in the clinical trials have caused significant optimism. Better
knowledge of the genesis and course of the neurodegenerative illnesses may assist quick
diagnosis and target selection, which should allow early therapy for some of these diseases.
As research continues in optimizing transgene design, transport, and vectors, the prospects of
gene therapy for neurodegenerative illnesses will likely grow even brighter (Martier, and
Konstantinova, 2020).

 Infectious Disease Prevention

Gene therapy may be applied to boost the body’s immunological response to infectious
illnesses. Gene therapy for infectious disorders needs the introduction of genes engineered to
selectively block or restrict the gene expression or function of gene products, such that the

13
reproduction of the infectious agent is inhibited or limited. In addition to this internal
intervention, gene therapy may be utilized to interfere in the propagation of the infectious
agent at the extracellular level. This might be done by prolonged production in vivo of a
secreted inhibitory protein or by activation of a particular immunological response (Li et al.,
2020).

 Individualized Medicine

Gene therapy may be adapted to an individual’s genetic composition, allowing for

individualized treatment strategies. This method is especially effective in situations with
different genetic variables, including cancer or cardiovascular problems. The technique,
predicated on developments in gene research and data analytics, promises transformational
prospects for illness treatment and might upend the way medicine historically has been
conducted. Rather than classify individuals together under broad categories of disorders,
precision medicine tries to customize prevention, diagnosis, and therapy to a person’s unique
biochemical composition (Das et al., 2015).

 Ex Vivo vs. In Vivo Approaches

Gene therapy may be done using two major techniques — ex vivo and in vivo. Ex vivo gene
therapy involves removing and changing the patient’s cells outside the body before returning
them, whereas in vivo treatment directly targets cells inside the body. In vivo gene therapy
works through the help of a vector, which directly inserts functional copies of a gene into
target cells to treat a mutated or missing gene. Ex vivo gene therapy works by genetically
modifying a patient’s stem cells, which then replace target cells that have a missing or
malfunctioning gene. Today, ex vivo gene therapy is most frequently applied to
hematopoietic stem cells (HSCs); this is used to treat blood and immunological diseases as
well as genetic diseases that affect tissues and organs that can be reached by blood cells. Both
treatments provide distinct options and may be selected depending on the individual
condition and therapeutic aims (Martier, and Konstantinova, 2020).

14
  Advancements in Delivery techniques

Ongoing research focuses on developing gene delivery techniques, such as viral vectors and
nanoparticles, making them safer and more efficient. Enhanced delivery systems will
increase the uses of gene therapy. Viral vectors are the most extensively utilized delivery
vehicles for gene therapy in vivo and in vitro owing to their high transfection efficiency and
sustained transgene expression. With the advent of nanotechnology, innovative Nano carriers
are progressively replacing viral vectors, emerging better performance in Gene therapy
(Schmidt et al., 2023).

15
2.5 Emerging Technologies in Gene Therapy

 CRISPR Gene Therapy

The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and
CRISPR-associated (Cas) proteins has broadened the applications of genetic research in
hundreds of labs throughout the world and is altering our approach to gene therapy.
Traditional gene therapy has aroused some concerns, since its dependence on viral vector
delivery of therapeutic transgenes may produce both insertional oncogenesis and
immunogenic toxicity. While viral vectors remain a critical delivery mechanism, CRISPR
technology presents a reasonably easy and effective alternative for site-specific gene editing,
obliviating several problems expressed by classical gene therapy. Of the CRISPR/Cas
systems, CRISPR/Cas9 is the most developed and commonly utilized technology for
contemporary genome editing (Uddin et al., 2020) (Asmamaw and Zawdie, 2021).

The bacterial CRISPR locus was Initially reported by Francisco Mojica and subsequently
recognized as a critical part in the adaptive immune system in prokaryotes. The locus
comprises of snippets of viral or plasmid DNA that previously infected the microbe (later
named “spacers”), which were located between an array of short palindromic repeat
sequences. Later, Alexander Bolotin found the Cas9 protein in Streptococcus thermophilus,
which unlike other known Cas genes, Cas9 was a big gene that encoded for a single-effector
protein with nuclease activity. They additionally identified a similar sequence in the target
DNA next to the spacer, subsequently termed as the protospacer adjacent motif (PAM)—the
sequence essential for Cas9 to detect and attach its target DNA. Later investigations found
that spacers were translated to CRISPR RNAs (crRNAs) that direct the Cas proteins to the
target location of DNA. Following investigations found the trans-activating CRISPR RNA
(tracrRNA), which forms a duplex with crRNA that jointly lead Cas9 to its target DNA. The
potential application of this technology was simplified by introducing a synthetic combined
crRNA and tracrRNA construct dubbed a single-guide RNA (sgRNA). This was followed by
research showing effective genome editing by CRISPR/Cas9 in mammalian cells, hence
raising the prospect of applying CRISPR/Cas9 in gene therapy (Uddin et al., 2020)

CRISPR/Cas9 is a simple two-component system utilized for successful targeted gene

editing. The first component is the single-effector Cas9 protein, which includes the
endonuclease domains RuvC and HNH. RuvC cleaves the DNA strand non-complimentary to
the spacer sequence while HNH cleaves the complementary strand. Together, these domains

16
create double-stranded breaks (DSBs) in the target DNA. The second component of efficient
targeted gene editing is a single guide RNA (sgRNA) bearing a scaffold sequence which
facilitates its attachment to Cas9 and a 20 base pair spacer sequence complementary to the
target gene and next to the PAM region. This sgRNA leads the CRISPR/Cas9 complex to its
designated chromosomal site. The editing mechanism then depends on one of two
endogenous DNA repair pathways: non-homologous end-joining (NHEJ) or homology-
directed repair (HDR). NHEJ happens far more often in most cell types and includes random
insertion and deletion of base pairs, or indels, at the cut site (Asmamaw and Zawdie, 2021).
This error-prone method frequently leads in frameshift mutations, often causing a premature
stop codon and/or a non-functional polypeptide. This route has been especially effective in
genetic knock-out investigations and functional genomic CRISPR screenings, but it may also
be useful in the clinic in the setting where gene disruption gives a therapeutic potential. The
second approach, which is particularly tempting to use for therapeutic applications, is the
error-free HDR pathway. This route includes utilizing the homologous portion of the unedited
DNA strand as a template to fix the damaged DNA, resulting in error-free repair.
Experimentally, this route may be utilized by supplying an external donor template with the
CRISPR/Cas9 machinery to allow the desired change into the genome (Uddin et al., 2020)

 Base Editing

Treating human genetic illnesses has provided a substantial and enduring challenge to the
medical community with existing treatments being restricted by their targeting breadth,
accuracy, and safety. In recent years, the introduction of Base Editors (Bes) has
revolutionized the genome editing industry, enabling a precise and effective therapeutic
approach for repairing single nucleotide mutations responsible for more than half of human
genetic illnesses without inflicting unintentional DNA damage (Petraitytė et al., 2021). Since
its creation in 2016, Bes have been effectively employed in both research and clinical settings
to treat various human genetic illnesses, including sickle cell disease and amyotrophic lateral
sclerosis. Cytosine Bes (CBEs) and adenine Bes (ABEs) are the two primary kinds of Bes,
consisting of a cytosine deaminase (e.g. rat APOBEC1) or an adenine deaminase (TadA)
linked to a catalytically deficient Cas effector, respectively. CBEs deaminate cytosine to
uracil within an editing window, which is interpreted by endogenous cellular repair
machinery as thymine, accomplishing C-to-T base pair conversions. Similarly, ABEs

17
deaminate adenine to inosine, which is interpreted as guanine by cellular repair machinery,
accomplishing A-to-G base pair conversions (Rees et al., 2021), (Petraitytė et al., 2021).

In the four years after their development, major advancements were made to Bes boosting
both editing efficiency and therapeutic usefulness. In the case of CBEs, the addition of a
uracil glycosylase inhibitor (UGI) to prevent the uracil intermediate from reverting to
cytosine, the addition of a C-terminal nuclear localization signal (SV40 NLS) to ensure
transport to the nucleus, and the replacement of the catalytically dead Cas9 with a Cas9
nickase (nCas9) to nick the non-edited strand and preserve the edited nucleotide led to
cytosine BE2 and BE3, each with improved editing efficiencies. Further enhancements were
accomplished by inserting an extra UGI and modifying the amino acid linker between the
nCas9 and the rAPOBEC1 cytosine deaminase to increase subunit flexibility, culminating in
the production of BE4. Although discoveries before 2020 enhanced BE editing efficiency,
possible dangers related with Cas-dependent and Cas-independent genomic and
transcriptome off-target editing remain a serious obstacle for practical deployment (Rees et
al., 2021).

 Prime Editing

Prime editing is a gene editing technology that can accomplish targeted tiny insertions,
deletions, and base swaps in a precise fashion. This seems pretty substantially like previous
CRISPR approaches. The consequences of prime editing have been attained earlier in various
methods. The capacity to remove bases is a characteristic of knockouts, whereas the ability to
insert certain nucleotides in a precise way is the notion behind knock-ins. What differentiates
prime editing unique from regular CRISPR is that it permits focused editing without causing
double-stranded DNA breaks. Moreover, tailored insertions may be done without the
requirement for donor DNA templates. Looking to merely modify bases? No issue. Prime
editing improves the restricted breadth of existing base editing skills (4 potential
combinations C>T, G>A, T>C, and A>G) to all 12 combination swaps (Liu et al., 2022).

Prime editing is an all-in-one solution with minor enhancements in the CRISPR process that
effect the end result—a classic sum-is-larger-than-the-parts issue. Similar to CRISPR, prime
editing needs the presence of a Cas endonuclease and a single guide (sg) RNA. However,
given the principle of prime editing is to edit sequences without causing a double-stranded

18
break, both components are somewhat adjusted. Instead of standard Cas9, this approach
employs Cas9 nickase—a type of Cas9 that nicks the DNA rather than creating double-strand
breaks—fused to a reverse transcriptase. This Cas9 fusion is referred known as a prime editor
(PE) (Liu et al., 2022).

 Epigenome Editing

The epigenome (meaning ‘above the genome’) is a system of reversible markers controlling
how the DNA is read, translated and utilized. DNA encodes the fundamental building blocks
of life and the epigenome determines which blocks are available for active utilization. The
molecular machinery of the epigenetic system may selectively package certain portions of
DNA away, making them inaccessible and less active. The technology may also unwind the
DNA to expose it for active usage. Epigenetic editing, often termed genetic tuning, actively
manipulates epigenetic machinery to impact transcription. Orchestrating the naturally-
occurring conductors of gene expression may modify the functional states of cells and
rebalance gene expression. This modality is fundamentally unique from the ‘molecular
scissors’ method normally linked with gene editing. While epigenetic editing and nuclease-
based gene editing employ comparable machinery to send their effectors to the DNA,
epigenetic editing is a fully ‘no-cut’ procedure. Instead of slicing into the DNA like a
nuclease, epigenetic editing modifies the epigenomic markers decorating the DNA, leaving
the base coding unchanged (Atlante et al., 2020).

This technique allows epigenetic editing unparalleled control of gene expression, enabling
people to fine-tune gene output. This accuracy has tremendous therapeutic implications since
altering the epigenetic imprint of a cell alters its transcriptional profile and, eventually, its
biological activity. Epigenetic editing is a daring approach created from the desire to increase
the possibilities of genetic medicine. But what makes it unique from other techniques? The
approach is unusual because it retains the original DNA intact, has excellent multiplexing
capabilities, and hence enables considerable flexibility in gene output. These advantages
enhance the potential spectrum of treatment targets accessible to precision medicine beyond
those of traditional therapeutic technique (Rittiner et al., 2022).

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  RNA Therapies

RNA-based therapies are developing as a potent platform for the treatment of numerous
ailments. Currently, the two main categories of nucleic acid therapeutics, antisense
oligonucleotides and small interfering RNAs (siRNAs), achieve their therapeutic effect
through either gene silencing, splicing modulation or microRNA binding, giving rise to
versatile options to target pathogenic gene expression patterns. Moreover, continuing
research intends to broaden the breadth of RNA-based therapeutics to incorporate more
complex nucleic acid templates, such as messenger RNA, as shown by the first licensed
mRNA-based vaccination in 2020. The rising number of authorized sequences and current
clinical studies has generated substantial attention in the chemical development of
oligonucleotides and nucleic acids as pharmaceuticals, particularly with the FDA approval of
the first siRNA treatment in 2018. As a consequence, a diversity of creative ways is
developing, demonstrating the potential of RNA as one of the most significant therapeutic
tools in the drug design and development pipeline (Halloy et al., 2022). It took 20 years of
study in nucleic acid medicinal chemistry to completely develop synthetic nucleic acids as
efficient RNA-targeting medicines (RTDs). These have now become a highly busy sector of
drug discovery and development in the pharmaceutical business and a total of 14 RTDs have
been licensed to far, with many more in clinical trials. Currently, commercialized RTDs are
either single-stranded antisense oligonucleotides (ASOs) or double-stranded small interfering
RNAs (siRNAs) engaging a messenger RNA (mRNA) target via Watson–Crick base pairing.
However, nucleic acids in their original form are particularly vulnerable to nuclease
destruction and display limited cellular absorption. As a consequence, RTDs on the market all
feature chemical changes at the backbone, base, or sugar level to increase their biological
stability and optimize their pharmacokinetics qualities (Bege and Borbás, 2022).

Single-stranded oligonucleotides (ssONs) contain three primary types of therapeutics:

RNase-H active oligonucleotides, steric-blocking oligonucleotides, and aptamers. RNase-H
active and steric-blocking oligonucleotides that bind an RNA target in a sequence-specific
way have been named antisense oligonucleotides (ASOs). On one side, RNase-H active
oligonucleotides bind a cognate pre-mRNA or mature mRNA sequence, which initiates
RNase-H binding and cleavage of the mRNA target. This technique has been utilized for
protein knockdown and also for the knockdown of harmful RNA species, such as those
prevalent in repeat-expansion disorders. On the other hand, steric-blocking oligonucleotides
do not degrade their mRNA target but, rather, mask a homologous region upon hybridization
20
and impede further binding by RNA-binding proteins and intracellular RNA species, thereby
preventing protein translation ( Scharner and Aznarez, 2021).

Small interfering RNAs are developing as a novel class of therapeutics that offers
considerable potential for the treatment of genetically specified illnesses by targeting disease-
causing mRNAs for destruction. Their advantages over conventional drugs include: (i) ease
of design rationally achieved based on sequence information and straightforward screening,
leading to drug candidates within short periods of time; (ii) the ability to target disease genes
previously considered ‘undruggable’; and (iii) unprecedented potency and duration of effect.
However, therapeutic effectiveness is based on their efficient transport to sick tissues (Halloy
et al., 2022).

 Zinc-finger nucleases (ZFNs)

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes produced by connecting a zinc
finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains may be
tailored to target certain desired DNA sequences and this allows zinc-finger nucleases to
target unique sequences within complicated genomes. By taking advantage of native DNA
repair machinery, these compounds may be utilized to accurately modify the genomes of
higher species. Alongside CRISPR/Cas9 and TALEN, ZFN is a major technology in the area
of genome editing (Cantos et al., 2014).

Using ZFNs to change endogenous genes has always been a tough endeavor owing largely to
the issue of producing zinc finger domains that target the targeted region with adequate
specificity. Improved techniques of creating zinc finger domains and the availability of ZFNs
from a commercial provider now put this technology in the hands of growing numbers of
researchers. Several organizations are also exploring different kinds of engineered nucleases
including engineered homing endonucleases and nucleases based on engineered TAL
effectors. TAL effector nucleases (TALENs) are especially fascinating since TAL effectors
seem to be relatively straightforward to construct and TALENs may be utilized to target
endogenous loci in human cells. But to far no one has documented the isolation of clonal cell
lines or transgenic creatures using such chemicals. One kind of ZFN, known as SB-728-T,
has been studied for possible utility in the treatment of HIV (Wallis et al., 2020)

21
2.6 Challenges in Gene Therapy

 Gene delivery and activation

For other illnesses, gene therapy will function only if we can deliver a normal gene to a big
number of cells—say several million—in a tissue. And they have the proper cells, in the
correct tissue. Once the gene reaches its target, it must be activated, or switched on, to
generate the protein it encodes. And once it’s switched on, it must stay on; cells have a
history of shutting down genes that are excessively active or showing other strange
behaviors. Introducing modifications into the inappropriate cells Targeting a gene to the
relevant cells is critical to the success of any gene therapy treatment. Just as crucial, however,
is making sure that the gene is not integrated into the incorrect cells. Delivering a gene to the
incorrect tissue would be inefficient, and it might create health concerns for the patient. For
example, poor targeting might integrate the therapeutic gene into a patient’s germline, or
reproductive cells, which eventually create sperm and eggs. Should this happen, the patient
would carry the inserted gene to his or her offspring. The repercussions would vary,
depending on treatment (Gonçalves and Paiva, 2017).

 Short-lived Nature

Before gene therapy may become a lasting remedy for a disorder, the therapeutic DNA
injected into target cells must be functional and the cells holding the therapeutic DNA must
be stable. Problems in integrating therapeutic DNA into the nuclear genome and the rapidly
dividing nature of many cells prohibit it from achieving long-term effects. Patients need
repeated treatments ( Li et al., 2020).

 Immune response

Any time a foreign item is introduced into human tissues, the immune system is prompted to
combat the intruder. Our immune systems are highly adept at fighting off invaders such as
germs and viruses (Yatim and Lakkis, 2015). Stimulating the immune system in a manner
that lowers gene therapy efficacy is conceivable. The immune system’s heightened reaction
to viruses that it has encountered previously diminishes the efficacy to repeated treatments.
An unexpected immune reaction might cause severe sickness or even death (Freitas et al.,

22
2022). Gene-delivery vectors must be able to circumvent the body’s natural monitoring
mechanism. One approach researchers avoid generating an immune reaction is by delivering
viruses to cells outside of the patient’s body. Another is to give patients medications that
temporarily weaken the immune system during therapy. Researchers utilize the lowest
amount of virus that is effective, and wherever feasible, they select vectors that are less likely
to activate an immune response (De Haan et al., 2021)

 Problems with viral vectors

Viral vectors pose the dangers of toxicity, inflammatory reactions, and gene control and
targeting difficulties (Ghosh et al., 2020).

 Multigene disorders

Some widely occurring ailments, such as heart disease, high blood pressure, Alzheimer’s
disease, arthritis, and diabetes, are impacted by changes in numerous genes, which
complicate gene therapy (Guo et al., 2022).

 Disrupting key genes in target cells

A good gene treatment is one that will endure. Ideally, an inserted gene will continue acting
for the duration of the patient’s life. For this to happen, the inserted gene must become a
permanent part of the target cell’s genome, generally by integrating, or “stitching” itself, into
the cell’s own DNA. But what happens if the gene weaves itself into an incorrect position,
damaging another gene. This occurred in two gene therapy studies intended at treating
children with X-linked Severe Combined Immune Deficiency (SCID). People with this
condition have essentially little immune defense against germs and viruses. To evade diseases
and sickness, they must dwell in a fully germ-free environment (Shakiba et al., 2021)

Between 1999 and 2006, researchers tried a gene therapy treatment that would restore the
activity of a critical gene, gamma c, in cells of the immune system. The therapy looked quite
beneficial, restoring immunological function to majority of the youngsters who got it. But
subsequently, 5 of the youngsters got leukaemia, a blood cancer. Researchers observed that

23
the newly transplanted gamma c gene had sewn itself into a gene that ordinarily helps control
the pace at which cells divide. As a consequence, the cells started to divide out of control,
developing leukaemia. Doctors treated 4 of the patients effectively with chemotherapy, but
the fifth died. This terrible episode revealed crucial safety issues, and researchers have now
devised better techniques to deliver genes. Some modern vectors contain capabilities that aim
DNA integration to specified “safe” regions in the genome where it won’t create issues. And
genes delivered to cells outside of the patient may be examined to discover where they
integrated, before they are reintroduced to the patient (Shahryari et al., 2021).

 Insertional mutagenesis

If the DNA is integrated in a sensitive region in the genome, for example in a tumor
suppressor gene, the treatment might generate a cancer. This has happened in clinical studies
for X-linked severe combined immunodeficiency (X-SCID) patients, in which hematopoietic
stem cells were transduced with a corrected transgene using a retrovirus, and this led to the
development of T cell leukaemia in 3 of 20 individuals. One proposed method is to introduce
a functioning tumor suppressor gene to the DNA to be incorporated. This may be
troublesome as the longer the DNA is, the harder it is to incorporate into cell genomes.
CRISPR technology enables researchers to conduct considerably more accurate genetic
modifications at specified spots (Cavazzana et al., 2016).

 Cost

Alipogene tiparvovec or Glybera, for example, with a cost of $1.6 million per patient, was
reported in 2013, to be the world’s most costly medicine. Developing a novel therapy—
including bringing it through the clinical trials required for regulatory approval— is
exceedingly costly. With a small number of patients to recoup those expenditures from,
innovators may never gain money from treating such uncommon genetic illnesses. And other
people may never be able to pay them. Some disorders that can be treated with gene therapy,
such as cancer, are significantly more frequent. However, many potential gene therapy
procedures are tailored to each patient. For example, a patient’s own cells may be taken out,
changed with a therapeutic gene, and returned to the patient. This tailored technique may
prove to be incredibly beneficial, but it’s also pricey. It comes at a far greater price than

24
pharmaceuticals that can be made in quantity, which can rapidly repay the expense of their
research (Ylä-Herttuala, 2015).

 Commercial viability

Many genetic illnesses that might possibly be cured with gene therapy are exceedingly
uncommon, some affecting only one person out of a million. Gene therapy might be life-
saving for many patients, but the enormous expense of creating a medicine makes it an
unattractive proposition for pharmaceutical corporations (Razi Soofiyani et al., 2013).

25
2.7 Ethical Considerations in Gene therapy

 Off-Target Mutation

The most evident ethical argument especially from the National Institute of Health (NIH)
opposing GGE is the off-target impact. Off-target gene mutation might possibly result in
insertional mutagenesis and gene mutation. Bioethicists and researchers say that genome
editing is new and unexpected technology, and little is known about gene control and
processes of embryonic development; consequently, the implications of germline treatment
may be catastrophic (Zhang et al., 2020). Despite the fact that CRISPR/Cas shows to be an
effective technique for therapeutic somatic application, it has not reached the level to be
exploited in human genome editing for clinical reproductive reasons. Therefore, the apparent
long-term impacts cannot be dismissed. Genome editing done on human embryos has a
substantial risk of producing pathologic disorders and impairments that might permanently
harm the patient and the progeny. Although the specificity of Cas9 targeting is closely
regulated, possible off-target cleavage activity might still occur in DNA sequences and has
been proven in prior investigations. Nevertheless, integrated viral vectors like retrovirus,
lentivirus, and even adeno-associated viruses might transfer a gene of interest into a non-
targeted area of the host genome which can likely result in insertional mutagenesis (Ishii and
Beriain, 2019). A research in an animal model suggests that the integration of AAV into
chromosome 19 might likely result in genotoxic consequences, resulting to neoplastic
changes that are prone to cancer growth. In addition, off-target integration has been
documented in lentiviral vector systems (LV), one of the principal delivery vehicles because
to its strong tissue tropism and long-term expression of the transgene. However, revised
methodologies have been devised to enhance and optimize LV systems for efficient and
precise gene delivery (Blattner et al., 2020).

 Genetic Mosaicism

In CRISPR germline gene therapy, the CRISPR/Cas vector is introduced shortly after
fertilization so that each consecutive cell emerging from cleavage is genetically changed.
However, the vector may survive and transcribe, making it feasible to further introduce the
Cas protein into portions of previously altered cells and perhaps induce another cleavage,
resulting to mosaicism. Some cells may ultimately acquire edits that are different from those

26
of other cells, resulting to changes in gene copy quantity, causing skin, brain, and heart
diseases, and compromising embryo development (Li et al., 2020). In a research, substantial
levels of mosaicism were discovered following germline editing of a model bovine embryo
utilizing the Cas9 system. This result indicates the probability of its emergence in human
embryos if left uncontrolled. Furthermore, the technical technique of evaluating mosaic
mutations in an altered embryo may be useless, since the tiny number of cells picked for
testing may not contain a mosaic mutant cell. In the summer of 2019, the possible impact of
mosaicism coming from the therapeutic use of germline editing was considered by the US
National Academy of Medicine, the US National Academy of Sciences, and the Royal
Society of medicine. The lack of clear evidence from experts that mosaic mutation has not
occurred in a range of cell and tissue types of early-stage human embryo editing, as well as
the inability of the technology to validate that a particular edit is correct and devoid of mosaic
mutation could make it difficult for the public to support the application (Mehravar et al.,
2019).

 Informed Consent

Following the first gene therapy fatality documented in a clinical study in September 1999,
the informed choice about participation in a clinical trial has attracted various concerns. It is
advised that participants undertaking gene therapy clinical trials must be adequately informed
on the possible dangers and advantages connected with treatment to offer them with ample
knowledge on which to decide to participate or not without pressure. A research by the
National Human Genome Research Institute (NHGRI) advocated the necessity and relevance
of informed permission in CRISPR somatic genome editing after surveying individuals with
sickle cell disease. Inasmuch as gene treatments promise future change by healing many
incurable illnesses, the apparent advantages of the technology should not outweigh the
challenges that the patients may experience in recognizing long-term risks. Although somatic
gene therapy fulfills the requirement for informed consent, germline embryo editing raises a
more challenging regulatory question, that is, whether agreement of a future generation is
necessary and, if so, who should express consent since embryos cannot consent to germline
intervention (Sheppard et al., 2016). Moreover, the extent of authority over the embryo by
the prospective parents and practitioners raises ethical debate, whether parents will be the
only autonomous entity to make decisions for their unborn babies or will this be seen as

27
usurping the interests of future generations who are unable to consent at the time of the
decision. Due to too many unknowns, it is unclear what information would be necessary or
accessible to effectively advise potential parents of hazards, particularly those for future
generations. This provides a substantial issue in getting informed consent. As additional gene
treatments for incurable hereditary disorders enter the consent clinic, a discussion on ethics
should be started so that these issues can be discussed in a clear, fair, and balanced manner,
rather than allowing any particular profession to make the final decision on where the ethical
limits should be drawn. It is an inescapable reality that any study which may eventually show
to be a breakthrough should totally fulfill the ethical norms of informed consent (Ansah,
2022).

 Enhancement and Eugenics

Genetic enhancement or improvement is also a real worry concerning the use of gene
therapy. Enhancement gene therapy is modifying genes to enhance the traits of an individual
according to the interests of the person. Genetic treatment, on the other hand, includes
modifying defective genes to prevent or cure illnesses. A famous example of enhancement
treatment is the injection of recombinant human growth hormone (rhGH) into children of low
stature to boost the development rate and ultimate height. However, the injection of rhGH
into youngsters of average height in an effort to grow them taller may perhaps raise ethical
concerns. Furthermore, sportsmen depend on human recombinant erythropoietin (EPO) for
improvement (Gonçalves and Paiva, 2017). The EPO hormone is used to promote the
synthesis of red blood cells that are utilized to treat renal dialysis and anemia. However,
athletes who do not have any health concerns seek EPO treatment in an effort to boost
performance in competitive sports where muscles demand a lot of oxygen. Inasmuch as
certain enhancement procedures are deemed ethically immoral as it moves from the natural,
the line between enhancement and treatment may be a contextual problem and must be
properly recognized. An enhancing application may be therapeutic and vice versa. The
improvement of the height of short individuals whose condition is a consequence of human
growth hormone insufficiency, as well as, boosting the skin colour of patients suffering from
vitiligo signal a treatment enhancement. This shows that genetic treatment and augmentation
may have shared features. Moreover, augmentation might possibly lead to eugenics
(Schoener and Borger, 2019). CRISPER/Cas9 presents the promise of modifying the

28
germline to choose human qualities like as beauty, character, body shape, and intellect. This
makes it feasible to develop evolutionary people and advance the human race. In 2015, the
UNESCO International Bioethics Committee cautioned on the eugenic hazards of germline
treatments. The committee indicated that the inclusion of gene editing methods with gene
therapy may perhaps transform the therapeutic application to racial betterment. Hence, the
equal dignity of all human beings may be transformed and ultimately reinvigorate eugenics.
To restrict the use of technology, an intervention aiming at modifying the human genome
may be undertaken only for preventative, diagnostic, or therapeutic objectives, and any effort
to attain this goal should be outlawed (Locke, 2020). Furthermore, the scope of human
condition to which gene therapy is relevant should be clearly defined and carefully controlled
to make people aware of illnesses and states of sickness that need experimental therapies.
This may resolve concerns regarding equitable accessibility while reducing nontherapeutic
characteristics augmentation. Scientific researchers should clearly state the goal of any
applied or basic research involving CRISPR/Cas editing; either the research is to provide a
therapeutic solution, to generate preliminary data for the development of human genome
editing applications, or to just improve the expression of certain traits for non therapeutic
purposes. These differences are crucial in the sense that even if one opposes human
enhancement treatment, there are vital uses of CRISPR/Cas editing that do not serve that
objective. Nonetheless, it is vital to highlight that differentiating eugenics from therapy could
be challenging. For example, it is frequently argued whether boosting the immune system by
gene and immunotherapeutic techniques constitutes eugenics or not. As a consequence, a
case-by-case study is necessary to answer several difficulties. In truth, eugenics is anchored
in a societal construct which justifies discrimination and injustice against individuals who are
genetically unsuited. Therefore, it is necessary to highlight that gene therapy, when put in the
correct environment, has the ability to remove birth defects and fatal illnesses (Ayanoğlu et
al., 2020).

29
 CONCLUSION AND RECOMMENDATIONS

In conclusion, the future of gene therapy is an intriguing frontier in the domain of medical
research, providing a multiplicity of prospects and concurrently posing complicated
obstacles. This subject has shown the great potential to change healthcare by applying genetic
therapy in the treatment of genetic problems, treating cancer, and customizing medicine.
However, it has also underlined the importance for ethical concerns, equal access, and strong
safety measures. The route ahead will demand a careful balance between scientific progress
and ethical responsibility. In an age distinguished by tremendous scientific achievements, this
topic provides a look into a future when gene therapy has the potential to alter medicine and
healthcare as we know it. Understanding the options it brings, from addressing genetic
illnesses to tailored therapies, is crucial. Equally crucial is understanding and resolving the
ethical and practical difficulties that come with this strong technology.

30
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