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Drug Delivery to the Immune System: Immunotherapies and Vaccines 459
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Zhang F, Ni Q, Niu G, Chen X (2017)
Chapter 20
Unlocking the Potential of Gene Therapy: Principles
and Therapeutic Applications
Vijay Kumar
Abstract
Genetic diseases pose significant threats to human health, prompting the rapid emergence of gene therapy
as a potential solution. This innovative approach utilizes viral and non-viral particles to transport DNA or
RNA into target cells, offering promising treatment avenues for conditions like cancer and genetic
disorders. However, the development of these particles is complex and time-consuming, often resulting
in limited yields of high-quality products. Ensuring the superiority of gene delivery systems requires
meticulous assessment of various factors, including particle quantification, characteristics, purity, potency,
safety, and stability. Gene therapy revolves around modifying or replacing faulty genes responsible for
diseases with healthy ones. Advances in molecular biology, particularly genetic engineering, have facilitated
easier manipulation of genes. Genes serve dual functions: directing protein synthesis and regulating protein
production. Alterations in genes can lead to dysfunctional proteins, causing diseases. Gene therapy aims to
rectify this imbalance by inserting normal genes to compensate for abnormal ones or by selectively reversing
mutations. Diseases like cystic fibrosis, muscular dystrophy, and diabetes are potential targets for gene
therapy. By repairing faulty genes at the molecular level, this approach holds immense promise for treating
various disorders, including cancer and cardiovascular diseases. Effective gene delivery systems are crucial
for this endeavor, necessitating a thorough understanding of the interaction between targeting cells and
delivery systems.
Key words Adenovirus, Gene delivery system, Lentivirus, Retrovirus
1 Introduction
Genetic diseases critically intimidate human wellbeing and have
forever been one of the unruly conditions facing humanity. Gene
therapy is a hastily rising field that uses viral and non-viral particles
to carry DNA or RNA to a patient’s target cells to care for difficult
diseases, such as cancer and genetic diseases. The development of
such particles is intricate, may need years, and results in diverse
products with little yields of premium particles. Quantification of
particle, characteristics, purity, potency, safety, and stability are
required to guarantee gene delivery system superiority. The
461
462 Vijay Kumar
Fig. 1 Structure of different viral vectors
Fig. 2 Characteristics of different viral vectors
essential standard of gene therapy engaged the modification of
genes and exchanging the faulty or non-working gene, which is
mainly accountable for the basis of the disease, with the preferred
gene to treat the diseases.
The organization of DNA was worked by Watson and Crick in
1953, and Arber, Nathans, and Smith worked on DNA restriction
enzymes, which led to the fast development in the field of genetic
engineering. Gene therapy provides modern medicine with new
insights that seemed impossible 20 years ago. Development in
molecular biology and, particularly, molecular medicine is now
altering the fundamentals of clinical medicine. A range of viral
(Figs.
1 and 2) and non-viral potentials are existing for fundamental
and clinical research. Gene transfer could be accomplished through
in vivo and ex vivo methods (Fig. 3).
Unlocking the Potential of Gene Therapy: Principles and Therapeutic Applications 463
Fig. 3 In vivo versus ex vivo gene delivery
Developments in molecular biological knowledge such as
genetic engineering have made scientists to maneuver genes easier.
A gene is a linear series of DNA that codes for certain protein
1]. Proteins carry out a variety of necessary functions in the
[
body. DNA is made up of four bases: adenine, guanine, cytosine,
and thymine. These bases are structured to make the genes
2]. When genes are changed, the altered proteins are incapable
[
to do their usual function, resulting in diseases. Genes are mostly
associated with two sorts of function—shaping the arrangement of
various proteins and governing where, when, and in what quantity
each protein is made [
to counteract an abnormal one and replace faulty genes through
targeted reverse mutation. Researchers initiated researching gene
therapy in 1980, and the first gene therapy was done in 1990 for
correcting severe combined immunodeficiency [
therapy was termed as gene replacement therapy. Genetic disorders
like cystic fibrosis, muscular dystrophy, and diabetes could be treated by gene therapy [
Scientists h
disorders by altering and repairing the disease-causing gene. Gene
therapy is one of the important avant-garde remedial technologies
developed with genetic engineering and gene cloning techniques
5]. Gene therapy can revamp or even substitute the disease-
[
causing genes at the molecular level, repairing faulty protein.
After years of advancement, gene therapy has shown immense
possibility in curing important disorders caused by genetic abnormalities such as cancer, acquired immunodeficiency syndrome, and
cardiovascular diseases.
Corrected
cells to stimulate gene expression [
consequent to supply a patient’s somatic cells with corrected
genetic molecules for synthesizing precise therapeutic proteins to
amend genetic diseases. In order to get a working model of gene
3]. Gene therapy aims to insert a normal gene
1]. Initially, gene
4].
een investigating the explanation to genetic
ave b
genetic molecules are delivered to the nuclei of host
2, 3]. Gene therapy has the
464 Vijay Kumar
Fig. 4 Requirements for being an optimum vector
Fig. 5 Requirements for successful gene therapy
delivery system, it must necessitate the full understanding of interface between targeting cell and gene delivery system. The gene
delivery systems are made of three constituents, such as a plasmidbased gene expression system, a gene that codes a specific therapeutic protein, and a gene delivery system that manages the delivery of
the gene to a precise position in the patient [
3, 4]. A good gene
delivery system necessitates the modified genetic molecule to stay
stable within the host cells [
5, 6].
Gene delivery systems are categorized as: viral, non-viral, and
combined hybrid systems. Viral-mediated gene delivery systems are
made of viruses that are altered to be replication-deficient, nevertheless which can deliver DNA for expression. Adenoviruses, retroviruses, and lentiviruses are commonly used as viral gene delivery
vectors [
7]. Important features of a suitable vector, requirement for
successful gene therapy, and difficulties in gene therapy are represented in Figs.
4, 5,
and
6.
Unlocking the Potential of Gene Therapy: Principles and Therapeutic Applications 465
2 Materials
Fig. 6 Difficulties in gene therapy
2.1 Equipment
Descriptions of particular equipment are given; however, any
model of comparable capability can easily be substituted.
1. Biosafety Cabinets: Airstream
®
Class I Biological Safety Cabinet for providing protection for human resources and the
adjacent environment, but not the equipment itself.
2. Labculture
®
G4 Class II Type A2 Biological Safety Cabinet for
providing protection for human resources, the adjacent environment, and the equipment itself.
3. Airstream
®
Class III Biological Safety Cabinet for providing
the highest level of protection for human resources, the adjacent environment, and the equipment itself.
4. Cell-IQ™ Series 8.1 cu.ft.
CO
Incubator (MCO-230AI-
2
CUVLG-PA) for providing the best environment for growing
and maintaining microbiological cultures and cell cultures.
They control internal temperature, humidity, and carbon dioxide levels so that even the most susceptible cultures are capable
to thrive.
5. Pharmaceutical Refrigerator (MED 520 PRO-ACTIVE) for
storing a variety of cell and gene cultures at low temperatures.
These cultures and products need to be kept in constantly
refrigerated conditions in order to be fully safe and effective.
6. NuAire Blizzard
NU-99729VFT ultra-low freezer for storing
various cell and gene cultures in cold temperatures. It is
466 Vijay Kumar
significant that these freezers stay at their required temperature
because dropping too low or rising too high could compromise
the cultures’ viability.
7. Sorvall X4F R Pro Centrifuge for separating a variety of components of a fluid. In cell and gene therapy applications, it is
typically used to isolate strains of cells or DNA.
2.2 Reagents and Solutions
3 Methods
3.1 Adenovirus
1. MAXgene™ GMP Transfection Reagent solution
(Polysciences).
2. MAXgene™ GMP Transfection Reagent powder
(Polysciences).
3. Transporter 5™ Transfection Reagent (Polysciences).
4. PEI MAX™—Transfection Grade Linear (Polysciences).
Adenovirus (AdV) is an unenveloped double-stranded DNA virus
with an icosahedral nucleocapsid arrangement. Because of the large
range of Adenovirus hosts, simple refinement, genetic stability, big
foreign gene holding capacity, and ability to transfect DNA into
many cell types, this viral vector is most commonly used in gene
therapy.
Adenoviru
ses a
re one of the biggest and most intricate viruses,
whose organization was studied with cryo-electron microscopy and
X-ray diffractometry. It was found that crystal structures of an Ad
proteins consisted of fiber knob, shaft, domains, penton base,
hexon, and cysteine protease. Ad capsid is made up of 252 sub-units
called capsomeres, which consist of 240 hexon proteins and 12 penton bases. Every 12 capsid angles include penton bases draped by
5 hexons. The penton base works as a clip for the fiber protein,
which appears as an antenna. Fiber is a homotrimer, where three
similar polypeptides connect in the same direction, and which consists of three structurally and functionally unlike domains:
(i) N-terminus, which connects the fiber to the penton base;
(ii) C-terminus, which is accountable for fastening to the receptor;
and (iii) shaft, which differs in length as per the serotype of the virus
8, 9]
.
[
First explanation
of Adenoviruses took place in the early 1950s.
Adenoviruses were first isolated from human adenoid tissue
10]. In the last seven decades, a number of diverse serotypes
[
have been cultured and described. Adenovirus became very relevant
after establishing the biology of AdV and its ability to successfully
deliver the viral genome to the target cells. More notably, as AdV
was not oncogenic and the genomes of usual AdV were simple to
change, the creation of recombinant AdV was done. In the
Unlocking the Potential of Gene Therapy: Principles and Therapeutic Applications 467
perspective of gene delivery, serotypes 5 and 2 of the subgroup C
have been utilized mainly as their organization and biology are well
characterized and reagents required for creating recombinant are
available. AdVs of subgroup C can originate in minor to mild
pulmonary infections and may be associated with conjunctival
problems [
11].
For being doing well delivery of genetically modified DNA to
the nucleus, viruses must assist cell-specific attachment, endocytosis
internalization, transmission from endocytic vesicles to cytosol,
release into cytoplasm, translocation from one end of the nuclear
envelope to the other, and lastly expression of the delivered gene
[
12]. Direct injection by inhalation is the simplest form of viral
delivery. Nonetheless, as the virus will multiply from the injection
area, a large dose is requisite to get therapeutic efficacy. The multiplication of the virus from the injection site affects the local efficacy
and immune response. Adenoviral vectors can be used via distinct
alteration of coat proteins. There is a comparatively high level of
protein expression after transduction. Adenoviruses have many
expected qualities that permit them to be utilized as a vector for
gene therapy. Non-enveloped viruses can be preserved in lyophilized condition within a flacon tube or capsule; they can be shipped
with no cold chain; they have high transduction efficacy in cells; and
they can create 104 virus particles per infected cell [
13]. Adeno-
viruses attain appropriate transduction all the way through a high
level of expression and happen to be useful in in vivo conditions
[
14]. Adenoviruses are one of the main promising methods for
good efficacy in in vivo gene therapy. Adenoviruses are few of the
main efficient vectors for gene delivery; nonetheless, they have
certain important limitations. Few target cells have low adenoviral
receptors and consequently need a large dose of vector to cause
target-cell cytotoxicity. Furthermore, non-discriminating tropism
can lead to transduction of untargeted cells [
15]. The gravest
difficulty in the application of Ad vectors is their propensity to
cause high immune and inflammatory reactions at heavy
doses [
16].
3.2 AdenoAssociated Virus (AAV)
AAV is a parvovirus classified under the genus Dependovirus. It was
initially found as an impurity of laboratory preparations of adenovirus. AAV is a non-enveloped single-stranded DNA virus with
superior biological characteristics, genetic constancy, good gene
transduction competence, and extensive use. Nonetheless, the
packaging limit of AAV is small, the creation procedure is complex,
and the production is expensive, so the use of AAV vectors has some
limitatio
Presently, there are three ways for standardizing the
ns.
targeting of AAV infection: genetic modification, targeted regulation at the transcription level, and covalent coupling alteration on
capsid protein. Genetic modification is mainly used to give the
targeting capacity of AAV vectors.
468 Vijay Kumar
Six serotypes of AAV classified under AAV2 are mostly used for
gene-transfer studies [
17]. AAV2 cell entry is regulated by fastening
to heparin sulfate proteoglycans and αvβ5 integrin; fibroblast
growth factor receptor-1 (FGFR-1) may possibly also be
concerned. The sharing of these molecules on numerous diverse
cell types can clarify the extended in vivo expression subsequent to
AAV treatment seen in the renal, brain, skeletal muscle, pulmonary
tissue, and hematopoietic stem cells. AAV vectors could not bring
forth adverse immune or inflammatory responses. The main body
response that could have an undesirable effect is the creation of
antibodies against the vir us. Gene therapy with AAV vectors has
wide future uses for curing Duchenne muscle disease DMD.
3.3 Retroviral Vectors (RV)
The family Retroviridae includes several viruses that have prospective usefulness for gene therapy. After attachment and access into
host cells, viral enzymes mediate reverse transcription and incorporation of the virus genome into the host-cell chromatin. Retroviral vectors have the capability for stable incorporation and permit
continuing expression so that supposedly a single dose could have a
lifetime remedial. The gamma-retroviruses are not able to infect
dormant, non-dividing cells. Nonetheless, this problem can be
mitigated by the use of lentiviral vectors. RV vectors might also
be applied in the delivery of toxic genes to tumor cells, which are
vigorously dividing.
The capability
of retrovirus-based gene delivery vectors to
transmit recombinant genetic material was initially worked on in
the early 1980s [7, 18]. Retroviruses incorporate with host genome
to synthesize viral proteins that are obtained during gene delivery
18]. Retroviral vectors have the ability to carry DNA up to 8 kb
[
[
16].
Moloney murine leukemia virus (MMLV) species are the
normally used retroviruses. A virion nucleus is made up of round
gag-encoded capsid proteins. Capsid proteins are layered with
gag-coded nucleocapsid protein. The nucleus has pol-coded
enzymes, reverse transcriptase, and integrase. Simple retroviruses
encode gag (group-specific antigen), pro (protease), and pol (polymerase) genes; complex retroviruses furthermore encode a large
number of other related genes. Retroviruses were the first viruses to
be adapted for gene delivery. Retroviruses have also been exten-
19].
sively utilized in the scientific experiment of gene therapy [
The
majority of retroviral vectors used in scientific experiments are
based on the Moloney murine leukemia virus (MMLV). Moloney
murine leukemia virus is an extensively characterized vir us. The best
retroviral vector for gene therapy should be cell-specific, precisely
synchronized, and secure. Efficacy of delivery is vital, as it will
decide the efficiency of gene therapy [
19]. In order to go into a
host cell, retroviral vectors exercise the communications among
cellular receptors and virally coded proteins [
19].