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183]

Review Article

Gene Therapy and its Applications

Alka Bansal1, Ravi Prakash2, Swati Agarwal3, Uma Advani1*
1
Department of Pharmacology, SMS Medical College, Jaipur, Rajasthan, India, 2Department of Medicine, RUHS Hospital, Jaipur, Rajasthan, India,
3
Department of Obstetrics and Gynaecology, District Women Hospital, Bareilly, Uttar Pradesh, India

Abstract
Gene therapy is the treatment of abnormal or mutated genes present in cells through the addition of healthy genes or replacement/deletion/
site‑specific modification of faulty genes. Deoxyribonucleic acid, messenger ribonucleic acid (RNA), small interference RNA, microsomal
RNA and antisense oligonucleotides are the genetic materials implicated in gene therapy. They are inserted into the diseased cells using viral
or non‑viral vectors through an in vivo or ex vivo transduction. Gamma retrovirus, lentivirus, herpesvirus, adenovirus and adeno‑associated
virus are common viral vectors, while transposons, cationic polymers, dendrimers and cell‑penetrating peptides or liposomes are common
non‑viral vectors. Allologous or autologous T cells, haematopoietic stem cells and chimeric antigen receptor T cells are used for ex vivo gene
transduction. Conventional gene therapy of inserting new genetic material shows toxicity such as off‑target effects, altered immune responses,
inflammatory reactions and possible oncogenic transformation in the recipient. Newer gene editing techniques such as zinc‑finger nuclease,
transcription activator‑like effector nucleases and clustered regularly interspaced short palindromic repeats allow the site‑specific correction or
control of expression of mutated genes present in cells. Until August 2020, 23 gene‑based medicines received approval from drug regulatory
agencies in various countries and 362 were in development. Single‑gene disorders have shown encouraging results, but evidence of using
gene therapy in polygenic and common age‑related diseases is still required. Recently, the horizon of gene therapy widened to include COVID
vaccines and as an adjunct to chemotherapy. If we could overcome its limitations such as immunogenicity, mutagenicity and high costs, gene
therapy can be the medicine of the next generation.

Keywords: Chimeric antigen receptor T cells, clustered regularly interspaced short palindromic repeats, COVID‑vaccines, gene therapy,
vectors in gene therapy

Introduction cases and mortality recorded by gene therapy, it was shelved for
some years.[3] With the unfolding of the whole human genome
Edward Tatum 1966 proposed wild (healthy) genes can be
at the beginning of the 21st century and related advances in
inserted into the somatic cells of the human body through
technology, expectations to treat rare and familial diseases
vectors as a treatment modality for certain diseases.[1] The
through it were revoked.[4]
foremost evidence of the use of gene therapy dates back
to 1988 when Mr. Jesse Geilsinger, an 18‑year‑old patient Single‑gene disorders such as muscular dystrophies, cystic
received adenoviral vector‑based gene therapy for X‑linked fibrosis, alpha‑1 antitrypsin deficiency, Huntington’s disease,
ornithine transcarbamylase (OTC) deficiency. Although he lysosomal storage diseases, chronic granulomatous disease,
did not survive long, the appropriateness and practicality OTC deficiency, junctional epidermolysis bullosa and
of gene therapy were proved. Today it has emerged as one haemophilia have shown encouraging results but pieces
of the most promising fields in health. The Food and Drug of evidence for the use of gene therapy in polygenic and
Administration (FDA) approved the first gene therapy that age‑related diseases, namely obesity, type 2 diabetes, heart
was administered on 14 September 1990 in the US, when failure and renal failure, are still awaited.[4,5] Twenty‑three
Ashanti DeSilva was successfully treated for severe combined
immunodeficiency caused by adenosine deaminase (ADA) Address for correspondence: Dr. Uma Advani,
deficiency ADA.[2] Later on, due to setbacks of two leukaemia Department of Pharmacology, SMS Medical College, Jaipur ‑ 302 001,
Rajasthan, India.
E‑mail: drupadvani@gmail.com
Received: 07‑07‑2021 Revised: 11‑02‑2023 Accepted: 09‑03‑2023 Available Online: 26-04-2023

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DOI:
10.4103/JME.JME_65_21 How to cite this article: Bansal A, Prakash R, Agarwal S, Advani U. Gene
therapy and its applications. J Med Evid 2023;4:46-56.

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Bansal, et al.: Gene therapy and its applications

gene‑based medicines have received approval from the drug

regulatory authorities worldwide and around 362 are in the
development phase. They are regulated as other biological
products.[5,6]
According to the FDA definition, gene therapy products
‘mediate their effects by transcription and/or translation of
transferred genetic material and/or by integrating into the
host genome and that are administered as nucleic acids,
viruses or genetically engineered microorganisms’. Typically,
deoxyribonucleic acid (DNA), messenger ribonucleic
acid (mRNA), small interference RNA (siRNA), microsomal
RNA (miRNA) and anti‑sense oligonucleotides (ASO) are the
genetic materials used to treat specific gene functions or turn
off a gene responsible for disease or disorder development.[7]
Turning off or the reduction of the expression of a gene using
siRNA is termed gene silencing.[8]
Figure 1: In‑vivo and ex vivo methods of gene therapy
Gene therapy, also known as ‘living therapy’, may be used to
target both somatic and germ cells but prevalent therapies target
somatic cells only. Somatic cell gene therapy is non‑inheritable complex for the same. Second‑generation CART therapy has
in contrast to germ cell gene therapy.[9] Conventional gene additional co‑stimulating molecules (like CD28) necessary
therapy includes ex vivo transduction of genetically modified for signal transduction. It shows marked proliferation of T
allologous or autologous T cells, haematopoietic stem cells. Third‑generation CART has two stimulatory chains of
cells (HPSCs) transplantation and chimeric antigen receptor CD28 and CD134 or CD137. Later generations demonstrate
T cells (CARTs) as well as direct in vivo transfer of genetic augmented benefit through Akt/protein kinase B intracellular
material into the target tissues of a patient. However, newer signalling pathway which regulates the cell cycle for
gene editing techniques such as zinc‑finger nuclease (ZFN), growth and has a longer‑lasting effect when compared to
transcription activator‑like effector nucleases (TALEN) the second‑generation CART.[14‑16] The 4th generation of
and clustered regularly interspaced short palindromic CART is more effective in solid tumour treatment due to
repeats (CRISPRs) allow the correction of defective genes to the secretion of cytokines (Interleukin‑12) in the target
prevent the development of or cure a particular disease.[10] The cells, thereby enhancing the T‑cell response and host innate
gene therapy system consists of three components: a gene that immunity resulting in the eradication of antigen‑negative
expresses essential therapeutic peptides, a plasmid‑based gene cancer cells [Figure 2].[17] However, neurological events and
encoding system to regulate the activity of the gene in the target cytokine release syndrome (CRS) presenting as high fever, and
tissue and a gene delivery system to administer the encoding flu‑like symptoms are the serious systemic adverse responses
gene and its regulatory plasmid to host tissue.[11] of CART therapy. Both CRS and neurological events can be
life‑threatening. FDA has approved tocilizumab and steroids
to treat CART‑cell‑induced CRS in patients 2 years old and
Ex Vivo and In Vivo Gene Therapy above. Other side effects are an increased risk of infections,
Depending on the site of healthy gene transduction, gene hypotension, acute kidney injury and hypoxia.[16]
therapy can be ex vivo or in vivo type [Figure 1].[12,13] For ex vivo
transduction, cells are first harvested from a donor (autologous In vivo gene therapy is the direct transfer of the desired genetic
or allologous), then they are genetically modified and expanded material into the target cells (without harvesting) of the body
using growth factors before being reinjected into the body for through viral or non‑viral‑based vectors to either replace,
treatment.[12,13] activate, silence or edit the affected gene.[18,19]

When autologous T cells or other immune cells (such as natural

killer cells, dendritic cells or macrophages) are reprogrammed Viral and Non‑Viral Vectors of Gene Therapy
to make them capable to identify and kill or modify target Viral vectors
cells, it is called CART therapy. First‑generation CART cells Viruses are capable of invading almost all cells of human,
are formed by the attachment of a single chain fragment animal, plant and bacterial origin and directing the host
variable (scFv) of antibodies to a transmembrane domain and machinery to their instructions. It makes them an ideal
an intracellular signalling unit called chain CD3 zeta. Thus, the candidate for gene therapy. Gene transfer through viral vector
active scFv element of monoclonal antibody (mAb) increases is called transduction. However, they can cause exacerbated
the recognition of the specific epitope on target cells and immune responses and genome manipulation. Retrovirus,
activates the modified T cells. Hence, the immunity is activated lentivirus, herpes‑virus, adenovirus (AV), adeno‑associated
without depending on molecules from the histocompatibility virus (AAV) or plasmids are used as viral vectors [Table 1].[18,20]

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Figure 2: CAR‑T cell structure with generations. CAR‑T cell structure has one scfv and a CD3 joined by a hinge. First generation CARs have only one
intracellular signal component CD3ζ; second‑ generation CARs have additional one costimulatory molecule and the third generation of CARs have
two costimulatory molecules. The Fourth‑generation CAR T‑cells can activate the intracellular transcription factor to induce cytokine production and
the fifth‑generation of CARs uses gene editing to inactivate the TRAC gene, leading to the removal of the TCR alpha and beta chains. Adopted from
Zhao L et al.[17] scfv: Single chain fragment variable, CAR: Chimeric antigen receptor

Table 1: Retroviral vectors for gene therapy

Retrovirus Lentivirus Herpesvirus Adenovirus Adeno‑associated Plasmid
Provirus RNA RNA RNA DNA DNA DNA
Capacity ~9 kB ~10 kB ~30 kB ~30 kB ~4.6 kB Unlimited
Integration into recipient genome Yes Yes Yes No Extremely rare No
Duration of Transgene expression Long Long Transient Transient Long in post‑mitotic cell Transient
Pre‑existing immunity in the recipient No No Yes Yes Yes No
Germ‑line transmission May occur Yes No No May Occur No
Adverse effects Insertional Insertional Inflammatory Inflammatory Inflammatory No
mutagenesis mutagenesis response response response (mild)
~: Approximately, RNA: Ribonucleic acid, DNA: Deoxyribonucleic acid

Some of these viruses stably integrate into the host genome advantage of the capacity to carry larger therapeutic genes (up
while others remain separate and perform independently to 37 kb in size), showing long‑lasting transgene expression
without integrating with parent cells. An ideal gene vector and lesser immunogenicity.[20,21]
should be very specific, highly efficient, non‑familiar to the AAVs constitute the largest category (14%–28%) of
immune system, and should have high scalability.[19] virus‑based gene therapy. It is naturally replication‑defective
AV is a double‑stranded DNA virus. It consists of penton and and can replicate only in the presence of a helper virus (AV).
hexon subunits. The hexon subunit forms the viral capsid and AAV vectors are usually non‑pathogenic in nature, mainly
carries antigenic motifs, while the penton subunit represents the target neural and muscle tissue, and can infect dividing and
fibre and knob domains required for infection. The fibre knob non‑dividing cells. Their multiple serotypes are available and
domain initiates AV infection by binding to various proteins AAV‑based gene therapy is comparatively safer with low
present on the host cell surface‑expressed glycoproteins, immunogenicity, and ease of manufacturing but can only
coxsackieviruses and AV receptors. The interaction between the package DNA sequences up to 5 kb. As the AVs and AAV do
arginine‑glycine‑aspartate sequence of the fibre penton subunit not normally integrate into the host genome, vector DNA will
and cell surface integrins promotes viral particle endocytosis remain episomal and will be eliminated when the cell divides
and the completion of viral infection. First‑generation or dies.[21,22]
adenoviral vectors have partially deleted E1 or E3 genes Retroviruses are enveloped non‑icosahedral viruses with two
incapacitating them to replicate or display oncogenicity but copies of single‑stranded RNA. This single‑stranded RNA
have limitations of delivering genes <8 kb and exhibiting genome is retrotranscribed to DNA by the reverse transcriptase
strong immune response and off‑target expression (also known enzyme which is then integrated into the host genome with the
as a leaky expression). Second‑generation AV‑based vectors advantage of providing stable gene delivery. However, retroviral
were developed by deleting E2A, E2B and E4 from the genome vectors have the drawbacks of causing immunogenicity and
of the first‑generation AV vectors but they were not successful insertional mutagenesis. To overcome these limitations, the
due to leaky expression of viral proteins and rapid withering retrovirus is rendered replication‑defective viral particles by
of therapeutic gene expression. Third‑generation AV vectors chemicals or by transmitting replication machinery in separate
also called gutless or helper‑dependent AV vectors, have the plasmids (trans) before injecting into the patient’s body.[21]

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The members of the Retroviridae family include Moloney DNA/RNA/oligonucleotides through electrostatic binding.[23]
leukemia virus (MLV), a gammaretrovirus, having a simple The complex protects genomic material and enhances cellular
retroviral genome composed of gag, pol and env genes and uptake and intracellular transport. Nucleofection is the most
human immunodeficiency virus type‑1 (HIV‑I), a lentivirus, reliable and recommended electroporation‑based, non‑viral gene
having complex genome with additional vif, vpu, rev, tat and transfer technology that allows the direct introduction of DNA
vpr genes. MLV can act only on the proliferating cells while into live cell nucleic acids with greater transfection efficiency. The
HIV‑1 can act both on quiescent as well as proliferating cells. addition of polyethylene glycol (PEG) enhances the permeability
The gag, pol and env genes of retrovirus all together are known and retention of non‑viral gene vectors in tumours but it notably
as open reading frames (ORFs) placed between long terminal reduces the uptake of the DNA complex by individual cells. It is
repeats (LTR) required for the expression of therapeutic genes. called the ‘PEG dilemma’. To address this, a construct composed
While acting as cargo in gene therapy, ORF is removed and of a peptide‑based connector that bridges a phospholipid and PEG
the desired gene to be transferred is inserted between the and can be degraded in the tumour by a matrix metalloproteinase
LTR. The different generations of lentivirus lack one or more has been developed. With this approach, the intact non‑viral
of the additional genes making the expression longer‑lasting vector with the gene can enter a tumour through PEG, and
and without inflammation. Over time and with technological then the processed vector (i.e., without PEG) can enter cells.
advancement, now surface glycoproteins expressed on the Recently, it has also been found that factors secreted by cancer
retroviral envelope can be replaced with other glycoproteins cells activate cells in the stroma and the activated stromal
to improve transfer, targeting and broadening the range of cells arouse cancer cell proliferation and migration. Therefore,
retroviruses with lesser toxicity.[9,11,18,21,22] Characteristics of manipulation of the cancer microenvironment may lead to the
commonly used viral vectors are summarised in Table 1.[18,21] treatment of cancer. Gene therapy with non‑viral vectors is safer,
more effective and more economical. In this way, the availability
Non‑viral vectors (also called chemical vectors, both of effective non‑viral vectors could have a significant impact on
organic and inorganic types) the development of new therapeutics.[24,25]
Common non‑viral vectors are transposons, cationic polymers,
dendrimers and cell‑penetrating peptides or liposomes as shown
in Figure 3.[8,9,11] Gene transfer through non‑viral vector is called Newer Gene Editing Techniques
transfection. They carry and improve gene therapy by altering Newer techniques allow the correction of mutated genes both
functional characterisation to enhance endocytosis or acting on in vitro and in vivo. For editing a defective gene, the foremost
an extracellular matrix protein. An organic vector, i.e. cationic requirement is the identification of the exact site in the genome
lipid‑based vectors, cationic polymer‑based vectors and requiring correction. It is done by protein (addition or alteration
peptide‑based vectors, form complexes with negatively charged such as in ZFN and meganuclease technique, respectively) or
nucleic acid (like small guiding RNA [sgRNA] in CRISPR).
Then comes the role of genome splicing, i.e. the separation
of double‑stranded DNA using the nuclease enzyme.
Once separated, genes tend to repair themselves as in the
non‑homologous end‑joining technique but they are more
error‑prone. Therefore, at times, the defective gene is repaired
using a homologous template, known as homology‑directed
repair.[20,26,27]Based on nucleases that break the nucleic acid,
four gene editing tools have been identified:
Meganucleases
They are sequence‑specific endonucleases that recognise
unique large (14–40 bp) target sites. This tool involves the
technical generation of fusion proteins from pre‑existing
meganuclease (MN) domains. MN specificity is enhanced by
the direct modification of protein residues in the DNA‑binding
domain. Despite low cytotoxicity, the complexity of
reengineering and low editing efficiency limits the use of
MNs.[20]
Zinc‑finger nucleases
In this tool, three to six zinc‑finger protein repeat domains
Figure 3: Vectors of gene therapy and factors affecting choice of vector. are fused on endonuclease Fokl artificially to form ZFNs.
Transfer of genetic material with viral vectors (transduction) is more FokI is a dimeric‑type IIS restriction enzyme isolated from
efficient and more immunogenic. Transfer of genetic material with non‑viral Flavobacterium okeanokoites. It recognises the 5′‑GGATG‑3′
vectors (transfection) is less efficient and low immunogenic sequence and introduces two single cuts 9nt away from the 3′

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Bansal, et al.: Gene therapy and its applications

end of its recognition sequence on the top strand and 13nt away domain of TALE effectors has 33–35 conserved amino acids.
from the 5′ end of the bottom strand sequence, complementary Except for locations 12 and 13, which are varied and exhibit a
to the first one. The role of zinc finger domains is to recognise substantial association with particular nucleotide recognition,
a trinucleotide DNA sequence on the desired target. Since each repeat is similar. FokI endonuclease does not specifically
each zinc finger can bind to three base pairs of DNA, so 9 target the DNA cleavage domain. As a dimer, the FokI domain
and 18 base pairs on the target can be identified. The FokI requires two constructs that attach to specific DNA binding
endonuclease functions as a dimer. Thus, double‑stranded sites in the target genome. Better activity depends on the
DNA breaks occur only at the binding sites of the two ZFNs distance in amino acids between the FokI cleavage domain and
on opposite DNA strands [Figure 4].[26,27] ZFNs are not the TALE DNA binding domain. DNA double‑strand breaks
recommended because the design and selection of modified are brought about by TALEN, and the target cells eventually
zinc‑finger arrays are difficult and time‑consuming. respond with innate repair mechanisms.[27,28]
Transcription activator‑like effector nucleases Clustered regularly interspaced short palindromic repeats
Are fusion proteins made up of the FokI endonuclease and the and Cas‑associated proteins
bacterial TALE protein. The repeating unit of the DNA‑binding CRISPR is a heritable, adaptive immune system of bacteria

c
Figure 4: Gene editing techniques (a) ZFNs – A constructed FokI dimer formed by binding of two separate ZFNs to particular locations on opposing
DNA strands (in such a way that Fokl nuclease form dimer) cuts the target DNA (b) TALEN- Here bacterial TALE protein is fused with Fokl nuclease
in place of zinc fingers to form TALEN. TALEN is cheaper, easier to design technique with comparatively well-defined target specificities and faster
results than ZFN. (c) CRISPR Cas – The exact target DNA-editing site is identified by complementary base between the genomic DNA and sgRNA in
the CRISPR Cas9 system. This CRISPR Cas9 system also possess a tracrRNA, and loaded Cas9 nuclease, which then cuts the DNA at the recognized
target size. ZFNs: Zinc finger nucleases, TALENs: Transcription activator like effector nucleases, CRISPR Cas: Clustered regularly interspaced short
palindromic repeats and Cas associated proteins

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that allows the retention of memory of previous infections destroys the tumour cells by multiplicating in those specific
and defends against re‑infection from similar pathogens. In cells to lyse them (suicide genes). Sometimes, it functions by
it, the invader’s DNA known as mobile genetic elements such inducing a robust antitumor immune response by the release of
as bacteriophages, transposons or plasmids are integrated cell debris and viral antigens and then the body itself becomes
into the bacterial genome as ‘spacer’ sequences in between capable to get rid of culprit cells (immunostimulatory genes).
palindromic repeats. These ‘spacer’ sequences are transcribed At other times, it delivers anti‑angiogenesis genes that interfere
into short RNA sequences which act as sgRNA for Cas with the blood supply of growing tumour cells. Moreover, it can
endonuclease to complementary sequences of DNA. Hence, also work by introducing wild‑type tumour suppressor genes
CRISPR‑Cas has Cas protein endonuclease, site‑specific into tumour cells lacking them, thereby promoting tumour
sgRNA and tracrRNA (RNA to transfer/transcript) with or cell apoptosis.[20,34]
without repair DNA template containing wild‑type sequence.
Cas makes site‑specific DNA breaks that are then repaired by Administration Techniques of Gene Therapy
host DNA repair machinery to restore the wild‑type allele.
Viral and non‑viral vectors of gene therapy are administered
In the absence of a repair template, truncation can occur
by physical and chemical means. Physical methods involve
resulting in null mutations. Therefore, to increase the targeting
needle injection, microinjection, electroporation, gene gun (to
approach, a repair template containing the mutation of interest
shoot tissue with gold or tungsten particles that are coated
is introduced. The repair matrix is composed of a DNA motif
with DNA), ultrasound, hydrodynamic delivery and magnetic
called the protospacer adjacent motif (PAM). Each Cas protein
microparticles. Physical methods are used for viral as well as
subtype has a specific PAM sequence e.g. 5’‑NGG‑3’ for
non‑viral gene therapy. Chemical methods involve the use of
standard Cas9. DNA breaks are performed by Cas9 nucleases,
cationic detergents (calcium phosphate to enhance the entry
resulting in double‑strand breaks in the case of the wild‑type
of genetic material by increasing the pore size) and artificial
enzyme and single‑strand breaks when mutant Cas9 variants
phospholipid vesicles called lipoplexes. Mainly chemical
called nickases are used. This single‑strand break, also known
methods are used for non‑viral vectors.[34-36]
as prime editing, has been developed recently in 2019. This
method appears to have very high fidelity for editing small
DNA changes (point mutations or small deletions) such as Applications of Gene Therapy
those responsible for sickle cell disease or a common variant Gene therapy has a wide range of potential applications
in cystic fibrosis.[29‑32] Emmanuelle Charpentier, and Jennifer including cancer, haemophilia, hypercholesterolaemia,
Doudna, received the Nobel prize in chemistry in 2020 for neurodegenerative diseases and more. To date, FDA has
the discovery and development of the genome editing tool approved many products with genetic modifications besides
CRISPR‑Cas9.[29] cord blood‑based cell therapy. Some of them, namely one
CRISPR‑Cas systems based on effector Cas proteins can be OV‑based therapy, two AAV vector‑based therapies and three
classified into two main classes, further divided into six types autologous CAR T‑cell therapies,[37‑41] have been discussed in
and several subtypes. In class 1 CRISPR‑Cas systems (types detail as follows.
I, III and IV), the effector module consists of multi‑protein Talimogene laherparepvec is cell‑based oncolytic viral therapy
complexes, whereas class 2 systems (types II, V and VI) use useful in patients with unresectable cutaneous, subcutaneous
only one effector protein. The most common subtype CRISPR/ nodal lesions in recurrent melanoma. In this therapy, modified
Cas9 can be guided to virtually any DNA sequence by changing live‑attenuated herpes simplex virus (HSV1) viruses are used to
the sequence of the guide RNA (sgRNA) to match the DNA target tumour cells. HSV‑1 JS1 strain is altered by deleting the
sequence of interest. [30,32,33] CRISPR/Cas technology has ICP34.5 gene to attenuate the natural neuro‑virulence of HSV‑1
certain advantages over ZN and TALEN. It uses non‑bulky and deleting the ICP47 gene to permit antigen presentation
single‑guide RNA for DNA sequence recognition and hence for earlier and increased expression of US11 before injecting
is more specific. In addition, multiple guide RNAs can be into the recipient. The increased expression of the US11 gene
employed for multiplexed gene targeting.[33,34] CRISPR has results in increased replication of ICP34.5 deleted HSV‑1 in
been used for the treatment of sickle cell disease, β‑thalassemia tumour cells without any loss of tumour selectivity. Thus,
and hereditary transthyretin amyloidosis.[29] these modified viruses replicate in cells and cause tumour
cell lysis (oncolysis) but have no disease‑causing capacity.
Pharmacodynamics of Gene Therapy Replicated viral progeny infects neighbouring tumour cells
and destroys them also. In addition, two human Granulocyte
There are multiple mechanisms by which modified cells
macrophage colony stimulating factor (GM-CSF) genes are
and gene therapy work. In cases of deficiency diseases,
inserted into the virus promoting more GM‑CSF production
addition/replacement/correction by healthy genes increases
which activates the antigen‑presenting cells leading to a
the production of required proteins. Newer methods (miRNA,
systemic antitumor immune response.
ASO) allow the control of the expression of mutant genes also
in case of overexpression or underexpression. In malignancy, Voretigene neparvovec used in retinal dystrophy and
the use of oncolytic virus (OV)‑based therapy selectively onasemnogene abeparvovec used in spinal muscular

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atrophy are two AAV vector‑based therapies. Voretigene SARS‑CoV‑2 whereas the BNT162b1 RNA vaccine
neparvovec‑rzyl is a non‑replicating AAV serotype 2, which encodes the receptor binding domain of the SARS‑CoV‑2
has been genetically modified to express the human retinal spike protein. ZyCoV‑D vaccine is a DNA plasmid
pigment epithelium 65 (RPE65) transgene. RPE deficiency vaccine expressing SARS‑CoV‑2 spike protein
is responsible for blindness in 15% of cases. Voretigene iv. Protein‑based vaccines: Protein subunits (extracted from
neparvovec restores the visual cycle by providing functional the virus and purified) and virus‑like particles are injected
retinoid isomerohydrolase, a 65‑kD protein expressed in the in protein‑based vaccines. Examples are Novavax and
RPE. AdaptVac.[43,44]
Spinal motor neuron (SMN) proteins found all over the body are
essential for the maintenance and function of motor neurons. Challenges of Gene Therapy
In the absence of sufficient functional SMN protein, motor Viral vector‑based gene therapy shows toxicity due to off‑target
neurons die, which leads to debilitating and often fatal muscle effects, immunogenicity and inflammatory reactions in the host
weakness called SMA. For SMA, the only FDA‑approved drug whereas non‑viral vector‑based therapy has limited penetration
is nusinersen (an antisense oligonucleotide). Onasemnogene and efficacy. High cost and possible oncogenic transformation
abeparvovec is a biologic consisting of an AAV9 viral capsid are other challenges. Gene therapy may be ineffective because
containing the SMN1 transgene and a synthetic promoter. cancer cells remain undetected by evolving escape mechanisms
Upon administration, the AAV9 viral vector delivers her SMN1 and reducing the expression of tumour antigens on their surface.
transgene to affected motor neurons, resulting in increased In addition, they can also initiate immune cell inactivation and
SMN protein synthesis. release substances into the microenvironment to promote
tumour cell proliferation and survival. The transduction
Tisagenlecleucel, axicabtagene ciloleucel and brexucabtagene
efficiency of oncolytic adenoviral vectors (especially
autoleucel are autologous CAR T‑cell‑based therapies used in
serotype 5) that induce autophagy‑associated immunogenicity
acute lymphoblastic leukaemia, diffuse large B‑cell lymphoma
is compromised by the prevalence of neutralising antibodies
and mantle cell lymphoma, respectively. Second‑generation and
in the human population. Multiple‑gene‑based diseases cannot
3rd‑generation CAR‑T cell therapy have additional co‑stimulators
be treated effectively using gene therapy to date.[45,46]
along with genetically‑modified T cells to improve efficacy and
target selection. Since the CD19 antigen is also present in normal
B‑cells, and they will also destroy those normal B cells that Novel Perspectives
produce antibodies, there may be an increased risk of infections To overcome the challenges, increase the prospects of gene
for a prolonged period besides other mentioned side effects. therapy and use it as an adjunct in combination with other
therapies, various advanced steps have been taken or are under
Gene‑based products permitted by approval agencies of
development.[46‑49] These include
different countries are given in Table 2.[20,29,34,42]
1. Use of human telomerase reverse transcriptase
In recent times, the pandemic of COVID‑19 has been caused by promoters to restrict replication of an AV vector to
a single‑stranded spherical RNA virus with different structural telomerase‑positive cancer cells
proteins: Spike, envelope, membrane and nucleocapsid 2. Gene‑directed enzyme prodrug therapy consists of
proteins. Gene‑therapy‑based following four types of COVID introducing genes that encode enzymes capable of
vaccines, developed on a larger scale in a relatively shorter converting prodrugs to cytotoxic drugs. Non‑toxic
time proved as a boon against it. prodrugs can thus be administered in high doses
i. Viral vector vaccines: In these vaccines, non‑replicating with no untoward effects and converted in situ to the
AV vector is combined with the structural spike protein cytotoxic drug where it is needed (i.e., in the tumour
of severe acute respiratory syndrome coronavirus and its immediate environment). This strategy consists
2 (SARS‑CoV‑2). These AVs are simian AV vector of using gene therapy to better utilise conventional
ChAdOx1 (in Oxford–AstraZeneca and Covishield), chemotherapy. One such example is the direct injection
AV type Ad5 (in CanSino Biological Inc. and Beijing of the gamma‑retroviral vector encoding the enzyme
Institute of Biotechnology), Ad26 COV2S (in Janssen cytidine deamidase (vocimagene amiretrorepvec; Toca
Pharmaceutical) and both Ad26 and Ad5 (in Gamaleya’s 511) into the glioblastomas in patients to convert the
Sputnik V) 5‑flucytosine to 5‑fluorouracil, which has antineoplastic
ii. Whole‑cell viral vaccines: Here whole modified (weakened) activity increasing the target‑based precision. Similarly,
or completely inactivated virus is used to produce HSV thymidine kinase renders the virus‑affected cells
vaccines e.g. Vero cell and BBV152 (Covaxin, indigenous susceptible to the antiviral drug ganciclovir
vaccine of India) 3. Tr a n s d u c t ion of a va r ia nt of me t hylg u a n i ne
iii. Nucleic acid vaccines (RNA and DNA): In this variety methyltransferase in HPSC (HSCs) to allow them to
of vaccines, instead of the whole virus, only its nucleic survive high doses of chemotherapy drugs metabolised
acid is inserted. Moderna and NIAID are mRNA‑based by this enzyme system (e.g., temozolomide or carmustine)
vaccines that encode the surface glycoprotein of 4. Developing and using engineered CAS‑chimeras to

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Table 2: Human gene products approved by various regulatory agencies till August 2020
Human gene product Genetic material Indication Mechanism of action
approval authority and year
Vitravene (Fomivirsen) ASO CMV retinitis in an HIV‑positive Gene silencing by targeting the IE‑2 mRNA
US FDA 1998 patient molecule which encodes a protein required
EMA 1999 (withdrawn in 2006 for CMV replication
as, after HAART, CMV retinitis
due to HIV got extremely rare)
Gendicine (rAd‑p53) (Ad5) with human wild‑type Advanced stages and grades Antitumor properties by initiating apoptotic
China FDA 2003 gene p53 of head and neck squamous pathways, suppressing DNA repair and cell
cell cancer, malignant glioma, proliferation
ovarian cancer and hepatic cell
carcinoma
Macugen (Pegaptanib) A polynucleotide aptamer Neovascular AMD (requires Targets VEGF165 isoform and inhibits
US FDA 2004 repeated administration so vascularisation
aflibercept, ranibizumab and
bevacizumab drugs are preferred)
Oncorine (rAd5‑H101) China Oncolytic recombinant ad5 Refractory nasopharyngeal The removal of the E1B 55K gene inhibits Ad
FDA 2005 (rAd5‑H101) virus with a cancer, lung, liver, pancreatic viral proliferation in normal cells and allows
deletion in the E1B 55K gene cancer, malignant pleural and proliferation only in p53‑deficient host cancer
peritoneal effusion cells leading to oncolysis of tumour cells
Rexin G (Mx‑dnG1) Replication‑incompetent Metastatic solid tumors Retroviral vector binds to abnormal signature
Philippine FDA 2007 retroviral vector with wild (pancreatic, breast, proteins in the tumour cell that increase
US FDA 2010 cyclin G1 osteosarcoma) vector concentration in tumour cells.
Cyclin‑G synthesises cytocidal dnG1 proteins
that block the cell cycle in the G1 phase and
promote apoptosis of cancer cells
Neovasculgen (Pl‑VEGF165) Plasmid DNA encoding human Atherosclerotic peripheral arterial VEGF triggers angiogenesis as well as
Russia 2012 VEGF 165 under the control of disease enhanced endothelial renovation via the
a CMV promoter induction of nitric oxide and prostacyclin
molecules from endothelium
Glybera (Alipogenetiparvovec) AAV with an LPL gene variant For Lipoprotein lipase deficiency Increased production of lipoprotein lipase
Approved by EMA in 2012 and (LPLD) treatment required in fat metabolism
withdrawn in 2017
Kynamro (Mipomersen) ASO Adjunct therapy for homozygous Mipomersen acts by reducing the synthesis
US FDA 2013 familial hypercholesterolemia of ApoB 100 in the hepatocytes and thereby
(causes serious liver toxicity and decreasing LDL‑C, TC and non‑HDL‑C
reactions at weekly s.c injection
so used in controlled conditions)
Strimvelis (GSK‑2696273 cDNA sequence for adenosine ADA‑SCID patients. 15% of all Increase the expression of functional human
EMA 2016 deaminase enzyme in SCID patients ADA. Pre‑conditioning with low‑dose
autologous hematopoietic busulfan is required
stem/progenitor (CD34+)
enriched cells transduced ex
vivo with a retroviral delivery
system
Exondys51 (Eteplirsen) PMO Duchenne muscular dystrophy PMO works by skipping exon 51 in the
US FDA 2016 (DMD); 13% cases mutated DMD gene. This increases the
expression of functional dystrophin protein
Spinraza (Nusinersen) Modified 2’‑O‑2 methoxyethyl Spinal muscular atrophy (SMA) Targets intron 7 on the SMN2 hnRNA,
US FDA 2016 phosphorothioate ASO modulating alternative splicing by
EMA 2016 increasing the inclusion of exon 7 in the
final processed RNA. It increases the levels
of functional SMN protein in the central
nervous system
Defitelio (Defibrotide) Combination of primarily Hepatic SOS/VOD with renal or Aptamers (and adenosine receptor
US FDA 2016 single‑stranded oligo DNAs pulmonary dysfunction following agonists) on vascular endothelial cells
EMA 2016 obtained from porcine the cytoreductive treatment show antithrombotic, thrombolytic,
mucosa tissue by controlled before HSCT anti‑inflammatory and anti‑ischemic
depolymerisation properties
Talimogene Laherparpevec HSV 1 oncolytic virus with Solid tumours‑ melanoma and Tumour lysis and induce an antitumour
(Imlygic) y‑34.5 and a 47 gene deletion pancreatic immune response
US FDA 2016

Contd...

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Table 2: Contd...
Human gene product Genetic material Indication Mechanism of action
approval authority and year
Zalmoxis Allogenic T cells encoding Haematopoietic stem cell In the case of GVHD initiation, ΔLNGFR
EMA 2016 LNGFR and HSV‑TK‑suicide transplantation‑induced graft expression cassette was used as the selection
gene versus host disease marker of engineered transplanted T cells
‑retroviral vector transduced and HSV‑TK Mut2‑mediated tyrosine kinase
expression provides suicide gene induction
by converting prodrug ganciclovir to a toxic
triphosphate form administration. It kills the
donor T cells expressing HSV‑TK and GVHD
control
Invossa (TissueGene‑C or Contains a 3:1 mixture Symptomatic and persistent knee Upregulated
tonogenchoncel‑L) ratio of non‑transformed Osteoarthritis (OA) TGF β1 improves function and pain
South Korea 2017 and retrovirally transduced relief with structural refinement and
allogenic chondrocytes that anti‑inflammatory action
upregulate TGF β1
Yescarta (axicabtagene Autologous CAR T‑cell therapy Non‑Hodgkin lymphoma, all (not It binds with CD19protein on lymphoma cell,
ciloleucel) targeting CD 19, retroviral for primary or CNS lymphoma) enhance activation and proliferation of CAR
US FDA 2017 vector transduced T‑cells to kill cancer cells
Kymriah (tisagenlecleucel) Autologous CAR T‑cell‑based Relapsed B‑cell ALL in children After binding to target CD19‑expressing
US FDA 2017 therapy targeting CD 19, and young adult patients (up to cells, the activated tisagenlecleucel CAR will
lentiviral vector transduced 25 years old) initiate the antitumour activity through the
CD3 domain. The intracellular costimulatory
domain augments the antitumour reaction and
ensures the durability of CAR T‑cells
Luxturna (voretigene AAV2 transduced a normal Inherited retinal dystrophy Gene product isomer hydrolase transforms
Neparvovec‑rzyl US FDA 2017 copy of the RPE65 gene the transretinyl esters to 11‑cis‑retinal, which
EMA 2018) is the natural ligand and chromophore of the
opsins of rod and cones photoreceptors
Patisiran (Onpattro) RNAi encapsulated Hereditary transthyretin‑mediated After entering the cell, transthyretin mRNA
US FDA 2018 in aliposome to target amyloidosis (hATTR; also is cleaved by patisiran leading to a decrease
EMA 2019 transthyretin mRNA known as FAP. It affects the liver, in circulating transthyretin protein which
peripheral nerves, heart, kidney reduces transthyretin‑mediated amyloidosis
and GIT)
Zolgensma (Onasemnogene cDNA of human SMN gene in SMN disease in paediatric Healthy SMN gene expression checks disease
Abeparvovec) non‑replicating AAV9 patients<2 years of age progression through the maintenance of SMN
US FDA 2019 protein
Zynteglo (Betibeglogene Autologous CD34+cells β‑TDT who do not have a β0/ Restores the β‑globin protein in patients
autotemcel) encoding βA‑T87Q‑globin β0 genotype, for whom HSC by adding normal copies of the modified
EMA 2019 gene cells using lentiglobin transplantation is appropriate β‑globin gene into the
viral vector BB305 but a HLA‑matched related HSC
donor is not available
Givlaari (Givosiran) RNAi Acute hepatic porphyria Acts to decrease ALAS1 mRNA in the liver,
US FDA 2019 which reduces the buildup of aminolevulinic
acid and porphobilinogen
Tecartus (Brexucabtagene CAR‑T For relapsed or refractory mantle CD19 targeting and T‑cell‑mediated cell
autoleucel) cell lymphoma death. BTK inhibition is mediated via its
FDA 2020 effect on a non‑receptor tyrosine kinase
EMA 2020
US FDA: United States Food and drug administration, CMV: Cytomegalovirus, ASO: Antisense oligonucleotide, DNA: Deoxyribonucleic acid, RNA:
Ribonucleic acid, mRNA: Messenger RNA, AMD: Age related macular degeneration, VEGF: Vascular endothelial growth factor, rAd: recombinant
adenovirus, AAV: Adeno associated viruses, LPL: Lipoprotein lipase, LPLD: LPL deficiency, ADA: Adenosine deaminase, ADA SCID: ADA deficient severe
combined immunodeficiency, PMO: Phosphomorpholidate morpholino oligomer, DMD: Duchenne muscular dystrophy, SMA: Spinal muscular atrophy,
SMN: Spinal motor neuron, SOS/ VOD: Sinusoidal obstruction syndrome/ veno occlusive disease, HSCT: Hematopoietic stem cell transplantation, HSV TK:
Herpes simplex virus thymidine kinase, OA: Osteoarthritis, TGF β1: transforming growth factor β1, CART: Chimeric antigen receptor T cells, RNAi: RNA
interference, RPE: Retinal pigment epithelium, TDT: Transfusion dependent thalassemia, FAP: Familial amyloid polyneuropathy, HSC: Haematopoietic
stem cell, HLA: Human leucocyte antigen, IE: Immediate early, ALAS1: Aminolevulinic acid synthase 1, HIV: Human immunodeficiency virus, TC: Total
cholesterol, HDL C: High density lipoprotein cholesterol, LDL C: Low density lipoprotein cholesterol, BTK: Bruton tyrosine kinase, hnRNA: Heterogeneous
nuclear RNA, EMA: European medicines agency, HAART: Highly active antiretroviral therapy, GVHD: Graft versus host disease, LNGFR: low-affinity
nerve growth factor receptor , GIT: Gastrointestinal tract

improve target specificity, reducing the half‑life or lower 5. Antibody gene transfer is an investigational approach
exposure by using other variants to recombinant mAb therapy that uses a gene therapy

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construct to produce the mAb inside the patient rather than diverse as metabolic, protein and enzyme‑related hereditary
administering the antibody directly from outside (i.e., giving and non‑heredity genetic disorders, cancer‑like acquired
the patient the antibody DNA rather than the protein) genetic diseases and viral infections such as AIDS. While still
6. C R I SPR i n h i bit io n (C R I SPR i) a n d C R I SPR in the developing phase, 23 gene‑therapy‑based products have
activation (CRISPRa) are strategies to change the level been approved and marketed successfully and many more are
of protein by controlling the expression of nucleic acids in the pipeline. Although some had to be withdrawn, it was
at the transcriptional level without changing the DNA mainly because of economic reasons and not safety issues.
sequence Hence, by overcoming the limitations such as immunogenicity,
7. Bacterial‑mediated gene transfer (Bactofection): Instead mutagenicity and high costs, gene therapy can be a medicine
of the virus, bacteria have been tried in gene therapy. of the next generation.
This is based on evidence that some bacteria specifically
target tumour cells and cause gene silencing or inhibit
Acknowledgment
The authors are also thankful for the constructive input
RNA interference (RNAi). In addition, intracellular
provided by Dr. Rupa Kapadia, Senior Professor and Head
bacteria such as Salmonella spp., Listeria monocytogenes,
of the Department of Pharmacology, SMS Medical College,
Shigella flexneri, Bifidobacterium longum, Escherichia
Jaipur.
coli and Yersinia enterocolitica have been used to transfer
plasmids, prodrug‑converting enzymes, and cytotoxic Financial support and sponsorship
drugs into the target cells. Nil.
Conflicts of interest
Ethical and Scientific Issues in the Gene (and There are no conflicts of interest.
Stem Cell) Therapy
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