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 Table of Contents  
Year : 2023  |  Volume : 4  |  Issue : 1  |  Page : 46-56

Gene therapy and its applications

1 Department of Pharmacology, SMS Medical College, Jaipur, Rajasthan, India
2 Department of Medicine, RUHS Hospital, Jaipur, Rajasthan, India
3 Department of Obstetrics and Gynaecology, District Women Hospital, Bareilly, Uttar Pradesh, India

Date of Submission07-Jul-2021
Date of Decision11-Feb-2023
Date of Acceptance09-Mar-2023
Date of Web Publication26-Apr-2023

Correspondence Address:
Dr. Uma Advani
Department of Pharmacology, SMS Medical College, Jaipur - 302 001, Rajasthan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JME.JME_65_21

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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

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

How to cite this URL:
Bansal A, Prakash R, Agarwal S, Advani U. Gene therapy and its applications. J Med Evid [serial online] 2023 [cited 2023 Jun 7];4:46-56. Available from: http://www.journaljme.org/text.asp?2023/4/1/46/374725

  Introduction Top

Edward Tatum 1966 proposed wild (healthy) genes can be inserted into the somatic cells of the human body through vectors as a treatment modality for certain diseases.[1] The foremost evidence of the use of gene therapy dates back to 1988 when Mr. Jesse Geilsinger, an 18-year-old patient received adenoviral vector-based gene therapy for X-linked ornithine transcarbamylase (OTC) deficiency. Although he did not survive long, the appropriateness and practicality of gene therapy were proved. Today it has emerged as one of the most promising fields in health. The Food and Drug Administration (FDA) approved the first gene therapy that was administered on 14 September 1990 in the US, when Ashanti DeSilva was successfully treated for severe combined immunodeficiency caused by adenosine deaminase (ADA) deficiency ADA.[2] Later on, due to setbacks of two leukaemia cases and mortality recorded by gene therapy, it was shelved for some years.[3] With the unfolding of the whole human genome at the beginning of the 21st century and related advances in technology, expectations to treat rare and familial diseases through it were revoked.[4]

Single-gene disorders such as muscular dystrophies, cystic fibrosis, alpha-1 antitrypsin deficiency, Huntington's disease, lysosomal storage diseases, chronic granulomatous disease, OTC deficiency, junctional epidermolysis bullosa and haemophilia have shown encouraging results but pieces of evidence for the use of gene therapy in polygenic and age-related diseases, namely obesity, type 2 diabetes, heart failure and renal failure, are still awaited.[4],[5] Twenty-three 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]

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 in contrast to germ cell gene therapy.[9] Conventional gene therapy includes ex vivo transduction of genetically modified allologous or autologous T cells, haematopoietic stem cells (HPSCs) transplantation and chimeric antigen receptor T cells (CARTs) as well as direct in vivo transfer of genetic material into the target tissues of a patient. However, newer gene editing techniques such as zinc-finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPRs) allow the correction of defective genes to prevent the development of or cure a particular disease.[10] The gene therapy system consists of three components: a gene that expresses essential therapeutic peptides, a plasmid-based gene encoding system to regulate the activity of the gene in the target tissue and a gene delivery system to administer the encoding gene and its regulatory plasmid to host tissue.[11]

  Ex Vivo and In Vivo Gene Therapy Top

Depending on the site of healthy gene transduction, gene 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 or allologous), then they are genetically modified and expanded using growth factors before being reinjected into the body for treatment.[12],[13]
Figure 1: In-vivo and ex vivo methods of gene therapy

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When autologous T cells or other immune cells (such as natural killer cells, dendritic cells or macrophages) are reprogrammed to make them capable to identify and kill or modify target cells, it is called CART therapy. First-generation CART cells are formed by the attachment of a single chain fragment variable (scFv) of antibodies to a transmembrane domain and an intracellular signalling unit called chain CD3 zeta. Thus, the active scFv element of monoclonal antibody (mAb) increases the recognition of the specific epitope on target cells and activates the modified T cells. Hence, the immunity is activated without depending on molecules from the histocompatibility complex for the same. Second-generation CART therapy has additional co-stimulating molecules (like CD28) necessary for signal transduction. It shows marked proliferation of T cells. Third-generation CART has two stimulatory chains of CD28 and CD134 or CD137. Later generations demonstrate augmented benefit through Akt/protein kinase B intracellular signalling pathway which regulates the cell cycle for growth and has a longer-lasting effect when compared to the second-generation CART.[14],[15],[16] The 4th generation of CART is more effective in solid tumour treatment due to the secretion of cytokines (Interleukin-12) in the target cells, thereby enhancing the T-cell response and host innate immunity resulting in the eradication of antigen-negative cancer cells [Figure 2].[17] However, neurological events and cytokine release syndrome (CRS) presenting as high fever, and flu-like symptoms are the serious systemic adverse responses 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 above. Other side effects are an increased risk of infections, hypotension, acute kidney injury and hypoxia.[16]
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

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In vivo gene therapy is the direct transfer of the desired genetic material into the target cells (without harvesting) of the body through viral or non-viral-based vectors to either replace, activate, silence or edit the affected gene.[18],[19]

  Viral and Non-Viral Vectors of Gene Therapy Top

Viral vectors

Viruses are capable of invading almost all cells of human, animal, plant and bacterial origin and directing the host machinery to their instructions. It makes them an ideal candidate for gene therapy. Gene transfer through viral vector is called transduction. However, they can cause exacerbated immune responses and genome manipulation. Retrovirus, lentivirus, herpes-virus, adenovirus (AV), adeno-associated virus (AAV) or plasmids are used as viral vectors [Table 1].[18],[20] Some of these viruses stably integrate into the host genome while others remain separate and perform independently without integrating with parent cells. An ideal gene vector should be very specific, highly efficient, non-familiar to the immune system, and should have high scalability.[19]
Table 1: Retroviral vectors for gene therapy

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AV is a double-stranded DNA virus. It consists of penton and hexon subunits. The hexon subunit forms the viral capsid and carries antigenic motifs, while the penton subunit represents the fibre and knob domains required for infection. The fibre knob domain initiates AV infection by binding to various proteins present on the host cell surface-expressed glycoproteins, coxsackieviruses and AV receptors. The interaction between the arginine-glycine-aspartate sequence of the fibre penton subunit and cell surface integrins promotes viral particle endocytosis and the completion of viral infection. First-generation adenoviral vectors have partially deleted E1 or E3 genes incapacitating them to replicate or display oncogenicity but have limitations of delivering genes <8 kb and exhibiting strong immune response and off-target expression (also known as a leaky expression). Second-generation AV-based vectors were developed by deleting E2A, E2B and E4 from the genome of the first-generation AV vectors but they were not successful due to leaky expression of viral proteins and rapid withering of therapeutic gene expression. Third-generation AV vectors also called gutless or helper-dependent AV vectors, have the advantage of the capacity to carry larger therapeutic genes (up to 37 kb in size), showing long-lasting transgene expression and lesser immunogenicity.[20],[21]

AAVs constitute the largest category (14%–28%) of virus-based gene therapy. It is naturally replication-defective and can replicate only in the presence of a helper virus (AV). AAV vectors are usually non-pathogenic in nature, mainly target neural and muscle tissue, and can infect dividing and non-dividing cells. Their multiple serotypes are available and AAV-based gene therapy is comparatively safer with low immunogenicity, and ease of manufacturing but can only package DNA sequences up to 5 kb. As the AVs and AAV do not normally integrate into the host genome, vector DNA will remain episomal and will be eliminated when the cell divides or dies.[21],[22]

Retroviruses are enveloped non-icosahedral viruses with two copies of single-stranded RNA. This single-stranded RNA genome is retrotranscribed to DNA by the reverse transcriptase enzyme which is then integrated into the host genome with the advantage of providing stable gene delivery. However, retroviral vectors have the drawbacks of causing immunogenicity and insertional mutagenesis. To overcome these limitations, the retrovirus is rendered replication-defective viral particles by chemicals or by transmitting replication machinery in separate plasmids (trans) before injecting into the patient's body.[21]

The members of the Retroviridae family include Moloney leukemia virus (MLV), a gammaretrovirus, having a simple retroviral genome composed of gag, pol and env genes and human immunodeficiency virus type-1 (HIV-I), a lentivirus, having complex genome with additional vif, vpu, rev, tat and vpr genes. MLV can act only on the proliferating cells while HIV-1 can act both on quiescent as well as proliferating cells. The gag, pol and env genes of retrovirus all together are known as open reading frames (ORFs) placed between long terminal repeats (LTR) required for the expression of therapeutic genes. While acting as cargo in gene therapy, ORF is removed and the desired gene to be transferred is inserted between the LTR. The different generations of lentivirus lack one or more of the additional genes making the expression longer-lasting and without inflammation. Over time and with technological advancement, now surface glycoproteins expressed on the retroviral envelope can be replaced with other glycoproteins to improve transfer, targeting and broadening the range of retroviruses with lesser toxicity.[9],[11],[18],[21],[22] Characteristics of commonly used viral vectors are summarised in [Table 1].[18],[21]

Non-viral vectors (also called chemical vectors, both organic and inorganic types)

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 transfection. They carry and improve gene therapy by altering functional characterisation to enhance endocytosis or acting on an extracellular matrix protein. An organic vector, i.e. cationic lipid-based vectors, cationic polymer-based vectors and peptide-based vectors, form complexes with negatively charged DNA/RNA/oligonucleotides through electrostatic binding.[23] The complex protects genomic material and enhances cellular uptake and intracellular transport. Nucleofection is the most reliable and recommended electroporation-based, non-viral gene transfer technology that allows the direct introduction of DNA into live cell nucleic acids with greater transfection efficiency. The addition of polyethylene glycol (PEG) enhances the permeability and retention of non-viral gene vectors in tumours but it notably reduces the uptake of the DNA complex by individual cells. It is called the 'PEG dilemma'. To address this, a construct composed of a peptide-based connector that bridges a phospholipid and PEG and can be degraded in the tumour by a matrix metalloproteinase has been developed. With this approach, the intact non-viral vector with the gene can enter a tumour through PEG, and then the processed vector (i.e., without PEG) can enter cells. Recently, it has also been found that factors secreted by cancer cells activate cells in the stroma and the activated stromal cells arouse cancer cell proliferation and migration. Therefore, manipulation of the cancer microenvironment may lead to the treatment of cancer. Gene therapy with non-viral vectors is safer, more effective and more economical. In this way, the availability of effective non-viral vectors could have a significant impact on the development of new therapeutics.[24],[25]
Figure 3: Vectors of gene therapy and factors affecting choice of vector. Transfer of genetic material with viral vectors (transduction) is more efficient and more immunogenic. Transfer of genetic material with non-viral vectors (transfection) is less efficient and low immunogenic

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  Newer Gene Editing Techniques Top

Newer techniques allow the correction of mutated genes both in vitro and in vivo. For editing a defective gene, the foremost requirement is the identification of the exact site in the genome requiring correction. It is done by protein (addition or alteration 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:


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 are fused on endonuclease Fokl artificially to form ZFNs. FokI is a dimeric-type IIS restriction enzyme isolated from Flavobacterium okeanokoites. It recognises the 5′-GGATG-3′ sequence and introduces two single cuts 9nt away from the 3′ end of its recognition sequence on the top strand and 13nt away from the 5′ end of the bottom strand sequence, complementary to the first one. The role of zinc finger domains is to recognise a trinucleotide DNA sequence on the desired target. Since each zinc finger can bind to three base pairs of DNA, so 9 and 18 base pairs on the target can be identified. The FokI endonuclease functions as a dimer. Thus, double-stranded DNA breaks occur only at the binding sites of the two ZFNs on opposite DNA strands [Figure 4].[26],[27] ZFNs are not recommended because the design and selection of modified zinc-finger arrays are difficult and time-consuming.
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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Transcription activator-like effector nucleases

Are fusion proteins made up of the FokI endonuclease and the bacterial TALE protein. The repeating unit of the DNA-binding domain of TALE effectors has 33–35 conserved amino acids. Except for locations 12 and 13, which are varied and exhibit a substantial association with particular nucleotide recognition, each repeat is similar. FokI endonuclease does not specifically target the DNA cleavage domain. As a dimer, the FokI domain requires two constructs that attach to specific DNA binding sites in the target genome. Better activity depends on the distance in amino acids between the FokI cleavage domain and the TALE DNA binding domain. DNA double-strand breaks are brought about by TALEN, and the target cells eventually respond with innate repair mechanisms.[27],[28]

Clustered regularly interspaced short palindromic repeats and Cas-associated proteins

CRISPR is a heritable, adaptive immune system of bacteria that allows the retention of memory of previous infections and defends against re-infection from similar pathogens. In it, the invader's DNA known as mobile genetic elements such as bacteriophages, transposons or plasmids are integrated into the bacterial genome as 'spacer' sequences in between palindromic repeats. These 'spacer' sequences are transcribed into short RNA sequences which act as sgRNA for Cas endonuclease to complementary sequences of DNA. Hence, CRISPR-Cas has Cas protein endonuclease, site-specific sgRNA and tracrRNA (RNA to transfer/transcript) with or without repair DNA template containing wild-type sequence. Cas makes site-specific DNA breaks that are then repaired by host DNA repair machinery to restore the wild-type allele. In the absence of a repair template, truncation can occur resulting in null mutations. Therefore, to increase the targeting approach, a repair template containing the mutation of interest is introduced. The repair matrix is composed of a DNA motif called the protospacer adjacent motif (PAM). Each Cas protein subtype has a specific PAM sequence e.g. 5'-NGG-3' for standard Cas9. DNA breaks are performed by Cas9 nucleases, resulting in double-strand breaks in the case of the wild-type enzyme and single-strand breaks when mutant Cas9 variants called nickases are used. This single-strand break, also known 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 those responsible for sickle cell disease or a common variant in cystic fibrosis.[29],[30],[31],[32] Emmanuelle Charpentier, and Jennifer Doudna, received the Nobel prize in chemistry in 2020 for the discovery and development of the genome editing tool CRISPR-Cas9.[29]

CRISPR-Cas systems based on effector Cas proteins can be classified into two main classes, further divided into six types and several subtypes. In class 1 CRISPR-Cas systems (types I, III and IV), the effector module consists of multi-protein complexes, whereas class 2 systems (types II, V and VI) use only one effector protein. The most common subtype CRISPR/Cas9 can be guided to virtually any DNA sequence by changing the sequence of the guide RNA (sgRNA) to match the DNA sequence of interest.[30],[32],[33] CRISPR/Cas technology has certain advantages over ZN and TALEN. It uses non-bulky single-guide RNA for DNA sequence recognition and hence is more specific. In addition, multiple guide RNAs can be employed for multiplexed gene targeting.[33],[34] CRISPR has been used for the treatment of sickle cell disease, β-thalassemia and hereditary transthyretin amyloidosis.[29]

  Pharmacodynamics of Gene Therapy Top

There are multiple mechanisms by which modified cells and gene therapy work. In cases of deficiency diseases, addition/replacement/correction by healthy genes increases the production of required proteins. Newer methods (miRNA, ASO) allow the control of the expression of mutant genes also in case of overexpression or underexpression. In malignancy, the use of oncolytic virus (OV)-based therapy selectively destroys the tumour cells by multiplicating in those specific cells to lyse them (suicide genes). Sometimes, it functions by inducing a robust antitumor immune response by the release of cell debris and viral antigens and then the body itself becomes capable to get rid of culprit cells (immunostimulatory genes). At other times, it delivers anti-angiogenesis genes that interfere with the blood supply of growing tumour cells. Moreover, it can also work by introducing wild-type tumour suppressor genes into tumour cells lacking them, thereby promoting tumour cell apoptosis.[20],[34]

  Administration Techniques of Gene Therapy Top

Viral and non-viral vectors of gene therapy are administered by physical and chemical means. Physical methods involve needle injection, microinjection, electroporation, gene gun (to shoot tissue with gold or tungsten particles that are coated with DNA), ultrasound, hydrodynamic delivery and magnetic microparticles. Physical methods are used for viral as well as non-viral gene therapy. Chemical methods involve the use of cationic detergents (calcium phosphate to enhance the entry of genetic material by increasing the pore size) and artificial phospholipid vesicles called lipoplexes. Mainly chemical methods are used for non-viral vectors.[34],[34],[35],[36]

  Applications of Gene Therapy Top

Gene therapy has a wide range of potential applications including cancer, haemophilia, hypercholesterolaemia, neurodegenerative diseases and more. To date, FDA has approved many products with genetic modifications besides cord blood-based cell therapy. Some of them, namely one OV-based therapy, two AAV vector-based therapies and three autologous CAR T-cell therapies,[37],[38],[40],[41] have been discussed in detail as follows.

Talimogene laherparepvec is cell-based oncolytic viral therapy useful in patients with unresectable cutaneous, subcutaneous nodal lesions in recurrent melanoma. In this therapy, modified live-attenuated herpes simplex virus (HSV1) viruses are used to target tumour cells. HSV-1 JS1 strain is altered by deleting the ICP34.5 gene to attenuate the natural neuro-virulence of HSV-1 and deleting the ICP47 gene to permit antigen presentation for earlier and increased expression of US11 before injecting into the recipient. The increased expression of the US11 gene results in increased replication of ICP34.5 deleted HSV-1 in tumour cells without any loss of tumour selectivity. Thus, these modified viruses replicate in cells and cause tumour cell lysis (oncolysis) but have no disease-causing capacity. Replicated viral progeny infects neighbouring tumour cells and destroys them also. In addition, two human Granulocyte macrophage colony stimulating factor (GM-CSF) genes are inserted into the virus promoting more GM-CSF production which activates the antigen-presenting cells leading to a systemic antitumor immune response.

Voretigene neparvovec used in retinal dystrophy and onasemnogene abeparvovec used in spinal muscular atrophy are two AAV vector-based therapies. Voretigene neparvovec-rzyl is a non-replicating AAV serotype 2, which has been genetically modified to express the human retinal pigment epithelium 65 (RPE65) transgene. RPE deficiency is responsible for blindness in 15% of cases. Voretigene neparvovec restores the visual cycle by providing functional retinoid isomerohydrolase, a 65-kD protein expressed in the RPE.

Spinal motor neuron (SMN) proteins found all over the body are essential for the maintenance and function of motor neurons. In the absence of sufficient functional SMN protein, motor neurons die, which leads to debilitating and often fatal muscle weakness called SMA. For SMA, the only FDA-approved drug is nusinersen (an antisense oligonucleotide). Onasemnogene abeparvovec is a biologic consisting of an AAV9 viral capsid containing the SMN1 transgene and a synthetic promoter. Upon administration, the AAV9 viral vector delivers her SMN1 transgene to affected motor neurons, resulting in increased SMN protein synthesis.

Tisagenlecleucel, axicabtagene ciloleucel and brexucabtagene autoleucel are autologous CAR T-cell-based therapies used in acute lymphoblastic leukaemia, diffuse large B-cell lymphoma and mantle cell lymphoma, respectively. Second-generation and 3rd-generation CAR-T cell therapy have additional co-stimulators 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 produce antibodies, there may be an increased risk of infections for a prolonged period besides other mentioned side effects.

Gene-based products permitted by approval agencies of different countries are given in [Table 2].[20],[29],[34],[42]
Table 2: Human gene products approved by various regulatory agencies till August 2020

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In recent times, the pandemic of COVID-19 has been caused by a single-stranded spherical RNA virus with different structural proteins: Spike, envelope, membrane and nucleocapsid proteins. Gene-therapy-based following four types of COVID vaccines, developed on a larger scale in a relatively shorter time proved as a boon against it.

  1. Viral vector vaccines: In these vaccines, non-replicating AV vector is combined with the structural spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These AVs are simian AV vector ChAdOx1 (in Oxford–AstraZeneca and Covishield), AV type Ad5 (in CanSino Biological Inc. and Beijing Institute of Biotechnology), Ad26 COV2S (in Janssen Pharmaceutical) and both Ad26 and Ad5 (in Gamaleya's Sputnik V)
  2. Whole-cell viral vaccines: Here whole modified (weakened) or completely inactivated virus is used to produce vaccines e.g. Vero cell and BBV152 (Covaxin, indigenous vaccine of India)
  3. Nucleic acid vaccines (RNA and DNA): In this variety of vaccines, instead of the whole virus, only its nucleic acid is inserted. Moderna and NIAID are mRNA-based vaccines that encode the surface glycoprotein of SARS-CoV-2 whereas the BNT162b1 RNA vaccine encodes the receptor binding domain of the SARS-CoV-2 spike protein. ZyCoV-D vaccine is a DNA plasmid vaccine expressing SARS-CoV-2 spike protein
  4. Protein-based vaccines: Protein subunits (extracted from the virus and purified) and virus-like particles are injected in protein-based vaccines. Examples are Novavax and AdaptVac.[43],[44]

  Challenges of Gene Therapy Top

Viral vector-based gene therapy shows toxicity due to off-target effects, immunogenicity and inflammatory reactions in the host whereas non-viral vector-based therapy has limited penetration and efficacy. High cost and possible oncogenic transformation are other challenges. Gene therapy may be ineffective because cancer cells remain undetected by evolving escape mechanisms and reducing the expression of tumour antigens on their surface. In addition, they can also initiate immune cell inactivation and release substances into the microenvironment to promote tumour cell proliferation and survival. The transduction efficiency of oncolytic adenoviral vectors (especially serotype 5) that induce autophagy-associated immunogenicity is compromised by the prevalence of neutralising antibodies in the human population. Multiple-gene-based diseases cannot be treated effectively using gene therapy to date.[45],[46]

  Novel Perspectives Top

To overcome the challenges, increase the prospects of gene therapy and use it as an adjunct in combination with other therapies, various advanced steps have been taken or are under development.[46],[47],[48],[49] These include

  1. Use of human telomerase reverse transcriptase promoters to restrict replication of an AV vector to telomerase-positive cancer cells
  2. Gene-directed enzyme prodrug therapy consists of introducing genes that encode enzymes capable of converting prodrugs to cytotoxic drugs. Non-toxic prodrugs can thus be administered in high doses with no untoward effects and converted in situ to the cytotoxic drug where it is needed (i.e., in the tumour and its immediate environment). This strategy consists of using gene therapy to better utilise conventional chemotherapy. One such example is the direct injection of the gamma-retroviral vector encoding the enzyme cytidine deamidase (vocimagene amiretrorepvec; Toca 511) into the glioblastomas in patients to convert the 5-flucytosine to 5-fluorouracil, which has antineoplastic activity increasing the target-based precision. Similarly, HSV thymidine kinase renders the virus-affected cells susceptible to the antiviral drug ganciclovir
  3. Transduction of a variant of methylguanine methyltransferase in HPSC (HSCs) to allow them to survive high doses of chemotherapy drugs metabolised by this enzyme system (e.g., temozolomide or carmustine)
  4. Developing and using engineered CAS-chimeras to improve target specificity, reducing the half-life or lower exposure by using other variants
  5. Antibody gene transfer is an investigational approach to recombinant mAb therapy that uses a gene therapy construct to produce the mAb inside the patient rather than administering the antibody directly from outside (i.e., giving the patient the antibody DNA rather than the protein)
  6. CRISPR inhibition (CRISPRi) and CRISPR activation (CRISPRa) are strategies to change the level of protein by controlling the expression of nucleic acids at the transcriptional level without changing the DNA sequence
  7. Bacterial-mediated gene transfer (Bactofection): Instead of the virus, bacteria have been tried in gene therapy. This is based on evidence that some bacteria specifically target tumour cells and cause gene silencing or inhibit RNA interference (RNAi). In addition, intracellular bacteria such as Salmonella spp., Listeria monocytogenes, Shigella flexneri, Bifidobacterium longum, Escherichia coli and Yersinia enterocolitica have been used to transfer plasmids, prodrug-converting enzymes, and cytotoxic drugs into the target cells.

  Ethical and Scientific Issues in the Gene (and Stem Cell) Therapy Top

Gene therapy is often looked upon as a future panacea for severe illness in subjects and prospective offspring. However, certain ethical and scientific issues require considerable deliberation. For example, in the case of germline cell therapy and human embryonic stem cell, informed consent and autonomy are at stake as the embryo that has the potential to develop into life cannot participate in the decision and if used either the embryo's viability is lost or the future is uncertain. That is why their use is either prohibited or highly restricted. Similarly, for induced pluripotent stem cells and mesenchymal stem cell-based gene therapy, beneficence and non-maleficence of ethics need to be weighed with great caution due to unintentionally increased chances of toxicity, off-targeting and tumorigenicity at times. Gene (and stem cell) therapy also raises concerns about distribution justice as it is a costlier treatment.[50],[51]

The scientific issues related to the selection of appropriate vector, disease modality for treatment, target tissue selection, standardisation of therapy, the decision to prefer it over available treatments and the guidelines regarding the regulation of developing, producing, storage, disposal of gene and stem cell therapy products are also perplexing. Moreover, as per the definition of gene therapy, it should include nucleic acid which jeopardises the use of ZFN and TALEN like newer editing techniques.[52],[53] However, in the present scenario, a balanced approach to judiciously differentiate between the essentiality of gene therapy and augmentation by gene therapy after weighing the benefits and associated risks is advocated. Further consensus and dynamic strategies on ethical and scientific issues are required before gene therapy takes the forefront in treating grave illnesses.

  Conclusion Top

Gene therapy gives hope to permanently cure diseases as diverse as metabolic, protein and enzyme-related hereditary and non-heredity genetic disorders, cancer-like acquired genetic diseases and viral infections such as AIDS. While still in the developing phase, 23 gene-therapy-based products have been approved and marketed successfully and many more are in the pipeline. Although some had to be withdrawn, it was mainly because of economic reasons and not safety issues. Hence, by overcoming the limitations such as immunogenicity, mutagenicity and high costs, gene therapy can be a medicine of the next generation.


The authors are also thankful for the constructive input provided by Dr. Rupa Kapadia, Senior Professor and Head of the Department of Pharmacology, SMS Medical College, Jaipur.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Li B, Gao N, Zhang Z, Chen QM, Li LJ, Li Y. Historical and clinical experiences of gene therapy for solid cancers in China. Genes (Basel) 2017;8:85.  Back to cited text no. 1
Wilson JM. Lessons learned from the gene therapy trial for ornithine transcarbamylase deficiency. Mol Genet Metab 2009;96:151-7.  Back to cited text no. 2
Edelstein ML, Abedi MR, Wixon J, Edelstein RM. Gene therapy clinical trials worldwide 1989-2004 – An overview. J Gene Med 2004;6:597-602.  Back to cited text no. 3
Pareek CS, Smoczynski R, Tretyn A. Sequencing technologies and genome sequencing. J Appl Genet 2011;52:413-35.  Back to cited text no. 4
Ma CC, Wang ZL, Xu T, He ZY, Wei YQ. The approved gene therapy drugs worldwide: From 1998 to 2019. Biotechnol Adv 2020;40:107502.  Back to cited text no. 5
Carvalho M, Sepodes B, Martins AP. Regulatory and scientific advancements in gene therapy: State-of-the-art of clinical applications and of the supporting European regulatory framework. Front Med (Lausanne) 2017;4:182.  Back to cited text no. 6
Zhou W, Wang X. Human gene therapy: A scientometric analysis. Biomed Pharmacother 2021;138:111510.  Back to cited text no. 7
Parashar D, Rajendran V, Shukla R, Sistla R. Lipid-based nanocarriers for delivery of small interfering RNA for therapeutic use. Eur J Pharm Sci 2020;142:105159.  Back to cited text no. 8
Goswami R, Subramanian G, Silayeva L, Newkirk I, Doctor D, Chawla K, et al. Gene therapy leaves a vicious cycle. Front Oncol 2019;9:297.  Back to cited text no. 9
Motta I, Ghiaccio V, Cosentino A, Breda L. Curing hemoglobinopathies: Challenges and advances of conventional and new gene therapy approaches. Mediterr J Hematol Infect Dis 2019;11:e2019067.  Back to cited text no. 10
Mali S. Delivery systems for gene therapy. Indian J Hum Genet 2013;19:3-8.  Back to cited text no. 11
[PUBMED]  [Full text]  
Kumar SR, Markusic DM, Biswas M, High KA, Herzog RW. Clinical development of gene therapy: Results and lessons from recent successes. Mol Ther Methods Clin Dev 2016;3:16034.  Back to cited text no. 12
Kaji EH, Leiden JM. Gene and stem cell therapies. JAMA 2001;285:545-50.  Back to cited text no. 13
Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci U S A 1989;86:10024-8.  Back to cited text no. 14
Parham P. Antigen recognition by T lymphocytes. In: Parham P, editor. The Immune System. 3rd ed. Nova York: Garland Science; 2009. p. 113.  Back to cited text no. 15
Wilkins O, Keeler AM, Flotte TR. CAR T-cell therapy: Progress and prospects. Hum Gene Ther Methods 2017;28:61-6.  Back to cited text no. 16
Zhao L, Cao YJ. Engineered T cell therapy for cancer in the clinic. Front Immunol 2019;10:2250.  Back to cited text no. 17
Linden R. Gene therapy: What it is, what it is not and what it will be. Estud Av 2010;24:31-69.  Back to cited text no. 18
Shirley JL, de Jong YP, Terhorst C, Herzog RW. Immune responses to viral gene therapy vectors. Mol Ther 2020;28:709-22.  Back to cited text no. 19
Gonçalves GA, Paiva RM. Gene therapy: Advances, challenges and perspectives. Einstein (Sao Paulo) 2017;15:369-75.  Back to cited text no. 20
Shahryari A, Saghaeian Jazi M, Mohammadi S, Razavi Nikoo H, Nazari Z, Hosseini ES, et al. Development and clinical translation of approved gene therapy products for genetic disorders. Front Genet 2019;10:868.  Back to cited text no. 21
Misra S. Human gene therapy: A brief overview of the genetic revolution. J Assoc Physicians India 2013;61:127-33.  Back to cited text no. 22
Akhtar S. Non-viral cancer gene therapy: Beyond delivery. Gene Ther 2006;13:739-40.  Back to cited text no. 23
Mohammadinejad R, Dehshahri A, Sagar Madamsetty V, Zahmatkeshan M, Tavakol S, Makvandi P, et al. In vivo gene delivery mediated by non-viral vectors for cancer therapy. J Control Release 2020;325:249-75.  Back to cited text no. 24
Hidai C, Kitano H. Nonviral gene therapy for cancer: A review. Diseases 2018;6:57.  Back to cited text no. 25
Suleiman AA, Saedi WY, Muhaidi MJ. Widely used gene editing strategies in cancer treatment a systematic review. Gene Reports 2021; 22:100983.  Back to cited text no. 26
Zhang HX, Zhang Y, Yin H. Genome editing with mRNA encoding ZFN, TALEN, and Cas9. Mol Ther 2019;27:735-46.  Back to cited text no. 27
Available from: https://www.ptglab.com/news/blog/crispr-cas9-talens-and-zfns-the-battle-in-gene-editing/. [Last accessed on 2021 Jun 15].  Back to cited text no. 28
Frangoul H, Altshuler D, Cappellini MD, Chen YS, Domm J, Eustace BK, et al. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. N Engl J Med 2021;384:252-60.  Back to cited text no. 29
Zhang S, Wang J, Wang J. One-day TALEN assembly protocol and a dual-tagging system for genome editing. ACS Omega 2020;5:19702-14.  Back to cited text no. 30
Hille F, Charpentier E. CRISPR-Cas: biology, mechanisms and relevance. Philosophical transactions of the royal society B: biological sciences 2016;371:20150496.  Back to cited text no. 31
Pickar-Oliver A, Gersbach CA. The next generation of CRISPR-Cas technologies and applications. Nat Rev Mol Cell Biol 2019;20:490-507.  Back to cited text no. 32
Jain S, Shukla S, Yang C, Zhang M, Fatma Z, Lingamaneni M, et al. TALEN outperforms Cas9 in editing heterochromatin target sites. Nat Commun 2021;12:606.  Back to cited text no. 33
Belete TM. The current status of gene therapy for the treatment of cancer. Biologics 2021;15:67-77.  Back to cited text no. 34
Wang W, Li W, Ma N, Steinhoff G. Non-viral gene delivery methods. Curr Pharm Biotechnol 2013;14:46-60.  Back to cited text no. 35
Li Y, Li LJ, Wang LJ, Zhang Z, Gao N, Liang CY, et al. Selective intra-arterial infusion of rAd-p53 with chemotherapy for advanced oral cancer: A randomized clinical trial. BMC Med 2014;12:16.  Back to cited text no. 36
Russell L, Peng KW. The emerging role of oncolytic virus therapy against cancer. Chin Clin Oncol 2018;7:16.  Back to cited text no. 37
Liu Y, Chen X, Han W, Zhang Y. Tisagenlecleucel, an approved anti-CD19 chimeric antigen receptor T-cell therapy for the treatment of leukemia. Drugs Today (Barc) 2017;53:597-608.  Back to cited text no. 38
Jacobson CA, Farooq U, Ghobadi A. Axicabtagene ciloleucel, an anti-CD19 chimeric antigen receptor T-cell therapy for relapsed or refractory large B-cell lymphoma: Practical implications for the community oncologist. Oncologist 2020;25:e138.  Back to cited text no. 39
Ramlogan-Steel CA, Murali A, Andrzejewski S, Dhungel B, Steel JC, Layton CJ. Gene therapy and the adeno-associated virus in the treatment of genetic and acquired ophthalmic diseases in humans: Trials, future directions and safety considerations. Clin Exp Ophthalmol 2019;47:521-36.  Back to cited text no. 40
Rao VK, Kapp D, Schroth M. Gene therapy for spinal muscular atrophy: An emerging treatment option for a devastating disease. J Manag Care Spec Pharm 2018;24:S3-16.  Back to cited text no. 41
Available from: https://www.ema.europa.eu'pr oduct-information. [Last accessed on 2021 Jun 23].  Back to cited text no. 42
Gupta M, Kapoor I. An update on coronavirus disease-19 vaccines. J Med Evid 2021;2:24.  Back to cited text no. 43
  [Full text]  
Patel MK, Bergeri I, Bresee JS, Cowling BJ, Crowcroft NS, et al. Evaluation of post-introduction COVID-19 vaccine effectiveness: Summary of interim guidance of the World Health Organization. Vaccine 2021;39:4013-24.  Back to cited text no. 44
Uusi-Kerttula H, Hulin-Curtis S, Davies J, Parker AL. Oncolytic adenovirus: Strategies and insights for vector design and immuno-oncolytic applications. Viruses 2015;7:6009-42.  Back to cited text no. 45
Cring MR, Sheffield VC. Gene therapy and gene correction: Targets, progress, and challenges for treating human diseases. Gene Ther 2022;29:3-12.  Back to cited text no. 46
Davidsohn N, Pezone M, Vernet A, Graveline A, Oliver D, Slomovic S, et al. A single combination gene therapy treats multiple age-related diseases. Proc Natl Acad Sci U S A 2019;116:23505-11.  Back to cited text no. 47
Schäfer R. Advanced cell therapeutics are changing the clinical landscape: Will mesenchymal stromal cells be a part of it? BMC Med 2019;17:53.  Back to cited text no. 48
Flomenberg P, Daniel R. Overview of gene therapy, gene editing, and gene silencing. UpToDate, Waltham, MA 2019. https://www.uptodate.com/contents/overview-of-gene-therapy-gene-editing-and-gene-silencing [Last accessed on 2021 Jun 23].  Back to cited text no. 49
King NM, Perrin J. Ethical issues in stem cell research and therapy. Stem Cell Res Ther 2014;5:85.  Back to cited text no. 50
Volarevic V, Markovic BS, Gazdic M, Volarevic A, Jovicic N, Arsenijevic N, et al. Ethical and safety issues of stem cell-based therapy. Int J Med Sci 2018;15:36-45.  Back to cited text no. 51
Mourby M, Morrison M. Gene therapy regulation: Could in-body editing fall through the net? Eur J Hum Genet 2020;28:979-81.  Back to cited text no. 52
Sivagourounadin K, Ravichandran M, Rajendran P. National guidelines for gene therapy product (2019): A road-map to gene therapy products development and clinical trials. Perspect Clin Res 2021;12:118-25.  Back to cited text no. 53
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2]


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