Listicle

Gene Therapy – Insights Into Current

Applications and Future Prospects
By Neeta Ratanghayra

Gene therapy is the administration of genetic material to modify or manipulate the expression of a
gene product or to alter the biological properties of living cells for therapeutic use. The advent of next-
generation technologies has led to a revival in gene therapy research with path-breaking therapies
approved after years of hard work.1

Several in vivo gene therapies, oligonucleotide-based therapies and cell therapies are now approved and
available. These therapies treat diverse disorders such as inherited blindness, neuromuscular disease
and cancer.2 More than 4000 gene therapy clinical trials are in various phases of development with
majority of the trials focused on somatic-alteration diseases, mainly cancer.3

This listicle will explore the different approaches for developing gene therapies and the latest
applications of gene therapies in modern medicine.

In vivo and ex vivo approaches

The two main approaches used in clinical gene therapy are direct in vivo administration and ex vivo gene
therapy. In vivo gene therapy involves the direct introduction of a vector containing the therapeutic genetic
material into the patient. In the ex vivo approach, the patient’s cells are taken out of the body and then
transduced by a vector in culture to incorporate the therapeutic gene. The gene-modified cells are then
transplanted back into the patient.4

Both approaches utilize a vector to deliver the genetic material, but there are different types of vectors
to choose from. Adenoviral vectors are the mainstay of in vivo strategies owing to their high transfection
efficiency. However, problems relating to antigenicity and short-term expression of transgenes can
restrict their use.

Last-generation adenovirus vectors, or gutless adenovirus, can be used in place of the traditional
adenoviral vector as the associated in vivo immune response is highly reduced compared to first- and
second-generation adenovirus vectors. Last-generation adenovirus vectors are also associated with high
transduction efficiency and tropism.5

Lentiviral vectors are another extensively used gene delivery vectors. Lentiviral vectors can integrate
into both dividing and nondividing cells. They also enable long term expression of transgenes and lack
antigenicity, which makes them a unique and ideal tool.6

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Ex vivo approaches offer the advantage of fully characterizing the genetically modified cells and selecting
them for desired phenotypes before using them for therapy. The property of the cells may also be chosen
to enhance the therapeutic potential.

Gene therapy techniques

1. Gene replacement therapy

In gene replacement therapy, a fully functioning gene is introduced to replace a mutated gene either
directly in vivo or through ex vivo cell therapy.7 Gene replacement therapy is a viable strategy that can be
used in monogenic diseases resulting from a single defective gene on the autosomes. It has been used in
diseases such as cystic fibrosis, hemophilia and spinal muscular atrophy.

2. Gene addition for complex disorders and infectious diseases

Gene replacement is not feasible for disorders caused by the combinatorial effect of multiple genes and
environmental factors. These include disorders such as cancer and heart disease as well as infectious
diseases. In such complex disorders, gene addition can be used. Gene addition involves incorporating
functional copies of a gene to address the underlying genetic cause. Gene addition strategies in complex
disorders require a deep understanding of the disease mechanisms.8

Gene addition strategies have shown promising results in several cancer clinical studies. One such
example is the use of replication-competent herpes simplex virus vectors that replicate specifically in
actively dividing tumor cells.9

3. Gene knockdown by RNA interference

Gain-of-toxicity mutations lead to the production of toxic gene products that require gene knockdown
strategies to treat the disorder. The selective silencing of target genes in specific cell types by RNA
interference (RNAi) is a powerful approach to tackle such mutations.10

RNAi is a biological process by which double-stranded RNA induces sequence-specific gene silencing by
targeting mRNA for degradation. RNAi has been widely used as a tool for knocking down the expression
of individual genes post-transcriptionally.11 The first RNAi based therapy was approved in 2018, nearly 20
years after the researchers first discovered the technique. 12

4. Gene editing to introduce targeted changes in the host genome

Genome editing technologies, via engineered or bacterial nucleases, have paved the way to directly
targeting and modifying genomic sequences. Genome editing can be achieved in vitro or in vivo by
delivering the gene-editing machinery in situ, which can add, ablates and “corrects” genes as well as
perform other highly targeted genomic modifications. Genome editing tools such as zinc-finger nucleases
(ZNFs), TALENs, and CRISPR/Cas9 are promising tools that are being used to manipulate genomes with
unprecedented precision.13

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Challenges with gene therapy

1. Immune response to gene delivery vectors and products of foreign transgenes

Immune response to gene delivery vectors and products of foreign transgenes is a major obstacle in
gene therapy. The engineering of modified AAV capsids that evade pre-existing neutralizing antibodies,
methods for temporary clearing of antibodies from circulation and immunosuppression regimens are
some possible ways to deal with the immune response.2

2. Inefficient gene transfer and lack of viral specificity

Inefficient gene transfer by viral vectors is another challenge. Even vectors with very high transduction
efficiency in vitro can fail to produce significant infection rates when applied to clinical trials.6,14

Another limitation is the lack of viral specificity, which may lead to cells in the vicinity of the target cells
being infected. The use of tissue-specific promoters has partially addressed this issue, however, tissue-
specific promoters cannot be applied to all disease states. There may also be problems with promoter
“leakage” from endogenous viral sequences.

3. Issues with controlled gene expression

Controlled gene expression is another overarching issue. For gene therapy to succeed as a strategy, it is
important that the gene is correctly regulated. The ability to switch transgenes on and off is critical not
only when the therapy is no longer needed but also in the case of the development of adverse side effects
to the treatment.15

4. Safety issues

Administration of a virus can result in inflammation or active infection. The administration of retrovirus,
which incorporates randomly into the genome, can also result in insertional mutagenesis and malignant
transformation.6

Future prospects
During the last five years, gene therapy has experienced substantial progress for a multitude of
indications. Among the clinical trials conducted or currently in progress, most trials have focused on
cancer. The other indications include monogenic, infectious and cardiovascular diseases.16

Approved therapies lay a foundation for future treatments

Approved gene therapies form the basis on which treatments for many other conditions can be
developed.2 For example, the success of in vivo AAV gene transfer to the human retina and central
nervous system has facilitated the development of AAV-based therapies to treat hemophilia.17 Likewise,
early technology development in ex vivo lentiviral and retroviral gene transfer to T cells has enabled
therapy development for sickle cell disease18 and beta-thalassemia. 19

Non-viral nanoparticles for gene delivery

Substantial progress has been made in the engineering and profiling of non-viral nanoparticles for
gene delivery. Lipid-based nanoparticles, quantum dots, carbon nanotubes, magnetic nanoparticles,
silica nanoparticles and polymer-based nanoparticles are some non-viral, multifunctional

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nanoparticles explored in gene therapy. Nanoparticles are being studied in gene delivery strategies
such as combinational gene therapy, image-guided gene delivery, optically-trackable and optically-
activated gene therapy.20

Nanoparticles offer the potential to circumvent detection by the immune system. They also enhance gene
stability, enable protection from nuclease degradation and allow improved targeting.

Next-generation editing technologies

To improve success with gene therapy, several next-generation editing technologies are being explored.
These techniques are seen to improve specificity, accuracy, efficiency, and applicability to different
classes of disease.

• Base editing and prime editing have enabled the precise alteration of genomic sequences in the
absence of DNA breaks and without the reliance on the activity of endogenous DNA repair pathways.21

• RNA editing involves targeting disease-linked mutations in RNA. This enables alterations in gene
expression without necessitating permanent changes to genomic sequences; a relatively safe and
efficient option.22

Another innovation is the use of epigenome editing technologies. The epigenome is dysfunctional in many
diseases and disorders. Thus, the ability to precisely edit the epigenome holds promise for enhancing our
understanding of how epigenetic modifications function and enabling the manipulation of cell phenotypes
for research or therapeutic purposes.

Functional genomics

Another field that will dramatically impact the space of gene therapy is functional genomics. Studies
and therapeutic interventions have focused almost exclusively on genes. However, ~ 98% of our genome
comprises non-coding DNA that harbors epigenetic regulators responsible for > 90% of susceptibility to
common disease. Exploring the non-coding component of the genome will pave the way to a whole new
area of disease biology and therapeutic target discovery.2

The promise of gene therapy

Gene therapy offers great promise in treating disease for which ineffective modalities exist, or no options
are available. The traditional strategies to deliver genetic material are highly variable, and innovative,
insightful techniques continue to emerge. The field is progressing rapidly, and varied approaches are
being studied in several clinical trials for hard-to-treat disease, including rare genetic conditions.

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References
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