Gene therapy involves the introduction of foreign genomic materials into host cells to trigger clinical benefits.1 Over the years, scientists have developed numerous viral and non-viral vectors to deliver genetic material into cells. The choice of a vector used in a gene therapy depends on multiple factors, including the type, amount of genetic material to be delivered, and route of administration.
This article discusses the mechanisms, advantages, and limitations of different viral and non-viral vectors used in gene therapy.
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Viral Vectors: Mechanisms and Applications
In gene therapy, the unique ability of viruses to survive and replicate in host cells is exploited to deliver genes or genetic materials to cell target sites.2 Different viruses follow variable mechanisms to transfer genetic material into host cells. In the case of Lentiviruses and Retroviruses, transgenes are incorporated into the viral DNA, and the modified DNA is injected into the viral vector.3
Once the viral vector reaches the target site, it binds to a specific receptor present on the cell membrane of the target cells and infiltrates the host cell. Post-cellular internalization, the vector is packed into endosomes.
An acidic breakdown of these endosomes releases the capsid containing modified DNA, which migrates toward the nucleus and attaches to nuclear pores to enter the nucleus.
The modified gene gets integrated into the genome of the target cell. Following this event, transcription and translation lead to the formation of the protein of interest that induces gene expression. However, in the case of adenoviruses, they simply deliver the genetic material into the cytoplasm or nucleus, and transgene expression occurs from there.
In 1999, the first successful clinical trial on gene therapy indicated the potential of viral vectors for the treatment of severe immunodeficiency disorder.4 According to a report published in The Journal of Gene Medicine in 2017, more than 68% of the clinical trials on gene therapy use viral vectors, including retrovirus, adenovirus (types 2 and 5), herpes virus, human foamy virus (HFV), and lentivirus. The main advantage of using viral vectors is their ability to cross cellular barriers and protect transgenes from biological degradation efficiently.5
Adenoviruses serve as vectors for a wide variety of therapies, including gene delivery, assessment of hereditary disorders, and regenerative therapy.6
Retroviruses and Lentiviruses have been used for the treatment of diseases caused by anomalies in a single gene and not a segment of a genome.7 Smallpox vaccine has been developed using a virus belonging to the poxvirus family.
Viral Vectors Overview
Non-viral Vector: Mechanisms and Applications
Non-viral vectors are synthetically developed to provide significant gene expression without inducing unwanted inflammatory and immune reactions.3 These vectors are primarily developed via physical and chemical methods. Physical methods include microinjection, electroporation, sonoporation, needle injection, and gene gun, while chemical methods involve certain inorganic materials, polymer and lipid-based systems.
The majority of chemical systems are cationic, which enables the combining of negatively charged DNA via electrostatic interactions. This interaction induces an overall positive charge on the vector-gene complex, which binds to the negatively charged molecules of the cellular membrane, leading to cellular internalization. Following this, they deliver the transgene to the target site.
Although physical methods, such as microinjection, can directly inject transgenes into the cellular target site, they have several limitations, including the need for individual cell manipulation and low-level gene expression and gene persistence.
Scientists use the jet injection method in multiple gene therapy applications, including the development of genetic vaccines and targeting suicide genes to promote antitumor therapy. This method has been used for the treatment of many skin diseases.
The key advantage of this technique is minimal side effects except a few including local inflammation or hyperthermia and minor bleeding at the injection site. The electroporation method has been used for in vitro nucleic acid delivery and for the treatment of skin and liver tumors.8
Engineered polymers containing desired characteristics exhibit improved gene delivery efficacy. Generally, cationic polymers are used for the formation of polyplexes for gene delivery, such as poly 2-N-dimethylaminoethyl methacrylate (PDMAEMA) and poly-l-lysine (PLL).
Scientists design synthetic biodegradable polymers with desirable chemical structures and inert properties that enable highly specific gene therapy.9
A gelatin-based nanocarrier has been developed to deliver a specific RNA called STAT6 siRNA to A459 cancer cells to inhibit STAT6 gene expression. This nano-based non-viral vector enabled efficient gene silencing and destruction of the A459 cancer cells.10
Lipid nanoparticle vectors have been recently used to develop vaccines to prevent infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causal agent of the recent pandemic called coronavirus disease 2019 (COVID-19).
What does the Future of Gene Therapy Look Like?
Viral vs Non-viral Vector
Viral and non-viral vectors are developed using different strategies, and each has its advantages and limitations in clinical applications. For instance, despite numerous benefits, the main limitations of non-viral vectors have been their high vulnerability to intracellular and extracellular barriers, lower expression of the transgene, and reduced transfection ability.3
The availability of different types of viruses with variable morphology, size, natural tropism, and genetic materials (DNA or RNA) offers scientists greater opportunity to select a specific type as per their requirement for specific gene therapy.
Despite being used in several gene therapies, the inherent infective nature of viruses triggers safety concerns.
Genetic engineering strategies focussed on inhibiting the natural toxicity of a virus and promoting inactivation have effectively reduced side effects without compromising their efficacy.
Other limitations of viral vectors are associated with their vulnerability to immunogenicity, poor targeting potential, and high production cost.
In contrast to viral vectors, non-viral vectors are relatively less toxic, versatile, non-immunogenic, cost-effective, easy to manufacture, and stable. Furthermore, the high loading capacity of non-viral vectors enhances their significance in gene delivery research.
Many non-viral vectors transfer large amounts of genetic materials into cells without triggering unfavorable immune responses.
The advancements in gene therapy have not only revolutionized medicine but also opened new doors for commercial applications, driving significant investments from biotech and pharmaceutical companies. With the rise of personalized medicine, gene therapy is paving the way for breakthrough treatments in rare genetic disorders, oncology, and regenerative medicine.
Companies are actively developing and patenting novel viral and non-viral vector technologies to enhance efficacy, safety, and scalability. The recent success of mRNA-based vaccines, which leverage non-viral lipid nanoparticle vectors, has further accelerated interest in gene-based therapeutics.
As regulatory frameworks evolve and production costs decrease, the commercialization of gene therapy is set to expand rapidly, offering lucrative opportunities for biotech startups, pharmaceutical giants, and investors looking to capitalize on the next frontier of medical innovation.
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Conclusions
Gene therapy has the potential to cure diseases, such as Parkinson’s disease and lysosomal storage disorders, that cannot be treated through conventional treatments. The advent of viral and non-viral vectors has revolutionized gene therapy.
Scientists select a specific vector based on the nature of the gene to be delivered and the characteristics of the vector. Continued research on vectors could lead to the discovery of efficient viruses and nonviral materials with higher gene transfection ability and fewer side effects.
References
- Malech HL, Garabedian EK, Hsieh MM. Evolution of Gene Therapy, Historical Perspective. Hematol Oncol Clin North Am. 2022;36(4):627-645. doi: 10.1016/j.hoc.2022.05.001.
- De Haan P, Van Diemen FR, Toscano MG. Viral gene delivery vectors: the next generation medicines for immune-related diseases. Hum Vaccin Immunother. 2021;17(1):14-21. doi: 10.1080/21645515.2020.1757989.
- Butt MH, Zaman M, Ahmad A, Khan R, Mallhi TH, Hasan MM, Khan YH, Hafeez S, Massoud EES, Rahman MH, Cavalu S. Appraisal for the Potential of Viral and Nonviral Vectors in Gene Therapy: A Review. Genes (Basel). 2022;13(8):1370. doi: 10.3390/genes13081370.
- Fischer A, Hacein-Bey-Abina S. Gene therapy for severe combined immunodeficiencies and beyond. J Exp Med. 2020;217(2):e20190607. doi: 10.1084/jem.20190607.
- Lundstrom K. Viral Vectors in Gene Therapy. Diseases. 2018;6(2):42. doi: 10.3390/diseases6020042.
- Salauddin M, Saha S, Hossain MG, Okuda K, Shimada M. Clinical Application of Adenovirus (AdV): A Comprehensive Review. Viruses. 2024; 16(7):1094. doi.org/10.3390/v16071094
- Cooray S, Howe SJ, Thrasher AJ. Retrovirus and lentivirus vector design and methods of cell conditioning. Methods Enzymol. 2012;507:29-57. doi: 10.1016/B978-0-12-386509-0.00003-X.
- Young JL, Dean DA. Electroporation-mediated gene delivery. Adv Genet. 2015;89:49-88. doi: 10.1016/bs.adgen.2014.10.003.
- Chen CK, Huang PK, Law WC, Chu CH, Chen NT, Lo LW. Biodegradable Polymers for Gene-Delivery Applications. Int J Nanomedicine. 2020;15:2131-2150. doi: 10.2147/IJN.S222419.
- Youngren SR, Tekade RK, Gustilo B, Hoffmann PR, Chougule MB. STAT6 siRNA matrix-loaded gelatin nanocarriers: formulation, characterization, and ex vivo proof of concept using adenocarcinoma cells. Biomed Res Int. 2013;:858946. doi: 10.1155/2013/858946.
Further Reading
Noninvasive Ultrasound Method Enables Tracking of Gene Expression in Specific Brain Regions