Viral vectors are genetically modified viruses used to transfer genes without causing disease.

Why use viruses for gene therapy?

Viruses have evolved systems to transport their genome to the organism they infect. Once they enter the target cell, they hijack its biological machinery to express their genes and multiply. Scientists have used this mechanism to develop gene therapies and produce biologics. The process of delivering a gene using a viral vector is called transduction. Through transduction, the newly delivered genes can elicit the expression of therapeutic proteins essential for treating a patient's condition or correcting genetic anomalies to cure the underlying disease.

This article discusses common viral vectors for gene therapy, including their production, limitations and future potential.

Common Viral Vectors Used in Gene Therapy

Currently, many viruses are being studied for their potential use as viral vectors. These vectors can deliver genes of interest to targeted cells for permanent or temporary transgene expression. Some commonly studied viral vectors include retroviruses such as γ-retroviruses and lentiviruses, herpes simplex viruses, adenoviruses and adeno-associated viruses (AAVs).

Each of these viral vectors has unique advantages and disadvantages.

• Retroviral Vectors: These vectors integrate into the host genome through reverse transcriptase. They are widely used to deliver target or therapeutic genes during gene therapy. The vector is advantageous for stabilizing the gene of interest into the target cell genome. Small insert sizes and immunogenicity are known challenges.
• Lentiviral Vectors (LVVs): These vectors can effectively deliver genes to non-dividing and difficult-to-transduce cells. LVVs are coveted for their ability to facilitate stable gene expression. They have recently been utilized for the development of CAR T therapies. However, LVVs present a risk of unwanted off-target effects due to random genomic site integrations.
• Herpes Simplex Virus Vectors: These vectors can deliver a large amount of desired DNA to different host cells, including non-dividing cells and neurons. These vectors offer a high transgene dosage per infected cell and can accommodate large insert sizes. However, there are known challenges with cytotoxicity and the requirement for helper viruses.
• Adenovirus Vectors: Adenovirus vectors are highly versatile in delivering genes of interest to various cell types, making them valuable for in vitro and in vivo research. They are also easy to construct and capable of delivering large cargo sizes. However, they are not ideal for stable gene expression.
• Adeno-Associated Virus (AAV): The vector once considered a contaminant in adenovirus cultures has become a crucial tool for gene editing, silencing and replacement. It is widely used due to its high transduction efficiency across various cells, making it an ideal choice for gene therapy. However, certain factors must be considered before using AAVs in gene therapy, such as tropism concerns, limited cargo capacity, immunogenicity and manufacturing constraints.

Considerations in Viral Vector Production

The development process for viral vectors involves several key considerations independent of viral species. Designing a suitable viral vector determines its efficacy and function in gene therapy applications. Here are some considerations that should be accounted for during viral vector production:

• Viral vector selection: This requires in-depth comprehension of the in vivo delivery mechanism and cell type specificity. Each vector has its advantages and limitations.
• pDNA/plasmid creation: Plasmids are crucial components of viral vector production. This requires them to be free of impurities with consistent batch-to-batch quality. Plasmid design and development impacts downstream vector production and transgene expression.
• Choice of cell line: Cell lines can impact the overall timeline, cost and efficiency of viral vector production. Commonly used cell lines include HEK293, Sf9, BHK21 and MDCK, with different cell lines demonstrating various efficiencies in producing specific vectors.
• Cell expansion adherent vs. suspension: The decision to use adherent or suspension cell culture depends on the required amount of viral production. Adherent cells are less amenable to scaling up, while suspension cells may require more rounds of transfection optimization.
• Capsid biology: The characterization and engineering of capsids can reduce the risk of an immune response while improving the desired tissue tropism. However, the introduction of novel capsid biology can result in challenges related to purification and concentration. It is crucial to characterize and separate full capsids from partial or empty capsids that are considered impurities and do not contain genomic material. Sometimes, the technologies available to address a specific serotype or novel capsid biology may not be adequate.

Gene Therapy Current Challenges

Viral vectors have tremendous potential in gene therapy applications, yet the research gains don’t always translate in vivo.

Here are a few challenges surrounding gene therapy:

• Gene expression levels may not be sufficient for symptom treatment or disease cure due to delivery inefficiencies, incorrect gene copies or cell type limitations.
• Drug developers still struggle to eliminate off-target effects despite extensive risk research.
• Gene therapy may not be feasible for all diseases due to access limitations to disease sites.
• Treatment costs can be a significant obstacle for low-income patients, as they often remain prohibitively high.

Future Direction

While small molecule and monoclonal antibody therapies have been more extensively developed, gene therapy shows incredible potential to transform the treatment of genetic disorders such as cystic fibrosis, severe combined immunodeficiency and hemophilia. Despite its relative newness in the field, viral vectors are crucial in creating practical therapeutic approaches within the rapidly evolving landscape of gene therapy.

The emergence of innovative molecular tools such as miRNA and CRISPR/Cas9, along with vector engineering approaches, has opened up new possibilities for treating critical diseases. Vector design has also advanced significantly, with the development of tissue-specific and regulatable promoters, which allows for greater specificity in viral vectors and better control over gene expression. As a result, researchers are constantly exploring new avenues in gene therapy, with significant progress being made in vector technology.

Scientists and physicians will continue unraveling the complexities of genetic diseases in the coming decades, offering hope for a better future through gene therapy using viral vectors.

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