Viruses have unparalleled capability to transport their genome into host organisms, encouraging them to multiply and express proteins that hijack the host’s biological pathways. This mechanism has been exploited to develop viral vectors devoid of disease-causing genes. Viral vector design involves inserting custom genes into viral vectors, which promote the expression of therapeutic or functional proteins in the host. From this perspective, viral vectors are powerful delivery vehicles for gene therapies, correcting deficiencies in genetic disorders, such as muscular atrophy. They can also be used in vaccine development, exemplified by SARS-CoV-2 vaccines¹, where the transduced gene produces antigens to educate the host immune system.

Large-scale viral vector manufacturing makes delivering viral vector products to large populations possible. However, the process must adhere to strict Good Manufacturing Practices (GMP) to ensure safe and consistent gene expression profiles.

Types of Viral Vectors

Several viral vectors are leveraged for their transgene expression capacities and safety profiles.

Retroviral vectors

Retroviral vectors are developed by removing viral genes from retroviruses and inserting therapeutic genes. These vectors can integrate into the host genome to induce long-term gene expressions for treating chronic genetic disorders. Although retroviral vectors are very efficient in transducing dividing cells, e.g., epithelial or bone marrow progenitor cells, this is not the case for non-dividing cells, e.g., neurons and cardiomyocytes. This drawback, combined with their small cargo capacity, restricts the clinical applicability of retroviral vectors.

Adenoviral vectors

Adenoviral vectors are derived from adenoviruses that cause infections in many organs, including the lungs and intestines. These vectors are modified to insert functional genes, mainly for vaccine development. Some of the widely-used COVID-19 vaccines, such as Oxford-AstraZeneca¹, and Johnson & Johnson² are composed of adenoviral vectors, preferred due to their high transduction efficiency in various cells, rapid gene expression upon delivery, and large genetic capacities. However, they are not ideal for chronic conditions and genetic disorders, as gene expression is transient and subject to rapid immune clearance.

Lentiviral vectors

Lentiviral vectors are derived from lentiviruses, a subclass of retroviruses, with the human immunodeficiency virus (HIV) being the most well-known. They are versatile tools that can insert genes into difficult-to-transduce cells like neurons and stem cells. The stable and long-term expression profiles make them ideal candidates for gene therapies in β-thalassemia and sickle cell disease.^3,4 They were also used in ex vivo therapies, such as CAR-T, for cancer immunotherapy.⁵ On the other hand, their ability to integrate into the host genome poses critical risks of insertional mutagenesis.

AAV (Adeno-Associated Virus) vectors

AAV was initially discovered as a contaminant in adenovirus cultures, characterized by a nonenveloped structure and single-stranded DNA (ssDNA) cargo as opposed to adenoviruses with double-stranded DNA (dsDNA). Thanks to their non-pathogenic nature, AAVs have demonstrated safe and efficient gene delivery into the host nucleus, generating extra-chromosomal episomes, making them ideal for treating chronic conditions without altering the host genome. However, they are not conducive to the insertion of large genes. In addition, people previously exposed to AAVs in nature may develop an immune response, diminishing efficacy.⁶

Other emerging vectors

Alternative viral vectors have emerged to address bottlenecks in traditional viral vector technologies, improving cargo capacity, safety, immune response, and efficacy. Some examples include:

• Synthetic AAV Variants created by the addition of rational design and bioconjugation: They demonstrated an improved ability to cross the blood-brain barrier (BBB) and enhanced transduction in the central nervous system. ⁷
• Plant Virus-Based Vectors: They stand out for their superb safety profiles, as they do not evoke immunogenicity. ⁸
• Foamy Virus Vectors are non-pathogenic alternatives to retroviral vectors. They display improved transduction over a wide range of cells and a lower risk of integration into the genome. ⁹
• Hybrid viral vectors, such as adenovirus + AAV, exhibit more efficient and longer transduction with lower side effects than standalone vectors. ¹⁰
• Bacteriophage-derived vectors, derived from viruses that infect bacteria, are widely used in cancer therapies due to their low immunogenicity and cost-effective production. ¹¹
• Virus-like particles (VLPs) can be synthesized using viral structural proteins that self-assemble into a virus-like shape, entirely free of pathogenicity. They are safe and versatile tools for delivering various gene therapy modalities, including mRNA, siRNA, and CRISPR systems. ¹²

Viral Vector Production Process

Cell Line Development

Pharmaceutical-scale viral vector production requires reliable host cell lines. The choice of cell line depends on the type of viral vectors, the therapeutic goals, and the scalability needs.

• HEK293 cells are preferred for AAV and lentiviral vector production due to the ease of transfection, rapid growth, and capacity for plasmid replication. ¹³
• PER.C6 retinal cell lines are ideal for the large-scale production of adenoviral vectors. Janssen used them to expand the vectors for the COVID-19 vaccine. ¹⁴
• Vero cells are derived from the kidney cells of African green monkeys. They are widely used in continuous cell line development for vaccine manufacturing. ¹⁵

Viral Vector Packaging and Transfection Methods

Transfection methods introduce viral genes necessary for vector expression into host cell lines. Transfection can be transient or stable.

Viral vector packaging is a critical part of transfection. It is used to assemble the viral particles containing the gene of interest but lacking viral replication capability. Packaging is crucial to preventing the production of replication-competent viruses that may affect safety and effectiveness.

• Plasma DNA transfection involves preparing multiple plasmids for co-transfection. A transgene plasmid containing the gene of interest is co-transfected with packaging plasmids and helper plasmids. The former is required for initiating the expression of viral structural and enzymatic proteins, while the latter is crucial for the self-assembly of the viral vector. Although they are suitable for small-scale and transient viral vector production, lending themselves to research applications and preclinical studies, they suffer from batch-to-batch variability and high costs for large-scale production.
• The transgene, packaging gene, and other essential viral genes can be integrated into the cell line genome for stable cell line production, enabling consistent viral vector expression and paving the way for GMP-compliant and large-scale viral vector manufacturing. Although their development timeline is longer than plasmid DNA transfection, the payoff is much greater in cost, yield, and consistency.
• Electroporation can introduce viral DNA into cells that are hard to transfect by other methods. This method introduces electric pulses to the producer cell line to create temporary pores for viral DNA entry.
• Helper viruses are sometimes used in AAV production to temporarily activate AAV replication and assembly without integrating into the final vector product.

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Harvesting and Purification

Harvesting and purification are critical steps in viral vector manufacturing to ensure the elimination of contaminants and byproducts so that the vector product is biologically active and safe for clinical use. Harvesting involves lysing the cell culture and centrifuging to collect the supernatant containing the viral vectors. Further purification steps include:

• Ultracentrifugation: High-speed centrifugation to separate viral vectors from cellular debris based on particle density
• Chromatography: Separation of viral vectors from cellular debris by charge (ion exchange chromatography), size (size exclusion chromatography), and ligand binding affinity (affinity chromatography)
• Tangential Flow Filtration (TFF) is a purification method in which the feed flows parallel to the membrane to enable continuous separation of viral vectors. It is often used in conjunction with chromatography to optimize the viral vector titer.

Quality Control Measures

Importance of quality assurance in viral vector manufacturing

Quality control and assurance are mandatory in viral vector manufacturing to confirm clinical and commercial usability and regulatory compliance. Manufacturers must ensure batch-to-batch consistency and eliminate contaminated or ineffective vectors to promote patient safety.

Regulatory compliance and standards

FDA and EMA impose strict compliance standards to ensure that viral vector-based gene therapies are safe for administering. Key requirements include detailed QC documentation containing batch records and method validation for transfection, harvesting, and purification. Manufacturers must adhere to the guidelines released by the agencies.

Key tests for potency, purity, and identity

A wide range of tests are required as a part of QC documentation.

• Identity tests confirm the vector's potency and biological activity. Reporter assays, PCR, and NGS, can demonstrate the presence of the transgene, while ELISA, mass spectrometry, and immunoblotting can confirm viral protein expression.
• Physical titer helps measure viral particle concentration, vital for determining clinical dosing. Examples include optical density assays, high-performance liquid chromatography (HPLC), and real-time PCR-based assays.
• Functional titer tests measure the net viral particle concentration able to transduce target cells. Examples include viral plaque, endpoint dilution, and immunofluorescence foci assays.
• Purity tests, such as ELISA and transmission electron microscopy, reveal protein impurities from the producer cell lines. Furthermore, mass spectrometry can reveal the capsid content and detect process impurities, especially during AAV purification.
• Safety and sterility tests ensure the final product is free of potentially harmful impurities, such as endotoxins, mycoplasma, and replication-competent virus particles.
• Stability tests, which detect potential aggregation risks, ensure that the product retains integrity and quality throughout its shelf-life.

Challenges in Viral Vector Manufacturing

Producing viral vectors in large quantities for clinical applications is an intricate process subject to various challenges.

Bioprocess optimization is a key concern. Manufacturers must optimize bioreactors and closely monitor transfection, harvesting, and purification to achieve desired vector yields with uniform quality. Nevertheless, process optimization can be time-consuming and costly, delaying delivery to market.

The importance of quality assurance cannot be overstated. Variability in cell line expansion, transfection, and packaging may result in compromised viral vector products with unpredictable biological activities and gene delivery power. Contamination is one of the key reasons for variabilities. Residual plasmids, microbial contamination, or host cell DNA in the final product may pose health risks to patients. Real-time monitoring systems, closed-system bioreactors, and sterile conditions must be implemented to minimize contamination.

Finally, immunogenicity remains a hurdle in many viral vector applications due to pre-existing patient immunity or unintended antibody production and cytotoxic T-cell responses. Therefore, viral vector research must be encouraged to produce fewer immunogenic capsids, immunosuppression methods, and alternatives to traditional vectors.

References

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