Gene Therapy with Viral Vectors: Types, Mechanisms, and Single-Use Systems in the Manufacturing Chain

Gene therapy has moved from an experimental field to an established clinical reality. Viral vector-based drugs are now approved by regulatory authorities for diseases that until recently had no effective treatment. Behind each of these products lies a complex manufacturing chain with demanding technical and regulatory requirements, and one that is highly dependent on choosing the right production platforms.

What Is Gene Therapy and How Do Viral Vectors Act on the Genome?

A viral vector is a genetically modified virus engineered to eliminate its ability to replicate autonomously, turning it into a vehicle for genetic transfer. Depending on their nature, vectors can be integrating or non-integrating, and they are used in both in vivo strategies (administered directly to the patient to reach the target tissue) and ex vivo approaches, where cells are modified outside the body before being reinfused.

Gene therapy uses these vectors to introduce, silence, or edit specific sequences in target cells. The mechanism of action on the genome varies by vector type: integrating vectors (such as lentiviral or retroviral) stably incorporate the transgene into the host’s DNA, ensuring long-term expression but carrying a potential risk of insertional mutagenesis. Non-integrating vectors (such as adeno-associated viruses, AAV, or adenoviruses) maintain the therapeutic genome predominantly in episomal form, which reduces the risk but may compromise expression durability in tissues with high cell division rates.

Types of Viral Vectors in Gene Therapy: AAV, Lentiviral, Retroviral, and Adenoviral

Each vector type has a distinct technical profile that makes it more or less suitable depending on the indication, target tissue, need for stable integration, and required product volume.

• AAV vectors are the dominant platform for in vivo strategies. Their small size (20 nm), broad serotype-dependent tropism, ability to transduce both dividing and non-dividing cells, and low immunogenicity make them the reference vector for monogenic diseases. Their main limitation is packaging capacity, which is under 5 kb, requiring a compact design of the expression cassette.

• Lentiviral vectors stably integrate into the host genome, including non-dividing cells, making them particularly well-suited for ex vivo strategies involving hematopoietic stem cells or T lymphocytes. Their cargo capacity of up to 9 kb and the ability to express multiple transgenes from a single vector add to their versatility. They are the preferred platform for CAR-T therapies in hematological malignancies.

• Retroviral vectors stably integrate the transgene but only in actively dividing cells. They were the first vectors used in clinical gene therapy trials for severe combined immunodeficiencies. The risk of insertional mutagenesis has driven the development of third-generation vectors with self-inactivating elements.

• Adenoviral vectors offer very high transduction efficiency, broad tissue tropism, and a large cargo capacity of up to 37 kb. Expression is transient since they do not integrate into the genome, making them well-suited for vaccines and oncolytic therapies, although their immunogenicity limits applications requiring sustained expression.

In practice, AAV and lentiviral vectors account for the majority of active clinical development in advanced therapies. The former for their suitability in in vivo strategies for monogenic indications, the latter for their well-established role in ex vivo cell therapies where stable transgene integration is a requirement.

Table 1. Advantages and limitations of different viral vector classes. Adapted from: Hurnikova J, Venkatesan JK, Liu W, Madry H, Cucchiarini M, Petrovova E. Viral vector research in human gene therapy: Basic principles, alternative evaluation models, and clinical applications. SLAS Technol. 2026 May;38:100404

Stages of the Viral Vector Manufacturing Chain: Upstream, Downstream, and Formulation

The viral vector manufacturing chain is structured around three sequential stages:

1- In the upstream phase, the cell bank is expanded, producer cells are transfected, and the vector is propagated in the bioreactor. This is where the conditions that determine vector yield and quality are established: cell density, pH, dissolved oxygen, and temperature. The choice of culture format directly affects process scalability and the impurity profile that will need to be managed in later stages.

2- In downstream processing, vectors are clarified, concentrated, and purified through chromatography and tangential flow filtration. For AAV vectors, separating full capsids from empty ones is one of the most demanding steps, with a direct impact on product potency. For lentiviral vectors, purification must preserve vector infectivity throughout the process, requiring careful management of each transfer and concentration step.

3- Formulation and final fill-finish close the chain. In advanced therapies, this step requires strict aseptic conditions and cryopreservation to maintain product viability until administration to the patient.

Single-Use Systems for AAV and Lentiviral Vector Production: Bioreactors, Bags, and Selection Criteria

In current GMP environments, the dominant trend points toward single-use systems across all stages. Recent studies indicate that stainless steel can negatively affect AAV vector infectivity, reinforcing the case for implementing this strategy from the initial culture phase through to final fill-finish.

• Single-use bioreactors have become the preferred solution for scaling lentiviral and AAV vector production up to 2,000 L.

• Bioprocess bags for gene therapy in 2D and 3D configurations allow media, inocula, and harvests to be managed under sterile conditions, without environmental exposure and without the need for clean-in-place/steam-in-place (CIP/SIP) cycles.

• Pre-assembled assemblies and manifolds complete this circuit, adapting the flow to each phase and reducing opening points between product changeovers.

• In the formulation stage, automated closed-system filling platforms combine formulation and filling in a single step, preserving cellular viability and vector potency through to use.

Selecting single-use components for viral vector production cannot be based on operational criteria alone. Biological compatibility of the material with the vector and cell line must be demonstrated; the mechanical robustness of each component must ensure integrity throughout the entire process; and gene therapy regulations require that every component in the circuit can be qualified, documented, and traced lot by lot.

Integrating single-use systems across the full process also enables the design of more flexible multiproduct facilities, where switching vectors or indications does not require extended infrastructure revalidation periods.

At Bexen Bioservices, we offer solutions for the entire viral vector manufacturing chain: bioprocess bags in 2D and 3D configurations, configurable assemblies and manifolds, and automated filling systems compatible with cryobags and closed-system cryogenic vials. All our components are supplied with the technical documentation required for GMP environments.

If you’re developing or scaling a gene therapy process, let’s talk.

References

Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther. 2021 Feb 8;6(1):53. doi: 10.1038/s41392-021-00487-6

Challener CA. Single-Use Technologies Prove Effective for Viral Vector Process Development. BioPharm International. 2021;34(8):12–19. Available from: https://www.biopharminternational.com/view/single-use-technologies-prove-effective-for-viral-vector-process-development 

Hurnikova J, Venkatesan JK, Liu W, Madry H, Cucchiarini M, Petrovova E. Viral vector research in human gene therapy: Basic principles, alternative evaluation models, and clinical applications. SLAS Technol. 2026 May;38:100404. doi: 10.1016/j.slast.2026.100404