Ask a Scientist: Are Adenoviral Vectors Still Used in Gene Therapy?

An early workhorse in gene therapy research, this delivery system still offers certain advantages despite some key challenges. Delve into some of the reasons why this system remains relevant today.

Adenoviral vectors were among the earliest developed and most widely used vector types.¹ The decade from the early 1990s through the early 2000s saw a rapid surge in gene therapy studies in the literature, driven largely by adenoviral vectors. Many of these studies, employing adenoviral vectors in gene delivery strategies, advanced to clinical trials. However, the data from these trials generally showed a lack of effectiveness. This failure to recapitulate the efficacy seen in preclinical studies was due in part to the highly immunogenic nature of adenoviruses, which limited efficacy in patients. More problematic, this immunogenicity caused undesirable side effects, leading to a patient’s death in one unfortunate case. These challenges dampened the initial enthusiasm for gene therapy overall and prompted the search for alternative platforms.

Today, adeno-associated vectors AAVs, which offer relatively durable gene expression with lower immunogenicity, have become the preferred vector system for gene therapy.  However, adenoviral vectors remain useful in certain settings. Adenoviral vectors offer a larger packaging capacity than AAVs, have a variety of targeting options, and offer the potential for robust gene expression in a broad range of cell types. Their primary drawback, strong immunogenicity, can nonetheless be harnessed for good in vaccine or immunotherapy applications.¹ In recent years, a few examples have demonstrated the continued value of adenoviral vectors for gene delivery. The best illustration of this surfaced during  the COVID-19 pandemic, when  several adenoviral vector-based vaccine products were quickly brought to market, with millions of doses produced globally.² More recently, in 2022, the product Adstiladrin was approved by the FDA for the treatment of bladder cancer, becoming the first adenoviral-based gene therapy approved in the US, though other ad-based gene therapies had previously been approved in other countries.¹ 

What is the current state of manufacturing, upstream and downstream?

There is a great variety of adenoviral vectors, and likewise a variety of ways to manufacture them.^3–5 At laboratory scales, most vectors are produced in flasks using adherent cells. Purification may be done by lysis and clarification alone or can include density gradient ultracentrifugation if higher purity is needed. Large-scale manufacturing can be organized into upstream and downstream activities. Upstream production includes the initial steps, from thawing a vial of a master cell bank (MCB) and expanding the culture to an appropriate scale, to infecting the cells with a master virus bank (MVB).⁶ Upstream production may involve adherent culture using stacked flasks or a fixed-bed bioreactor, depending on scale or suspension culture, typically in a stirred-tank bioreactor. In either system, cultures may be fed at intervals or continuously by perfusion. Of the two options, suspension culture is more commonly selected because it is generally regarded as more suitable for commercially relevant scales. Downstream production involves several purification steps, beginning with cell harvest and lysis.⁷ The resulting lysate is clarified to remove cell debris, and then the material undergoes a progression of filtration, concentration, buffer exchange, and chromatography steps (generally two, anion exchange followed by size exclusion). A final sterile filtration step is included before the vector material is vialed as drug product.  

How do you select a production cell line?

The selection of a production cell line is one of the most important decisions to be made during manufacturing, because the cells are a key determinant of upstream productivity. The earliest adenoviral vectors were manufactured in HEK293 cells, which are still widely used for research production. Over the years, many cell lines have been developed for adenoviral vector manufacturing, including the 911 cell line and multiple variants of HEK293 and HeLa cells.⁸ However, most of these lines are stably transduced with the adenoviral E1A gene, potentially allowing for recombination events to generate replication-competent adenoviruses. Cell lines such as CAP and Per.C6 were developed to reduce this risk. Regardless of which cell line is selected, it must be noted that the production cells influence product testing as well.

What are some considerations for oncolytic and non-Ad5 systems?

Most adenoviral vectors are replication-deficient and based on serotype 5 (Ad5); thus, much of the literature on manufacturing focuses on this type. However, there is considerable interest in oncolytic (conditionally replicating) and non-Ad5 vectors as well.⁹ Non Ad5 systems include human viruses of other serotypes like Ad35 or Ad26, chimeric vectors (Ad5/35, etc.), and animal adenoviruses. There are several special considerations for manufacturing these vector types, starting with the selection of a production cell line. Cell lines used for the production of typical Ad5 vectors may not be suitable for other adenoviral vector types. For example, cell lines containing the Ad5 E1A gene (such as HEK293) should be avoided for production of oncolytic vectors due to the potential for recombination events. Thus, the A549 cell line is often used instead. Also, for any non-Ad5 vector (or capsid-modified Ad5 vector), the multiplicity of infection (MOI) may need to be optimized during upstream production.

Downstream process optimization may be needed as well, since any differences in the capsid from Ad5 may alter the overall charge of the viral particles, potentially impacting chromatography conditions. 

How do we test these products?

Adenoviral vectors, like other vector products, must be extensively tested to confirm purity, safety, identity, quality, and strength/potency before they can be administered to patients. Many of these tests are compendial (like pH and sterility) while others are product-specific (potency). Some tests are linked to the production method. For example, tests for process-related residual impurities, such as host-cell protein (HCP) and host-cell DNA (HCD), are typically cell line-specific. Viral titer, or concentration, is a key measurement used to assess productivity and determine dose levels. Generally, both particle titer and infectious titer are assessed. Particle titer methods can include HPLC or genome-based PCR. Infectious titer methods include TCID50, PFU, FFU, etc. Particle titers are typically higher than infectious titers by 10-fold or more, due to the presence of defective particles in any given batch. The ratio between these values provides some indication of product quality, with the FDA recommending a ratio of no more than 30:1 between physical and infectious particles.

What are common challenges in manufacturing?

Some challenges are relevant to vector manufacturing in general. For instance, the procurement of GMP-quality MCBs and MVBs can be time-consuming and costly if they are not immediately available. Certain other materials (like chromatography columns) can also have long lead times. Regarding the manufacturing process itself, achieving sufficient upstream productivity is always a potential challenge, since some constructs are inherently more difficult to produce than others.¹⁰ Usually, this can be mitigated by carefully optimizing upstream parameters such as the cell density, the MOI, and the timing of the harvest. However, in some cases, the gene of interest (GOI) may be toxic to the cells. For these constructs, it may be necessary to produce them using an inducible expression system. The TRiPAdeno¹¹ and T-REx™ (ThermoFisher) systems are two examples of this, both of which rely on engineered cell lines carrying bacterial gene regulatory elements responsive to tryptophan and tetracycline, respectively. 

In the TRiP (Transgene Repression in vector Production) system, the production cells stably express tryptophan RNA-binding attenuation protein (TRAP) while the vector carries a TRAP-binding sequence (TBS). In the presence of excess tryptophan, TRAP binds to the TBS and blocks gene expression. Similarly, in the T-REx^™ system, the cells stably express the tetracycline repressor while the vector carries a CMV promoter with Tet operator 2 (TetO2) sequences that the repressor binds to. In the presence of tetracycline, the repressor detaches from the TetO2 sites, and gene expression is turned on (or expression is able to proceed from the CMV promoter in the absence of the repressor). 

In these systems, the expression of the GOI is repressed during upstream production, allowing the vector to be propagated while avoiding GOI toxicity.  During downstream production, aggregation can be an issue, which can be mitigated by optimizing salt concentrations and using non-ionic detergents. Another challenge is particle heterogeneity, or the separation of full capsids from empty and partially full capsids. This can be done by ultracentrifugation, although ultracentrifugation becomes increasingly cumbersome at larger scales. Chromatography can also be used, provided that the conditions are sufficiently optimized. To address all of the above challenges, it is helpful to start with a well-vetted platform process, allowing optimization efforts to be focused on a few discrete steps.

Conclusions

Adenoviral vectors were at the forefront of gene therapy research for many years and were the predominant gene delivery system in early clinical trials. While there have been challenges and setbacks along the way, adenoviral vectors remain a valuable tool for gene delivery. Having found new life as a platform for vaccines and oncolytic virotherapy, they are now delivering real patient benefits and clinical approvals. Along with these clinical successes, numerous manufacturing hurdles have been overcome, and several methods now exist to produce these vectors efficiently in large quantities. Stay tuned as this platform continues to evolve and find application in new therapeutic areas.

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James Cody, PhD, is an Associate Director for Technical Sales and Evaluations for Charles River’s Cell and Gene Therapy CDMO business unit. Focusing on viral vector manufacturing, he serves as a technical liaison between the commercial team and operational subject matter experts, helping to align client CMC needs with site capabilities. James has published over 20 scientific and industry articles and gives frequent webinar and conference presentations, covering topics related to the key considerations and challenges in viral vector manufacturing. 

James joined Charles River in 2021 with the acquisition of Vigene Biosciences, having previously transitioned to a CDMO business development role in 2018. Previously, he was a research scientist for four years, specializing in infectious diseases and oncology at two small companies in the Rockville, MD area. James obtained a Ph.D. and completed postdoctoral training at the University of Alabama at Birmingham, developing adenoviral and HSV vectors for cancer gene therapy.
Earlier this year, James contributed the chapter “Manufacturing and upscaling” to Adenoviral Vectors for Gene Therapy, 3rd Edition (Elsevier), edited by David T. Curiel and Alan L. Parker. This article provides a brief overview of some key topics covered in greater detail in the chapter.

References:
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2.    Mendonça SA, Lorincz R, Boucher P, Curiel DT. Adenoviral vector vaccine platforms in the SARS-CoV-2 pandemic. NPJ Vaccines. 2021 Aug 5;6:97.
3.    Cody, James. Chapter 26 - Manufacturing and upscaling. In: Adenoviral Vectors for Gene Therapy [Internet]. 3rd ed. Academic Press (Elsevier); 2025. p. 777–829. Available from: https://doi.org/10.1016/B978-0-323-89821-8.00026-7
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8.    Kovesdi I, Hedley SJ. Adenoviral Producer Cells. Viruses. 2010 Aug 16;2(8):1681–703.
9.    Ungerechts G, Bossow S, Leuchs B, Holm PS, Rommelaere J, Coffey M, et al. Moving oncolytic viruses into the clinic: clinical-grade production, purification, and characterization of diverse oncolytic viruses. Mol Ther Methods Clin Dev. 2016 Apr 6;3:16018.
10.    Dormond E, Perrier M, Kamen A. From the first to the third generation adenoviral vector: what parameters are governing the production yield? Biotechnol Adv. 2009;27(2):133–44.
11.    Maunder HE, Wright J, Kolli BR, Vieira CR, Mkandawire TT, Tatoris S, et al. Enhancing titres of therapeutic viral vectors using the transgene repression in vector production (TRiP) system. Nat Commun. 2017 Mar 27;8:14834.