Balancing AAVs Versus LLVs

Published on: 
BioPharm International, BioPharm International, August 2023, Volume 36, Issue 8
Pages: 13–14

Burgeoning CGT modalities bring promise and challenges.

Review articles are quick to outline the tremendous progress and consolidated gains accrued during the past 40 years for cell and gene therapies (CGT). Because they are retrospectively oriented, review articles are not always adept at identifying means to further optimize performance. For example, of the approximately 1000 known adeno-associated virus (AAV) variants, “…our knowledge of AAV’s infection pathway is based on only a few serotypes. The prevailing understanding is that serotypes bind to different, or have differential affinities to an array of primary cell surface glycoprotein receptors and secondary receptors” (1).

Pharmaceutical research energy is now being focused on studies to elucidate if the lower performance of therapies for some racial minorities or gender are based on these serotype differences (2).By studying these less understood serotypes, it may be possible to uncover a mechanism that not only works for more patients, but might just work better per se. This could be an opportunity similar to gene editing with Cas9 proteins, which was admittedly partly driven by a desire to find paths around patent claims. Today, NgAgo protein gene editing systems (argonaute family proteins) are able to cut DNA at a specific site without requiring guide RNA. And relevant to CGT, mini-Cas9 are derived from staphylococcus aureus bacterium, which are small enough to fit inside these viruses, alongside the increasingly popular Cpf1 (clustered regularly interspaced short palindromic repeats [CRISPR] from Prevotella and Francisella, previously known as Cas12a) (3).

Working through and around viral baggage

When immersed in a topic-specific scientific paper (not a review paper), it quickly becomes clear how enormous a difference AAV2 binding to heparan sulfate proteoglycans is, compared with, say, binding to angiotensin-converting enzyme 2 (ACE2) and the SARS-CoV-2 spike protein, which enhances COVID-19 virulence (2). Indeed, the spike protein for COVID-19 was initially rumored to have been a lab-engineered version of the HIV spike protein, atypical of coronavirus, and not a naturally occurring variant.

Harnessing a virus to alter a patient’s genetic code as a therapy will always present natural obstacles cojoined to natural fear. CGT has a deep foundational knowledge accrued during the study of HIV and similar pandemics. Harnessing these viruses brings along some of the problems they create for a human’s complex immune system responses. Most people carry neutralizing antibodies (NAbs) against one or more of the commonly used vectors and capsids, thus greatly reducing efficacy for those individuals.

According to a study, racial minority and gender differences were a determining factor in efficacy. The study concluded, “We suggest that capsid selection for drug development take into consideration AAV seroprevalence among the patient population while avoiding sampling bias” (4). Understanding the underlying biology in subpopulations may lead to more highly optimized treatments.

Naiara Tejados, head of Communi-cations, VIVEBiotech, says, “Regarding adverse immune responses, compared to other vector types, only a small portion of the population has preexisting antibodies to lentiviral vectors [LLVs]. As a result, patients who receive CGTs based on lentiviral vectors will have low risk of experiencing immediate immune reactions.”

Hanna Dreja, Upstream Subject Matter Expert at Astrea Bioseparations, summarizes her perspective on balancing CGT use applications saying, “Lentiviral vectors (LVV) are widely used in cell therapy, specifically for autologous treatments, where a patient’s own cells are infected with viral particles and then reintroduced into the body for chimeric antigen receptor T Cells (CAR-T) and stem cell therapy. Conversely, AAV vectors are directly administered to patients to deliver therapeutic genes.” When it comes to examining performance against immune response, Dreja goes on to say, “While the immune response to LVV is generally less concerning, repeated AAV injections can lead to side effects and reduced therapy effectiveness, due to the presence of neutralizing antibodies. However, it is important to note that LVV carries the added risk of integrational mutagenesis. Despite these concerns both systems are good solutions for long-term and stable expressions of the desired genes.”


High cost and optimization factors

Manufacturing CGTs at a large scale poses significant challenges. Automation and improved process analytics that provide data-driven decision making leads to improved efficiency and cost reductions. According to Tejados, “the main advantage of allogenic gene-modified cell therapy products is the decrease in manufacturing costs. Manufacturers in the field of gene and cell therapy are making great efforts to lower the costs of advanced therapies, and obviously allogeneic therapies are more accessible,” she says. Tejados also points to some customer projects using intermediate-scale bioreactors that have a small footprint that reduces costs at development stages.

Dreja states that, “Recent advancements in single-use units and automation have made it possible to reproducibly generate these particles consistently, although at a high cost. Inducible packaging cell lines offer an intriguing alternative to transient transfection protocols, as they eliminate the need for large quantities of expensive plasmid DNA.” Tejados points to recent successes with stable producer cell lines, where there is obvious industry excitement mixed with some hesitation.

Luca Peterfay, process development scientist II at Charles River, however, emphasized that the generation of the stable producer cells is an additional step prior to the initiation of production, “…and this step can potentially increase preclinical timelines by several months. The generation of stable producer cells is a complex and time-consuming procedure, which entails tedious cell-line development due to the numerous clonal isolation steps followed by extensive screening procedures,” she explained (5).

For the constitutive approach, Peterfay also noted, “… due to replacement of the VSV-G with a low-cytotoxicity alternative envelope, titers may not be as high as those achieved via transient transfection, and transduction efficiency can also be lower. The limitation in titers can be compensated by longer processing times and multiple harvests; however, due to the reduced transduction efficiency higher doses might be required to achieve the required therapeutic effect in patients,” she said (5).

Innovations driving advances

Developing a purification system that is effective and simple is crucial as LVVs in particular are delicate molecules and suffer under processing sheer forces and stresses and, therefore, benefit from minimal handling. “Even more effective and gentle approaches, which eliminate contaminants, will be key for future production at scale,” Dreja suggests. “For example, next-generation purification technologies, such as nanofibers, present an interesting solution for LVV and AAV purifications, offering an open structure and a large surface area that is easily accessible to viral vectors. The nanofiber technology has the potential to reduce processing times by relying on convection-based mass transfer rather than diffusion,” she concludes.


1. Bulcha, J.; Wang, Y.; Ma, H.; Tai, P.; and Gao, G. Viral Vector Platforms within the Gene Therapy Landscape. Signal Transduction and Targeted Therapy. 2021, February Springer Nature Group.
2. Mengstie, M. Viral Vectors for the in Vivo Delivery of CRISPR Components: Advances and Challenges. Frontiers in Bioengineering and Biotechnology, May 2022 Frontiers.
3. Clausen, T.; Sandoval, D; Spliid, C. et al. SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2. Cell. 2020, September 12;183(4):1043-1057.e15. Elsevier DOI: 10.1016/j.cell.2020.09.033
4. Khatri, A.; Shelke, R.; Guan, A.; Somanathan, S. Higher Seroprevalence of Anti-Adeno-Associated Viral Vector Neutralizing Antibodies Among Racial Minorities in the United States, Hum. Gene. Ther. 2022 April 33(7-8). Doi: 10.1089/hum.2021.243
5. Challener, C. State of Producer Cell Development for Lentiviral Vector Manufacturing. BioPharm Internat. 2023 36(7).

About the author

Chris Spivey is the editorial director of BioPharm International.

Article details

BioPharm International
Vol. 36, No. 8
August 2023
Pages: 13–14


When referring to this article, please cite it as Spivey, C. Balancing AAVs Versus LLVs. BioPharm International, 2023, 36 (8) 13–14.