What’s driving the trending preference for gammaretroviral vectors in C&GT?

As we enter a new era set to be dominated by cell and gene therapies (C&GTs), increasing numbers of developers are driving towards the use of gammaretrovirus (also referred to as retrovirus) as tools in their product development. In this blog, we examine the reasons behind the rising popularity of retroviral vectors amongst those in the biopharma space – from improvements in safety to the advantages offered in scalability. For a more in-depth analysis of the changing role of retroviral vectors in C&GTs, download the “Gammaretrovirus: Overcoming development and manufacturing challenges” whitepaper.

In the past two decades, once unimaginable possibilities achievable by therapeutics started to become a reality as a result of increasing advancements in the capabilities of C&GTs. The previously limited treatment options for diseases and disorders caused by genetic abnormalities – often due to missing or mutated genes – are now expanding, with C&GTs offering the potential for long-term benefits.

Originally used to target rare diseases, continued innovations in molecular engineering techniques have broadened C&GT applications. Indications being targeted by gene therapies in the development pipeline range from cardiovascular diseases to metabolic conditions and respiratory diseases (1). These gene therapies are most commonly directed toward cancer indications. In fact, of the 19 currently approved gene therapies (including genetically modified cell therapies) globally, 12 target cancers (1).

With over 1000 gene therapies currently in the development pipeline and rising numbers of biopharma companies realizing their potential, growth in the C&GT space is unlikely to decelerate. This is reflected in the global outlook of the gene therapy market, which is predicted to grow at a compound annual growth rate (CAGR) of 39.62% between 2021 and 2027, rising from USD 4.99 billion to USD 36.92 billion (2).

Gammaretrovirus as a tool for CAR-T therapies

The most common technology used in the pipeline of genetically modified cell therapies – and one of the key drivers in market growth – is CAR-T cell therapies. By modifying patients’ extracted T cells to introduce a gene encoding a chimeric antigen receptor (CAR), these cells can specifically recognize cancer cells and elicit a targeted immune response for their destruction once reintroduced into the patient.

Producing this powerful technology has predominantly relied on the use of viral vectors to introduce the CAR gene. Gammaretroviral vectors are one of the most commonly used vectors in this application, with five currently FDA-approved C&GTs generated using this tool.

Retroviral vectors are well-suited for the delivery of CAR genes to T cells due to their large cassette capacity (up to 10 kb) and ability to integrate transgenes into the host cell genome, enabling stable long-term expression.

Driving the trend towards a preference for retroviral vectors

The popularity of retroviral vectors in the biopharma space as a tool for C&GT production has been steadily rising. As well as having inherent characteristics that make them excellent tools for delivering genes into target cells, they also offer a number of benefits as compared with other viral vectors when it comes to their development and manufacturing. The potential advantages of using retroviral vectors in gene therapies have been further expanded through careful molecular engineering.

1) Improved safety

One of the key reasons that retrovirus is seeing an upsurge in use in the C&GT space is the rise in therapies that do not involve patient cells that produce progeny. Previously, safety concerns around retrovirus’ propensity to integrate the transgene into the host genome at sites close to oncogene promoters had limited their use in gene therapies, being linked to an increased risk of cancer development (3-5).

As mature T cells have been demonstrated to be less prone to oncogene transformation into a malignant phenotype as compared with other cells like hematopoietic stem cells upon gammaretrovirus transduction (6), retrovirus is ideal for use in CAR-T therapy production.

Advances in genetic engineering and safety principles employed in the lentiviral vector field have also been applied to retroviral vectors, further reducing concerns surrounding safety. Self-inactivating (SIN) retroviral vectors have modifications that prevent enhancer and promoter sequences in long terminal repeats (LTR) from activating adjacent cellular genes following transgene integration, reducing the likelihood of cancer development.

2) Stable producer cell lines

During upstream processing, the development of stable producer cell lines can offer many benefits as compared to relying on transient transfection methods. As well as reducing the costs of using clinical-grade plasmids and transfection agents, viral particles produced by stable cell lines are often of greater quality. This is because they will be near-homologous, ultimately decreasing potential batch-to-batch variability and providing cleaner harvests. Stable producer cell lines also offer greater scalability as compared with transient transfection methods and can be used for small-scale or large-scale production according to the quantities required.

Production of stable producer cell lines can be a challenge when using viral vectors that exhibit cytotoxicity. For example, in lentiviral production, some viral glycoproteins and HIV-1 genes are cytotoxic to the producer cell, limiting continuous expression (7). Developers therefore often rely on transient transfection for lentivirus manufacturing. On the other hand, the generation of stable producer cell lines is more common for retroviral vectors, which do not exhibit these cytotoxicity challenges.

However, it is important to remember that producing a stable producer cell line can rely on whether a SIN transfer plasmid is used or not. This is because stable cell lines can be difficult to produce in the presence of a self-inactivating U3 in the 3’LTR (8).

3) A relatively simple purification process
As well as offering advantages during upstream development, retroviruses also have distinct benefits in downstream processing. As they are most often used for therapeutic ex vivo applications, purification is generally less extensive for retrovirus as compared with adeno-associated virus or lentivirus in early clinical trials.

Although the exact downstream processing strategy will depend on the characteristics of the virus – particularly how it is pseudotyped – filtration is commonly the first step in retroviral downstream processing. If required, additional purification steps could include tangential flow filtration (TFF) to remove host cell proteins (HCPs) and anion exchange chromatography.

Looking forward

With CAR-T therapies representing 49% of genetically modified cell therapies in the development pipeline at present (1), the prevalence of retrovirus as a tool for introducing genetic material to these cells can be expected to continue to rise. Examining the benefits that it can offer developers, from scalability through stable cell line generation to relatively simple downstream processing requirements, it is clear why there is a growing trend toward their use.

However, finding experts with the experience and expertise to overcome the potential challenges involved in their production can be difficult. With many biopharma companies previously preferring to rely on lentivirus tools, those with the required expertise in the retrovirus area are scarce.

As a viral vector contract development manufacturing organization with expertise in retroviral and lentiviral vectors, Genezen can support C&GT developers in challenges they might encounter on their journey to market.

To learn more about how Genezen could support your next gammaretrovirus project, download the “Gammaretrovirus: Overcoming development and manufacturing challenges” whitepaper.

References
1. American Society of Gene + Cell Therapy. Gene, Cell & RNA Therapy Landscape Q1 2022 Quarterly Data Report
2. https://www.businesswire.com/news/home/20220201006057/en/36.92-Billion-Cell-Gene-Therapy-Market—Global-Outlook-Forecasts-2022-2027—ResearchAndMarkets.com
3. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1 [published correction appears in Science. 2003 Oct 24;302(5645):568]. Science. 2003;302(5644):415-419.
4. Howe SJ, Mansour MR, Schwarzwaelder K, et al. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J Clin Invest. 2008;118(9):3143-3150.
5. Braun CJ, Boztug K, Paruzynski A, et al. Gene therapy for Wiskott-Aldrich syndrome–long-term efficacy and genotoxicity. Sci Transl Med. 2014;6(227):227ra33.
6. Newrzela S, Cornils K, Li Z, et al. Resistance of mature T cells to oncogene transformation. Blood. 2008;112(6):2278-2286.
7. Nie Z, Phenix BN, Lum JJ, et al. HIV-1 protease processes procaspase 8 to cause mitochondrial release of cytochrome c, caspase cleavage and nuclear fragmentation. Cell Death Differ. 2002;9(11):1172-1184.
8. Maetzig T, Galla M, Baum C, Schambach A. Gammaretroviral vectors: biology, technology and application. Viruses. 2011;3(6):677-713.