Designing the plasmids that will be transfected into producer cells in gammaretrovirus (also referred to as retrovirus) development is a critical step that will ultimately determine transgene incorporation efficiency. The safety and efficacy of the cell and gene therapy (C&GT) end-product are also closely tied to the initial plasmid design.

In this blog, we explore the role of pseudotyping in delivering the clinical benefits of C&GTs, as well as how decisions made at this stage in development can impact the gammaretroviral vector manufacturing processes.

Plasmid design and envelope glycoproteins

In retrovirus production, plasmids carrying the essential genes for vector production and the transgene of interest (TOI) are transfected into producer cell lines. The plasmid design techniques implemented typically aim to improve viral titer during upstream processing and improve safety through the removal of unnecessary viral sequences. At this stage, retroviral vector developers will also pseudotype the virus, helping to control cell specificity during transduction.

Viruses in the family Retroviridae, such as retrovirus and lentivirus, have a very simple ssRNA genome which includes long terminal repeats (LTRs) and gag, pol, and env genes. In current practice, these genomic elements are split into three plasmids before transfection into the producer cell. By separating trans and cis elements onto different plasmids, recombination can be minimized and safety improved as a result.

The three plasmids typically constructed include:

1) A transfer plasmid containing the TOI and a packaging signal (psi)
2) An envelope plasmid for envelope protein (env) expression
3) A packaging plasmid containing the essential trans element gag-pol, encoding Gag-polypeptide (a core structural protein) and polymerase (for viral replication)

The envelope glycoproteins in the envelope plasmid will be transcribed and translated by the producer cell machinery before being delivered to the cell membrane. As an enveloped virus, retrovirus will obtain its envelope from the host cell upon budding, therefore gaining the envelope glycoprotein on release.

Choosing an envelope protein

The cellular tropism (the preferred type of target cell) of the retroviral vector will be dependent on the envelope glycoproteins. Interaction and subsequent infection of the target host cell (patient cells) rely on the efficiency of the envelope glycoprotein to specifically bind to receptors on the cell membrane and mediate fusion. These glycoproteins will have specificity to different receptors presented on different cell types.

By pseudotyping vectors – designing plasmids to include genes for chosen envelope glycoproteins – developers can control the cell specificity during transduction, acting to either broaden or narrow the range of cell types targeted. Transduction activity can also be improved by pseudotyping. For example, pseudotyping with particular chimeric envelope protein GALV-Env proteins can improve T cell and B cell transductions.

Glycoproteins commonly used for retrovirus pseudotyping include but are not limited to:

 

The impact of pseudotyping on viral vector manufacturing

Although pseudotyping will help control retrovirus transduction into the target patient cells, it is important to remember that the presence of envelope proteins can make viral vector manufacturing more complex in numerous ways.

Cellular toxicity

Some viral envelope proteins, including VSV-G, are toxic to most mammalian cells due to overexpression, hindering cell growth and impacting proliferation during upstream processes; this can ultimately reduce the viral titer.

Additionally, these envelope glycoproteins can mean that the production of stable cell lines is a particular challenge, as cells will not be able to produce the viral vectors continuously due to the inherent cytotoxicity. As a result, the development of stable producer cell lines will require controlled, inducible expression of the envelope protein to achieve suitable viral titers.

Stability

Retroviruses are very sensitive to a variety of physical and chemical conditions, including pH, temperature, conductivity, and shear forces. Shear forces during techniques such as ultracentrifugation, for example, can damage molecules on the surface of the viral vector, including envelope proteins.

Pseudotyping with alternative envelope proteins can be used to achieve higher virus particle stability. Retroviral vectors pseudotyped with VSV-G often outperform those with influenza envelope proteins, appearing to be more resistant to shear, improving stability.

Another stability issue that can arise if the retrovirus pseudotype is not considered carefully is vector clumping. This is a common occurrence with foamy-pseudotyped viruses during sterile filtration in downstream processing, potentially resulting in product loss. Although using surfactants can reduce clumping, the impact they could have on patient cells transduced ex vivo should be assessed.

Adsorption

Following sterile filtration, the final fill will often take place as part of aseptic processing using single-use closed systems. The materials used throughout the final-fill step – including the chosen final product bags and/or vials – must be compatible with vector stability. Adsorption of VSV-G pseudotyped viral vectors to glass vials has previously been reported (1). For these vectors, alternative containers such as polymer bags and flexible bio-containers will be better suited.

Key lessons

Having such an extensive impact on all subsequent processes, plasmid design and pseudotyping retroviruses must be performed carefully to achieve the best results in C&GT therapies, as well as the highest yields following processing.

Without assessing how the different types of envelope glycoprotein could affect processes downstream, issues surrounding cytotoxicity, stability, and adsorption could arise, putting yields and safety at risk. Avoiding these problems necessitates plasmid design expertise and an approach to development that encourages communication between all teams.
As a contract development and manufacturing organization (CDMO) with expertise in viral vector manufacturing, Genezen can help navigate the challenges of pseudotyping in your next gammaretrovirus. For more information download the “Overcoming challenges in gammaretroviral vector development and manufacturing” whitepaper or get in touch today.

References
Higashikawa F, Chang L. Kinetic analyses of stability of simple and complex retroviral vectors. Virology. 2001;280(1):124-131.