March 29, 2023

Introduction

As cell and gene therapies continue to provide unprecedented advancements in treating diseases, lentiviruses (LVs) have emerged as the go-to vectors, delivering accurate genetic material to target cells to treat a wide range of medical conditions. However, their effectiveness and cytotoxicity depend largely on a crucial step required to target specific cells— pseudotyping.

To rapidly progress C&GTs and make them a reality, researchers must carefully consider their approaches in LVV design, most notably aspects around plasmid selection. In this blog post, we’ll delve into the importance of pseudotyping LVs and explore the various approaches to achieve optimal results while providing high yields.

Pseudotyping Lentiviruses

Derived from human immunodeficiency virus (HIV), a type of lentivirus, these vectors typically contain a wild-type envelope with a natural tropism for CD4-expressing cells (such as CD4+ T cells).

For C&GTs, the virus is pseudotyped with alternative envelope glycoproteins in order to target cells lacking CD4 receptors. Pseudotyping can therefore be used to increase transduction efficiency for certain cell types (including T cells, B cells, hematopoietic cells, neuronal cells, hepatocytes, and lung epithelial cells).

The most commonly pseudotyped envelope protein, and widely considered the gold standard, is the VSV-G glycoprotein. This glycoprotein has a broad tropism and interacts with low-density lipoprotein receptors (LDL-R), which are common in most cell types.

During downstream processing, LVs pseudotyped with VSV-G often outperform those with influenza envelope proteins, appearing to be more resistant to shear during ultracentrifugation. However, they also have the disadvantage being linked to cytotoxicity and can be inactivated by the complement system (proteins that enhance immune responses) in human serum. Resting lymphocytes used in CAR-T therapies also express the LDL-R at low levels. This means that T cells must be activated before transduction.

Pseudotyping must therefore be carefully considered to achieve optimal results in C&GTs, while returning the highest yields throughout processing.

Improving Plasmid Design

Plasmids must be designed to ensure efficient incorporation of the transgene into the host genome while also ensuring safety. With improvements in design techniques, both titer and safety can be increased while removing unnecessary and potentially burdensome viral sequences. Developers must consider not only the design of the plasmid containing the transgene of interest, but the “packaging plasmids” as well.

By encoding the genes for packaging on different plasmids, there is a reduced risk of generating replication-competent lentivirus (RCL) that could lead to patient infection with replicating viruses. In current practice, three to four plasmids are used:

• A self-inactivating transfer plasmid containing the transgene flanked by HIV-1 long terminal repeats (LTRs) with a rev response element
• A packaging plasmid containing the essential trans element Gag-pol encoding Gag-polypeptide (a core structural protein)
• A plasmid for envelope protein expression
• A plasmid with the HIV-1 rev (encoding reverse transcriptase), if not encoded elsewhere

It is also important that the plasmid containing the transgene is self-inactivating to avoid enhancer and promoter sequences in the LTRs activating adjacent cellular genes following integration. If the adjacent genes are proto-oncogenes, intact lentiviral LTRs can lead to their activation and could cause cells to become cancerous.

As a result, these vectors will have a deletion in the 3’LTR which is transferred into the 5’LTR after one round of reverse transcription. As the viral promoter elements are inactivated by this action, production of the fully-formed virus after transfection into the host cell is prevented. Using this method also helps to avoid oncogene activation.

The recommended use of certain downstream processes will depend on the pseudotype of the LV, as certain pseudotypes can be sensitive to particular physico-chemical conditions. As a result, the pseudotype must be carefully considered when designing the plasmids.

One of the main challenges currently being faced by LV manufacturers currently is that many plasmids are licensed by companies. This can lead to roadblocks throughout commercialization and should be considered early in the lifecycle of the project.

Conclusion

LVV pseudotyping is a crucial step in the production process of lentiviruses. By understanding the interactions between LVs, their cells, and different pseudotypes, successful designs of the vector can be developed with minimal risk. It is imperative to create a plasmid design that is safe and effective for its intended purpose. Taking these considerations into account during every stage of your LVV project will ensure a safe and highly targeted product that meets your specific C&GT needs.

Partnering with Genezen

When undertaking the challenge of commercial-scale LV development and manufacturing, it is important to find the support and expertise needed to overcome and solve problems that could arise.

As a viral vector contract development and manufacturing organization with expertise in lentiviral and gamma retroviral vectors, Genezen can support you in facing challenges ahead.

To discover how Genezen we can help with your next LV project, contact us today: genezen.com/get-in-touch/