Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

Lentiviral vectors have been a staple in molecular biology for over three decades. Widely used across various research applications, they have become indispensable tools for manipulating cells and organisms. They can be used for a variety of research purposes, like creating stable cell lines, delivering CRISPR/Cas9 to cells for genome editing, and introducing inducible systems into cells, among many other uses. Lentiviral vectors can be produced easily, without the need for time-consuming purification processes, and are powerful, due to their ability to integrate into the host cell’s genome. However, sometimes inte-great-ion isn’t so great. In this post, we’ll cover how standard lentiviral vectors work with regards to integration, and how researchers have modified them to prevent this integration. 

Lentiviruses evolved to integrate

Viral genome integration into a host’s genome isn’t uncommon. Many naturally occurring viruses occasionally integrate their genomes into those of the cells they infect. These are typically DNA viruses, such as adeno-associated virus, herpesviruses, hepatitis B virus, adenoviruses, and human papillomavirus (HPV), to name a few (Desfarges & Ciuffi, 2012). One family of viruses, retroviridae, require integration for their life cycle to continue. They rely on a viral protein called reverse transcriptase to convert their RNA genomes into DNA to be integrated into the chromosome of the cell. This integrated viral genome is then part of the cellular DNA… forever! 

This can be an advantage for researchers looking to generate long-lasting changes in cells. However, for some applications, integration might be considered a double-edged sword with unintended consequences. In this blog, we’ll take a deep dive into integrase-deficient lentiviral vectors, how they work, and why you might choose them over integrating lentiviral vectors. 

Before we jump into integrase-deficient lentiviral vectors, let’s briefly revisit the basic biology of lentiviral vectors (for a more detailed explanation, check out Addgene's Lentiviral Guide). To produce lentiviral vectors, the lentiviral genome is distributed across multiple plasmids. For simplicity, we’ll use the second-generation system as an example. 

• The lentiviral transfer plasmid encodes your insert of interest, flanked by long terminal repeat (LTR) sequences, which facilitate integration of this portion into the host genome.
• The packaging plasmid contains Gag, Pol, Rev, and Tat genes. Specifically for our knowledge here, the Pol gene encodes three separate enzymes that are proteolytically processed from the larger protein: PR (protease), RT (reverse transcriptase), and IN (integrase). These viral packaging genes are required for the production of lentiviral particles. 
• The envelope plasmid typically encodes the vesicular stomatitis virus G protein (VSV-G), which facilitates viral entry into the host cell. 

Transfecting these three plasmids into cells enables the production of lentiviral vectors that are competent for one round of infection (AKA transduction in the viral vector world). Once inside the host cell, the viral payload is integrated into the host genome via the viral proteins packaged during production (read our blog post on the retroviral life cycle for a more detailed description). Since the integrated portion does not contain any of the viral packaging genes, the natural cycle of the lentiviral vector ends here and no viral progeny are produced (Figure 1). 

Figure 1: Comparison of the life cycle of a lentiviral vector with an integrase-deficient viral vector. Created with BioRender.com.

What are integrase-deficient lentiviral vectors?

Now let’s turn to integrase-deficient lentiviruses (IDLVs). As the name suggests, these lentiviruses lack a functioning integrase, the enzyme responsible for inserting viral DNA into the host genome. Without a working integrase, IDLVs can still deliver their genetic payload to the cell but cannot integrate it into the host genome (Figure 1). IDLVs were first discovered by researchers investigating HIV integrase mutants in the early 1990s (Leavitt et al., 1996), and since then, numerous variants have been developed with different mechanisms to inactivate the integrase. 

The IDLV life cycle

When IDLVs are delivered to cells, the viral life cycle initially proceeds as usual. Reverse transcription of the transgene occurs in the cytoplasm, then the resulting double-stranded DNA transgene is incorporated into a large complex of both viral and cellular proteins and trafficked to the nucleus. Since the integrase is non-functional and yet the linear proviral DNA is poised for integration, two types of circular episomes can be generated through either homologous recombination or non-homologous end joining (see Figure 2). 

Figure 2: Schematic representation of integration process and episomal forms of non-integrating transgenes. Created with BioRender.com; adapted from Banasik and McCray, 2010.

These episomes are not maintained in dividing cells because they lack an origin of replication (Banasik and McCray, 2010). However, IDLV genomes are transcriptionally active, leading to transient expression of gene products (Wu et al., 2004). Over time, the expression diminishes as the episomes are lost through cycles of cell division, though the rate of loss greatly depends on cell turnover.

Why use integrase-deficient lentiviral vectors?

There are several reasons why you might choose an IDLV over a lentiviral vector:

1. No risk of insertional mutagenesis

Lentiviral vectors tend to integrate into transcriptionally active regions of the genome (Schröder et al., 2002), thus increasing the risk for insertional mutagenesis. This integration could lead to an undesirable interruption of a critical gene. Because the integration of lentiviral vectors is random, and the integration site will be different for each transduced cell, there’s no telling which cells may harbor these unwanted interruptions. Insertional mutagenesis is especially concerning in clinical settings, as evidenced by retroviral gene therapy trials for X-linked severe combined immunodeficiency (SCID-X1) in the early 2000s. These trials unfortunately resulted in several patients developing T-cell acute lymphoblastic leukemia due to insertional mutagenesis (Howe et al., 2008).

2. Transient expression of transgenes/shRNAs

If you want your cells to express a protein or nucleic acid for only a limited time, IDLVs are ideal. As the episomes are lost during cell division, the expression of the delivered payload is gradually reduced. For example, after using CRISPR/Cas9 to edit a gene, the expression of Cas9 is no longer necessary. However, Cas9 can be toxic if continuously expressed (Fu et al., 2013). Using IDLVs ensures transient expression without the risks associated with prolonged expression.

3. Stable expression in non-dividing cells

IDLVs can also be useful for transducing non-dividing cells, as they avoid the risk of insertional mutagenesis while still allowing for stable expression of the transgene. This makes IDLVs an attractive choice for certain types of clinical or basic research applications.

Considerations for IDLV use

If you’re reading this and thinking “Gee, I’d love to give these a try myself,” look no further! You can package your own lentiviral vector using an integrase-deficient packaging plasmid available at Addgene: psPAX2-D64V. This construct has the D64V mutation, a single amino acid change (from aspartic acid to valine at position 64) which is sufficient to render the integrase non-functional without affecting the other steps of transduction. 

The process for producing your own IDLVs is nearly identical to that of regular lentiviral vectors. The only difference is that you would use the integrase-deficient packaging plasmid instead of the standard packaging plasmid. For more details on how to package lentiviral vectors, check out Addgene’s Lentiviral Guide and our lentiviral production protocol. 

One thing to keep in mind is that titering these IDLVs may require a slightly different protocol than regular lentiviral vectors. This is because most titering methods rely on isolating genomic DNA from transduced cells and quantifying the integrated viral DNA along with a reference cellular gene. When titering IDLVs, there is no integrated piece to measure! Instead, two commonly employed methods for titering IDLVs include detecting vector particle components or performing quantitative PCR titration of vector DNA in samples harvested 24 hours post-transduction (Wanisch and Yáñez-Muñoz, 2009). 

Now that you know how IDLVs could be useful in your own research, we wish you the best of luck with any experiments involving IDLVs. And remember:

You can never trust a lentivirus with secrets...

Because they tend to insert themselves into every conversation!

This blog post was written by Addgenie Rebecca Hutcheson.

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References and resources

References

Banasik, M. B., & McCray, P. B., Jr (2010). Integrase-defective lentiviral vectors: progress and applications. Gene Therapy, 17(2), 150–157. https://doi.org/10.1038/gt.2009.135. PubMed PMID: 19847206

Desfarges, S., & Ciuffi, A. (2012). Viral Integration and Consequences on Host Gene Expression. Viruses: Essential Agents of Life, 147–175. https://doi.org/10.1007/978-94-007-4899-6_7. PubMed Central PMCID: PMC7120651

Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander, J. D. (2013). High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology, 31(9), 822–826. https://doi.org/10.1038/nbt.2623. PubMed PMID: 23792628 PubMed Central PMCID: PMC3773023

Howe, S. J., Mansour, M. R., Schwarzwaelder, K., Bartholomae, C., Hubank, M., Kempski, H., Brugman, M. H., Pike-Overzet, K., Chatters, S. J., de Ridder, D., Gilmour, K. C., Adams, S., Thornhill, S. I., Parsley, K. L., Staal, F. J., Gale, R. E., Linch, D. C., Bayford, J., Brown, L., Quaye, M., Kinnon, C., Ancliff, P., Webb, D. K., Schmidt, M., von Kalle, C., Gaspar, H. B., Thrasher, A. J. (2008). Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. The Journal of Clinical Investigation, 118(9), 3143–3150. https://doi.org/10.1172/JCI35798. PubMed PMID: 18688286 PubMed Central PMCID: PMC2496964

Leavitt, A. D., Robles, G., Alesandro, N., & Varmus, H. E. (1996). Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection. Journal of Virology, 70(2), 721–728. https://doi.org/10.1128/JVI.70.2.721-728.1996. PubMed PMID: 8551608 PubMed Central PMCID: PMC189872

Schröder, A. R., Shinn, P., Chen, H., Berry, C., Ecker, J. R., & Bushman, F. (2002). HIV-1 integration in the human genome favors active genes and local hotspots. Cell, 110(4), 521–529. https://doi.org/10.1016/s0092-8674(02)00864-4. PubMed PMID: 12202041

Wanisch, K., & Yáñez-Muñoz, R. J. (2009). Integration-deficient lentiviral vectors: a slow coming of age. Molecular Therapy : the Journal of the American Society of Gene Therapy, 17(8), 1316–1332. https://doi.org/10.1038/mt.2009.122. PubMed PMID: 19491821 PubMed Central PMCID: PMC2835240

Wu Y. (2004). HIV-1 gene expression: lessons from provirus and non-integrated DNA. Retrovirology, 1, 13. https://doi.org/10.1186/1742-4690-1-13. PubMed PMID: 15219234 PubMed Central PMCID: PMC449739

Yew, C.T., Gurumoorthy, N., Nordin, F., Tye, G.J., Wan Kamarul Zaman, W.S., Tan, J.J., Ng, M.H. (2022). Integrase deficient lentiviral vector: prospects for safe clinical applications. PeerJ, 10, e13704. https://doi.org/10.7717/peerj.13704. PubMed PMID: 35979475 PubMed Central PMCID: PMC9377332

More resources on the Addgene blog

• Viral Vectors 101: The Retroviral Life Cycle
• Quick Guide to All Things Lentivirus

More resources on addgene.org

• Lentiviral Guide  
• Popular lentiviral vectors