Viral Vector Analysis

Hello! This week, I did research on viral vectors for delivering guide RNA (gRNA). To recap, gRNAs are 20-nucleotide sequences recognized by Cas9 nickase to initiate cuts in DNA sequences. I analyzed 3 different types of vectors this week, so I would like to provide my findings on these viral vectors and my conclusion on what I plan to use.  

Adenoviruses and AAVs 

Adenoviruses (AdVs) are non-enveloped viruses known to cause infections of the upper respiratory tract and infect organs like the brain and bladder. AAVs, also known as adeno-associated viruses, are similar to AdVs in that both are non-enveloped viruses; however, AAVs can be modified for gene therapy purposes to prevent infection from occurring compared to AAVs. Both AdVs and AAVs generally have a broad tropism, which means that they can infect a wide variety of cell types. AdVs are expected to have higher immunogenicity compared to AAVs because the infection part of the virus is not removed in AdVs. Another unusual (and limiting) aspect of AAVs is that they only can package DNA up to 4.7 kb. This proves to be inconvenient for the theoretical treatment I am designing since the gRNAs and template plasmids are all expected to exceed 4.7 kb.  

hCMVs  

Human cytomegaloviruses (hCMVs) are viruses known to infect all cell types and cause infection of all organs. It belongs to the β-herpes virus family. This virus has not been used frequently for gene therapy and is still in a study for genetic treatment. Although this virus has a broad tropism, it can launch systemic infections in humans if the infection becomes serious. In the colon, infection by hCMVs can cause colitis, and inflammation of the colon, which can impair digestion and lead to blood poisoning and sepsis. This leads to higher immunogenicity as a whole. However, one major advantage of hCMVs is their packaging size limit, which is about 250 kb. This will allow for the packaging of the template plasmid and gRNA comfortably into one virus upon packaging.  

Lentiviruses 

Lentiviruses are a small subset of retroviruses that are known to cause slowly progressive human diseases. This virus is also known to be used in the gene therapy world, especially in the field of the central nervous system. This virus also has a broad tropism and can infect all cell types. Lentiviruses have a medium packaging capacity of ~10 kb. In addition to this, lentiviruses are known to exhibit a higher immunogenicity due to their ability to cause increased infection in the body. This can be limited by modifying the integrase protein of the virus, which is involved in integrating viral DNA into the host genome. This virus, known as the integrase-deficient lentivirus, takes care of immunogenicity and allows it to be used for gene therapy. 

Conclusion 

In conclusion, I decided to choose a member of the lentivirus family, the integrase-deficient lentivirus (IDLV) as my viral vector of choice. This is due to the following reasons: broad tropism, reduced immune response, and average packaging size. Although IDLVs only have a 10 kb packaging size limit, it is something I can work around by dividing my treatment into 4 IDLVs (2 for gRNAs and 2 for their corresponding templates). Another reason is that AdVs and hCMVs are known for integrating viral DNA into the host genome, which can lead to cell death, increased infection, and sepsis in some cases. The specific reason why I (and also my mentor) decided not to choose hCMVs is that hCMVs can cause colitis or inflammation of the colon due to increased immune response by the body. This can be problematic in HNPCC patients as they are dealing with colon cancer polyps added to the inflammation. This means that not only their digestion of food is impaired, but their ability to fight infections is also impaired. The latter is crucial as cancer can exploit the immune system of its resources to prevent cell death. Therefore, my analysis has led me to choose the IDLV as my viral vector to proceed with for this treatment. 

Sources: 

1. Bulcha, Jote T., et al. “Viral Vector Platforms within the Gene Therapy Landscape.” Signal Transduction and Targeted Therapy, vol. 6, no. 1, Feb. 2021, doi:https://doi.org/10.1038/s41392-021-00487-6.
2. “Definition of LENTIVIRUS.” Www.merriam-Webster.com, www.merriam-webster.com/dictionary/lentivirus.  
3. Heuman, Douglas, et al. “Cytomegalovirus Colitis: Background, Pathophysiology, Etiology.” EMedicine, July 2021, https://emedicine.medscape.com/article/173151-overview#:~:text=Systemic%20CMV%20disease%20is%20characterized,of%20this%20acute%20systemic%20illness.    
4. Lundstrom, Kenneth. “Viral Vectors in Gene Therapy.” Diseases, vol. 6, no. 2, May 2018, p. 42, doi:https://doi.org/10.3390/diseases6020042.  
5. Naso, Michael F., et al. “Adeno-Associated Virus (AAV) as a Vector for Gene Therapy.” BioDrugs, vol. 31, no. 4, July 2017, pp. 317–34, doi:https://doi.org/10.1007/s40259-017-0234-5.  
6. Whitley, Richard J. “Herpesviruses.” Nih.gov, University of Texas Medical Branch at Galveston, 2013, www.ncbi.nlm.nih.gov/books/NBK8157/#:~:text=Herpesviruses%20are%20divided%20into%20three.  
7. Ye, Lele, et al. “Functional Profile of Human Cytomegalovirus Genes and Their Associated Diseases: A Review.” Frontiers in Microbiology, vol. 11, Sept. 2020, doi:https://doi.org/10.3389/fmicb.2020.02104.