Democratizing Gene Therapy Through Viruses

Reading time: 5 minutes

Kelsey Woodruff

Hematopoietic stem cells (HSCs) are a rare population of cells that have the capacity to differentiate into one of the many types of blood cells that keep your body healthy. Through a complex series of signaling events and carefully timed cell divisions, these cells repopulate the entire blood compartment. When this process goes awry – either through aging or infection – it can have dire consequences for the body and lead to diseases like HIV, sickle cell disease, Fanconi anemia, and hemophilia. Fortunately, scientists have found ways to treat and sometimes cure some of these diseases through HSC transplant [1]. This process involves finding a compatible bone marrow donor or isolating HSCs from the patient, growing more of these cells in culture, and injecting them back into the patient (Figure 1). In cases where the HSCs are isolated from the patient, these cells can be gene-edited while in culture to correct the mutation leading to their disease. After the edited cells are reintroduced into the patient, they can differentiate and divide to fully repopulate a healthy blood system! While this treatment is powerful and has benefited countless patients, it still comes with many limitations. Bone marrow matching is extremely difficult, so matched donors are hard to come by, especially for minoritized groups. Isolating HSCs is also challenging because they are such a rare cell population in the body. Expanding them in culture without differentiation into other blood cells or losing the ability to divide is another hurdle. Finally, the entire process presents many barriers to patients. Few centers in the United States are qualified to perform such a procedure because they lack adequate resources and expertise, so some patients must travel to receive this life-saving healthcare [2]. Transplants leave patients with compromised immune systems, making them vulnerable to potentially life-threatening pathogens. Finding ways to expand access to this procedure and reduce the risk it poses to patients would revolutionize HSC gene therapy. Fortunately, the Kiem lab in Fred Hutch’s Translational Sciences and Therapeutics Division is doing just that.

Justin Thomas and Kurt Berckmueller spearheaded a project that can potentially allow for HSC gene editing to happen in the patient’s body [3]. This project involves engineering lentiviruses to target the rare HSC population that gives rise to the entire blood system. Once introduced, these viruses enter the cell and deliver a “payload” into the cell’s DNA. This payload edits specific genes that have gone awry to cause a patient’s disease. By stopping these mutations at the source (the stem cell), the group hopes these gene-edited cells can give rise to a normal hematopoietic system.

As exciting as these results are, completing this project was no easy feat. Engineering viruses that are specific enough to target only the desired HSCs while still being potent enough to efficiently enter these cells required the group to overcome significant obstacles in designing and validating the particles. First, the team collaborated with the Strong Lab to design single chain variable fragments (scFvs), or truncated antibodies, targeting CD90, a blood antigen exclusively marking the HSC subset the group is interested in. Once these CD90-targeting viruses were validated, the group had to delete the native cell entry machinery normally expressed by the viruses. This step was crucial to ensure the viruses did not produce off-target gene editing effects. However, because the normal cell entry machinery is gone in the engineered viruses, traditional titration methods to validate the viruses are impossible to use. The group turned to a novel hydrodynamics-based approach to titrate and validate their engineered viral particles to overcome this obstacle. “What we introduce in this paper is a physical type of titering where you’re not looking at the functionality, but instead looking at the physical attributes of the particles,” says Thomas. Once these technical challenges were overcome, it was time to put the newly engineered viruses to the test: Could they actually infect and deliver gene edits to HSCs?

To test this, the group used the engineered virus to transduce cells in a dish. They found that viruses expressing only the CD90-targeting scFvs could efficiently enter and transduce the cells, although they were less efficient than the wild type virus. Furthermore, the cells transduced with the CD90-targeting virus performed just as well as cells transduced with wild type viruses in a primary colony forming cell (CFC) assay, an experimental technique used to determine the proliferative capacity of hematopoietic cells. After the primary CFC, the group took colonies from both groups and performed a secondary CFC. They saw that the CD90-targeted cells performed better than the wild type group. This indicates that the engineered viruses target cells with a greater proliferation and expansion potential, a result with exciting implications for future work in vivo (Figure 2). 

To further validate the specificity of the engineered viral particles, the CD90-targeting particles were used to transduce cells lacking the CD90 antigen. If the engineered viruses are truly specific for CD90-expressing cells, they should see very little transduction in cells lacking CD90. Luckily, that is exactly what they saw. This indicated that the new CD90-targeting scFv was truly specific for CD90, making the viral particles viable for potential patient use.

Through the development of these engineered particles, the Kiem lab hopes to make HSC gene therapy accessible to people in minoritized groups and resource-limited countries. “The goal is to democratize the technology,” says Thomas, “Hematopoietic stem cell gene therapy is up to the imagination. Once you prove that this technology works, it can be applied to so many different disorders and diseases.”

Edited by Melanie Padalino

References

1. Hatzimichael, E., & Tuthill, M. (2010). Hematopoietic stem cell transplantation. Stem Cells and Cloning: Advances and Applications, 3, 105–117. https://doi.org/10.2147/SCCAA.S6815
2. Gratwohl A., Baldomero H., Aljurf M., et al. Hematopoietic Stem Cell Transplantation: A Global Perspective. JAMA. 2010;303(16):1617–1624. doi:10.1001/jama.2010.491
3. Berckmueller, K., Thomas, J., et al. (2023). CD90-targeted lentiviral vectors for HSC gene therapy. Molecular Therapy, 31(10), 2901–2913. https://doi.org/10.1016/j.ymthe.2023.08.003
4. MS Australia. (2023). What is autologous hematopoietic stem cell transplant?. MS Australia. https://www.msaustralia.org.au/ahsct/autologous-haematopoietic-stem-cell-transplant/ 

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