Gene therapy, and in particular, therapy involving genetically modified stem cells, is one of the most promising methods for treating genetic disease and defects, and has therefore become a popular focus of research and development. In recent years, using adenoviral vectors to deliver modified genes has become standard practice. Adeno-associated viruses (AVVs) are easy to modify, and have the inherent capability of being able to transfer genetic information into a variety of host cell types.
The most common drawback of this type of gene editing is that AVV transduction efficiency can vary. Part of that variability lies in the methodology and choice of vector, but no small part is due to donor variability. The same gene-modified AVV can have significantly different transduction efficiencies in stem cells derived from different donors. Variable transduction efficiency can limit the effectiveness of gene therapy since the same dose of cells would be carrying varying doses of the therapeutic gene. The research team at the University of Florida decided to investigate strategies for overcoming donor variability and improving overall transduction efficiency. Their strategy involved modifying both viral vectors and transduction methodology to produce the desired effect, more efficient gene transfer, and better transgene expression.
The scientists set up a detailed comparative study examining a series of commonly utilized AAVs and transduction conditions. Human hematopoietic stem cells (HSCs) are notoriously difficult to transduce. However, AAVs are the vector of choice for this cell type because they carry a lower risk of creating unintended genetic mutations. Based on previous studies, the researchers decided to modify the outer capsule of the AAVs in a way they believed would make it more likely for the vector to be taken up by the host cell.
After modifying two tyrosine residues on the outer surface of the virus capsule, the researchers observed a significant increase in transduction efficiency. They then proceeded to evaluate how this modification affected transduction efficiency in the ten most common AAV serotypes. Results showed that modification of AAV6 produced the greatest improvement in transduction efficiency.
Following this portion of the study, the research team wanted to see whether they could modify transduction conditions to produce even greater improvements in efficiency. During the course of their research, they had observed that higher cell density correlated with higher transduction efficiency. Transduction efficiency was studied in a common immortalized blood cancer cell line (K562) over a range of cell densities to see whether this observation could be repeated, with the results analyzed by flow cytometry.
The scientists found that increasing K562 cell density up to 10 million cells/ml dramatically increased both transduction efficiency and transgene expression. Transgene expression also correlated with increased viral genome copy number. The experiments were repeated in two primary human HSCs, one of which expressed a surface receptor and co-receptors reported to facilitate vector binding and entry by the AAV vector they were using, and the other of which did not. Results showed that increased cell density only increased transduction efficiency and transgene expression in the cell line carrying the appropriate AAV receptor/co-receptor combination. This information gave the research group important information about the relationship between transduction conditions and donor cell gene expression that they hope to pursue in future research.
The challenges posed by low transduction efficiency are not trivial. Low levels of transduced gene delivery, limited transgene expression, and cytotoxicty can combine to create a perfect storm in which the efficacy of a potential therapeutic is severely impacted. Since the described research was completed, scientists have looked more deeply into the reasons behind donor cell variability [2, 3], and found that cellular innate immunity in donors impacts stem cell “fitness’ for transduction. This strongly suggests that donor screening can potentially be used to improve the efficiency of modified gene delivery, not just for AAV vectors and stem cells, but for other cell types and delivery systems as well.
Our research team at Excellos believe that in-depth donor characterization helps companies choose the best possible starting material for their cell and gene therapy programs. Please contact us to learn more.
- Ling, C., et al. (2016) High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells by AAV6 Vectors: Strategies for Overcoming Donor-Variation and Implications in Genome Editing. Sci Rep 6, 35495. https://doi.org/10.1038/srep35495
- Kajaste-Rudnitski, A., & Naldini, L. (2015). Cellular innate immunity and restriction of viral infection: implications for lentiviral gene therapy in human hematopoietic cells. Human gene therapy, 26(4), 201–209. https://doi.org/10.1089/hum.2015.036
- Piras F, Kajaste-Rudnitski A. Antiviral immunity and nucleic acid sensing in haematopoietic stem cell gene engineering. Gene Ther. 2021;28(1-2):16-28. doi:10.1038/s41434-020-0175-3