New gene therapy strategy for the treatment of Wiskott-Aldrich Syndrome

by Luyanda Hlongwa

Gene therapy is one of the most convincing therapies for primary immunodeficiencies, despite many issues and concerns. Primary immunodeficiencies are rare genetic disorders that lead to immune system insufficiencies. Wiskott-Aldrich syndrome (WAS) is one such disease. WAS is caused by mutations in the WAS gene resulting in defects in haematopoietic cells (cells from which blood cells develop). This leads to the development of defective immune cells. WAS is mainly treated by a haematopoietic stem cell transplant. However, donor mismatch complications call for an alternative. That is where gene therapy comes in; different strategies have been explored. One of the most prominent ones is using a self-inactivating Lentivirus vector. Among other issues, such as genotoxicity, one of the most significant disadvantages of this strategy is that while it has a robust correction of T cell abnormalities, it seems to fail in other lineages, including platelets. Platelets are cells in our blood essential for blood clotting. In this paper, the authors developed CRISPR/Cas9 gene editing platform to knock in a therapeutic WAS gene to counteract these disadvantages. They hoped that this strategy would confer correction of all known disease-causing mutations. CRISPR/Cas9 uses a guide RNA matching the target sequence to enhance its gene editing functions. The guide RNA unwinds and binds the target. The enzyme Cas9 introduces a double-strand break which can be repaired by the cell’s internal mechanism, resulting in mutations rendering the gene dysfunctional or replaced by a new introduced functional gene.

The authors first designed a guide RNA (gRNA) that guides the cas9 enzyme to the target gene, tested it, and selected the best performing gRNA. They then created an AAV6 vector that contained a GFP reporter, which will produce fluorescence if expressed, confirming that the vector worked. This vector was tested in Haematopoietic stem and progenitor cells (HSPCs), and the GFP reporter was successfully integrated and showed no deterioration in terms of cell viability. The authors further wanted to show that their protocol can restore functional WAS expression in HSPCs from WAS patients and in cells derived from those HSPCs. They replaced the GFP reporter in the AAV6 vector with a functional WAS gene and subjected the HSPCs from these patients to the protocol. Using digital drop PCR and flow cytometry they confirmed the expression of WAS gene in these cells, they also showed that there was increased WAS expression compared to using the Lentivirus vector strategy. They then differentiated the manipulated cells into macrophages and platelets and assessed functional correction. The authors found that both macrophages and platelets had increased WAS expression compared to lentivirus vector transduced cells and the expression was comparable to the wild type. This showed that this protocol not only does better than the lentiviral vector but also corrects the function of the macrophages and platelets which the lentiviral vector could not do. Activated T cells from WAS patients were also subjected to this protocol to demonstrate that it can correct intrinsic T cell defects. They found that indeed the T cell-intrinsic defects were corrected.  The authors went further to investigate whether this would work in vivo; to do this, they transplanted the manipulated cells into immunodeficient mice. They found that their strategy restored the semi-physiological expression of WAS gene but was better compared to the lentiviral vector strategy.

This is a significant advancement in primary immunodeficiency gene therapy as it offers not only a safer alternative to the lentiviral vector strategy but can fix other issues that the lentiviral vector strategy cannot. This strategy has been tested in mice, not humans the evidence suggests it is a viable and good alternative.  The authors have demonstrated the safety and efficacy of their strategy however they advise that further and deeper assessment of genotoxicity may be required as the protocol is moved from bench to bedside.


RAI, R., ROMITO, M., RIVERS, E., TURCHIANO, G., BLATTNER, G., VETHAROY, W., LADON, D., ANDRIEUX, G., ZHANG, F., ZINICOLA, M., LEON-RICO, D., SANTILLI, G., THRASHER, A. J. & CAVAZZA, A. 2020. Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott – Aldrich Syndrome. Nat Commun, 11, 4034.

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