®

Hematopoietic Stem Cell Electroporation for HDR-Mediated Correction of

an X-Linked Blood Disorder
By Marianna Romito, Ph.D. and Caroline Beckett

Background: Wiskott-Aldrich Syndrome (WAS) is an X-linked recessive primary immunodeficiency affecting between 1 in 50,000 to 1 in 250,000 live births
Research Spotlight

worldwide. Mutations in the WAS gene cause abnormal immune system function, thrombocytopenia, and an increased incidence of autoimmune malignancies. WAS
pathophysiology is linked to impaired activity of the WAS protein (WASp), expressed in hematopoietic stem and progenitor cells (HSPC) and their lineages. Gene
therapy clinical trials have used a lentiviral vector (LV) with a fragment of the endogenous WAS promoter in HSPCs to upregulate WASp expression and restore immune
function in most patients. Unfortunately, viral vector treatment brings a potential risk of genotoxicity, a significant concern for WAS patients already predisposed to
lymphoid malignancy. Furthermore, viral vectors do not restore physiological WAS expression in all hematological cells. Homology Directed Repair (HDR)-mediated
transgene knockin enables more precise control over site integration and copy number relative to viral vector delivery. MaxCyte® electroporation (EP) enables a non-
lentiviral-based therapy to correct all known WAS disease-causing mutations.

Rationale: This work shows the first therapeutically

relevant restoration of WASp expression in healthy
and patient-derived HSPCs and differentiated cells.
The MaxCyte electroporation facilitates the delivery
of CRISPR-Cas9 ribonucleoproteins (RNP) followed by
transduction with the Adeno-Associated Virus 6 (AAV6)
donor vector for efficient, scalable and safe, functional
correction of the WAS defect. Importantly, MaxCyte
Flow Electroporation® technology empowers the clinical
translation of this innovative approach to a broad range
of other HSPC blood disorders.

Technical Approach: This study demonstrates

that precise CRISPR-Cas9 genome editing of WAS
mutations is possible in up to 60% of HSPCs without
impairing cell viability or differentiation potential.
Gene correction is achieved by site-specific knockin
of a codon-optimized cDNA at the WAS locus. When
RNP electroporation was followed by transduction
with the AAV6 donor vector, up to 61% of HSPCs
showed positive targeted integration of therapeutic WAS cDNA in patient- inflammatory complications in addition to ameliorating symptoms such as
derived HSPCs, with no significant decrease in cell viability compared to thrombocytopenia. The potential to engineer millions of ex vivo corrected
mock-targeted HSPCs. The observed rates of targeted integration met or HSPCs in a single process may unlock a universal treatment option for WAS
exceeded the standards set at small scale during optimization studies. patients.
Using the MaxCyte GTx TM
instrument, the optimized gene editing protocol References:
was seamlessly scaled up for clinical use from 1.25 to 20 million cells. Rai, R., Romito, M., Rivers, E. et al. Targeted gene correction of human hematopoietic stem
cells for the treatment of Wiskott - Aldrich Syndrome. Nat Commun. 11, 4034 (2020).
https://doi.org/10.1038/s41467-020-17626-2
Conclusions: MaxCyte® electroporation of gene editing tools offers
Worth, A. J. & Thrasher, A. J. Current and emerging treatment options for Wiskott-Aldrich
several advantages over past treatment strategies for WAS patients. HDR- syndrome. Expert Rev. Clin. Immunol. 11, 1015–1032 (2015).
mediated transgene integration provides controlled genetic engineering Aiuti, A. et al. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich
avoiding the genotoxicity of semi-random lentiviral integration. Superior syndrome. Science. 341, 1233151 (2013).

gene correction and higher WASp expression were detected in several
hematopoietic and mature cell lineages. The reconstitution of WASp expression
demonstrated here could reduce the risks of autoimmunity and persistent
Braun, C. J. et al. Gene therapy for Wiskott-Aldrich syndrome–long-term efficacy and
genotoxicity. Sci. Transl. Med. 6, 227ra233 (2014).
Notarangelo, L. D., Miao, C. H. & Ochs, H. D. Wiskott-Aldrich syndrome. Curr. Opin. Hematol. 15,
30 –36, https://doi.org/10.1097/MOH.0b013e3282f30448 (2008).

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