Background: Gene therapy is a promising approach for treating monogenic diseases such as hemophilia A (HA). Considering the heterogeneity of FVIII mutations, a “gene addition” approach to knock-in a functional fVIII transgene would be more efficient compared to gene editing, and CRISPR/Cas9 can be used to achieve location-specific insertion of a fVIII transgene into a genomic safe harbor with less off-target effects in comparison to lentiviral transduction, which can cause insertional mutagenesis.

Aims: To insert the bioengineered fVIII transgene “lcoET3” into the AAVS1 site in human placental stem cells (PLCs) and human liver-derived endothelial cells (HLECs) and assess and compare their subsequent levels of FVIII production and to confirm integration at the AAVS1 site and check for off-target effects.

Methods: CRISPR/Cas9-mediated gene addition was achieved with co-transfection of the plasmids pCas-Guide-AAVS1 and pAAVS1-puro-EF1a-lcoET3. Genomic insertion, mRNA expression, and clotting activity was assessed using PCR, RT-qPCR and aPTT assay, respectively.

Results:

RT-qPCR results show fold change in expression of lcoET3. Delivery of lcoET3 through a lentiviral vector resulted in robust expressionFigure 1

aPTT assay shows PLCs transduced with lentivirus (LV) encoding lcoET3 secreted significantly more FVIII than CRISPR/Cas9-engineered PLCs and control PLCsFigure 2

RT-qPCR with lcoET3-specific primers confirmed expression of the bioengineered fVIII transgene in both HLECs and PLCs (Figure 1). aPTT assay demonstrated that the CRISPR/Cas9-engineered PLCs secreted significantly more FVIII:C than engineered HLECs and control PLCs (Figure 2). However, lentivirus transduced cells produced the highest levels of expression and protein. NGS to confirm successful insertion at the AAVS1 site and off-target effect analysis is currently underway.

Conclusions: The levels of FVIII:C produced by PLCs following CRISPR/Cas9-mediated knock-in of the bioengineered lcoET3 fVIII transgene were sufficient to be in the therapeutic range, showing its potential as a cell-gene delivery platform to treat HA. The ability to successfully edit HLECs in vitro represents an important first step to establishing the feasibility of using CRISPR/Cas9 to mediate gene-editing in HLECs in vivo, achieving FVIII expression within the cells that serve as the natural site of synthesis of this protein and thereby correcting HA.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/targeted-crispr-cas9-mediated-gene-addition-to-a-safe-harbor-in-placental-cells-for-the-treatment-of-hemophilia-a/