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Targeted homology-directed repair in blood stem and progenitor cells with CRISPR nanoformulations

Abstract

Ex vivo CRISPR gene editing in haematopoietic stem and progenitor cells has opened potential treatment modalities for numerous diseases. The current process uses electroporation, sometimes followed by virus transduction. While this complex manipulation has resulted in high levels of gene editing at some genetic loci, cellular toxicity was observed. We have developed a CRISPR nanoformulation based on colloidal gold nanoparticles with a unique loading design capable of cellular entry without the need for electroporation or viruses. This highly monodispersed nanoformulation avoids lysosomal entrapment and localizes to the nucleus in primary human blood progenitors without toxicity. Nanoformulation-mediated gene editing is efficient and sustained with different CRISPR nucleases at multiple loci of therapeutic interest. The engraftment kinetics of nanoformulation-treated primary cells in humanized mice are better relative to those of non-treated cells, with no differences in differentiation. Here we demonstrate non-toxic delivery of the entire CRISPR payload into primary human blood progenitors.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Sequence data are available for download through the National Center for Biotechnology Information (BioProject ID: PRJNA529681).

Code availability

The bioinformatics pipeline used to analyse sequencing reads is available via GitHub (https://github.com/FredHutch-CGT/FredHutch_CGT_Gene_Edit_1).

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

We thank the healthy donors who submitted to mobilization and leukapheresis collection. We thank the laboratory of M. Zhang at the University of Washington for access to and use of Nanosizer equipment. We extend a special thanks to H. Crawford for assistance in manuscript preparation, and J. Chen and C. Ironside for excellent support in animal studies. This work was primarily supported by funds to J.E.A. from the Fred Hutch including Development and Evergreen awards, and the Hartwell Foundation. This research was also funded in part through a pilot study award to R.S. from the NIDDK Cooperative Center of Excellence in Hematology grant U54 DK106829. All shared resources used in this study were supported by the NIH/NCI Cancer Center Support Grant P30 CA015704. H.-P.K. is a Markey Molecular Medicine Investigator, the inaugural recipient of the José Carreras/E. Donnall Thomas Endowed Chair for Cancer Research and the Fred Hutch Endowed Chair for Cell and Gene Therapy.

Author information

R.S. and J.E.A. designed the study. R.S., G.S.-H., J.L.R., K.G.H. and O.H. performed the experiments, and generated data and figures. S.K. isolated primary human CD34+ cells from donor products. R.S. and J.E.A. analysed data. R.S., G.S.-H., S.K., K.G.H., O.H. and J.E.A. reviewed and interpreted data. H.-P.K. funded K.G.H. and O.H, served as the IACUC protocol Principal Investigator and provided the Cas9 protein used in the study. R.S. and J.E.A. funded the study and wrote the manuscript. All authors reviewed and edited the final manuscript.

Competing interests

H.-P.K., O.H. and J.E.A. received licensing revenue from Rocket Pharmaceuticals for research unrelated to this manuscript. K.G.H. is employed by and holds equity in Nohla Therapeutics. The other authors declare no competing interests associated with this work. J.E.A. and R.S. are co-inventors on US patent WO2018226762A1 entitled ‘Genomic safe harbors for genetic therapies in human stem cells and engineered nanoparticles to provide targeted genetic therapies.’.

Correspondence to Jennifer E. Adair.

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Fig. 1: Layer-by-layer conjugation of CRISPR components onto AuNPs.
Fig. 2: AuNP/CRISPR can deliver CRISPR components to the nucleus of HSPCs.
Fig. 3: Optimization of HDR conditions and optimal editing dosage.
Fig. 4: AuNP/CRISPR nanoformulations carrying Cpf1 outperform Cas9 in terms of HDR.
Fig. 5: AuNP treatment enhanced HSPC engraftment in neonatal immune-deficient mice.
Fig. 6: Persistent editing levels after engraftment.