From the bench to Hutch services: Refining gene-editing methods for HSPCs used in pre-clinical applications

From the Paddison lab, Human Biology Division

Functionally relevant changes to cellular genetic code were first observed by exposing cells to radiation or genotoxic chemicals to introduce random mutations. It was later shown that precise genome editing required breaks in the double-stranded DNA at the specific sites to be changed. The practice of targeted genome editing was achieved in yeast cells and mouse models in the 1970s and 1980s, and now there are three classes of nucleases being used to initiate site-specific mutagenesis by cutting double stranded DNA at defined sequences: zinc-finger nucleases (ZGNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated proteins (CRISPR/Cas) – history of genome editing. The Paddison lab in the Human Biology Division at Fred Hutch has honed the use of CRISPR/Cas technology to perform precise gene-editing of one or more genes in primary human stem cells used for pre-clinical and clinical applications. In their recent publication in PLoS One, they report achieving high editing efficiency of greater than 90% and rapid turnaround time for in vivo applications.

In 2012, Jinek et al. reported that CRISPR/Cas9-dependent gene editing was guided by RNA that was complementary to the genomic DNA targeting sequence to initiate targeted DNA breaks. Despite the routine use of CRISPR/Cas9 in gene editing techniques in the subsequent decade, certain cell types continued to be especially challenging to edit. Dr. Dan Kuppers, a talented staff scientist in the Paddison lab provided some insight into the use of this technology in primary human stem cells, a donor cell used for generating ‘humanized’ mice among other applications. Dr. Kuppers commented, “Highly penetrant genetic manipulation of human CD34+ hematopoietic stem and progenitor cells (HSPCs) has been difficult and laborious to obtain. This has been due to a variety of factors including standard methods of transgene delivery, such as lentiviral transduction and [DNA] transfections that typically have at best an efficiency of 50%. Additionally, HSPCs can only be propagated in culture for a limited time span before they begin to lose their differentiation potential, typically necessitating the use of more time-consuming enrichment methods such as flow sorting,” a method that uses fluorescence labeling to sort cells into fluorescence negative versus positive pools. “Furthermore, extended in vitro culture reduces the engraftment potential of the cells for in vivo studies” as in the generation of humanized mice.

HSPCs from human donors can be transplanted into human recipients to treat many diseases including sickle cell disease, HIV/AIDs and several cancers. Transplantation of these cells into immune-deficient mice can constitute a human-like immune system in this rodent model and are referred to as ‘humanized’ mice. Since these cells have clinical and pre-clinical applications, being able to edit their genomes has significant implications for studying and treating diseases in which HSPC transplantation is performed. Reported editing efficiencies using Lonza’s 4D nucleofection technology—a previously accepted standard—to deliver guide RNAs and Cas9 protein range from 15-90% in HSPCs and vary by 40-80% editing efficiency between human donors. To optimize gene-editing of HSPC genomes from multiple donors, the researchers first tested different electroporation settings for efficient nucleofection of cells with Cas9 and guide RNAs to knockout the CD8 gene. Electroporation involves pulsing electrical currents through cells to increase membrane pore size and enable uptake of extracellular factors such as CRISPR/Cas9 proteins and RNAs for genome editing. By modifying the proprietary electroporation settings, the researchers increased gene-specific editing using a two-guide RNA cutting approach. This significantly enhanced knockout efficiency to 92% compared to 36% for the recommended Lonza program setting for these cells. Importantly, both settings resulted in greater than 90% cell viability. Efficient editing was consistent for three different donor’s cells and maintained when extended to target three separate genes for CRISPR/Cas9-mediated knockout. With these enhanced knockout efficiencies, the researchers wanted to shorten the duration of HSPCs growth in culture media before transplantation into mice with the goal to improve engraftment. “With the optimized nucleofection method we report, we’re able to generate HSPCs with greater than 90% gene KO and ready for in vivo use within 8-hours of thawing [the donor cells],” shared Dr. Kuppers. Additionally, the humanized mice showed near complete knockout of targeted CD33 gene in their blood and bone marrow myeloid cells.

Diagram of the optimized workflow for efficient CRISPR/Cas9 RNP genome editing of human donor HSPC cells for immediate transplantation into immune-deficient mice to generate a ‘humanized’ immune system.
Diagram of the optimized workflow for efficient CRISPR/Cas9 RNP genome editing of human donor HSPC cells for immediate transplantation into immune-deficient mice to generate a ‘humanized’ immune system. Image taken from primary publication

This demonstration of improved gene-editing performance is broadly relevant. Dr. Kuppers added, “We are currently working on utilizing the optimized conditions to improve lower efficiency CRISPR/Cas9 applications, such as targeted mutagenesis and tagging of endogenous loci.” In addition to these applications, the Paddison lab has also “been able to knockout multiple genes in human HSPCs, including ones associated with marking of mRNAs with N6-methyladenosine, a mark that can affect mRNA localization, translation, or turnover,” stated Dr. Kuppers. “This mark is a requirement for erythroid lineage formation and likely has roles in multiple hematopoietic-associated cancers.” As another example, “The Rongvaux lab is currently using the approach to identify key regulators of HIV infection in mice with humanized immune systems. In addition, we are now starting to use the approach to create human mutation-specific HSPC models of hematopoietic progenitor-associated diseases, including myeloid dysplastic syndrome.” The Paddison lab has prioritized sharing this optimized method. “We are now offering a CD34+ [HSPC] gene editing service as part of the Co-operative Center for Excellence in Hematology at the Hutch,” commented Dr. Kuppers. The published data and expansion of these findings into a Hutch-wide resource are impressive accomplishments and reinforce the collaborative efforts of this research center to find cures for human diseases.

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The spotlighted research was funded by the National Institutes of Health, the Fred Hutchinson Cancer Center Immunotherapy Integrated Research Center, and the Bezos family.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Anthony Rongvaux and Patrick Paddison contributed to this work.

Kuppers DA, Linton J, Ortiz Espinosa S, McKenna KM, Rongvaux A, Paddison PJ. 2023. Gene knock-outs in human CD34+ hematopoietic stem and progenitor cells and in the human immune system of mice. PLoS One. 18(6):e0287052.