Gene editing has the potential to cure genetic immune disorders, malignancies, and infectious diseases by changing the DNA of blood stem cells known as haematopoietic stem and progenitor cells (HSPCs). However, current methods of editing DNA can be toxic to the cells or result in malignancies after DNA is inserted into the wrong place. The laboratory of Dr. Jennifer Adair (Clinical Research Division) has generated a new method of delivering gene editing machinery with the potential for large scale clinical application using gold nanoparticles and CRISPR.
CRISPR works by targeting specific DNA sequences with the help of a guide RNA. CRISPR then cuts the DNA creating an opportunity for repair using a new synthetic DNA sequence as a template. While the CRISPR complex composed of proteins and nucleic acids is a malleable tool for editing the genome, it cannot pass through cell membranes on its own, and it must be delivered with its guide RNA and a synthetic DNA for complete activity. Typically, cells are pulsed with electricity to open pores in the membranes in a process known as electroporation. Unfortunately, the damage electroporation causes is not always repaired, resulting in the loss of many cells in the process. Moreover, getting all of the CRISPR machinery into cells with electroporation requires large amounts of CRISPR, RNA guide and synthetic DNA; or multiple steps, such as first electroporating CRISPR and guide, then using an engineered virus particle to introduce the synthetic DNA sequence.
Dr. Adair and her team sought out a better method for carrying CRISPR into cells and found gold. Gold nanoparticles are composed of clusters of gold atoms and can uniformly bind to the RNA, DNA, and protein molecules required for CRISPR to function, resulting in predictable delivery. Gold nanoparticles can also be incredibly small and are well suited for slipping into cells efficiently without electroporation. Dr. Reza Shahbazi, a postdoctoral researcher in the Adair lab, led a study testing gold nanoparticles loaded with CRISPR (AuNP/CRISPR) published in Nature Materials. When the authors added them to HSPCs, they found that AuNP/CRISPR edited the target DNA without harming the viability of the cells.
The authors then used AuNP/CRISPR to modify a gene important to HIV protection, CCR5. CCR5 on T cells can bind to HIV, helping the virus to enter the cell. The authors used AuNP/CRISPR to mutate CCR5 in HSPCs with a naturally-occurring, HIV-resistant version. This new CCR5 would pass from HSPCs into progenitor T cells and render them resistant to HIV. In order to validate that AuNP/CRISPR activity is not restricted to any part of the genome, they went on to use AuNP/CRISPR to mutate a second site on another chromosome in HSPCs which provides natural relief from symptoms of red blood cell disorders such as Sickle Cell Disease and thalassemia. Both of these disease categories impact tens of millions of patients worldwide, most of whom live in resource-limited parts of the world. “We show that a single nanoparticle can passively deliver everything needed for CRISPR-mediated gene editing into blood stem and progenitor cells,” Dr. Adair said. “Because this method does not require electroporation or engineered virus particles, it presents a transformative step in making gene therapy easier, and consequently more accessible to patients who need it most.”
The authors next tested the potential of nanoparticle treated HSPCs to engraft in a humanized mouse model. Incredibly, AuNP/CRISPR treated HSPCs exhibited higher levels of engraftment compared to mock treated HSPCs. Dr. Adair said in this study that they“demonstrate a method that not only is nontoxic, but it appears that nanoparticle treatment of blood stem and progenitor cells might actually improve cell fitness for transplantation. Understanding the cell biology underlying this improvement is part of ongoing research in the lab.”
Dr. Adair also commented that they are looking to improve the levels of gene editing they were able to achieve in this study. In addition, they are interested in targeting of the nanoparticles to specific cell types, which could enable direct in vivo delivery.
This work was supported by Fred Hutchinson Cancer Research Center, the NIDDK Cooperative Center of Excellence in Hematology, and the National Institutes of Health.
UW/Fred Hutch Cancer Consortium members Hans-Peter Kiem and Jennifer Adair contributed to this work.
Shahbazi R, Sghia-Hughes G, Reid JL, Kubek S, Haworth KG, Humbert O, Kiem HP, Adair JE. 2019. Targeted homology-directed repair in blood stem and progenitor cells with CRISPR nanoformulations. Nat Mater. 2019 May 27;. doi: 10.1038/s41563-019-0385-5. [Epub ahead of print] PubMed PMID: 31133730.