Research
Engineering genome editing enzymes for cell-targeted delivery
CRISPR-based genome editing technology has rapidly transformed biomedical research and shows great promise for the development of novel therapeutic applications. Enzymes such as Cas9 (represented above) already contain several powerful properties: binding to a specific region of the genome and performing a precise cut at that site (left, above). This provides the foundation for therapies that may soon be able to correct the genetic defects that give rise to disease. But for this promise to be fully realized, therapeutic enzymes must be delivered to cells safely and efficiently. Our laboratory is working to develop genome-editing enzymes that are readily internalized by cells (bottom right, above). Furthermore, we strive to perform targeted delivery of these enzymes, maximizing precision in correcting specific cells, tissues, or organs (top right, above). A first step towards realizing this approach has been published in Rouet et al. JACS 2018 (see summary below, under Publications).
Improving access to genetic therapies
Recent advances in genetic and cell-based technology have demonstrated the transformative power of next-generation therapeutics. However, these treatments are incredibly expensive, and are often only available at a handful of cutting-edge research hospitals. Tragically, there are millions of people in the developing world who would benefit from such treatments, but deployment is currently impractical. The Wilson Lab’s efforts are motivated by the pressing need to improve access to life-changing technologies. A central goal of our work is to find affordable and straightforward new ways to genetic medicines, enabling cures globally.
Cell-targeted delivery
To direct Cas9 to liver cells, for example, we rely on a receptor that is distinct to hepatocytes, the key functional cells of the liver. By tethering Cas9 to a small molecule that is recognized by the liver-specific receptor, we can induce efficient uptake of Cas9 into the cells. However, this represents only half of the journey to an edited cell: if no further action is taken, the Cas9 will move from endosomal cellular compartments to be digested in the cell’s lysosomes. To help Cas9 complete its journey, we include delivery peptides that facilitate endosomal escape, allowing efficient genome editing.
Delivery targeted to immune cells
Because of the proven clinical power of immune cell-derived therapies, we are interested in engineering Cas9 to target T cells for therapeutic editing. Current work has relied on ex vivo editing, which involves removal of the patient’s cells, modification of the cells in a laboratory setting, and return of the cells to the patient. In contrast, we hope to enable in vivo editing of a patient’s cells within the bloodstream, following a simple intravenous administration. Using a strategy analogous to the liver-targeted approach described above, we are tethering Cas9 to a molecule that can specifically recognize T cells, driving selective delivery even in the presence of other white blood cells. Having achieved reliable cell targeting, we are now working to enable endosomal escape and efficient genome editing.
Publications
Wilson lab
Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes
Foss et al.; Nature Biomedical Engineering [PDF] [SI]
Here we report PERC: peptide-mediated RNP delivery for CRISPR engineering, which we applied to primary human T cells. This approach represents an enticing alternative to electroporation, which is cumbersome, toxic, and has a substantial impact on cell state. In contrast, PERC allows hardware-independent T cell engineering that relies on just three components: the RNA & protein that constitute the CRISPR enzyme, plus a synthetic peptide that facilitates intracellular delivery. Efficient knock-ins can be performed using PERC if the template DNA is carried by an AAV vector. We showed that this approach works for nucleases Cas9 and Cas12a, as well as an adenine base editor. Our work culminated in the generation of multi-edit CAR-T cells that showed excellent anti-tumor potency in a mouse model. If you’re interested in trying PERC, our how-to-page is intended to help you get started, with guidelines on procuring the reagents you’ll need.
A traceless linker for aliphatic amines that rapidly and quantitatively fragments after reduction
He et al.; Chemical Science 2020 [PDF]
This work reflects a collaboration between the Wilson lab and the Murthy and Bankiewicz labs. We report the use of a novel chemical linker that allows coating of the surface of CRISPR-Cas9 with functional groups (e.g. peptides that promote intracellular deliery; PEG molecules that can enhance tissue distribution or immune evasion) and is bioreducible, restoring a native and fully-functional Cas9 after transit to the cytosol. Figure 3d demonstrates that this approach can increase the spread of Cas9 throughout the brain of a mouse, addressing a key hurdle in enabling therapeutic genome editing of the brain.
The Daunting Economics of Therapeutic Genome Editing
Ross Wilson & Dana Carroll; CRISPR J. 2019 [PDF]
We offer a perspective on the exceptionally high prices of genetic medicines, and the consequences for widespread deployment of next-generation therapeutics. We discuss the origins of these high prices and propose advances that might help enable more affordable and accessible next-generation therapies.
Clinical Applications of CRISPR-based Genome Editing and Diagnostics
Dana Foss, Megan Hochstrasser, Ross Wilson; Transfusion 2019 [PDF]
We review CRISPR’s transition into the clinic, via ex vivo therapies and emerging diagnostic tools.
Engineering CRISPR-Cas9 RNA–Protein Complexes for Improved Function and Delivery
Romain Rouet, Lorena de Oñate, Jie Li, Niren Murthy, Ross Wilson, CRISPR J. 2018 [PDF]
We summarize recent progress in the engineering of Cas9 RNA-protein (RNP) complexes for cellular delivery, improved function, and more.
Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell Type Specific Gene Editing
Rouet et al.; J Am Chem Soc. 2018 [PDF] [supp. info]
Chemical modification allows a Cas9 RNA-protein (RNP) complex to be selectively taken into cells bearing a liver-associated receptor. This demonstrates the feasibility of using molecular targeting to specify which cells Cas9 will edit, potentially for therapeutic use in vivo.
The Promise and Challenge of In Vivo Delivery for Genome Therapeutics
Ross Wilson & Luke Gilbert; ACS Chem Biol. 2018 [PDF]
A review of progress towards genome editing that can cure, prevent, or treat disease, along with a summary of the hurdles that remain.
Emerging Strategies for Genome Editing in the Brain
Dana Foss & Ross Wilson; Trends Mol Med. 2018 [PDF]
This Spotlight article describes nanoparticle-driven delivery of Cas9 RNP enzymes and compares/contrasts this approach to virally mediated delivery.