Like a delivery driver navigating crowded city streets, a gene-therapy-toting lipid nanoparticle faces a gauntlet of potential detours on its journey toward a cell's nucleus.
First, there's entering the cell's plasma membrane; then navigating around organelles like the Golgi apparatus, mitochondria and endoplasmic reticulum — all destinations that can errantly absorb the particle's payload, rendering it ineffective at best or harmful at worst.
And that's all before the particle even enters the nucleus and successfully makes a genetic change.
As part of an effort to improve nonviral genetic editing technologies, with particular interest in treating genetic eye diseases, a group of University of Wisconsin–Madison researchers developed a platform to screen nearly all genes in the human genome — more than 19,000 genes — linking disruption of individual genes to improved genetic outcomes in human cells. The work could provide a roadmap for identifying potential detours in different cell types when gene editors like CRISPR-Cas9 are applied to cells in the lab or to tissues in the body. Such knowledge could inform the design of new strategies, such as pre-treatments to temporarily mute those genes, to enhance the effectiveness of gene therapies, and even mRNA vaccines and immunotherapies.
The researchers, led by Biomedical Engineering Professor Krishanu Saha and representing the Wisconsin Institute of Discovery, the McPherson Eye Research Institute, the School of Medicine and Public Health, the Waisman Center and the School of Pharmacy, published their results in a paper in the journal Nature Communications.
"We essentially knock out each gene one by one in different cells. It's a massive undertaking if you were to try to do that in 19,144 different cell cultures, for instance," says Saha. "Here we do it in essentially a few dishes of cells that have 100 million cells in them. Then we use a precise sequencing strategy to find which genes matter the most, essentially the needles in this genomic haystack."
Over the course of the six-year project, the researchers culled a list of 19,144 genes down to 26 that, when knocked out, increased the editing efficiency by the Cas9 gene editing protein. They further trimmed their list to six, then validated the top two and tested the effects of knocking them out on editing efficiency in human model cell systems. Depleting either of the two genes (GJB2 and BET1L) improved editing efficiency by more than six times using base editors, so called "CRISPR 2.0," which chemically change individual bases in DNA rather than making cuts in the genome like the traditional CRISPR-Cas9 editor does.
How and why these particular genes impede editing remains an open question — and a larger one for research groups across the field to explore.
"After we add Cas9, if we see more of the proper edits in a cell population where a candidate gene has been knocked down, then that means the gene is somehow blocking the gene editing process," says Saha lab alumna Shivani Saxena (PhD BME '24), now a postdoctoral researcher at the University of California, San Francisco and one of two first authors on the paper. Meha Kabra, a research scientist in the UW-Madison School of Medicine and Public Health's Department of Pediatrics, is the other.
When testing their approach on patient-derived retinal cells carrying the genetic eye disease Leber congenital amaurosis (LCA), the researchers found that inhibiting the top two genes from their screening increased editing efficiency by more than three-and-a-half times.
Saha and fellow study authors David Gamm (professor of ophthalmology and visual sciences), Bikash Pattnaik (associate professor of pediatrics) and Shaoqin "Sarah" Gong (Vilas Distinguished Professor of ophthalmology and visual sciences) are the UW–Madison project leads of the National Institutes of Health-funded CRISPR Vision Program , applying nonviral gene-editing technologies to treating inherited eye diseases like LCA. This consortium recently developed a personalized CRISPR drug on demand last year.
The other genes flagged by the screen could be potentially more relevant to other cell types and diseases, Saha notes, while the group's broader approach could inform delivery strategies for emerging mRNA cancer vaccines, CAR T-cell therapies and more.
"My hope is that the field picks up the methodology and helps understand what cellular mechanisms, pathways, are important for the delivery of really anything that could be packaged into lipid nanoparticles," says Saha. "And in that way, I believe the impacts could be beyond just genome editing."
Krishanu Saha holds the Retina Research Foundation Kathryn and Latimer Murfee Chair through the McPherson Eye Research Institute.
Funding for this research came from the National Institutes of Health (grants U19NS132296 and R35 GM119644), the McPherson Eye Research Institute chairs and professorships and the Sandra Lemke Trout Chair in Eye Research, and Research to Prevent Blindness.
Other UW-Madison-affiliated authors on the paper include Amr Abdeen, Diana Marcela Tabima Martinez, Divya Sinha, Piper A. Rawding, Min Zhu, Ruosen Xie, Tanvi Kulkarni, Giovanni M. Hanstad and Maria A. Fernandez Zepeda.
