NEW YORK – Researchers from the University of Pennsylvania have developed a new, gentler method of delivering CRISPR-Cas genome editing enzymes to mammalian primary cells, which could help produce cell-based therapies.
Described in a proof-of-concept paper published last week in Nature Biotechnology, the Peptide-Assisted Genome Editing (PAGE) system uses cell-penetrating peptides found in HIV and influenza to shepherd Cas9 or Cas12 proteins into cells. A peptide from HIV helps the editing enzyme enter cells via endosomes, while the influenza HA2 peptide helps with endosomal escape, which frees the editing enzyme so it can go to the nucleus.
"This is really exciting new technology that allows for a softer, more efficient way to genetically edit immune cells — including, potentially, immune cells used as therapeutics," Marcela Maus, director of the cellular immunotherapy program at Massachusetts General Hospital, said in an email.
Popular methods for introducing CRISPR-Cas systems to cells, such as electroporation or viral vectors, have yields as low as 10 percent when used with tumor-infiltrating immune cells, such as T cells. With PAGE-CRISPR, cells take up CRISPR-Cas complexes in the same manner that they might take up nutrients from their environment.
"The biggest advantage is that we can edit really fragile cells," said John Wherry, director of the Institute for Immunology at the UPenn's Perelman School of Medicine and a senior author of the paper. "We get super-high viability and really efficient editing in cells that would otherwise die when you try to edit them."
In the paper, the researchers reported cell survival rates as high as 98 percent after editing with modified Cas12 enzymes and editing efficiency in the range of 75 percent to 98 percent. Moreover, the method is fast, taking as little as 30 minutes.
The paper is indicative of great strides being made in the field of CRISPR-based cell engineering. Those editing efficiencies "would have been thought to be unrealistic by many just five years ago," said Krishanu Saha, an expert on chimeric antigen receptor (CAR) T-cell manufacturing at the University of Wisconsin-Madison.
In addition to electroporation, viral vector, and lipid nanoparticle-based CRISPR-Cas delivery, several other protein-based schemes have been tried, the authors noted. One approach uses negatively charged carrier molecules, including green fluorescent protein, and cationic lipids to draw in the CRISPR-Cas complex. Other cell-penetrating peptide-based methods, including polyarginine peptides and nuclear localization sequences, have enabled editing in mammalian cell lines, but "these methods were generally inefficient in primary cell types," the authors wrote.
They hypothesized that a combination of the HIV trans-activator of transcription (TAT) and influenza A virus's hemagglutinin (HA2) peptides would help get the genome editing machinery to where it needed to be. "Viruses have figured out the tricks of the immune system long before scientists have," Wherry said, adding that the final TAT-HA2-Cas proteins were optimized through "low-throughput" screening.
Preexisting immunity against the viral components of the system "may limit its use for in vivo delivery," the authors noted, but shouldn't limit ex vivo editing. Further research is needed to evaluate the ability of the edited cells to provoke an immune response when reintroduced to a tissue.
The paper did not address alternative methods to introduce DNA payloads into cells, Saha noted, where electroporation and viral vectors are also used. The genome editing shown in the paper was limited to gene knock-out. Wherry said the team is working at adapting PAGE-CRISPR for knock-in experiments. Also, though not shown in the paper, it can be coupled with base editing and prime editing methods.
The publication did show multiplex editing, an important consideration for creating allogenic CAR T cells, which could be used as an off-the-shelf therapy. Autologous cell therapy, on the other hand, edits a patient's cells and reintroduces them.
PAGE-CRISPR has "enormous potential to facilitate novel CAR T cells," Maus said.
Though it could also be useful in editing other fragile cell types such as neurons, tissue stem cells, or endothelial cells, the most immediate potential application is in developing cell therapies.
Wherry and his coauthors have applied for a patent on the method and have founded a startup, called NextPage, to explore its commercialization.
The company is only "at the very earliest stages," Wherry said. "We're still working to get it financed." One possibility is to create a microfluidic device that leverages the method's speed to edit patient cells at the point of care.
"You could envision doing [editing of autologous cell-based therapies] with the patient never leaving an infusion suite," he said.