Next-generation gene editing uses cell-penetrating peptides 2023

Utilizing virus-based protein fragments, scientists have created a highly effective new gene-editing technique. The method could be used to enhance existing cancer and other disease-treating cell and gene therapies.

The use of CRISPR technology to modify genes in a straightforward and efficient manner has revolutionized biomedical research and treatment and improved our understanding of the genetic basis of disease.

CRISPR is an immune system component of bacteria that can cleave DNA; it has been repurposed as a gene-editing tool. Cas is a CRISPR-associated protein (Cas) that is attached to a guide RNA designed to match the gene being edited. The guide RNA directs Cas to the target gene, where it functions as “molecular scissors” to cut the offending DNA.

PAGE is used for next-generation gene editing with cell-penetrating peptides.

However, despite all of its advantages, the technology has difficulty accessing primary cells, which are cells extracted directly from living tissue or organs and grown in the laboratory. T cells, which are part of the immune system, are primary cells.

After discovering that some viruses use protein fragments – peptides – to enter cells, researchers from the University of Pennsylvania investigated whether this method could be used to introduce CRISPR gene-editing technology into primary cells more effectively.

“Current methods of delivering CRISPR-Cas systems into cells, which include the use of carrier viruses and electric pulses, are ineffective for cells taken directly from patients (termed primary cells),” said co-corresponding author Shelley Berger.

Additionally, these methods typically result in the death of a significant number of the cells they are applied to, and they can even result in widespread unintended alterations in gene activity.

Cell-penetrating peptides make PAGE suitable for next-generation gene editing.

The researchers used peptides to direct CRISPR-Cas9 and Cas12a molecules into the nuclei of primordial human and mouse cells, where the majority of the cell’s DNA resides. They discovered that combining two modified peptides, one found in HIV and one in influenza viruses, with CRISPR-Cas molecules resulted in nearly 100 percent gene-editing efficiency, was non-toxic, and did not alter gene expression.

The researchers have dubbed their novel technique peptide-assisted genome editing, or PAGE, and believe it could be particularly useful in T cell-related therapies, such as chimeric antigen receptor (CAR) T cell therapy, which employs a patient’s modified immune cells to treat blood cancer.

“This new approach has the potential to be a major enabling technology for engineering cellular therapies,” stated E. John Wherry, one of the study’s co-corresponding authors.

In addition to its use in cell and gene therapies, PAGE has, according to researchers, many other applications.

“The simplicity and power of the peptide-assisted concept suggests that it could potentially be adapted in the future for the delivery into primary cells of other genome-editing proteins, or even protein-based drugs,” said co-corresponding author Junwei Shi.