Better gene editing methods could power the next generation of cell and gene therapies

(Nanowerk News) New approaches to cell genetic engineering promise significant improvements in speed, efficiency, and reduction of cellular toxicity compared to current methods. This approach could also power the development of advanced cell therapies for cancer and other diseases, according to a study from researchers at the Perelman School of Medicine at the University of Pennsylvania.

In research appearing in Natural Biotechnology (“Efficient engineering of human and mouse primary cells using peptide-assisted genome editing”), the researchers found that protein fragments used by some viruses to help them enter cells can also be used to introduce CRISPR-Cas gene-editing molecules into cells and DNA-containing nuclei with unusually high efficiency and low cellular toxicity.

Scientists hope this new technique is very useful for modifying T cells and other cells from the patient’s own body to carry out cell therapy. One such application is CAR T (chimeric antigen receptor T cell) therapy, which uses specially modified immune cells from a patient to treat cancer. T cells—a type of white blood cell—are taken from patients and reprogrammed to find and attack cancer cells when they are reintroduced into the bloodstream.

The first FDA-approved CAR T-cell therapy was developed at Penn Medicine, and received Food & Drug Administration approval in 2017. There are now six FDA-approved CAR T-cell therapies in the United States. The therapies have revolutionized the treatment of certain B-cell leukemias, lymphomas, and other blood cancers, leaving many patients who previously had little hope of long-term remission.

“This new approach—built on Penn Medicine’s history of cell and gene therapy innovation—has the potential to become a major enabling technology for engineered cellular therapies,” said co-senior author E. John Wherry, PhD, Richard and Barbara Schiffrin President’s Distinguished Professor. and chair of Systems of Pharmacology & Translational Therapies at Penn Medicine.

The CRISPR-Cas molecule originates from ancient bacterial antiviral defenses, and is designed to precisely remove DNA at the desired location in the cell’s genome. Some CRISPR-Cas-based systems combine deletion of old DNA with insertion of new DNA for versatile genome editing. This approach can be used to replace faulty genes with repaired ones or to delete or modify genes to improve cellular function. Some systems can also add genes that confer novel properties on CAR T cells such as the ability to recognize tumors or withstand the harsh tumor microenvironment that normally depletes T cells.

Although the CRISPR-Cas system is already widely used as a standard laboratory tool for molecular biology, its use in modifying patient cells to make cell-based therapies is limited—partly because CRISPR-Cas molecules have difficulty entering cells and then into the cell nucleus which contains DNA.

“Current methods for introducing the CRISPR-Cas system into cells, which include the use of a carrier virus and an electrical pulse, are not efficient for cells taken directly from the patient (so-called primary cells). These methods also usually kill many cells they are used in, and can even cause widespread unwanted changes in gene activity,” said co-senior author Shelley L. Berger, PhD, Daniel S. Och University Professor in Cell and Developmental Biology and Genetics and director of the Penn Epigenetics Institute.

In the study, the researchers explored using small virus-derived protein fragments, called peptides, to more efficiently drive CRISPR-Cas molecules through the outer membrane of primary human cells and into their nucleus. Specifically, the researchers found that the combined combination of two modified peptides — one found in HIV and one in the influenza virus — can be mixed with CRISPR-Cas molecules to insert them into primary human or mouse cells and their nuclei with efficiency of up to nearly 100 percent, depending in cell type—with almost no toxicity or changes in gene expression.

The team demonstrated an approach, which they call PAGE (peptide-assisted genome editing), for several types of cell therapy envisioned including CAR T-cell therapy.

In addition to its potential use in cell and gene therapy, the authors note that the PAGE approach has broad application in basic scientific research. The inefficiency of the standard CRISPR-Cas cell penetration method means that gene editing to create mouse disease models usually requires a time-consuming multi-step process to produce transgenic mice—to incorporate the gene editing machinery into their DNA. In contrast, PAGE with high efficiency and low toxicity enables fast, efficient and direct gene editing in ordinary laboratory mice.

“The simplicity and power of the peptide assist concept suggests that it could potentially be adapted in the future for delivery to primary cells of other genome editing proteins, or even protein-based drugs,” said co-senior author Junwei Shi, PhD, assistant professor of Cancer Biology and member of Penn Epigenetics. Institute and the Abramson Family Cancer Research Institute.

The study is a collaboration that includes the laboratories of co-author Penn Rahul Kohli, MD, PhD, a professor of Infectious Diseases and Biochemistry and Biophysics, and co-author Gerd Blobel, MD, PhD, Professor Frank E. Weise III of Pediatrics and co-director of the Institute of Epigenetics .