PASTE, New Gene-editing Tool, May Lead to ‘Blanket’ CF Gene Therapy
Tool can insert large DNA sequences into genome, addressing variety of mutations
A novel gene-editing tool that allows scientists to “drag-and-drop” sequences of DNA into the genome, without inducing large cuts, could aid in treating genetic diseases like cystic fibrosis (CF), a study reported.
The technology, aptly called PASTE (for programmable addition via site-specific targeting elements), “expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways,” its researchers reported.
As such, PASTE could be particularly helpful in therapy work for diseases with large numbers of gene mutations. The tool’s potential in treating CF, caused by nearly 2,000 known mutations in the CFTR gene, is the team’s current focus.
“It’s a new genetic way of potentially targeting these really hard to treat diseases,” Omar Abudayyeh, a study co-senior author and gene therapy researcher with the Massachusetts Institute of Technology (MIT), said in a press release.
Gene editing without risky double-stranded breaks in DNA
The study, “Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases,” was published in the journal Nature Biotechnology.
Gene editing is a broad term for technologies that aim to alter the genetic code inside of a cell. Theoretically, gene editing could offer a functional cure for genetic disorders like CF. However, optimizing the efficacy and safety of these technologies is an ongoing challenge.
One common issue in gene-editing technologies is that of double-stranded breaks — a “cut” in DNA. Most gene-editing strategies rely on making one or more such breaks, then inserting a new genetic code when the cell’s repair machinery goes to fix the break.
But using double-stranded breaks as a tool for editing carries risks. In particular, it can cause off-target effects such as “indels,” where chunks of DNA are unintentionally either lost or inserted into the genome.
A team led by MIT researchers developed PASTE, a tool to insert large pieces of DNA into a cell’s genetic code without inducing double-stranded breaks.
“We wanted to work toward what gene therapy was supposed to do at its original inception, which is to replace genes, not just correct individual mutations,” Abudayyeh said.
The tool combines an integrase with a CRISPR-Cas9 system. Integrases are proteins that certain viruses use to insert their genetic material into the DNA of a cell that the virus is infecting.
In nature, integrases insert a genetic payload into a spot in the DNA where there is a specific genetic sequence, referred to as a “landing site.” This has historically made them difficult to use in a therapeutic context, since “landing sites” in the human genome are very specific.
The tool uses CRISPR-Cas9 — a gene-editing technology that was created by adapting molecular systems that bacteria use to defend against infecting viruses — to insert a landing site into the genome. The landing site has 46 pairs of nucleotides, the building blocks of the genetic code. Double-stranded breaks are avoided by basically first adding the landing site to one side of the double-stranded DNA molecule, then adding its complementary sequence on the other side.
Once the landing site is placed into the desired location in the genome, the integrase can add larger sequences of DNA without causing breaks. In this manner, PASTE can facilitate large pieces of DNA being “dragged-and-dropped” to a specific location.
“We think that this is a large step toward achieving the dream of programmable insertion of DNA,” said Jonathan Gootenberg, the study’s other co-senior author and a scientist at MIT’s McGovern Institute for Brain Research. “It’s a technique that can be easily tailored both to the site that we want to integrate as well as the cargo.”
Scientists tested the PASTE system in a battery of experiments, first in cell models and then in living mice, using the tool to alter the sequence of several different target genes. Results broadly showed that the system worked as intended: it was able to insert large sequences, up to about 36,000 nucleotide pairs long, with efficiencies between 5% and 60%.
These findings indicate that PASTE could effectively insert working copies for more than 99% of human protein-coding genes, the researchers said.
Potential for CF gene therapy addressing most mutations
Notably, the system showed similar or better efficiency than other technologies that work by inducing double-stranded breaks, and the frequency of indels was quite low.
“We see very few indels, and because we’re not making double-stranded breaks, you don’t have to worry about chromosomal rearrangements or large-scale chromosome arm deletions,” Abudayyeh said.
“We found no off-target activity with PASTE,” the team noted in the study.
This system could be applied regardless of the underlying disease-causing mutation, which is an advantage over many other approaches, the researchers said.
“Current genome-editing approaches for diseases such as cystic fibrosis … are limited, as systems must be tailored for specific mutations, requiring unique genome-editing therapies for each subset of the clinical population,” they wrote. “Programmable insertion of the [unmutated] gene at the endogenous location could address most potential mutations, serving as a blanket therapy.”
The scientists placed their technology online for other researchers to use.
“One of the fantastic things about engineering these molecular technologies is that people can build on them, develop and apply them in ways that maybe we didn’t think of or hadn’t considered,” Gootenberg said. “It’s really great to be part of that emerging community.”