Original story from University of Illinois Chicago (IL, USA).

A novel gene-editing technique that mimics natural gene activation may offer a more efficient way to engineer cells for advanced therapies. 

A breakthrough in CRISPR gene editing may allow scientists to more efficiently engineer cells for use in medical therapies, according to a new study led by UIC’s professor Brad Merrill. The team successfully tested a gene-editing technique that can program sequential edits in human cells, mimicking natural gene activation.

CRISPR is a gene-editing tool first described in the early 2010s that allows scientists to cut DNA at precise locations and either shut off genes or insert new ones at one or many sites on a cell’s DNA. It revolutionized gene editing with its precision and speed, and it can be used in medical therapies to fix mutated genes or create beneficial cells.

Initially, available CRISPR systems required scientists to make all these cuts at once. But in 2021, researchers in Merrill’s lab discovered a way to use CRISPR to edit at multiple distinct times. Building off of that discovery, Merrill and his former PhD student Ryan Clarke formed the biotech startup Syntax Bio (IL, USA), where the new study took place.

An organism’s genes are organized in relationships with one another, and often rely upon sequential relationships to function correctly.

“Genetics of cell differentiation work through sequences of events, kind of like baking a cake,” said Merrill, professor of biochemistry and molecular genetics at the College of Medicine. “You wouldn’t at one time combine the birthday candles, frosting, flour, eggs and then throw that whole mess into an oven.”

Rather, you mix wet ingredients together, then dry ingredients, then beat these two mixes together before baking and decorating.

“That’s analogous to how many relationships between genes work in your genome, to provide instructions to go from a stem cell to the functional cell type, like a blood cell or cell in the pancreas,” said Merrill.

Designing new gene delivery systems

Rice University researchers launch a project to engineer and test libraries of optimized gene therapy vectors designed to improve delivery of large DNA payloads.

From baking cakes to building cells

The team’s method involves special molecules called guide RNAs that ferry the CRISPR system to the proper cut location. The researchers build a daisy chain consisting of, for example, one DNA encoding an active guide RNA and nine DNA encoding inactive guide RNAs. The product of guide one turns on two by editing its DNA, then guide two turns on three and so on, until the sequence is completed. They call these special DNA encoding the guide proGuides.

“We just use the activity of one to convert the next one from inactive to an active state,” Merrill said. “We also figured out how to use it to activate endogenous genes in addition to conversion processes. So, we use the system to program cells by turning on certain genes in the right order that may normally happen during embryonic development.” The end product is the desired cell type.

Merrill and Clarke published these earlier results in the journal Molecular Cell. They formed Syntax Bio to refine and optimize proGuides and bring their use to market.

Creating life-saving therapies

In their new study, Merrill and the researchers presented a proof of concept using their optimized method in human-induced pluripotent stem cells. These are stem cells that have been reverted to an immature state, and thus has the ability to become any specialized cell, for example, a neuron or a blood cell.

“These cells are important for making cell-based therapies for regenerative medicine,” Merrill said. One such application is programming pancreatic beta cells for transplantation into people with type 1 diabetes, potentially eliminating their need for insulin injections.

Typically, researchers grow large quantities of induced pluripotent stem cells and move them through a series of cocktails of different solutions and culture conditions to get them to become a specialized cell. This approach can work well on a small scale, said Merrill. But it can be difficult to scale it up to create the large volume of cells needed for medical applications, he said.

Merrill and his team propose using their method for a more internal approach. By adding specific proGuides to stem cells, they can program them to turn into valuable cells. The team is now testing the method with different cell types, seeing what the right recipe is for each.

“Pancreatic beta cells are one cell type that we’re focusing on, because the need and the fact that with these cells you can have a huge effect on human health,” Merrill said.

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