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Lund University Team Reports on microRNA Vectors for Cell-specific Transgene Expression

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Building on work from other groups showing that microRNAs can be used to control vectors for cell-specific transgene expression, a team of Lund University researchers has published data demonstrating the use of miR-9 for targeted genetic modification in microglia cells.

In recent years, a number of studies have explored the use of miRNA-regulated vectors in transgene expression.

Essentially, complementary miRNA target sites are incorporated into a transgene cassette, which results in transgene mRNA degradation only in cells expressing the miRNA and de-targeted transgene expression.

According to Johan Jakobsson, a Lund University researcher and senior author of a recently published Nature Communications paper describing the new method, he and his colleagues were studying the activity of different miRNAs in the brain, but faced technical hurdles in pinning down their sources.

“There is one population of microRNA that populates the brain during early development and then stays in the brain throughout life,” he explained to Gene Silencing News. “But there is the possibility for cells to enter to the brain … during life.”

Although these two sets of miRNAs are believed to have different functions, “they are extremely difficult to separate [from each other] when you do functional studies,” Jakobsson said.

To address this, the scientists developed a generation of sensor mice using miR-regulated vectors and lentiviral transgenesis based on miRNA-9, which is highly expressed in the brain and has been linked to neuronal differentiation.

An analysis of the brains of the mice yielded “a quite surprising finding,” Jakobbson said — namely that the miRNA was active in virtually all brain cells except for microglia, a resident population of immune cells associated with canonical central nervous system responses to pathogens and brain injuries.

According to the paper, cells of a neuroectodermal origin, with the exception of a “few discrete neuronal populations,” display miR-9 activity in the mouse brain. Meanwhile, microglia, which constitute the largest population of non-ectodermal cells in the brain, do not.

“With this in mind, we hypothesized that miR-9-regulated vectors may constitute a useful tool to genetically modify specifically resident microglial cells in most brain regions,” the Lund investigators wrote.

“In theory, it's a very simple design,” Jakobbson said. “We have a vector that is regulated by this microRNA, so when the microRNA is present, it kills the vector. When it’s not there, the vector is active.

“Since we know this microRNA is not active in microglia, it will only be active there,” he added. “It basically exploits the cell’s own system.”

To test the approach, the researchers injected either miR-9-regulated vectors or green fluorescent protein-expressing control vectors into the striatum of adult rats, which were later sacrificed and analyzed.

Based on the expression patterns in the rats’ brains, the experiments showed that “miR-9 target sites are capable of repressing transgene expression in non-microglial cell types … allowing high-level transgene expression specifically in resident microglia,” according to the paper.

The miR-9 vector was then used to visualize and isolate activated resident microglia following excitotoxic insult — an experiment the team noted has traditionally been difficult to conduct using other genetic modification techniques given the similar profiles of resident microglia and immune cells that infiltrate the brain following injury or infection.

In the end, the researchers were able to successfully use the miR-9-regulated vectors to monitor and isolate the activated resident microglia from non-resident cells, supporting their potential in the study of microglial function in healthy and diseased brain tissue, they wrote.

In addition, the work further validates the use of vectors encompassing miRNA target sites as a tool to target transgene expression in certain cell populations, Jakobbson noted.

“The main advantage of [miRNA]-regulated vectors is the highly robust downregulation of transgene expression, which occurs when the regulating miRNA is present in the cell,” the researchers wrote in Nature Communications. “This advantage is combined with a strong housekeeping promoter in the vector.”

Such vectors therefore show “great promise in achieving cell-type-specific transgene expression, for either experimental studies or gene therapy purposes.”

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