Novel lab technique promotes RNAi technology

The technique has further thrust RNA interference into the spotlight as it has emerged as an increasingly important tool in studying how genes function in normal biological processes and in disease.

US scientists have combined the latest laboratory technologies in using RNA interference to manipulate human T cells. T cells are immune cells that circulate in the blood, with important roles in autoimmune diseases, infectious diseases and some cancers.

The research combined nucleofection, siRNA expression cassettes, and siRNA expression vectors to temporarily open a cell nucleus. Into the nucleus, researchers inserted a payload of DNA.

The researchers encased the DNA within an siRNA expression cassette (SEC), an inexpensive, quickly synthesized product that carries genetic sequences to regulate the gene activity that yields an siRNA.

After the researchers tested a variety of SECs to determine which is the most effective, they inserted the desired SEC into a vector, a biological agent that inserts itself into a target cell's nucleus more efficiently than an unaccompanied cassette.

The researchers first tested their approach by introducing a gene for green fluorescent protein into human T cells, and using siRNA to inhibit that gene's expression, and dim its fluorescent glow.

"T cells have previously been difficult to modify with interfering RNA, being more mobile than other cell types that typically remain stationary in cell cultures,"​ said study leader Terri Finkel, chief of Rheumatology at The Children's Hospital of Philadelphia.

"Our approach achieves results comparable to the conventional technique, which uses synthetic small interfering RNA but is very expensive and in short supply. We expect our technique to expand the toolbox for scientists doing research in immunology."​

Over the past few years, biomedical researchers have been investigating how they might eventually harness RNAi in new medicines. Another line of research uses RNAi as a research tool, investigating the functions of specific genes by studying what happens when RNAi temporarily silences them- a process calling "knocking down" the gene.

RNA interference (RNAi), which naturally occurs in cells, is a process in which brief RNA sequences, called small interfering RNA (siRNA) block signals from a particular gene.

This process, called gene silencing, inhibits the gene from carrying out its function of creating a protein or another gene product. The body often uses RNAi as a defense against the action of hostile viruses.

The next stage of the research involved taking the progress made and applying their approach to HALP, a gene naturally active in T cells. Finkel previously discovered and named HALP, an acronym for "HIV-associated life preserver."

The name suggests the gene has a role in prolonging HIV infection by helping HIV-infected T cells survive attack by the immune system.

Using siRNA and their laboratory techniques, the investigators succeeded in "knocking down," or decreasing gene expression by HALP.

Because their previous research strongly suggests that HALP promotes latent HIV infection, the new technique has a potential application to HIV treatment.

"The siRNA may represent a suicide vector: by knocking down HALP it may allow HIV-infected cells to self-destruct, thus eliminating a hiding place for the virus,"​ said Finkel.

"More broadly,"​ she added, "the technique could theoretically be directed against other immune-related diseases, by silencing harmful genes active in T cells."​

The research by Finkel's team aims to extend RNAi to a wider pool of researchers by making the technique less expensive and more widely available, as well as adapting it to T cells, a cell type previously intractable to such manipulation.

The research team, from The Children's Hospital of Philadelphia, discuss this study in the March 24 online edition of the >Journal of Immunological Methods.​