High-Throughput Mapping of Viral and Vector Interactome with siRNA Libraries

A new report, recently published in Nature, adds to a series of papers that use small interfering RNA (siRNA) libraries to identify viral host factors. In the study by König and colleagues, the influenza strain A/WSN/33 was made replication defective by replacing the haemagglutinin (HA) protein with a Renilla luciferase reporter. ¹ This vector was then subjected to a high-throughput transduction assay of the human lung epithelial A549 cell line that was transfected with an arrayed siRNA library designed to target more than 19,000 human genes. With luciferase expression as an end point, 295 cellular genes, for which knockdown of expression was not toxic to the cells, were found to reduce infectivity by 35% or more. Next, the investigators subjected these genes to several studies that were aimed to (1) confirm the hits from their initial screen and (2) functionally classify top-scoring genes as to whether they are involved in an entry or postentry step.

Of the initial 295 genes selected on the basis of reduced viral infection by at least two siRNAs per gene, some genes had been previously found to be important for influenza replication; however, most had not. In assays that used wild-type rather than the vectored influenza, 219 (of the 295) genes were confirmed to inhibit multiple-cycle replication.

The use of a genome-wide RNAi screen is not novel for elucidating viral transduction pathways, but this manuscript illustrates the power of the technology for influenza. The availability of data sets retrieved from other studies that focused on HIV, ^{2 –4} dengue, ⁵ West Nile, ⁶ and hepatitis C ⁷ viruses pinpoints common mechanisms across viruses. Indeed, 53 genes from this study were found important for other RNA viruses. Comparisons across functionally related gene groups highlight other aspects of similarity and dissimilarity; where cytoskeleton appears critical for all viruses studied in this and other studies, the nuclear transport machinery is implicated for influenza and HIV, but less so for the other viruses.

In the past, results from studies employing this methodology have been met with some controversy, particularly in the case of HIV, for which three independent studies have been performed. ^{2 –4} In a meta-analysis of two data sets, a startlingly low overlap was found between important genes that emerged from each independent screen. ³ Indeed, of the 295 genes pinpointed in one study, only 13 were found in another study. This very modest concordance was attributed by the authors to experimental and analytical differences; a higher degree of agreement is found when gene families are considered as a whole. ^3,4

The relevance for gene therapy vectors is substantial because recombinant vectors are often used in the initial screening. The host factors identified in these studies likely impact on gene transfer vectors. In fact, in the HIV-1 study by König and colleagues, ³ HIV was tested alongside Moloney retrovirus and adeno-associated virus, although the detailed results from these additional studies remain unavailable to date. (lhv)

The U.S. Healthcare Bill and Biomedical Research

The health insurance reform bill signed on March 23, 2010 by President Obama includes an assortment of measures that immediately benefit life science research. Among these is a provision to speed the translation and application of promising new treatments for diseases from the laboratory bench to a patient's bedside. The provision, championed by U.S. Senator Arlen Specter (D-Pa.), is known as the Cures Acceleration Network (CAN). CAN, as authorized, would award grants through the National Institutes of Health (NIH) to biotech companies, universities, and patient advocacy groups to bridge the chasm between a basic scientific discovery and its application as a new health treatment.

CAN grants of up to $15 million will be used to fund clinical trials and hasten the approvals process. Many groups for lack of adequate funding have eliminated or postponed clinical trials to treat such diseases as melanoma, cervical cancer, lupus, multiple sclerosis, diabetes, atherosclerosis, and non-Hodgkin's lymphoma.

With respect to industry, the bill has adopted a 12-year exclusivity term for biosimilars (i.e. generic versions of biological therapeutics), which for reasons of political expediency, was left intact despite the preference of the Obama Administration and generics manufacturers for a shorter brand monopoly. The bill also includes a Therapeutic Discovery Project Credit—a measure that gives tax credits (or grants for non-taxpaying companies) equivalent to half the cost of investments in “qualified therapeutic discovery projects” undertaken in fiscal 2009 or 2010. What qualifies is vague, but potentially the credit can apply to any preclinical, clinical, diagnostic or technology development undertaken by companies with <250 employees. The program is worth $1 billion ($500 million a year)—a large sum, although still only 11% of the annual R&D spending of public biotech companies with <250 employees ¹ . (sk)