Opportunistic bacterial and viral pathogens have evolved sophisticated mechanisms to subvert the human immune system. The majority of host proteins targeted by such pathogens remains poorly understood. RNAi represents a potent molecular tool for identifying host genes manipulated by such microbes.
Elizabeth Hong-Geller, Ph.D., staff scientist, bioscience division, Los Alamos National Laboratory, is performing studies to systematically identify host signaling pathways affected by Yersinia and other organisms.
“The Yersinia genus encodes three human pathogens, the worst of which is Y. pestis, the causative agent of bubonic plague. All members of this bacterial family attack the lymphoid system by injecting Yersinia outer proteins (Yop) into the cell cytoplasm. These proteins target multiple host signaling pathways and induce cell death. Mechanisms for this are not well characterized.”
Dr. Hong-Geller is working to decipher such mechanisms by utilizing RNAi. “In the last couple years, RNAi screens have begun to identify which of the host’s proteins are affected after infection. Our studies utilize a less virulent but related form of Yersinia called Y. enterocolitica.
“We initially infect mammalian cells with Yersinia and then use a commercial shRNA library to screen for NF-κB activity rescue. Yersinia infection normally downregulates NF-κB activity, which controls gene transcription involved in inflammation and is important to help clear the bacteria,” she continued.
“We monitor NF-κB expression using a high-throughput luminescent reporter assay system. After individual knock-down of ~750 kinases or kinase-related genes, we identified 19 genes needed to suppress NF-κB activity. We also identified two genes that restored the viability of infected cells.”
Dr. Hong-Geller also validated her results by taking the hits and double-checking them with siRNA constructs and stable shRNA cell lines. Additionally, they decided to assess available small molecule kinase inhibitors.
“We wanted to know which, if any, of these inhibitors might rescue NF-κB activation in infected cells. We were pleased to determine that in a subset of targeted genes, we could effectively rescue this activity.
“Our goals now are to extend these findings by delving into the basic mechanistic biology represented by this data. We will determine if the RNAi screen hits represent direct or indirect interactions between the pathogen and host.”
These studies may help elucidate the pathogenic mechanisms of Yersinia and related pathogens. “Our hope is that this information may serve as the basis for the design of new inhibitors for broad-spectrum host-derived therapeutics.”
Lipid Nanoparticles Delivery
Successful delivery of the payload is a key aspect for the successful use of RNAi therapeutics. Tekmira Pharmaceuticals utilizes lipid nanoparticles (LNPs) as a delivery technology platform. The uniformly constructed LNPs encapsulate their target-specific double-stranded siRNA molecules or other nucleic acids.
“Nucleic acids are relatively large in size and unstable in the blood compartment,” explained Ian MacLachlan, Ph.D., evp and CSO. “They do not readily diffuse across the membranes of target cells, and thus need an effective delivery vehicle.
“Our LNP technology protects the nucleic acid payload and allows us to control the circulation time in blood. Since it is a modular platform technology, there are literally thousands of lipid combinations and lipid or lipid:nucleic acid ratios that can be customized for specific delivery applications."
According to Dr. MacLachlan, the field is tackling these challenges and making headway.
“As we continue developing formulations that are increasingly clinically validated for increased potency and reduced toxicity, we are acquiring new understandings of how to improve the therapeutic index of these drugs.
“Recent research has provided important information about the structural basis for the chemical toxicities associated with high doses of lipid excipients and the nucleic acids themselves. Further, we are beginning to unravel how immune toxicities can manifest through unwanted engagement of either the innate or adaptive immune systems, and how these can be avoided.”
Tekmira’s LNP siRNA delivery platform (known as SNALP) has confirmed RNAi-mediated efficacy in several preclinical models including oncology and infectious and metabolic diseases. Aside from systemic delivery, the company also has made progress in nebulization-mediated delivery of siRNA into the respiratory tract.
“One recent example of progress is that we have received approval from the FDA to initiate Phase I clinical trials with our Ebola therapeutic, TKM-Ebola. This disease and those caused by other hemorrhagic fever viruses have no approved treatments.
“Our preclinical studies demonstrated that the Ebola therapeutic provided 100% protection from an otherwise fatal infection with Zaire Ebola virus in nonhuman primates.”
Dr. MacLachlan said he is pleased with how the field is progressing. “This is an incredibly exciting time. Tekmira expects that six or seven of our and partner Alnylam’s LNP-based products will have entered the clinic by the end of 2012. A few years ago, we would have said that we couldn’t be sure if RNAi would ever translate into therapeutics. But now, especially with the use of siRNAs, we are close to achieving that goal.
“The last few years have seen us improve our understanding on a number of fronts such as development of more potent siRNA and lipid chemistries, advantageous sequence design, and controlling responses of the immune system. At the same time we are seeing the LNP approach validated in the clinic. All of this has increased my confidence that RNAi therapeutics are on the horizon.”