Delivery continues to be the biggest hurdle facing the in vivo application of RNA interference, but the challenges of getting RNAi compounds into one type of tissue are not always the same as with another type. As such, many researchers are focusing their RNAi efforts on the so-called lowest hanging fruit, and oftentimes this means targeting the liver.
Such is the case with two researchers from Thomas Jefferson University and Indiana University who have recently been awarded two-year grants from the National Institutes of Health to advance their RNAi projects.
Biddanda Ponnappa, associate professor in Thomas Jefferson University’s department of pathology, anatomy, and cell biology, received a grant last month worth about $186,000 from the National Institute on Alcohol Abuse and Alcoholism for a project entitled “Targeting Kupffer Cells with Short Interfering RNA.”
Ponnappa noted in the grant’s abstract that alcohol-mediated liver injury is “greatly potentiated by endotoxins … that are potent inducers of inflammatory cytokines, such as tumor necrosis factor-alpha, interleukins … and prostanoids.”
To test whether siRNAs can be used to treat alcoholic liver disease, Ponnappa is exploring whether they can be used to mediate TNF-alpha production in Kupffer cells, which are specialized liver cells responsible for cleaning out bacteria, foreign proteins, and other debris.
“In our first objective, the efficacy of 21 base-pair [long] … siRNAs targeted to defined regions on TNF-alpha mRNA will be tested in primary cultures of rat Kupffer cells,” Ponnappa stated in the abstract. “Efficacies of siRNAs will be evaluated by measuring the extent of inhibition of lipopolysaccharide-induced production of TNF-alpha following transfection.”
After this, the most efficacious siRNAs will be tested in vivo. The siRNA will be encapsulated in pH-sensitive liposomes and “injected intravenously for sequestration, primarily by Kupffer cells and other macrophages,” Ponnappa stated in the grant’s abstract. Measurements of the siRNAs ability to block lipopolysaccharide-induced TNF-alpha production will be taken one, two, three, and five days after administration. Levels of TNF-alpha mRNA will also be monitored.
“Since the siRNA constructs will be delivered using pH-sensitive liposomes, which are designed to destabilize the endosomal membrane, it is expected that relatively low doses of siRNAs will be sufficient to deliver pharmacologically significant amounts of the siRNAs to the cytosol following sequestration by Kupffer cells [and] macrophages,” Ponnappa concluded. “It is expected that the liposomal system will deliver siRNAs at least [five to] 10 times more efficiently to Kupffer cells than [without] a delivery vehicle.”
The second grant, from the National Institute of Diabetes and Digestive and Kidney Diseases, is worth about $300,000 and was awarded to Nuria Morral, assistant professor in the molecular genetics division of the Indiana University School of Medicine.
Morral’s project, entitled “RNA Interference Using Gutless Adenoviral Vectors,” is focused on developing an RNAi system for finding drug targets for new type II diabetes therapies.
“To achieve glucose control and prevent diabetic complications [such as] retinopathy, cardiovascular disease, and nephropathy, among others … [diabetes] patients currently receive therapeutic agents that have limited efficacy and are associated with side effects,” Morral stated in her grant’s abstract. “Thus, there is a need to develop more efficient therapeutic agents.”
Morral noted that current technologies used to validate the role of genes in disease involve the development of animal models with null alleles for a target gene. “In some instances, absence of a gene is incompatible with normal development, leading to death in utero,” she stated. “In addition, certain studies need to knockdown gene function in an adult animal.”
To circumvent this issue, Morral is developing an RNAi system to down-regulate gene expression in the liver that uses a recombinant gutless adenovirus expressing an siRNA targeted to Srebp-1, a gene involved in fatty acid production and up-regulated in type II diabetes.
Morral stated in the abstract that she aims to “establish the optimal conditions to achieve complete Srebp-1 gene silencing in liver; determine the feasibility of expressing a siRNA molecule in the liver of an adult animal for multiple months and; validate the siRNA expression system as a tool to study gene function in an animal model of disease.”