A novel anti-viral delivery system
Lee W. Riley, University of California, Berkeley
This is a resubmission of an application proposing to develop and optimize viral anti-infectives that can be delivered in vivo without the use of viral vectors. This will be done as a proof-of-the-principle project using CMV as a target.
A major obstacle in the treatment of AIDS and viral opportunistic infections, such as the CMV-associated diseases, is the problem of drug-resistance. This problem is not likely to be resolved by the traditional use of anti-virals. A radically new approach is needed. The use of highly target-specific anti-sense nucleic acids may offer this approach. One such class of highly specific anti-sense molecule is a catalytic ribonucleoprotein complex called RNAse P, which cleaves the 5' leader sequence from tRNA precursors during 5' maturation of tRNAs. The advantage of RNAse P is that it can be custom designed to target any mRNA sequence in a viral pathogen in a site-specific manner through modification of its external guide sequence (EGS). This allows construction of a cocktail of RNAse P molecules containing the same core RNA catalytic domain but different target specific EGS. In this way, several different sequences in a virus can be targeted at once, which would minimize the selection of resistant mutants. The ability to target many targets simply by changing the EGS and retaining the same catalytic RNA is a major advantage over the use of other classes of RNA-based anti-infectives, such as siRNA, which is less target- specific.
One major obstacle that has to be overcome for RNA-based anti-infectives to be clinically applicable is their systemic delivery. Most studies to date have been done in vitro in cultured cells. Here, we propose to take advantage of a new 22-amino acid cell penetrating peptide (CPP) called Inv3, which was derived from a protein called mycobacterial cell penetrating protein (Mcep), located in the cell wall of Mycobacterium tuberculosis, the etiologic agent of tuberculosis. The PI's laboratory has constructed a plasmid designed to express any protein of interest in fusion with Inv3, and shown that such a protein can be delivered into cells with high efficiency. RNAse P is a unique ribozyme that has a protein component (P) that serves to stabilize the catalytic RNAse. This protein domain contains several hydrophobic regions that are highly diverse among RNAse P from different cell types. Such regions can, thus, be replaced with the 22-amino acid Inv3 sequence, which will allow the RNAse P (called RNAse P-Inv3) to enter cells on its own. This is a major distinction from other RNA-based anti-infectives, such as siRNA, which cannot be readily delivered systemically. This project seeks to optimize the delivery of this RNAse P-Inv3 to inactivate murine CMV in mice. The specific aims of this pilot study are to 1) re-engineer RNAse P so that it can be stably delivered into cells at high efficiency, and initially test for its ability to inactivate CMV in vitro, and 2) apply the optimized engineered RNase P to inactivate murine CMV in mice.
This project will be done in collaboration with Fenyong Liu, who has been studying the structure and function of ribozymes, as well as CMV pathogenesis in mice. A successful demonstration of this approach to deliver RNAse P in vivo to inactivate CMV could lead to its broader application to target any pathogenic virus, including HIV. Thus, while high risk, its successful outcome may engender a completely novel paradigm for viral anti-infective development.