The parasites of the genus Cryptosporidium are important causes of diarrheal diseases worldwide. However, there is currently no reliable drug treatment for cryptosporidiosis in compromised hosts. A major bottleneck for developing drugs and vaccines against Cryptosporidiosis is the lack of methods to study gene function in this parasite. Silencing of genes by RNAi interference is a powerful method to study gene function that has been widely used in the identification of targets for several pathogens. Unfortunately, as Cryptosporidium does not have the enzymes of the RNAi pathway, its genes cannot be silenced by standard siRNA technology. Thus, it is not amenable to High-throughput screening. To circumvent that problem we have developed a unique and fast strategy to knock down genes in Cryptosporidium by reconstituting the effector arm of the siRNA pathway. We have induced silencing in Cryptosporidium by transfecting parasites with hybrid complexes formed between recombinant human Argonaute (hAgo2) and Cryptosporidium-single strand RNA (ssRNA). A standardized method based in this strategy could be used to systematically study the function of genes of Cryptosporidium. The overall objective in this proposal is to optimize and standardize our novel method to silence genes in Cryptosporidium. A standardized method resulted from this proposal will be essential to scale this method for high throughput screening for target validation. Our long-term goal is to use reverse genetics to develop novel therapies against cryptosporidiosis.
The specific aims of this proposal are to:
Aim 1 : Optimize silencing in Cryptosporidium. 1a) Optimizing potency of ssRNAs using ssRNApools. We hypothesized that combinations of potent complexes used against a specific target, could act synergistically to enhance target silencing. 1b) Optimizing transfection of silencer complexes. We want to test the hypothesis that transfection with liposomes or novel cell-penetrating peptides will enhance the transfection.
Aim 2. Test for synergism of knocking down multiple targets in silencing pathways. We hypothesize that targeting multiple genes from a pathway will provide a clearer phenotype by overcoming redundancy. After completing of these studies, we will have in hand a rapid method to assess the function of genes in Cryptosporidium. This should greatly accelerate the identification of novel targets for drugs and vaccines for cryptosporidiosis.
Cryptosporidiosis is estimated to give rise to over 100 million cases of diarrheal disease worldwide causing an estimated 750,000 per year within the U.S. To date, there is no reliable- effective treatment; in this study we will develop a rapid method to elucidate the role of key genes during Cryptosporidium infection. This research will lead to discover novel targets useful to develop novel treatments against Cryptosporidiosis.
