Knockout Mice Models
Cell culture models provide valuable information concerning miRNA expression. The question is whether agents that inhibit the miRNA activity decrease activity so low that it is essentially the same as removing the gene. A low expression does not provide a true phenotype.
Gene targeting, or gene knockout, experiments are often used to inactivate single genes in mouse systems. Conditional knockout is based on a tissue-specific inactivation of the gene of interest, which can be achieved by means of a recombinase, such as CRE recombinase.
“Years ago, in collaboration with Brian Harfe, Ph.D., Cliff Tabin, Ph.D., and Phil Sharp, Ph.D., we created a conditional Dicer knockout mouse. These low-resolution studies informed us as to the overall importance for Dicer, a miRNA processing enzyme, in a given tissue. However, ablation of Dicer affects nearly all miRNAs and it is difficult, if not impossible, to identify which miRNA(s) are responsible for a given phenotype,” said Michael McManus, Ph.D., assistant professor, UCSF.
“So I started a challenging project to ablate 100 evolutionarily conserved noncoding RNA genes in the mouse, in the hopes that we might gain better insight into the roles for miRNA expression in a given tissue. We mutate the miRNA genes and then also do a gene replacement with a colorimetric reporter gene, the LacZ gene. Now we can see at a very granular level the specific cell types that are expressing that miRNA.”
The reagents have been released for public use and are available to investigators from the public repositories.
“Given the number of investigators exploring miRNAs in human diseases, it is only a matter of time before we see interesting studies that take advantage of this increased fantastic resource. The genome is a vast sea of uncharted territory, containing many noncoding RNAs with unknown function, and we would like to expand our project to the study of other noncoding RNAs,” concluded Dr. McManus.
Delivery and Targeting Hurdles
“Since the turn of the century when miRNAs were first implicated in human cancers, the field of miRNA research has grown exponentially,” said Christopher Cheng, Ph.D., postdoctoral researcher, Yale University. “Much of the research has involved elucidating the role of miRNAs in cancer, and we are now at a time when our understanding of miRNA biology will inform the development of new therapeutics. Due to their familiar chemical nature, there is a prevailing hope that miRNAs may be readily exploited as therapeutic tools by dovetailing off of established nucleic-acid delivery strategies.”
Two potential ways, with different delivery-system requirements, exist to use miRNAs as therapeutics. One is to deliver a precursor or synthetic tumor suppressor miRNA, and the other is to deliver an agent that inhibits endogenous oncogenic miRNA.
Delivery of tumor suppressor miRNAs can capitalize on established liposomal-based or nanoscale strategies for delivering siRNA, such as those pioneered by Tekmira or Alnylam.
Challenges abound. As with most nucleic-acid therapeutics in vivo, without an effective carrier, rapid degradation would occur. Delivery vehicles provide protection against degradation, but these carriers can be cleared from the bloodstream by the immune system and as a result, typically accumulate in the liver.
Cellular challenges emerge once the agents enter the cells of interest, usually through endocytosis. In the endocytotic pathway, which is one of the cell’s natural ways for taking up extracellular components, agents can eventually be degraded in lysosomes. Therefore, a delivery vehicle must be designed to escape the endocytosis pathway, which is no easy feat.
Unlike tumor suppressor miRNAs, miRNA inhibitors do not need to directly interact with the RNA interference machinery, which allows more freedom to employ chemical enhancements and modifications and opens up new delivery options for miRNA antagonists.
Targeting remains a main hurdle. Principles learned from studies over the last several decades to improve the delivery of therapeutics to tumors are now being applied to the miRNA field.
There is still a long way to go. Currently, drugs do not exist to target all of the identified mutations. For over 30 years, researchers have been trying to target TP53, the most mutated gene in cancer, and have yet to be successful.