Biological barriers impede the application of bio-macromolecule based drugs in the clinics . Small interfering RNA (siRNA) based drugs can be delivered to cells via endocytosis to silence the expression of disease causing genes [1, 2]. A major bottleneck in delivery, however, is the escape of siRNAs from endosomes to cytosol, where they load into the RNA induced silencing complex and degrade the target mRNA [2, 3]. Moreover, the mechanism of endosomal siRNA escape remains evasive to understanding.
Aiming to better understand and improve the siRNA escape from endosomes, our lab has previously performed a cell based high throughput assay (HTS) to identify small molecule siRNA delivery enhancers using two well established siRNA delivery systems: lipid nanoparticles and cholesterol-siRNAs from Alnylam Pharmaceuticals. We identified 52 enhancers showing specificity towards either delivery system and improving the efficiency by 2 to 5 fold. Interestingly, our analysis showed that compounds either act on the delivery system itself or the cell, by modulating the endocytic system .
Encouraged from our previous screen in HeLa cells, we are identifying new siRNA delivery enhancers (supported by the PathChooser program) in a mouse liver cell line using HTS for three reasons:
- There are multiple liver diseases that could be siRNA therapeutic targets such as hypercholesterolemia, hepatocellular carcinoma, amyloidosis etc. Although most siRNA delivery systems reach liver by default, the efficiency is limited by siRNA endosomal escape. Bypassing this limitation by increasing the siRNA dose leads to liver toxicity. Therefore, enhancing the siRNA endosomal escape will reduce the therapeutic dose and improve the therapeutic safety and efficiency.
- Liver is a reasonable target where delivery challenges such as siRNA endosomal escape can be easily overcome.
- Since our lab is specialized in various techniques and tools to study liver, further proof of principle studies in vitro and in vivo would be more suitable.
Additionally, this HTS is designed to induce hepatocyte polarity in FL83B-GFP cells using extracellular matrix and maintain them at a quiescence stage. This setup is more relevant to physiological conditions and is also screenable using high throughput technologies. This means, the transferability of this approach from in vitro to in vivo is expected to be more successful.
The pragmatic approach to overcome the endosomal siRNA escape challenge is to dissect this process at subcellular level. To dissect the mechanism of delivery enhancement by compounds , we developed a new in vitro assay that measures the siRNA escape quantitatively from purified early endosomes and the biophysical properties of endosomal membrane. Using this assay, we identified compounds that increase the siRNA escape from endosomes. The biophysical properties of membrane in the presence of compounds are under investigation to understand this process further.
The low endosomal escape is a major obstacle for clinical usage of other bio-macromolecules such as CRISPR-CAS9 components, antibodies and proteins. We hope that understanding the mechanisms and establishing methods to enhance the endosomal escape in our study will contribute to overcome the barriers of many such bio-macromolecules.
- Kanasty, R et al., D. Delivery materials for siRNA therapeutics. Nature Materials 12, 967-977 (2013).
- Gilleron, J. et al., Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nature Biotechnology 31, 638-646 (2013).
- Gilleron Jⱡ, Paramasivam Pⱡ et al., Identification of siRNA delivery enhancers by a chemical library screen. Nucleic Acids Research. 1-18, (2015).