"Hit-to-Lead" candidate drug discovery is based on the process of establishing at least 10 siRNA sequence designs for the same target genes using siRNA library screening followed by selection of the highest quality lead siRNAs. in vivo efficacy of the selected lead siRNAs is then determined in experimental animal models.
in vitro / in vivo assay development
- siRNA physicochemical character ID (size, pH, N:P Ratio etc.)
- siRNA transfection protocol optimization
- In vitro efficacy test (phenotype, cell death and aging, and cell cycle arrest)
- RT-qPCR (mRNA knock-down level)
- Western blotting (protein knock-down level)
- Animal experiment design (lead validation)
- Various modifications can be made to enhance in vivo stability.
- Sense (S) and antisense (AS) strand are partially modified and combined.
- Verification of the safety of siRNA derivatives in human serum is performed using the serum stability test.
- Analysis of siRNA derivatives superior to the lead substances using advanced assays, RT- qPCR, western blotting is conducted for validation of mRNA/protein level changes.
- Optimized siRNA candidate selection and animal experiments using candidate substances are performed.
- The siRNA pool is designed to reduce the off-target effect by using a lower concentration than the previously used concentration and to demonstrate superior therapeutic efficacy.