Antisense Oligonucleotides and RNA Interference - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 365

several siRNAs or shRNAs. Moreover, siRNAs or shRNAs that do not affect the tar-
get protein act as negative controls. Arguably, one could use a “scrambled” siRNA or
shRNA for this purpose. It is better to use such scrambled siRNAs, which are known
to enter the RNAi pathway effectively without any biological activity. Some exam-
ples of such target-validated RNAs are RNA-targeting luciferase, green fluorescent
protein, and other marker genes.
3. Work at the Lowest Possible Concentrations
siRNA effectiveness depends on its specificity toward the RISC enzyme complex. This
specificity is affected by the high enzyme-to-substrate ratio. Thus, siRNA concentra-
tion should be titrated to have a correlation between degree of suppression and pheno-
type outcome. In fact, 50% of the suppression of the expression has been observed in
some siRNAs, for example, in the Hela cell at a very low concentration of 500 pM.
4. Physical and Chemical Properties
Besides the design of siRNA and its cocktails, siRNA formulations modified as conju-
gates, complexes, or with delivery carriers must meet some criteria: ensuring reproduc-
ibility of reassembly of functional complexes, incorporation efficiency, zeta potential,
polydispersity, and no aggregation in 50% mouse/human serum, with chemical stabil-
ity data of the assembly for 30 days, can provide confidence in the formulation.
5. Activity in Cell-Based Assays
Final effectiveness of formulation can be predicted on the basis of specifications
designed for a particular formulation. To augment the triumph of final formula-
tion, many companies like Sirna Therapeutics have revealed some specifications.
Explicitly these state that there should be greater than 50% reduction in target
mRNA levels by target siRNA at concentrations 10 nM in media containing 10%
serum. But control siRNA at a concentration of 10 nM in media containing 10%
serum should reduce target mRNA levels less than 10%. There should be more than
a fivefold window between IC 50 of target gene silencing and IC 50 for reduction in
viability. Activity of the delivery system should be reported in at least three cell lines
relevant to the delivery system under evaluation. Targeting moiety in a delivery plat-
form should be validated for targeting by (1) using cell lines with differential expres-
sion of the targeted receptor and (2) using assemblies with “active” and “inactive or
mutant” targeting moieties.
6. In Vivo Performance in Mouse Models
In vivo performance of the siRNA-containing delivery assemblies should be evalu-
ated in suitable animal models. A comparison of a single intravenous dose at
1, 3, and 9 mg/kg for target siRNA-containing delivery assemblies with control
should be provided. Delivery platform with “targeting moeity” as well as assemblies