Researchers from TransDerm, which is developing RNAi-based treatments for skin disorders including pachyonychia congenita, last month published a report on the development of an in vivo imaging approach that can be used to rapidly screen and optimize siRNA delivery technologies.
Despite the promise of siRNA therapeutics, their use for skin diseases such as melanoma requires the development of more efficient delivery systems than are currently available, the team wrote in a paper appearing in Nucleic Acid Therapeutics.
Existing approaches, including intradermal injection and topical administration, provide “only partial inhibition of targeted genes, which may not be sufficient to achieve a therapeutic effect,” they noted. Meanwhile, methods for evaluating functional siRNA delivery such as RT-qPCR are “time-consuming and inefficient.
“The ability to intravitally image live animals for functional siRNA delivery in real time would facilitate rapid screening of delivery strategies without the need to process tissue samples post-mortem, allowing repeated imaging of the same mice, greatly reducing issues of mouse-to-mouse variability, and decreasing the number of mice required.”
As part of its ongoing effort to develop next-generation siRNA delivery approaches, TransDerm, along with collaborators from Stanford University, developed a dual fluorescent reporter xenograft melanoma model by intradermally injecting in mice human melanoma cells expressing tandem tomato fluorescent protein, or tdTFP, containing a small interfering RNA target site, as well as enhanced green fluorescent protein, or EGFP, for a normalization control, according to the paper.
The animals were then divided into two cohorts, with one receiving intratumoral injections of either siRNAs targeting the incorporated target site and complexed with a cationic lipid or complexed non-specific control siRNAs. The second cohort received the CBL3 siRNA treatment in some tumors, while other tumors were left untreated.
“As both EGFP and tdTFP signals increase as the tumor size enlarges regardless of treatment, tdTFP fluorescence signal was normalized to EGFP,” the researchers wrote in Nucleic Acid Therapeutics.
Following siRNA treatment, tumors showed decreased tdTFP expression relative to EGFP within 48 hours and continued for the duration of the treatment regimen. Overall, the team observed 65 percent knockdown of tdTFP relative to EGFP, with an 11 percent margin of error, quantified by in vivo imaging and 68 percent knockdown, with a margin of error of 10 percent, quantified by reverse transcription-quantitative polymerase chain reaction.
“No effect was observed with non-specific control siRNA treatment,” they noted.
The TransDerm researchers concluded that the xenograft model “should be useful for non-invasive systematic screening of putative melanoma target genes.” At the same time, it offers a platform on which siRNA delivery technologies can be screened and optimized.