The obstacles and challenges for in vivo RNAi delivery are very different than in an in vitro setting. In order to achieve successful knockdown, in vivo siRNA has to survive opsonization and degradation by nucleases, target particular cells, and traffic into the appropriate cell compartment. RNAi in vivo delivery holds wonderful promise for the medical science field. A significant amount of research is now underway to define the best method to deliver RNA effector molecules for in vivo experiments as efficiently and as safely as possible.
Guidelines for successful in vivo RNAi experiments.
Choosing RNAi effector molecules
siRNA vs. RNAi vectors
RNAi can be delivered using two different approaches—siRNA synthetic duplexes or siRNA expressed from plasmids or viral vectors (shRNA, miRNAi). siRNA are becoming the method of choice for the fast development of therapeutics. They are easy to use, easy to design, and easy to synthesize. siRNA can be rapidly identified and multiple genes can be targeted at the same time. With RNAi vectors, the expression will be steadier as a result of the possibility of stable integration of the plasmid into the genome, and they have the ability to target nondividing cells such as stem cells, lymphocytes and neurons. The drawbacks are the danger of oncogenic transformation from insertional mutagenesis, and unanticipated toxicity from long-term silencing of human genes and/or having high amounts of siRNA inside the cell (Grimm D. et al.: Nature 441: 537-541 (2006)).
More information siRNA synthetic duplexes or siRNA expressed from plasmids or viral vectors (shRNA and miRNAi).
Chemically modified vs unmodified siRNAs
When delivered in vivo, standard siRNA are rapidly degraded and cleared from plasma with a half-life of minutes (Layzer J. M. et al. 2004; Karlsen A.E. et al.: Biochem Biophys Res Commun 344, 406–415 (2006)). Chemical modifications that prolong siRNA half-life, without jeopardizing biological activity, are highly desirable for successful RNAi in vivo. Stealth RNAi duplexes are 25 base pairs in length, chemically modified, and blunt-ended. They offer higher stability in serum for longer lasting knockdown effects in cells (Figure 1). The modifications on the sense strand ensure that only the anti-sense strand is utilized by the RNA induced silencing complex (RISC) to inhibit a target RNA. This decreases the potential for off-target effects since the sense strand cannot cleave unintended targets. Additionally, siRNA has the potential to activate the innate immune response, setting off defense systems usually used to combat viruses. The presence of the chemical modifications in Stealth RNAi abolish the immunostimulatory response observed with some sequences.
Figure 1 - Stealth RNAi™ siRNA is stabilized against nuclease degradation in serum.
Unmodified 21-mer dsRNA sequence (left panel) and corresponding Stealth RNAi™ siRNA sequence (right panel) at 0, 4, 8, 24, 48, and 72 hours following incubation in 10% mouse serum. Following incubation samples were separated on a Novex® 15% TBE-Urea polyacrylamide precast gel and stained with methylene blue.
Choosing the purity
Invitrogen provides three levels of siRNA purity for in vivo RNAi research, as well as an optional endotoxin test.
This purity involves standard RNAi oligo synthesis followed by HPLC purification.
Advantage: Can be conjugated with dyes
Disadvantage: Does not include extra salt and solvent removal
DSL in vivo
This purity involves standard RNAi oligo synthesis followed by diafiltration to remove salts and solvents to a level <200µS and sterile filtration.
Advantages: Available at high scale (without custom order), offers important salt and solvent removal, most cost effective
Disadvantage: Cannot be modified with dyes
HPLC in vivo
This purity involves standard RNAi oligo synthesis followed by HPLC purification, diafiltration to remove salts and solvents to a level <200µS and sterile filtration.
Advantage: Highest purity available
Disadvantage: Least cost effective due to lower yields
Tracking delivered duplexes
To ensure that the siRNA has been delivered to targeted tissues, it is necessary to track the biodistribution of duplexes. The following three strategies have been used:
Stealth RNAi™ siRNA combined with BLOCK-iT™ Fluorescent control
Stealth RNAi™ siRNA of interest can be combined with the bright BLOCK-iT™ Fluorescent control without loss of activity (Figure 3). Use the BLOCK-iT™ RNAi Express for in vivo synthetics to select from among the various scales and modifications.
Directly-labeled Stealth RNAi™ siRNA
Stealth RNAi™ siRNA can be directly labeled Alexa Fluor® labeled without loss of activity. Use the in BLOCK-iT™ RNAi Express for in vivo synthetics.
Biotinylated Stealth RNAi™ siRNA
RNAi can be biotinylated and combined with either Streptavidin-Alexa Fluor® or Streptavidin-QDots. (Figure 4). Use the BLOCK-iT™ RNAi Express for in vivo synthetics.
MDA-MB-435 cells (human breast carcinoma) were transfected using Lipofectamine™ RNAiMAX. 24 hours post-transfection, fluorescent uptake was visualized and cells harvested for RNAi analysis.
Figure 4 - Biotinylated Stealth RNAi™ siRNA can be used to evaluate transfection efficiency of biotinylated Stealth RNAi™ siRNA with flow cytometry analysis.
Cells transfected with Stealth RNAi™ siRNA biotin, subsequently harvested, and labeled with either Streptavidin-AlexaFlour® 488 or Qdot®655 streptavidin conjugate.