RNAi Delivery

siRNA must be delivered to the target cell cytoplasm in its intact double-stranded form. The main delivery challenge is maintaining the stability of the 21-23 base oligonucleotide in the in vivo environment. 11  
There are several levels of delivery challenges:
1. siRNA’s are small, so are excreted easily by urine when administered directly into the bloodstream.
2. Doubled stranded siRNA’s are unstable in the blood, i.e. are susceptible to RNase degradation.
3. Nonspecific siRNA biodistribution.
4. siRNA’s must penetrate many barriers: endothelial wall, tissue barriers.
5. Cellular uptake of oligonucleotides is dependent on endocytosis
6. RNAi activity depends on an intact double-stranded siRNA after it has overcome all the aforementioned hurdles.

To increase stability, the oligo backbones may be chemically modified.12 Specifically, siRNAs were chemically stabilized with partial phosphorothioate backbone and 2'-O-methyl sugar modifications on the sense and antisense strands. This results in greater resistance to degradation by RNases. However, this does not resolve the problem of urine excretion.
To resolve the problem of urine excretion, siRNA’s must be carried by non-viral carriers (i.e. liposomes) and shRNA’s must be carried by plasmids or viral carriers.

Cationic lipids and polymers may serve as delivery vehicles via intravenous injection, forming complexes with negatively charged siRNA’s. Sorensen et al used cationic lipids containing anti- TNF-α siRNA’s to silence lipopolysaccharide (antigen)-induced TNF-α expression.13 Furthermore, ligand-targeted liposomes may specifically target disease-state cells. For example, in cancer models, an RGD peptide covalently grafted to a PEG grafted onto the liposome delivery vehicle has shown to specifically target integrins in tumor cells. In the context of asthma, chemokine ligands may be presented via an antibody-PEG arm on the liposome and deliver anti-iNOS siRNA to the epithelial cells which overexpress CCR3.

Delivery via the lung may be more optimal for asthma. Airway (nasal) delivery of siRNA, in formulations without cationic liposomes, has been seen in mouse models knocking down GAPDH and SARS coronavirus. Furthermore, antisense oligonucleotides, have been delivered as aerosols. It remains to be seen whether this can be achieved with siRNA’s.

Currently, SIRNA Therapeutics, a biopharmaceutical company based in Colorado is working on delivery strategies for siRNA for asthma. SIRNA is focusing on targeting the IL-4 receptor (IL-4 is responsible for goblet cell hyperplasia and mucus hypersecretion). They have proprietary technology that involves chemically modifying the siRNA oligonucleotides (i.e fluorinate the backbone). 14

Viral Carriers for shRNA’s   Recent work has shown that short hairpin RNA’s (shRNA’s) are more potent inducers of gene silencing than siRNA’s. shRNA’s are usually delivered via a viral vector.15 shRNA’s increase the persistence of siRNA’s which are limited to 3-7 days for direct siRNA administration in mammalian cells.16 Viral vectors permit the efficient delivery and stable expression of shRNA constructs in a variety of mammalian cells. 17 The three classes of viral vectors are: retroviral vectors, lentiviral vectors, and adenovirus vectors.