Mucosal Delivery of RNAi Therapeutics - RNA Interference from Biology to Therapeutics - page 104

transfection agent Mirus TKO in a RSV mouse model. In this study, viral titres were
reduced several logs after intranasal administration with no adverse or immunos-
timulatory effects observed [ 63 ]. Alvarez et al. likewise, was able to reduce RSV
titres (2.5-3 log reduction, 100 mg single dose) using the naked Alnylam siRNA
against RSV nucleocapsid gene (ALN-RSV01) after intranasal delivery in mice. By
RACE analysis of the ALN-RSV01 cleavage product, it was also confirmed that the
reduction of the viral titre was in fact an RNAi-mediated effect [ 76 ] . Recently, the
results from an Alnylam phase II clinical trial was published showing a 38% reduc-
tion of experimentally RSV infected test subjects receiving ALN-RSV01 (150 mg)
compared to subjects receiving a placebo [ 14 ]. A nasal spray was used to deliver the
PBS/ALN-RSV01 solution. As mentioned by the authors of the report, the induced
RSV infection in the study resulted in a mild to moderate upper respiratory tract
illness in the region the nasal spray is likely to reach. Studies are currently under-
way to evaluate the effect of ALN-RSV01 in naturally infected patients, and these
are likely to use aerosolised delivery methods in order to reach both the upper and
lower respiratory segments simultaneously. The success achieved in studies using
non-formulated siRNA is unexpected when one considers the polyionic nature of
the siRNA molecule that restricts cellular uptake. A possible explanation could be
loss of epithelial integrity due to infection that might allow entry of naked siRNA.
Nonetheless, at the time of writing, the Alnylam RSV programme is one of the most
advanced RNAi clinical trial programmes and the simplistic naked siRNA approach
could fulfil the clinical requirement of cost-effectiveness.
Whilst direct administration of naked siRNA to the mucosa has been extensively
used, the susceptibility of the duplex to serum nucleases makes intravenous (i.v.)
delivery less attractive. Modification of the siRNA backbone, however, is now stan-
dard to reduce serum degradation [ 77 ]. In a recent study, serum stability and silenc-
ing of enhanced green fluorescent protein (EGFP) in the bronchoepithelium of mice
have been demonstrated after i.v. administration of naked LNA modified siRNAs
[ 66 ]. Intravenous injections of naked LNA modified siRNA (five doses of 50 m g
siRNA) resulted in comparable reduction of EGFP (55% reduction) in the bron-
choepithelium as animals dosed intranasally with chitosan/siRNA particles (single
30 mg dose). Naked modified siRNA was less effective after intranasal dosing. The
authors suggest that the success of the naked modified siRNA to reach the lung
epithelium after i.v. injection might result from increased serum stability, allowing
for longer circulation time compared to unmodified siRNAs.

5.3.3 Nanoparticle Delivery

It is generally accepted that nanoparticle-based systems are needed to improve the
therapeutic potential of the siRNA despite the success of naked siRNA. The ability
to package high levels of siRNA into nanoscale carriers with a predisposition
to enter cells has promoted their use. Two prominent classes are polyplexes and
lipoplexes formed by self-assembly of polycations or cationic lipids with siRNA