In rodent experiments, in vivo transfection of siRNA has been achieved using a number of different vectors in a number of different organs. Surprisingly, even naked, synthetic siRNA has been successfully introduced and functioned in the cells of organs such as liver and kidney following hydrodynamic injection in mouse tail veins. However, naked synthetic RNA is degraded in serum unless it is modified for stabilization making this transfection route infeasible for clinical use. Cationic liposomes, polyethylenimine (PEI) vectors and atelocollagen are alternate efficient methods to deliver synthetic siRNAs by either topical or systemic injection. However, if synthetic siRNAs are used, RNAi will remain a transient phenomenon.
To achieve long-term, stable expression of siRNA, plasmid vectors with RNAi expression units driven by RNA polymerase III (Pol III) promoters U6 and H1 have been developed by several researchers (71
). Pol III transcribes significantly shorter RNAs than those transcribed by RNA polymerases I and II (pol II), with a short stretch of about 5 thymidine residues serving as the termination signal. The expression cassette contains an shRNA sequence of an inverted repeat of 19–29 nt separated by a short spacer sequence. The transcribed RNA molecule forms short hairpin RNA that is indistinguishable from siRNAs in terms of RNAi efficiency and mechanism ().
Figure 6 Scheme of RNA polymerase III (Pol III) driven, small hairpin RNA (shRNA) expression cassette. Pol III promoter (H1 or U6 promoter) is followed by DNA template that corresponds to the target sequence of the gene to be suppressed. The motif of inverted (more ...)
By using a RNA polymerase II promoter, Xia and colleagues developed an shRNA-expression plasmid with a modified CMV promoter, a downstream shRNA template with inverted repeat and a minimal polyA cassette. In this expression unit, the hairpin sequence was placed immediately adjacent to the transcription initiation site of the promoter (74
Ad and AAV vectors with a pol II or modified CMV shRNA expression unit have been demonstrated to induce in vivo
RNAi in neural tissues. In one study, an Ad vector with a modified CMV expression cassette of shRNA against eGFP was directly injected to the basal ganglia of transgenic mice expressing eGFP. Western blot analysis showed that eGFP expression was diminished in the injected hemisphere 5 d post injection (74
). The same group also injected an AAV vector with the H1-shRNA expression cassette into the cerebellum of a transgenic murine model of spinocerebellar ataxia type 1. Expression of shRNA targeting human ataxin1
was verified 10 d after the injection (51
). When they injected AAV.U6-shRNA vectors into the basal ganglia of a murine model of Huntington’s disease, shRNA expression against the human Huntington’s disease gene was detected 21 d post injection (52
). In these experiments using AAV vectors, a CMV-driven, eGFP expression unit was included in the chimeric viral constructs, and eGFP expression was detected in the injected sites up to 5 months later.
Lentiviral vectors with H1-shRNA expression units have been used to treat a mouse model of amyotrophic lateral sclerosis (ALS) that expresses a human SOD1
mutant. The SOD1
mutant causes a dominantly inherited form of ALS through a gain-of-function mechanism, and the lentiviral-mediated shRNA selectively targets the expression of the mutant gene. In two studies using these animals, lentiviral vectors were injected intraspinally (75
) or intramuscularly (utilizing retrograde axonal transport to motoneurons) (76
). Western blotting and immunofluorescence demonstrated a reduction in SOD1 mutant protein expression 15–50 d after spinal injection and an improvement was observed in motor neuron survival and motor ability 80–100 d after the spinal and intramuscular injection.