The advances made recently toward the in vivo applications of RNAi in vertebrate systems are critical towards developing siRNA as therapeutics [21–23]. These in vivo applications, however, do not yet apply to central nervous system function because siRNA do not readily cross the blood brain barrier (BBB) via systemic delivery. Because drugs may bypass the BBB by delivering them directly into the CSF, such delivery routes may be exploited in order to determine whether neurons and other cells in the nervous system may be amenable to RNAi by siRNA in vivo. Two recent reports used intrathecal delivery to prevent hypoxia-induced expression of brain derived neurotrophic factor in the spinal cord , or to knockdown the expression of the purinergic receptor subtype, P2X3, in sensory primary afferent . While both studies demonstrated that siRNA attenuated the intended gene target, the former was a short intervention (3.5 hr) while the experimental subject was under general anesthesia, whereas the latter required very high concentration and continuous infusion of siRNA (400 μg/day for up to 7 days) to elicit a modest knockdown of the target. It is not clear whether the high dose of siRNA needed to effect reflects an inefficient RNAi mechanism in neurons or whether the modulation of gene function cannot be sustained over time.
Our data show that in the presence of a suitable transfection agent, the siRNA to the DOR is highly effective at a low concentration of 2 μg, or 0.14 nmol, given once a day. This dose is 23 times lower than the amount of antisense ODN required to elicit a knock down of the DOR as previously published , and is substantially lower than the effective dose reported for the knock down of the P2X3 receptors in the DRG . The siRNA treatment delivered without the transfection reagent was without effect, suggesting that the transfection reagent significantly enhanced the uptake of the siRNA into target cells, as verified by the detection of fluorescence uptake of spinal cord and DRG tissues after injecting the tagged siRNA. This observation is consistent with recent evidence that the uptake of siRNA by neurons in culture is likely a key limiting step in the siRNA mediated gene silencing . The amount of RNA delivered as a bolus dose in transfection reagent tends to be limited by the solubility of the RNA. The transfection reagent used in the present study was i-Fect™, which is a cationic lipid mixture that has been optimized for the delivery of short oligonucleotides in vitro . This reagent was chosen for the present study because the RNA/lipid complexes remain in suspension at a fairly high concentration in a volume that is suitable for intrathecal delivery. A maximum of 2 μg of RNA can be given in a 10 μL injection volume. Should a daily dose of >2 μg is desired, the delivery can be adjusted by giving multiple doses.
A significant knockdown of DOR transcripts by siRNA treatment is consistent with the proposed mechanism of action of RNAi [25, 26]. In this regard, the knockdown of the DOR transcripts was observed in both the dorsal horn of the spinal cord as well as the lumbar DRG, demonstrating that the siRNA interferes with the synthesis of both the presynaptic and the postsynaptic populations of DOR, which is also highly consistent with the uptake pattern of the tagged siRNA. The effects of siRNA culminate in a significant reduction of DOR immunoreactivity and ligand binding capacity in the superficial laminae of the dorsal horn of the spinal cord where the functional receptors are predominantly located. The loss of functional DOR is evident by the loss of antinociceptive activity of the DOR selective agonist, DELT, given intrathecally. Together, these results justify our conclusion that we have established an effective method for delivering siRNA that interferes efficiently with the expression and function of target genes in both the peripheral nervous system (i.e., sensory primary afferent) and the central nervous system (i.e., spinal cord).
The use of a mismatch RNA confirms the specificity of the siRNA sequence for the DOR. The siRNA treatment had no effect on the expression or the function of the highly homologous receptor type, MOR, further supporting the target specificity of the siRNA employed here. Finally, the effect of the siRNA is fully reversible; thus the observed effects of the siRNA are specific to the use of siRNA, and the treatment paradigm does not precipitate any long-term effects due to toxicity such as motor impairment. This paradigm can be easily adjusted for dosage and duration of treatment, and is based on a well-established, relatively non-invasive method of drug delivery that has general applications for spinal and peripheral targets. The reagents required are minimal and economical, and can be adapted for other gene targets. Our findings support the hypothesis that siRNA can be effectively applied to modulate nervous system function. The significant knockdown of the target transcripts in both the DRG and the spinal cord conforms with the established mechanism of siRNA mediated gene silencing, and is consistent with the uptake of siRNA seen in both the peripheral neurons (i.e. DRG) and the central nervous system (i.e., spinal cord). The low dose of siRNA further suggests that the efficacy of RNAi depends critically on the efficient delivery of the siRNA to the target tissues. Unlike antisense oligodeoxynucleotides, siRNA may be delivered systemically . Thus, chemical modifications that enhance systemic stability and facilitate siRNA transport across the BBB or the uptake of siRNA by neurons would greatly advance the potential of siRNA as therapeutic.