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Lipophilic siRNAs mediate efficient gene silencing in oligodendrocytes with directCNS delivery
Qingmin Chen ⁎, David Butler, William Querbes, Rajendra K. Pandey, Pei Ge, Martin A. Maier, Ligang Zhang,Kallanthottathil G. Rajeev, Lubomir Nechev, Victor Kotelianski, Muthiah Manoharan, Dinah W.Y. Sah ⁎Alnylam Pharmaceuticals Inc., 300 Third Street, Cambridge, MA 02142, USA
⁎ Corresponding authors. Sah is to be contacted at Te617 551 8102. Chen, Tel.: +1 617 551 8385; fax: +1 6
Conjugation of small interfering RNA (siRNA) with lipophilic molecules has been demonstrated to enhancecellular uptake in cell culture and to produce efficient endogenous gene silencing in the liver after systemicadministration and in neurons after direct local injection. Here, we evaluated the in vivo delivery of siRNAsconjugated with different linkers to cholesterol by targeting CNPase (2′-3′-cyclic nucleotide 3′-phosphodiesterase) in oligodendrocytes. Cholesterol-conjugated siRNAs administered to the rat corpuscallosum by intraparenchymal central nervous system (CNS) infusion show improved silencing abilitycompared with unconjugated siRNA. Furthermore, conjugation of siRNA to cholesterol with a cleavabledisulfide linker appears to be beneficial for improving the potency of silencing of CNPase mRNA inoligodendrocytes in vivo. Taken together, these findings indicate that cholesterol-conjugated siRNAs areeffective for direct CNS delivery to oligodendrocytes, and that the biocleavable disulfide linker appears to bebeneficial for improving the potency of silencing of target mRNA in vivo.
RNA interference (RNAi) induced by small interfering RNAs(siRNA) is a highly efficient method for specific gene knockdownthat has been applied widely not only as a powerful tool for validationof targets implicated in disease, but also as the basis of noveltherapeutic drug candidates for the treatment of disease. However, invivo delivery of siRNA has been a key challenge for RNAi-basedtherapeutic applications. A wide variety of approaches including viral-vector based and non-viral delivery systems, such as liposomes,nanoparticles, lipophilic conjugates, polymers and cell penetratingpeptides [1–6] have been investigated to enhance target cell uptakeand silencing potency in vivo. A number of laboratories have reportedefficient knockdown of endogenous genes in several mammalianorgans (liver, lung, spleen, kidney, pancreas and muscle) withsystemic delivery, nasal instillation or local injection [7–11]. However,attempts to use RNAi to silence a desired gene in the brain havefocused on a specific cell type–neurons. Most of these studies usedviral delivery of short hairpin RNA (shRNA) to achieve efficientknockdown of endogenous or reporter genes in the brain [12–16].Wang et al. also succeeded in reducing huntingtin mRNA in newbornmice by using siRNA complexed with lipofectamine [17]. Althoughthese approaches appear to be effective for studies in the rodent
central nervous system (CNS), the lack of control over amount andtiming of delivery with the use of viral vectors, and potential toxicityof transfection agents restrict the use of these methodologies in theclinic.
siRNA conjugated to lipophilic moieties exhibit markedly im-proved cellular uptake in vitro [18], and conjugation of siRNAs tocholesterol enhances uptake in the liver and elicits robust RNAi, whichresults in silencing of endogenous genes in vivo [5,19]. More recentstudies suggest that neurons take up cholesterol-conjugated siRNAmore readily than unconjugated siRNA. The local injection ofcholesterol-conjugated siRNA targeting huntingtin achieved efficientknockdown of the target gene in neurons of the mouse CNS [20]. Werecently demonstrated that siRNAs simply formulated in saline arehighly effective in reducing target mRNA expression in oligodendro-cytes in vivo after direct CNS delivery, inhibiting an endogenousoligodendrocyte-specific gene in the rat as well as non-humanprimate CNS [21]. In the work described here, using directintraparenchymal CNS infusion, we evaluated the delivery of siRNAsconjugated to cholesterol to oligodendrocytes by targeting CNPase(2′-3′-cyclic nucleotide 3′-phosphodiesterase), which is expressedexclusively in the CNS by oligodendrocytes. Previous studies haveshown that disulfide-based conjugation aids the identification offormulations with high transfection potency, and facilitates receptor-mediated gene delivery [22]. For bioconjugation, the nature of thelinker used between the drug and the delivery-augmenting moietymay affect biological activity. The cleavable yet relatively stable natureof disulfide bonds renders this bond attractive in designing drug
ng in oligodendrocytes with direct CNS delivery, J.
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delivery systems. Successful application of disulfide-based bioconju-gation have included targeted delivery and enhanced cytosolicdelivery, improved pharmacokinetics and increased stability. There-fore, in the studies reported here, we used a reducible and cleavabledisulfide linker versus a non-reducible and non-cleavable alkyl linkerto prepare the cholesterol-conjugated siRNAs. With these twodifferent conjugates, as well as the unconjugated siRNA, the efficacyin vivo was investigated after direct infusion into the rat corpuscallosum. Here, we demonstrate that cholesterol-conjugated siRNAsare effective for direct CNS delivery to oligodendrocytes, andmoreover, that the cleavable disulfide linker improves potency in vivo.
2. Material and methods
2.1. Materials
All single-stranded RNAs were synthesized at Alnylam Pharma-ceuticals andwere characterized by ESI-MS and anion exchange HPLC.Single-stranded RNAs were synthesized as 19-mers with deoxythy-midine (dT) overhangs including 2′-O-Me substitutions (lower caseletters) on a subset of internal nucleotides, and phosphorothioate (s)linkages to provide stabilization against nucleases. siRNA sequencesand chemical modifications are as follows. CNPase siRNA (siCNP):antisense strand 5′-UCUCuAAGAGGUcAAGGCCdTsdT-3′, sense strand5′-GGccuuGAccucuuAGAGAdTsdT-3′; Cholesterol–CNPase–siRNA(siCNP–Chol): antisense strand 5′-UCUCuAAGAGGUcAAGGCCdTsdT-3′, sense strand 5′-GGccuuGAccucuuAGAGA-chol-3′; Cholesterol-mismatch-siRNA (siMM-Chol): 5′-UcACuAAcACGUcAACGCCdTsdT-3′,sense 5′-GGcGuuGAcGuGuuAGuGA-chol-3′; Disulfide–cholesterol–CNPase siRNA (siCNP–S–S–Chol): antisense strand 5′-UCUCuAAGAG-GUcAAGGCCdTsdT-3′, sense strand 5′-GGccuuGAccucuuAGAGA–S–S–Chol-3′; Disufide–cholesterol-mismatch-siRNA (siMM–S–S–Chol): 5′-UcACuAAcACGUcAACGCCdTsdT-3′, sense 5′-GGcGuuGAcGuGuuA-GuGA-S-S-chol-3′; Cholesterol–luciferase siRNA (siLUC–chol): anti-sense strand 5′-UCGAAGuACUcAGCGuAAGdTsdT-3′, sense strand 5′-cuuAcGcuGAGuAcuucGA-chol-3′. To generate siRNAs from RNA singlestrands, equimolar amounts of complementary sense and antisensestrands were annealed.
2.2. Direct CNS infusion in rats
Animal maintenance and surgical procedures conducted atAlnylam were approved by the Institutional Animal Care and UseCommittee. Male Sprague–Dawley rats were anesthetized withketamine/xylazine mixture before surgery, and then placed into astereotaxic instrument (Benchmark™ Digital Stereotaxic, myNeur-oLab). A 30 gauge osmotic pump infusion cannula (Plastics One,Wallingford, CN) was implanted into the right hemisphere, targetingthe corpus callosum (stereotaxic coordinates anteroposterior—(AP)0.7 mm relative to bregma, mediolateral (ML) 2.2 mm and dorsoven-tral (DV) 3.0 mm relative to skull surface; incisor bar 3.3 mm belowthe interaural line) (Fig. 2A). Osmotic pumps (10 µl/h flow rate, Alzet)containing PBS or siRNAs formulated in PBS were primed in 0.9%saline overnight at 37 °C according to the manufacturer's instructions,and then connected to the cannula and implanted subcutaneously. Amaximum concentration of 3 mg/ml siRNA administered to rats at aflow rate of 10 μl/h corresponds to a dose of 0.72 mg/day/rat. After 3–7 days of infusion, rats were euthanized and brains removed. FormRNA quantitation, coronal slices, 1 mm thick, through the rat brainfrom anterior to posterior were obtained using a brain matrix(Braintree Scientific, Braintree, MA) and corpus callosum ipsilateralto the infusion was dissected from each slice, snap-frozen in liquidnitrogen, and then stored at −80 °C until mRNA quantitation wasperformed.
Please cite this article as: Q. Chen, et al., Lipophilic siRNAs mediate efficControl. Release (2010), doi:10.1016/j.jconrel.2010.02.011
2.3. CNPase mRNA quantitation
The QuantiGene assay (Genospectra) was used to quantify mRNAlevels in tissue sample lysates according to the manufacturer'sinstructions (Panomics). CNPase mRNA levels were normalized toMBP mRNA levels and then further normalized to PBS.
2.4. Evaluation of tolerability of disulfide–cholesterol–CNPase siRNA
Sprague–Dawley rats were evaluated for tolerability of direct CNSinfusion of disulfide–cholesterol–CNPase siRNA. Direct CNS infusionwas performed as described above. Rats were monitored daily forsurvival, general health and body weight before and during infusion.Three days after infusion of PBS or 0.3, 1 or 3 mg/ml disulfide–cholesterol–CNPase siRNA into the corpus callosum at a flow rate of10 µl/h, the animals were perfused with 4% paraformaldehyde. Brainswere removed, fixed overnight in the same fixative, and thencryoprotected in 30% sucrose and cut into 40 μm thick sectionsencompassing the cannula site. Sections were stained with hematox-ylin and eosin (H&E) for light microscopic evaluation.
2.5. Statistical analyses
Statistical analysis of the data was performed using GraphPadPrismV4.0. The normalized CNPasemRNA levels in siRNA ormismatchvs. PBS-treated groups were compared by one-way ANOVA withDunnett's post-hoc test for significant difference between groups. Pvalues of less than 0.05 were considered significant.
3. Results
3.1. Specific knockdown of CNPase siRNA in rats by direct CNS infusion ofcholesterol-conjugated siRNAs
Our previous work showed that direct intraparenchymal infusionof naked CNPase siRNA results in uptake into oligodendrocytes, androbust suppression of CNPase mRNA levels [21]. Here, we evaluatedthe silencing effect of cholesterol-conjugated siRNAs with differentlinkers in rat corpus callosum. Cholesterol-conjugated siRNAs weresynthesized by linkage of cholesterol to the 3′ end of the sense strandvia a non-cleavable alkyl linker or a cleavable disulfide linker (Fig. 1).To evaluate silencing in oligodendrocytes with cholesterol-conjugat-ed siRNAs delivered by direct CNS infusion, we used a QuantiGeneassay to measure mRNA levels in rat corpus callosum (Fig. 2) ofCNPase, an oligodendroctye specific gene in the CNS, relative to MBPmRNA. As shown in Fig. 2B, after a 3 day infusion, rats treated withCNPase siRNA conjugated to cholesterol via a non-cleavable linkerexhibited significant knockdown of CNPase siRNA. Cholesterol-conjugated siRNA with a cleavable disulfide linker resulted in greaterreduction of CNPase mRNA (Fig. 2B). In contrast, all control groups,cholesterol-conjugated siRNAs targeting the non-mammalian geneluciferase (siLUC), or mismatch CNPase siRNAs (siMM), showed noCNPase mRNA suppression compared to the PBS-treated controlgroup (Fig. 2B), supporting the specificity of CNPase silencing by thecholesterol-conjugated CNPase siRNAs.
3.2. Improved and durable silencing of CNPase siRNA in rats bycholesterol-conjugated siRNA with cleavable linker
To further assess and optimize the efficiency of knockdown in thebrain using lipophilic siRNAs, we compared the silencing effects ofcholesterol-conjugated siRNAs with different linkers and unconjugatedsiRNA, andexamineddurabilityof silencingwith ansiRNAconjugated tocholesterol via a biocleavable disulfide linker. As shown in Fig. 3A,compared to the PBS-treated control group, animals treated withunconjugated siRNA did not show any silencing at an siRNA
ient gene silencing in oligodendrocytes with direct CNS delivery, J.
Fig. 1. Conjugation of siRNA to cholesterol with two different linkers. ConjugatedsiRNAs were synthesized by linkage of cholesterol to the 3′ end of the sense strand via anon-cleavable alkyl linker or a cleavable disulfide linker.
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concentration of 1 mg/ml. However, cholesterol-conjugated siRNAswith either cleavable or non-cleavable linkers produced substantialreduction of CNPase mRNA at this concentration. These results suggestthat siRNA conjugated to cholesterol is more potent than unconjugatedsiRNA in silencing CNPase mRNA in oligodendrocytes in vivo. Choles-terol-conjugated siRNAwith the cleavable disulfide linker showeddose-dependent silencing of CNPase and achievedmore suppression than thecholesterol-conjugated siRNAwith the non-cleavable linker at the samedoses (Fig. 3A). These data demonstrate that siRNA conjugated tocholesterol with a cleavable disulfide linker is beneficial for improvingpotency of silencing of CNPase mRNA in oligodendrocytes in vivo.
RNAi has been demonstrated to be durable in multiple tissues[9,23,24]. To evaluate the persistence of CNPase mRNA reduction aftersiCNP–S–S–Chol was infused into the rat corpus callosum, siCNP–S–S–
Fig. 2. Demonstration of effective delivery of cholesterol-conjugated CNPase siRNA to oligodendof brain slab shows the infusion site (asterisk), and region of corpus callosum (outlined in red)MBPmRNA, and then expressed relative to the PBS control group) after a 3 day infusion at 10 ul/Chol), siMM–S–S–Chol (mismatch siRNA for siCNP–S–S–Chol) or siLUC-chol (n=5–6 per groDunnett's post-hoc test; **pb0.01. (For interpretation of the references to color in this figure l
Please cite this article as: Q. Chen, et al., Lipophilic siRNAs mediate efficControl. Release (2010), doi:10.1016/j.jconrel.2010.02.011
Chol was infused for 3 days, and then after 1, 3 or 7 day recoveryperiods, CNPase mRNA levels were quantified. CNPase mRNA levelscontinued to be suppressed significantly even after a 3 day recoveryperiod, with no significant silencing remaining after 7 days ofrecovery (Fig. 3B).
3.3. Evaluation of tolerability of disulfide–cholesterol–CNPase siRNA innormal rat CNS
To evaluate the tolerability of siCNP–S–S–Chol, rats were evaluatedduring and after 3 day infusion of 0.3, 1 or 3 mg/ml siCNP–S–S–Cholinto the corpus callosum. All animals survived and gained weightduring treatment, and did not develop any abnormal behavior duringthe infusion period. At the end of the 3 day infusion, rats wereeuthanized and brains removed. From gross inspection, only ratsinfused with 3 mg/ml siCNP–S–S–Chol showed increased tissuedamage at the infusion site compared to the PBS-treated group.With H&E staining (Fig. 4), histological examination revealed nosignificant differences in brain areas adjacent to the cannulation sitein rats treated with siCNP–S–S–Chol at 0.3, 1 or 3 mg/ml (Fig. 4B, Cand D, respectively), compared to rats treated with PBS (Fig. 4A).
4. Discussion
RNAi therapeutics have great potential for treating neurologicaldiseases ranging from acquired disease, such as viral infections, topurely genetic disorders. The main challenges for successful applica-tion of RNAi are targeting of the relevant cell type, and promotion ofcellular uptake and release of siRNA into the cytoplasm in vivo.Conjugation to various ligands has been shown to improve thedelivery of siRNAs [25,26]. In particular, conjugation of siRNAs tocholesterol has been demonstrated to enhance cellular uptake in cellculture and achieve efficient endogenous gene silencing in cellsoutside of the CNS [5,27] and in neurons in the brain [20]. In thisstudy, local infusion of CNPase siRNAs conjugated to the lipophilicmolecule cholesterol was performed to evaluate delivery to anotherimportant cell type in the brain — oligodendrocytes. We demonstratefor the first time that siRNAs conjugated to a lipophilic molecule areefficiently delivered to rat oligodendrocytes in vivo and achievesignificant specific gene knockdown in these cells.
rocytes in rats with intraparenchymal infusion into the corpus callosum. (A) Coronal viewdissected for mRNA quantitation. (B) Quantitation of CNPase mRNA levels (normalized toh of PBS, or 3 mg/ml siCNP-Chol, siCNP–S–S–Chol, siMM-Chol (mismatch siRNA for siCNP-up). Data are shown as mean±SEM. Statistical analysis was done with one-way ANOVA,egend, the reader is referred to the web version of this article.)
ient gene silencing in oligodendrocytes with direct CNS delivery, J.
Fig. 4. Histology with H & E staining at and near infusion site after direct corpuscallosum infusion of siCNP–S–S–Chol. Coronal brain sections from rats infused with PBS(A), or siCNP–S–S–Chol at 0.3 mg/ml (B), 1 mg/ ml (C) or 3 mg/ml (D).
Fig. 3. Dose-dependence and durability of CNPase mRNA suppression by cholesterol-conjugated siRNA with non-cleavable or cleavable disulfide linker. (A) Comparison ofsiCNP, siCNP-Chol and siCNP–S–S–Chol at 1 mg/ml dose and dose-dependent silencingof CNPase mRNA after 3 day infusion at 10 ul/h of cholesterol-conjugated CNPase siRNAwith cleavable disulfide linker (siCNP–S–S–Chol) at 0.1, 0.3 or 0.6 mg/ml (n=5–6 pergroup). (B) Durability of CNPase mRNA suppression, assessed 1, 3 or 7 days followingtermination of 3 day infusion at 10 ul/hr of siCNP–S–S–Chol at 0.6 or 1 mg/ml (n=6 pergroup). Data are shown as mean±SEM. Statistical analysis was done with one-wayANOVA, Dunnett's post-hoc test; **pb0.01; *pb0.05.
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The present study shows that the reduction of target mRNAinduced by siRNAs conjugated to cholesterol is more efficient thanthat by unconjugated CNPase siRNA, indicating that cholesterol-conjugated siRNA can be delivered to oligodendrocytes by directlocal administration and that cholesterol conjugation may improvethe uptake of siRNA in these cells. This results are consistent withprevious studies showing that conjugation of siRNA to cholesterolenhances its uptake and results in silencing of a specific endogenousgene in liver after systemic administration [5,19]. Cholesterol-conjugated siRNAs also have been successfully delivered to lungand brain using local administration, producing reduction of thetarget gene [20,28]. Conjugation with cholesterol may enhancesiRNA uptake by receptor-mediated endocytosis or by an increasedmembrane permeability of the otherwise negatively charged RNA,resulting from increased hydrophobicity and cellular association dueto lipid conjugation. Recent reports demonstrate that cholesterol-conjugated siRNAs are taken up in vivo by a lipoprotein-dependentmechanism and that LDL-and HDL-receptors play key roles in thisuptake [19]. The presence of LDL receptors in rat brain has beendemonstrated by immunocytochemistry and expression of thesereceptors is higher in areas of white matter [29]. The expression ofLDL receptors by brain cells suggests that the central nervous systemhas a mechanism for lipid transport and cholesterol homeostasissimilar to that of other tissues and that the uptake of cholesterol-
Please cite this article as: Q. Chen, et al., Lipophilic siRNAs mediate efficControl. Release (2010), doi:10.1016/j.jconrel.2010.02.011
conjugated siRNAs in oligodendrocytes may occur via lipoproteinreceptors.
The disulfide bond (–S–S–) is usually formed from the oxidation oftwo thiol (–SH) groups. While it is relatively stable in the circulationand in the oxidizing extracellular space, this disulfide bond may beprone to rapid cleavage under a reductive intracellular environment[30,31], resulting in the formation of two thiols and separation of thecholesterol moiety from the siRNA. In normal and pathophysiologicalstates, a low concentration of glutathione (GSH) in body fluids (e.g.blood) and in extracellular spaces results in a relatively high redoxpotential, which stabilizes disulfides. In contrast, a high concentrationof GSH inside cells maintains a highly reducing environment insidecells [32]. The presence of a high redox potential gradient between theintracellular and extracellular environments can be leveraged forimproving intracellular delivery of a variety of bioactive moleculesincluding siRNA as well as DNA, antisense oligonucleotides, proteinsand low molecular weight drugs [33].
The disulfide linker demonstrates optimal characteristics withstability in plasma [34] and extracellular fluids such as CSF [35], but israpidly cleaved in rat brain in vitro [36] and in vivo [37]. Therefore, inoptimizing the delivery of cholesterol-conjugated CNPase siRNAs, weinvestigated the siRNA conjugated to cholesterol with a disulfidelinker. We demonstrated that a lipophilic siRNA conjugate with acleavable disulfide linker produced more than two times thesuppression of CNPase mRNA than a lipophilic siRNA conjugate witha non-cleavable linker when infused with the same dosing paradigm,and was well-tolerated at efficacious doses. This result suggests that
ient gene silencing in oligodendrocytes with direct CNS delivery, J.
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siRNA conjugated to cholesterol with a cleavable disulfide linker isbeneficial for improving potency of silencing of CNPase mRNA inoligodendrocytes in vivo. Using disulfide bonds to trigger the releaseof siRNA in the cytoplasm has also been investigated for non-viralgene delivery systems. Reducible cationic polymers exploiting areducible linker enhance specific RNAi by increasing the bioavailabil-ity of siRNA in the cytoplasm by a triggered activation mechanism[38]. Studies of PEI-based siRNA delivery demonstrated that theintracellular release of siRNA was accelerated by the intracellulardegradation of the carrier system due to disulfide bonds [39].Promising results from the in vivo application of disulfide bonds toliposomal delivery systems have also been recently reported [40].Chemical conjugation via disulfides enables the delivery of proteinand peptides as well as oligonucleotides [41–43]. The reduction of thedisulfide bond in these conjugates occurs quickly once the constructsare delivered to the reducing milieu of the cytosol [44]. The significantimprovement of efficacy with cholesterol-conjugated CNPase siRNAwith a cleavable disulfide linker over that with a non-cleavable linkersuggests that the conjugates are effectively taken up into oligoden-drocytes and that a cleavable linkage may increase the amount ofbioavailable siRNA in the cytoplasm.
In conclusion, our studies demonstrate that cholesterol-conjugat-ed siRNAs are effective in silencing oligodendrocyte specific genes inwhite matter. A cleavable disulfide linker is beneficial for improvingpotency of cholesterol-conjugated siRNAs which may be attributableto increased bioavailability of siRNA in the cytoplasm. Althoughfurther mechanistic studies are needed on the uptake of theselipophilic siRNAs by cells in the brain and their fate once inside thecell, it appears that lipophilic siRNAs are an efficient approach toproductive in vivo delivery of siRNAs to oligodendrocytes. Thisdelivery method is potentially useful for new therapeutic approachesfor CNS diseases and disorders with significant oligodendrocytepathology.
Acknowledgements
We thank JohnMaraganore and Barry Greene for helpful commentsand guidance, Maryellen Duckman for assistance with manuscriptpreparation, and KathyMills, Jeff Rollins, Mara Broberg, AmyDell, LauraCamera, Argenis Pena and David Mosher for technical assistance.
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