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. 2017 Jun 21;28(6):1758-1766.
doi: 10.1021/acs.bioconjchem.7b00226. Epub 2017 May 10.

Synthesis and Evaluation of Parenchymal Retention and Efficacy of a Metabolically Stable O-Phosphocholine-N-docosahexaenoyl-l-serine siRNA Conjugate in Mouse Brain

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Synthesis and Evaluation of Parenchymal Retention and Efficacy of a Metabolically Stable O-Phosphocholine-N-docosahexaenoyl-l-serine siRNA Conjugate in Mouse Brain

Mehran Nikan et al. Bioconjug Chem. .
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Ligand-conjugated siRNAs have the potential to achieve targeted delivery and efficient silencing in neurons following local administration in the central nervous system (CNS). We recently described the activity and safety profile of a docosahexaenoic acid (DHA)-conjugated, hydrophobic siRNA (DHA-hsiRNA) targeting Huntingtin (Htt) mRNA in mouse brain. Here, we report the synthesis of an amide-modified, phosphocholine-containing DHA-hsiRNA conjugate (PC-DHA-hsiRNA), which closely resembles the endogenously esterified biological structure of DHA. We hypothesized that this modification may enhance neuronal delivery in vivo. We demonstrate that PC-DHA-hsiRNA silences Htt in mouse primary cortical neurons and astrocytes. After intrastriatal delivery, Htt-targeting PC-DHA-hsiRNA induces ∼80% mRNA silencing and 71% protein silencing after 1 week. However, PC-DHA-hsiRNA did not substantially outperform DHA-hsiRNA under the conditions tested. Moreover, at the highest locally administered dose (4 nmol, 50 μg), we observe evidence of PC-DHA-hsiRNA-mediated reactive astrogliosis. Lipophilic ligand conjugation enables siRNA delivery to neural tissues, but rational design of functional, nontoxic siRNA conjugates for CNS delivery remains challenging.


Figure 1
Figure 1. Neuronal uptake and efficacy of PC-DHA-hsiRNA
(a) Cellular internalization of Cy3-labeled PC-DHA-hsiRNA in mouse primary cortical neurons. Cells incubated for 0, 24, or 48 h with PC-DHA-hsiRNAHtt. Images acquired with Leica DMi8 microscope (63X). Nuclei (Hoechst), blue; PC-DHA-hsiRNA (Cy3), red; NeuN (AlexaFluor 488), green (b) Primary mouse cortical neurons were incubated with 0–1.5 µM Htt-targeting compounds at concentrations shown for one week. Htt mRNA levels were measured using QuantiGene® (Affymetrix), normalized to a housekeeping gene, Hprt (Hypoxanthine-guanine phosphoribosyl transferase), and presented as percent of untreated control (n=3, mean ± SD). UNT—untreated; NTC—non-targeting control.
Figure 2
Figure 2. PC-DHA-hsiRNA shows enhanced distribution and retention in mouse striatum and cortex
(a) aCSF, Cy3-C7linker-hsiRNA, Cy3-DHA-hsiRNA, and Cy3-PC-DHA-hsiRNA were administered by unilateral intrastriatal injection (2 nmol, 25 µg). After 48 hours, fluorescent images were acquired with a Leica DMi8 tiling microscope using equivalent exposure and acquisition settings (10X). aCSF and C7linker-hsiRNAHtt images were publisher previously. (b) Tissue concentrations of animals injected with 2 nmol of non-Cy3-labeled unconjugated hsiRNA, C7linker-hsiRNA, DHA-hsiRNA, and PC-DHA-hsiRNA (n=8) measured by RNA hybridization from the ipsilateral (injected) striatum (b) or cortex (c). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Kruskal-Wallis one-way ANOVA with Dunn's post hoc analysis). aCSF – artificial cerebrospinal fluid (vehicle); n.s. - non-significant.
Figure 3
Figure 3. Huntingtin mRNA and protein are efficiently silenced by PC-DHA-hsiRNAHtt
aCSF, PC-DHA-hsiRNANTC (4 nmol, 50 µg), DHA-hsiRHAHtt, and PC-DHA-hsiRNAHtt (2 nmol, 25 µg) were unilaterally injected into the striatum of FVB/NJ mice (n = 8 per group). Punch biopsies (5 mg) of the striatum (a) and cortex (b) were collected after one week. Level of Htt mRNA was measured using QuantiGene® (Affymetrix), normalized to a housekeeping gene (Hprt), and presented as percentage of untreated control (mean ± SD). DHA-hsiRNAHtt efficacy was previously reported. (c,d) Protein expression levels of Huntingtin, Dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP32), and Glial fibrillary acidic protein (GFAP) levels in the ipsilateral striatum of wild-type (FVB/NJ) mice after a one-week treatment with PC-DHA-hsiRNANTC or PC-DHA-hsiRNAHtt (4 nmol, 25 µg). (n=5, mean ± SD). NTC—non-targeting control; aCSF—artificial cerebrospinal fluid; n.s. – nonsignificant (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Kruskal-Wallis one-way ANOVA with Dunn’s post hoc analysis).
Figure 4
Figure 4. DHA-hsiRNA and PC-DHA-hsiRNAHtt accumulation in mouse brain choroid plexus after systemic administration
(left panel) PBS (vehicle), Cy3-DHA-hsiRNAHtt, and Cy3-PC-DHA-hsiRNAHtt (two daily 20 mg/kg) were administered by intravenous injection in FVB/NJ mice (n=3 per group). After 48 hours, fluorescent images were acquired with a Leica DMi8 tiling microscope (63X). (right panel) Whole brain lysate measurement of Htt antisense strand accumulation following intravenous administration PBS, DHA-hsiRNAHtt, and PC-DHA-hsiRNAHtt (two daily 20 mg/kg) via PNA hybridization. PBS—phosphate-buffered saline.
Scheme 1
Scheme 1. Chemical structures of DHA-hsiRNA conjugates and derivatives
(a) Fully chemically stabilized (alternating 2´-fluoro, 2´-O-methyl substituted), hydrophobically modified siRNA (hsiRNA) containing a 5´-phosphate on the antisense strand and a 3´-DHA or phosphocholine-DHA conjugate attached via a carbon linker on the sense strand. Molecular models of hsiRNA represented to scale using PyMOL. The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC. (b) Chemical structures of 1-oleoyl-2-docosahexaenoyl phosphatidylcholine (a naturally esterified form of DHA) and sphingomyelin (an important lipid component of nerve cell membranes).
Scheme 2
Scheme 2. Synthetic route to PC-DHA-controlled pore glass (CPG)
Reagents and conditions: (a) 20% piperidine in DMF (2×15 min); (b) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, DIEA, DCM, 2 h, rt, 95%; (c) choline tosylate, ETT, MeCN, 2 h, rt, followed by mCPBA, 10 min, rt, 69%; (d,e) TFA in dry DCM (1:1), triisopropylsilane, 2 h, rt, then 10% diisopropylamine in MeCN, 1.5h, rt 74%(f) 3, BOP, HOBt, DMF, 2,4,6-collidine, rt, 12 h; (g) 20% piperidine in DMF (2×15 min), rt; (h) DHA, HATU, DMF, rt, 12 h

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