Diverse lipid conjugates for functional extra-hepatic siRNA delivery in vivo

Small interfering RNA (siRNA)-based therapies are proving to be efficient for treating liver-associated disorders. However, extra-hepatic delivery remains challenging, limiting therapeutic siRNA utility. We synthesized a panel of fifteen lipid-conjugated siRNAs and systematically evaluated the impact of conjugate on siRNA tissue distribution and efficacy. Generally, conjugate hydrophobicity defines the degree of clearance and the liver-to-kidney distribution profile. In addition to primary clearance tissues, several conjugates achieve significant siRNA accumulation in muscle, lung, heart, adrenal glands and fat. Oligonucleotide distribution to extra-hepatic tissues with some conjugates was significantly higher than with cholesterol, a well studied conjugate, suggesting that altering conjugate structure can enhance extra-hepatic delivery. These conjugated siRNAs enable functional gene silencing in lung, muscle, fat, heart and adrenal gland. Required levels for productive silencing vary (5-200 mug/g) per tissue, suggesting that the chemical nature of conjugates impacts tissue-dependent cellular/intracellular trafficking mechanisms. The collection of conjugated siRNA described here enables functional gene modulation in vivo in several extra-hepatic tissues opening these tissues for gene expression modulation. A systemic evaluation of a panel of conjugated siRNA, as reported here, has not previously been investigated and shows that chemical engineering of lipid siRNAs is essential to advance the RNA therapeutic field

Guanabenz (Wytensin) selectively enhances uptake and efficacy of hydrophobically modified siRNAs

One of the major obstacles to the pharmaceutical success of oligonucleotide therapeutics (ONTs) is efficient delivery from the point of injection to the intracellular setting where functional gene silencing occurs. In particular, a significant fraction of internalized ONTs are nonproductively sequestered in endo-lysosomal compartments. Here, we describe a two-step, robust assay for high-throughput de novo detection of small bioactive molecules that enhance cellular uptake, endosomal escape, and efficacy of ONTs. Using this assay, we screened the LOPAC (Sigma-Aldrich) Library of Pharmacologically Active Compounds and discovered that Guanabenz acetate (Wytensin), an FDA-approved drug formerly used as an antihypertensive agent, is capable of markedly increasing the cellular internalization and target mRNA silencing of hydrophobically modified siRNAs (hsiRNAs), yielding a approximately 100-fold decrease in hsiRNA IC50 (from 132 nM to 2.4 nM). This is one of the first descriptions of a high-throughput small-molecule screen to identify novel chemistries that specifically enhance siRNA intracellular efficacy, and can be applied toward expansion of the chemical diversity of ONTs

Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways

Efficient delivery of therapeutic RNA beyond the liver is the fundamental obstacle preventing its clinical utility. Lipid conjugation increases plasma half-life and enhances tissue accumulation and cellular uptake of small interfering RNAs (siRNAs). However, the mechanism relating lipid hydrophobicity, structure, and siRNA pharmacokinetics is unclear. Here, using a diverse panel of biologically occurring lipids, we show that lipid conjugation directly modulates siRNA hydrophobicity. When administered in vivo, highly hydrophobic lipid-siRNAs preferentially and spontaneously associate with circulating low-density lipoprotein (LDL), while less lipophilic lipid-siRNAs bind to high-density lipoprotein (HDL). Lipid-siRNAs are targeted to lipoprotein receptor-enriched tissues, eliciting significant mRNA silencing in liver (65%), adrenal gland (37%), ovary (35%), and kidney (78%). Interestingly, siRNA internalization may not be completely driven by lipoprotein endocytosis, but the extent of siRNA phosphorothioate modifications may also be a factor. Although biomimetic lipoprotein nanoparticles have been explored for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation

Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain

Applications of RNA interference for neuroscience research have been limited by a lack of simple and efficient methods to deliver oligonucleotides to primary neurons in culture and to the brain. Here, we show that primary neurons rapidly internalize hydrophobically modified siRNAs (hsiRNAs) added directly to the culture medium without lipid formulation. We identify functional hsiRNAs targeting the mRNA of huntingtin, the mutation of which is responsible for Huntington\u27s disease, and show that direct uptake in neurons induces potent and specific silencing in vitro. Moreover, a single injection of unformulated hsiRNA into mouse brain silences Htt mRNA with minimal neuronal toxicity. Thus, hsiRNAs embody a class of therapeutic oligonucleotides that enable simple and straightforward functional studies of genes involved in neuronal biology and neurodegenerative disorders in a native biological context

Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo

Abstract Small interfering RNA (siRNA)-based drugs require chemical modifications or formulation to promote stability, minimize innate immunity, and enable delivery to target tissues. Partially modified siRNAs (up to 70% of the nucleotides) provide significant stabilization in vitro and are commercially available; thus are commonly used to evaluate efficacy of bio-conjugates for in vivo delivery. In contrast, most clinically-advanced non-formulated compounds, using conjugation as a delivery strategy, are fully chemically modified (100% of nucleotides). Here, we compare partially and fully chemically modified siRNAs in conjugate mediated delivery. We show that fully modified siRNAs are retained at 100x greater levels in various tissues, independently of the nature of the conjugate or siRNA sequence, and support productive mRNA silencing. Thus, fully chemically stabilized siRNAs may provide a better platform to identify novel moieties (peptides, aptamers, small molecules) for targeted RNAi delivery

RNA-Pt Adducts Following Cisplatin Treatment of <i>Saccharomyces cerevisiae</i>

The numerous regulatory roles of cellular RNAs suggest novel potential drug targets, but establishing intracellular drug–RNA interactions is challenging. Cisplatin (<i>cis</i>-diamminedichloridoplatinum­(II)) is a leading anticancer drug that forms exchange-inert complexes with nucleic acids, allowing its distribution on cellular RNAs to be followed <i>ex vivo</i>. Although Pt adduct formation on DNA is well-known, a complete characterization of cellular RNA–Pt adducts has not been performed. In this study, the action of cisplatin on <i>S. cerevisiae</i> in minimal media was established with growth curves, clonogenic assays, and tests for apoptotic markers. Despite high toxicity, cisplatin-induced apoptosis in <i>S. cerevisiae</i> was not observed under these conditions. In-cell Pt concentrations and Pt accumulation on poly­(A)-mRNA, rRNA, total RNA, and DNA quantified <i>via</i> ICP-MS indicate ∼4- to 20-fold more Pt accumulation in total cellular RNA than in DNA. Interestingly, similar Pt accumulation is observed on rRNA and total RNA, corresponding to one Pt per (14,600 ± 1,500) and (5760 ± 580) nucleotides on total RNA following 100 and 200 μM cisplatin treatments, respectively. Specific Pt adducts mapped by primer extension analysis of a solvent-accessible 18S rRNA helix occur at terminal and internal loop regions and appear as soon as 1 h post-treatment. Pt per nucleotide accumulation on poly­(A)-mRNA is 4- to 6-fold lower than on rRNA but could have consequences for low copy-number or highly regulated transcripts. Taken together, these data demonstrate significant accumulation of Pt adducts on cellular RNA species following <i>in cellulo</i> cisplatin treatment. These and other small molecule–RNA interactions could disrupt processes regulated by RNA

Platinum-RNA Modifications Following Drug Treatment in <i>S. cerevisiae</i> Identified by Click Chemistry and Enzymatic Mapping

With the importance of RNA-based regulatory pathways, the potential for targeting noncoding and coding RNAs by small molecule therapeutics is of great interest. Platinum­(II) complexes including cisplatin (<i>cis</i>-diamminedichloroplatinum­(II)) are widely prescribed anticancer compounds that form stable adducts on nucleic acids. In tumors, DNA damage from Pt­(II) initiates apoptotic signaling, but this activity is not necessary for cytotoxicity (e.g., Yu <i>et al.</i>, 2008), suggesting accumulation and consequences of Pt­(II) lesions on non-DNA targets. We previously reported an azide-functionalized compound, picazoplatin, designed for post-treatment click labeling that enables detection of Pt complexes (White <i>et al.</i>, 2013). Here, we report in-gel fluorescent detection of Pt-bound rRNA and tRNA extracted from picazoplatin-treated <i>S. cerevisiae</i> and labeled using Cu-free click chemistry. These data provide the first evidence that cellular tRNA is a platinum drug substrate. We assess Pt­(II) binding sites within rRNA from cisplatin-treated <i>S. cerevisiae</i>, in regions where damage is linked to significant downstream consequences including the sarcin-ricin loop (SRL) Helix 95. Pt-RNA adducts occur on the nucleotide substrates of ribosome-inactivating proteins, as well as on the bulged-G motif critical for elongation factor recognition of the loop. At therapeutically relevant concentrations, Pt­(II) also binds robustly within conserved cation-binding pockets in Domains V and VI rRNA at the peptidyl transferase center. Taken together, these results demonstrate a convenient click chemistry methodology that can be applied to identify other metal or covalent modification-based drug targets and suggest a ribotoxic mechanism for cisplatin cytotoxicity

Efficient Gene Silencing in Brain Tumors with Hydrophobically Modified siRNAs

Glioblastoma (GBM) is the most common and lethal form of primary brain tumor with dismal median and 2-year survivals of 14.5 months and 18%, respectively. The paucity of new therapeutic agents stems from the complex biology of a highly adaptable tumor that uses multiple survival and proliferation mechanisms to circumvent current treatment approaches. Here, we investigated the potency of a new generation of siRNAs to silence gene expression in orthotopic brain tumors generated by transplantation of human glioma stem-like cells in athymic nude mice. We demonstrate that cholesterol-conjugated, nuclease-resistant siRNAs (Chol-hsiRNAs) decrease mRNA and silence luciferase expression by 90% in vitro in GBM neurospheres. Furthermore, Chol-hsiRNAs distribute broadly in brain tumors after a single intratumoral injection, achieving sustained and potent (\u3e45% mRNA and \u3e90% protein) tumor-specific gene silencing. This readily available platform is sequence-independent and can be adapted to target one or more candidate GBM driver genes, providing a straightforward means of modulating GBM biology in vivoMol Cancer Ther; 17(6); 1251-8. ©2018 AACRin vivo

Picazoplatin, an Azide-Containing Platinum(II) Derivative for Target Analysis by Click Chemistry

Despite the broad use of platinum-based chemotherapeutics, identification of their full range of cellular targets remains a significant challenge. In order to identify, visualize, and isolate cellular targets of Pt­(II) complexes, we have modified the chemotherapeutic drug picoplatin with an azide moiety for subsequent click reactivity. The new compound picazoplatin readily binds DNA and RNA oligonucleotides and undergoes facile post-labeling click reactions to alkyne-fluorophore conjugates. Pt-fluorophore click reactions in rRNA purified from drug-treated Saccharomyces cerevisiae demonstrate its potential for future <i>in vivo</i> efforts

Docosahexaenoic Acid Conjugation Enhances Distribution and Safety of siRNA upon Local Administration in Mouse Brain

The use of siRNA-based therapies for the treatment of neurodegenerative disease requires efficient, nontoxic distribution to the affected brain parenchyma, notably the striatum and cortex. Here, we describe the synthesis and activity of a fully chemically modified siRNA that is directly conjugated to docosahexaenoic acid (DHA), the most abundant polyunsaturated fatty acid in the mammalian brain. DHA conjugation enables enhanced siRNA retention throughout both the ipsilateral striatum and cortex following a single, intrastriatal injection (ranging from 6–60 μg). Within these tissues, DHA conjugation promotes internalization by both neurons and astrocytes. We demonstrate efficient and specific silencing of Huntingtin mRNA expression in both the ipsilateral striatum (up to 73%) and cortex (up to 51%) after 1 week. Moreover, following a bilateral intrastriatal injection (60 μg), we achieve up to 80% silencing of a secondary target, Cyclophilin B, at both the mRNA and protein level. Importantly, DHA-hsiRNAs do not induce neural cell death or measurable innate immune activation following administration of concentrations over 20 times above the efficacious dose. Thus, DHA conjugation is a novel strategy for improving siRNA activity in mouse brain, with potential to act as a new therapeutic platform for the treatment of neurodegenerative disorders