Can a branched divalent siRNA target the central nervous system

Yes, a branched divalent siRNA chemical scaffold recently introduced by Alterman et al. (2010) allows targeting the central nervous system with therapeutic siRNA.

Branched siRNA nanostructures offer improved resistance against exonucleases and suppress off-target gene silencing through selection of the passenger strand as the branching unit. According to Chandela & Ueno (2019), branched siRNAs form a compact assembly with a hydrodynamic diameter of 6.9 nm, compared with 2.8 nm for the duplex. Automated solid-phase oligonucleotide synthesis enables the production of branched siRNA using branched or trebling solid supports. Branched small interfering RNA (siRNA) nanostructures are designed to improve exonuclease resistance, reduce off-target effects, and maintain sequence-specific gene silencing. Branched siRNA assemblies can use a doubling or trebling solid-support branching unit on the passenger strand to form stable, higher-order architectures.

Chemical modification of the ribose sugar, backbone, and nucleobases is essential for therapeutic utility.

Backbone and sugar modifications allow the formulation of branched RNAs, enabling siRNA to evade barriers to systemic administration and regulate gene expression. Historically, the complexity and difficulty of synthesizing branching RNA units have restricted investigations into this type of pharmaceutics. Common ribose 2'-modifications are 2'-O-methyl (2'-OMe) and 2'-fluoro (2'-F), which can be used in alternating patterns to protect against endonucleases and attenuate TLR-mediated immune sensing while maintaining a C3'-endo A-form RNA conformation. Locked Nucleic Acids (LNA) and Ethylene-bridged Nucleic Acids (ENA) restrict sugar conformation to C3'-endo, increasing duplex melting temperature () and biostability. Backbone Modification utilizing phosphorothioate (PS) linkages by replacing a non-bridging oxygen atom with sulfur at the 3'- and 5'-ends of each strand resists exonuclease cleavage, increases non-specific plasma protein binding, and reduces renal clearance. Modifications such as mesyl-Phosphoramidate (μ) and phosphoryl-guanidine (PGA) modifications are increasingly explored to improve cellular uptake and pharmacokinetic profiles without compromising AGO2 loading. A 5'-vinyl phosphonate on the guide strand mimics a stable 5'-monophosphate, facilitating AGO2 recognition without being susceptible to cellular phosphatases.

In 1989, Horn and Urdea investigated chemical approaches for signal amplification in rapid nonradioisotopic DNA probe assays by synthesizing large branched single-stranded oligodeoxyribonucleotide polymers. This approach made amplification of target sequences possible with the help of a short hybridization probe (< 20 nucleotides) containing one label. These researchers showed that nucleoside phosphoramidite derivatives containing two protected primary hydroxyl functions can be incorporated into synthetic oligonucleotides as 'branching monomers'. Utilizing selective deprotection allowed the incorporation of multiple identical copies of additional oligonucleotides to form fork- or comb-like structures for use as signal amplification materials in nucleic acid hybridization assays.

Development of newer siRNA delivery platforms to expand tissue targeting

Recently, Alterman et al. (2019) introduced a divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system. Alterman et al. reported the design of a divalent siRNA (Di-siRNA) that supports potent, sustained gene silencing in the central nervous system (CNS) of mice and non-human primates following a single injection into cerebrospinal fluid. Di-siRNAs are composed of two chemically modified, phosphorothioate-containing siRNAs connected by a linker (see image below).

Alterman et al. showed that in mice, Di-siRNAs induced potent silencing of huntingtin, the causative gene in Huntington’s disease, by reducing mRNA and protein throughout the brain. The researchers noted that silencing persisted for at least six months, with the degree of gene silencing correlating with the accumulation levels or the guide strand in the tissue. Also, in Cynomolgus macaques, a bolus injection of Di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects. The researchers suggest that di-siRNAs may enable RNAi-based gene silencing in the CNS for the treatment of neurological disorders.

Chandela and Ueno (2019) also reported the synthesis and evaluation of novel, branched trident small interfering RNA nanostructures for sequence-specific RNAi activity. The researchers used trebling solid-supports for the synthesis of a trident siRNA constructs. The physical characterization of the branched siRNAs using Dynamic Light Scattering (DLS) observed a hydrodynamic diameter of 6.9 nm for the trident siRNA compared to the 2.8 nm diameter of the duplex siRNA, revealing the compactness of these nanostructures. In vitro experiments with stable HeLa-psiCHECK-2 cells expressing both Renilla and firefly luciferases investigated gene silencing effects of the siRNA constructs. The experiments observed that all alkyl halide base-modified trident siRNAs studied showed comparable activity, inhibiting gene expression by 78% in each case, whereas 81% inhibition was observed with an unmodified branched trident siRNA at a 10 nM concentration. The duplex siRNA also showed 78% inhibition, indicating that the RNAi activity was conserved with all structurally different moieties.

Established siRNA Delivery Platforms

Delivery across the hydrophobic cell membrane remains the primary engineering hurdle for siRNA drugs. Modern siRNA therapeutics rely on two primary approaches:

Current R&D Frontiers and Challenges

Presently, the expansion of target tissues beyond the liver to allow targeting the CNS, lung, muscle, and tumor tissues remains the main priority. To target the central nervous system (CNS) direct intrathecal delivery into the fluid-filled space under the protective layers covering the brain and spinal cord is investigated using lipophilic conjugates such as C16 (palmitic acid) conjugates. For targeting eye or lung tissues, local intravitreal injections or aerosolized inhalation to the lung epithelium are tested.

Further, conjugation of siRNA to peptide ligands, aptamers, or antibodies may soon allow targeting to specific surface markers such as transferrin receptor 1 (TfR1) for blood-brain barrier crossing. Off-target effects or silencing can be minimized via target site modification, for example by adding 2'-OMe at position 2. Removing unmodified ribofuranose may allow mitigation of immune activities.

References

Alterman JF et al. Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain. Mol Ther Nucleic Acids 4, e266 (2015). [PubMed: 26623938] [PMC]

Chandela A, Ueno Y. Design, synthesis and evaluation of novel, branched trident small interfering RNA nanostructures for sequence-specific RNAi activity. RSC Adv. 2019 Oct 23;9(59):34166-34171. [RSC, PMC]

Horn T, Urdea MS. Forks and combs and DNA: the synthesis of branched oligodeoxyribonucleotides. Nucleic Acids Res. 1989 Sep 12;17(17):6959-67. [PMC]