Extended Nucleic Acid (exNA): A Novel, Biologically Compatible Backbone that Significantly Enhances Oligonucleotide Efficacy in vivo

Abstract

Metabolic stabilization of therapeutic oligonucleotides requires both sugar and backbone modifications, where phosphorothioate (PS) is the only backbone chemistry used in the clinic. Here, we describe the discovery, synthesis, and characterization of a novel biologically compatible backbone, extended nucleic acid (exNA). Upon exNA precursor scale up, exNA incorporation is fully compatible with common nucleic acid synthetic protocols. The novel backbone is orthogonal to PS and shows profound stabilization against 3'- and 5'-exonucleases. Using small interfering RNAs (siRNAs) as an example, we show exNA is tolerated at most nucleotide positions and profoundly improves in vivo efficacy. A combined exNA-PS backbone enhances siRNA resistance to serum 3'-exonuclease by ~32-fold over PS backbone and >1000-fold over the natural phosphodiester backbone, thereby enhancing tissue exposure (~6-fold), tissues accumulation (4- to 20-fold), and potency both systemically and in brain. The improved potency and durability imparted by exNA opens more tissues and indications to oligonucleotide-driven therapeutic interventions.

Fig 1.

Chemical structure of extended Nucleic Acid (exNA) with methyl inserts (permanent structural modulation) and natural epigenetic modification with methyl adduct (removable by endogenous demethylase).

Fig 2.. Thermostability of rxU modified RNA duplex.

T_m measurements were carried out in buffer containing 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, 0.1 mM EDTA, and 1 μM duplex. ΔT_m^a is the difference in T_m value between rxU versus rU duplexes. ΔT_m^b is the difference in T_m value between fully complementary duplexes versus duplexes with a mismatch.

Fig. 3. Nuclease resistance of exNA modified oligonucleotides.

Stability of oligonucleotide in the presence of (A) Snake venom phosphodiesterase I (SVPD) and (B) bovine spleen phosphodiesterase II (BSP). Residual oligonucleotide length was monitored over time and quantified by HPLC. (dT): 2′-deoxy-thymidine, (mU): 2′-OMe-uridine, (ex-mU): 2′-OMe-exNA-uridine. “#” indicates phosphorothioate linkage, otherwise phosphodiester linkage.

Fig. 4. Position-dependent impact of exNA replacement on siRNA activity in vitro.

(A) Schematic structure of chemical modification used in siRNA. (B) Position dependent impact of exNA on siRNA efficacy. Silencing efficacy change was defined by 100 x [IC₅₀(Ctrl)-IC₅₀(exNA)/IC₅₀ (Ctrl)]. A value of ~0 or a positive value (colored in red) indicate exNA modification was fully compatible with Ago2 or enhanced efficacy, respectively, compared to corresponding control siRNAs. See Tables S4 and S5 for sequences, and Table S6 for all IC₅₀ values used to calculate efficacy changes in the figure. ^aPotency change calculated based on IC₅₀ by lipid-mediated uptake.

Fig. 5. Systemic delivery of DCA-conjugated siRNA with 3′-exNA modification.

FVB mice injected with 10mg/kg of DCA conjugated siRNA variants subcutaneously. (A) Schematic of the siRNA scaffolds used, (B) Graphical study outline. (C, D and E) siRNA antisense strand accumulation in (C) Plasma concentrations of antisense strand (left) and tabulated curve fitting parameters (right) at 5min, 15min, 30min, 1h, 3h, 6h, 9h and 24h post injection, (D) tissues at 2 weeks and (E) tissues at4 weeks post injection. (F) Htt mRNA level at 4 weeks post injection measured by Quantigene 2.0 assay. NTC: Non-targeting control. n=4 (C and D) and n=5 (E and F). Data represented as mean ± s.d. of individual animals. Statistical significance calculated for individual tissues using two-tailed unpaired t-test (D and E) or ordinary one-way ANOVA with multiple comparisons (F). Pairwise comparisons between PS and exNA-PS in (F) were performed using two-tailed unpaired t-test (*/# -p<0.05, **/## -p<0.01, ***/### -p<0.001, ****/#### -p<0.0001).

Figure 6.. Impact of 3′-exNA modification on siRNA potency in the CNS.

(A) Schematic of di-valent siRNA structure without and with 3′-exNA modification. (B) Brain region of interest. FC: Frontal cortex; Str: Striatum; Thal: Thalamus; Hipp: Hippocampus. (C) WT mice injected with 2.5 nmol/10 μL of PS (D55) or exNA-PS siRNAs (D56) targeting ApoE at 8 weeks of age. Levels of ApoE mRNA were evaluated using Quantigene at 1 month post injection. glmmTMB results: log(exNA effect)=−0.663, P=0.0255. (D) WT mice injected with 1.25nmol/10 μL of PS (D58) or exNA-PS siRNAs (D59) targeting Htt at 8 weeks of age. The levels of WT huntingtin protein in frontal cortex, striatum, thalamus, and hippocampus were evaluated using western blot (ProteinSimple) 1-month post injection. N = 5, one-way Anova with Tukey correction for multiple comparisons. glmmTMB results: log(exNA effect)=−0.519, P=0.0092. (E) YAC128-HD mice injected with 20nmol/10μL with PS or exNA-PS siRNAs targeting Htt at 8 weeks of age. The levels of WT huntingtin protein in frontal cortex, striatum, thalamus, and hippocampus were evaluated using western blot (ProteinSimple) six months following siRNA injections. N = 6, one-way Anova with Tukey correction for multiple comparisons. glmmTMB results: log(exNA effect)=−0.321, P=0.0498. For sequences of di-siRNAs used, see Table S8.

Scheme 1.

Synthesis of exNA phosphoramidites (7a and 7b). Reagent and conditions: (i) TBDMSCl, Imidazole/DMF, rt, overnight; (ii) 3% DCA/CH₂Cl₂, triethylsilane, rt, 1 h, 2a:88 % (2 steps), 2b: 84 % (2 steps); (iii) IBX/ CH₃CN, 85°C, 1.5 h; (iv) CH₃PPh₃Br, ^tBuOK, THF, 0°C then rt, overnight, 4a: 75% (2 steps), 4b: 67% (2 steps); (v) 9-BBN, THF, 0°C, overnight; (vi) NaBO₃ · 4H₂O, MeOH, THF, H₂O, 0°C then rt, overnight, 5a: 62% (2 steps), 5b: ND; (vii) DMTr-Cl, pyridine, rt, 2 h; (viii) 0.1 M TBAF, THF, rt, 1 h, 6a:93% (2 steps), 6b: 12% (3 steps); (ix) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, DIPEA/CH₂Cl₂, 0°C then rt, 0.5 h, 7a: 86%, 7b: 81%.