Targeted tissue delivery of RNA therapeutics using antibody-oligonucleotide conjugates (AOCs)

Abstract

Although targeting TfR1 to deliver oligonucleotides to skeletal muscle has been demonstrated in rodents, effectiveness and pharmacokinetic/pharmacodynamic (PKPD) properties remained unknown in higher species. We developed antibody-oligonucleotide conjugates (AOCs) towards mice or monkeys utilizing anti-TfR1 monoclonal antibodies (αTfR1) conjugated to various classes of oligonucleotides (siRNA, ASOs and PMOs). αTfR1 AOCs delivered oligonucleotides to muscle tissue in both species. In mice, αTfR1 AOCs achieved a > 15-fold higher concentration to muscle tissue than unconjugated siRNA. A single dose of an αTfR1 conjugated to an siRNA against Ssb mRNA produced > 75% Ssb mRNA reduction in mice and monkeys, and mRNA silencing was greatest in skeletal and cardiac (striated) muscle with minimal to no activity in other major organs. In mice the EC50 for Ssb mRNA reduction in skeletal muscle was >75-fold less than in systemic tissues. Oligonucleotides conjugated to control antibodies or cholesterol produced no mRNA reduction or were 10-fold less potent, respectively. Tissue PKPD of AOCs demonstrated mRNA silencing activity primarily driven by receptor-mediated delivery in striated muscle for siRNA oligonucleotides. In mice, we show that AOC-mediated delivery is operable across various oligonucleotide modalities. AOC PKPD properties translated to higher species, providing promise for a new class of oligonucleotide therapeutics.

Graphical Abstract

Figure 1.

AOC-mediated siRNA tissue delivery is antibody-dependent. Female CD-1 mice were treated with a single IV dose of antibody-siRNA conjugates; each composed of an antibody targeting either mouse ASGR or mouse TfR1 and an siRNA targeting Ctnnb1 mRNA at 3 mg/kg. GA, heart, and liver samples were collected 4 days post dose, and siRNA concentration was determined using stem-loop qPCR (normalized to tissue weight, mean ± SEM; N = 4). Statistical analysis was performed using two-way ANOVA with Bonferroni post hoc test. *Indicates statistical difference at P< 0.05.

Figure 2.

αASGR AOCs produce mRNA reduction in liver across multiple gene targets. Female CD-1 mice were treated with a single IV dose of AOCs composed of an antibody targeting mouse ASGR and an siRNA targeting either Hprt, Ctnnb1, or FVII mRNAs at indicated doses. A GalNAc conjugated to an siRNA targeting FVII mRNA was also evaluated following a single IV dose. Liver was collected 4 days post dose, and mRNA expression was analyzed by RT-qPCR. Hprt, Ctnnb1, and FVII mRNA expression was normalized to that of a reference gene, Ppib. Data are represented as percent of vehicle control (mean ± SEM; N = 4 for treated groups, N = 5 for vehicle groups). Statistical analysis was performed using one-way ANOVA with Dunnett's post hoc test. Statistical difference relative to vehicle control at P< 0.05 was observed for the αASGR-siHprt, αASGR-siFVII, GalNAc-siFVII, and αASGR-siCtnnb1 at all doses evaluated.

Figure 3.

αTfR1 AOCs produce mRNA reduction in muscle across multiple gene targets. Female CD-1 mice were treated with a single IV dose of AOCs composed of an antibody targeting mouse TfR1 and an siRNA targeting either a scrambled oligonucleotide sequence (Scr), Dmpk, Mstn, or Ssb mRNAs at indicated doses. Cholesterol (Chol) conjugated to an siRNA targeting Mstn mRNA was also evaluated. GA muscle was isolated 4 days post dose of the αTfR1-siMstn and 1 week post dose of the αTfR1-siDmpk and αTfR1-siSsb, and mRNA expression was analyzed by RT-qPCR. Dmpk, Mstn, and Ssb mRNA expression was normalized to that of a reference gene, Ppib. Data are represented as percent of vehicle control (mean ± SEM; N = 4 for treated groups, N = 5 for vehicle groups). Statistical analysis was performed using one-way ANOVA and Dunnett's post hoc test. Statistical difference relative to vehicle control at P< 0.05 was observed for all groups except αTfR1-siScr, αTfR1-siMstn at 0.1 mg/kg, and Chol-siMstn at 2 mg/kg.

Figure 4.

αTfR1-siMstn produced dose-dependent increases in siRNA tissue concentration and Mstn mRNA reduction in a broad panel of skeletal muscles and heart. Female CD-1 mice were treated with a single IV dose of αTfR1-siMstn at indicated doses. Muscle tissue was isolated 7 days post dose. mRNA expression was analyzed by RT-qPCR, and siRNA concentration was analyzed by stem-loop qPCR (data normalized to tissue weight). Mstn mRNA expression was normalized to that of a reference gene, Ppib. mRNA data are represented as percent of vehicle control (mean ± SEM; N = 4 for treated groups, N = 5 for vehicle group). Statistical analysis was performed using one-way ANOVA and Dunnett's post hoc test. Statistical difference relative to vehicle control at P< 0.05 was observed for all treatments except for 0.1 mg/kg dose in tibialis anterior, gastrocnemius, quadriceps, triceps, and 0.3 mg/kg in triceps.

Figure 5.

αTfR1-siSSB productively delivers siRNA to muscle and produces mRNA reduction in cynomolgus monkeys. Male cynomolgus monkeys of Cambodian origin were administered a single IV dose of SSB siRNA conjugated to an antibody targeting TfR1 at 6 mg/kg. Tissues were collected at 28 days post dose, and mRNA expression was analyzed by RT-qPCR, while siRNA tissue concentrations were determined using stem-loop RT-qPCR. SSB expression was normalized to reference gene activator of HSP90 ATPase activity 1 (AHSA1). mRNA expression (percent of vehicle control) and siRNA tissue concentration (normalized to tissue weight) data are represented as mean ± SEM (N = 3). Statistical analysis was performed using one-way ANOVA and Tukey's post hoc test. *Indicates significant difference relative to vehicle control at P< 0.05.

Figure 6.

αTfR1 AOCs conjugated to siRNA or ASO oligonucleotides produce mRNA silencing activity in muscle. Male C57BL/6 mice were treated with a single IV dose of AOCs each composed of an antibody targeting mouse TfR1 conjugated to either an siRNA (αTfR1-siDmpk; DAR = 1) or an ASO (αTfR1-asoDmpk; DAR = average 2.5) targeting Dmpk mRNA at indicated doses. Tissue samples were isolated 14 days post dose, and mRNA expression was analyzed by RT-qPCR. siRNA concentration was assessed by stem-loop qPCR, and ASO concentration was assessed by oligonucleotide probe hybridization electrochemiluminescent (ECL) assay (data normalized to tissue weight). Dmpk mRNA expression was normalized to that of a reference gene, Ppib. Data are represented as percent of vehicle control (mean ± SEM; N = 4 for treated groups, N = 5 for vehicle group). Statistical analysis for KD was performed using one-way ANOVA and Dunnett's post hoc test. Statistical significance relative to vehicle control at P< 0.05 was noted for αTfR1-siDmpk in muscle and for αTfR1-asoDmpk in muscle and liver at all doses evaluated. To compare the αTfR1-siDmpk- and αTfR1-asoDmpk groups, a two-way ANOVA with Sidak's post hoc test was performed, and statistical significance was observed at the 0.6 and 1.8 mg/kg dose groups with P< 0.05. αTfR1-asoDmpk (TC) in gastrocnemius at 0.6 mg/kg dose was below the limit of quantification. KD: mRNA knockdown; TC: oligonucleotide tissue concentration.