Conjugation of hydrophobic moieties enhances potency of antisense oligonucleotides in the muscle of rodents and non-human primates

Abstract

We determined the effect of attaching palmitate, tocopherol or cholesterol to PS ASOs and their effects on plasma protein binding and on enhancing ASO potency in the muscle of rodents and monkeys. We found that cholesterol ASO conjugates showed 5-fold potency enhancement in the muscle of rodents relative to unconjugated ASOs. However, they were toxic in mice and as a result were not evaluated in the monkey. In contrast, palmitate and tocopherol-conjugated ASOs showed enhanced potency in the skeletal muscle of rodents and modest enhancements in potency in the monkey. Analysis of the plasma-protein binding profiles of the ASO-conjugates by size-exclusion chromatography revealed distinct and species-specific differences in their association with plasma proteins which likely rationalizes their behavior in animals. Overall, our data suggest that modulating binding to plasma proteins can influence ASO activity and distribution to extra-hepatic tissues in a species-dependent manner and sets the stage to identify other strategies to enhance ASO potency in muscle tissues.

Figure 1.

Conjugation of hydrophobic moieties to PS ASOs modulates plasma protein binding and enhances activity in extra-hepatic tissues. (A) Binding curves and (B) dissociation constants of palmitate, tocopherol and cholesterol ASOs with albumin, HDL, LDL and HRG. NB = no binding (C) Structures of hydrophobic moieties conjugated to PS ASOs. Palm = palmitic acid, Toc = tocopherol, Chol = cholesterol, Blue = cEt and black = DNA nucleotides. (D) Comparison of protein binding profiles of palmitate, tocopherol and cholesterol conjugated PS ASOs in mouse, rat, monkey and human plasma by size exclusion chromatography. (E) All ASOs showed similar activity in 3T3-L1 cells for reducing MALAT-1 RNA when delivered by free uptake. (F) Conjugation of hydrophobic moieties enhances potency of a PS ASO gapmer targeting MALAT-1 RNA in the liver and heart. All errors are ±std. dev.

Figure 2.

Palmitate ASO-conjugates show enhanced potency in skeletal muscle and heart tissues in mice. (A) Mice (Balb-c mice, n = 4/group) were injected subcutaneously with 10, 20 or 40 mg/kg of A-dmpk, or with 5, 10 and 20 mg/kg of Palm-dmpk1, Palm-dmpk2 or Palm-dmpk3, once weekly for 4 weeks. Animals were sacrificed 48 hours after last injection and tissues were harvested. Reduction of DMPK mRNA in heart, quadriceps, gastrocnemius, tibialis anterior and diaphragm was quantified by qRT-PCR. (B) Structures and design of ASO conjugates tested. The boxed regions indicate potential sites of metabolic cleavage. (C) Dose required to reduce DMPK mRNA by 50% (ED₅₀) in various muscle tissues. Palm = palmitic acid, HA = hexylamino, HDO = 1,6-hexanediol, Blue = cEt and black = DNA nucleotides; All ASOs are full PS modified except underlined letters which are PO. All errors are ±std. dev.

Figure 3.

Palmitate ASO-conjugates show enhanced potency in skeletal muscle and heart tissues in rats. (A) Rats (SD, n = 4/group) were injected subcutaneously with 10, 30 or 60 mg/kg of A-dmpk, or with 3, 10 and 30 mg/kg of Palm-dmpk2, once weekly for 4 weeks. Animals were sacrificed 48 h after last injection and tissues were harvested. Reduction of DMPK mRNA in heart, quadriceps, liver, gastrocnemius, tibialis anterior and diaphragm was quantified by qRT-PCR. (B) Structures, sequence, design of ASO conjugates and ED₅₀ (mg/kg) in various muscle tissues. Palm = palmitic acid, HA = hexylamino, Blue = cEt and black = DNA nucleotides; All ASOs are full PS modified. (C) ASO accumulation in the heart, quadriceps and liver as determined by LCMS. All errors are ±std. dev.

Figure 4.

Palmitate ASO-conjugates show enhanced efficacy in skeletal muscle and heart tissues in monkeys. (A) Dosing frequency and schedule for ASO injections. (B) Cynomolgus monkeys (n = 4/group) were injected subcutaneously with 10 injections over 7 weeks of 10, 20 or 40 mg/kg of A-dmpk or Palm-dmpk2. Animals were sacrificed 48 hours after last injection and tissues were harvested. Reduction of DMPK mRNA in heart, quadriceps, liver, gastrocnemius, tibialis anterior and diaphragm was quantified by qRT-PCR. (C) Structures, sequence, design of ASO conjugates and ED₅₀ (mg/kg) in various muscle tissues. Palm = palmitic acid, HA = hexylamino, Blue = cEt and black = DNA nucleotides; All ASOs are full PS modified. (D) ASO accumulation in the heart, quadriceps, tibialis anterior and liver as determined by LCMS. For Palm-dmpk2, the majority of conjugate was metabolized in tissues to liberate A-dmpk. All errors are ±std. dev.

Figure 5.

Palmitate, tocopherol and cholesterol conjugation enhances ASO activity in quadriceps and heart tissues. (A) Comparison of ASO activity in quadriceps and heart after a single subcutaneous or intravenous injection of ASO-conjugates. Mice (Balb-c, n = 4/group) were injected once weekly for four weeks with 10 mg/kg of A-dmpk, Palm-dmpk2, Toc-dmpk or Chol-dmpk. Animals were sacrificed 48 h after the last dose and DMPK mRNA reduction in quadriceps and heart was determined using qRT-PCR. (B) Structures of ASO conjugates. (C) Comparison of ASO activity in quadriceps and heart after four intravenous injection of ASO-conjugates. Mice (Balb-c, n = 4/group) were injected once weekly for 4 weeks with 5, 10 or 20 mg/kg of A-dmpk, Toc-dmpk or Chol-dmpk. Animals were sacrificed 48 h after the last dose and DMPK mRNA reduction in heart, quadriceps and tibialis anterior was determined using qRT-PCR. (D) Sequence and design of ASO conjugates and ED₅₀ (mg/kg) in various muscle tissues. Palm = palmitic acid, Toc = tocopherol, Chol = cholesterol, HA = hexylamino, Blue = cEt and black = DNA nucleotides; All ASOs are full PS modified except the underlined nucleotides which are PO. All errors are ±std. dev.

Figure 6.

Tocopherol ASO-conjugates show enhanced activity in skeletal muscle and heart tissues in monkeys. (A) Dosing frequency and schedule for ASO injections. (B) Cynomolgus monkeys (n = 4/group) were injected intravenously with 10 injections over 7 weeks of 10, 20 or 40 mg/kg of A-dmpk or Toc-dmpk. Animals were sacrificed 48 hours after last injection and tissues were harvested. Reduction of DMPK mRNA in heart, quadriceps, gastrocnemius was quantified by qRT-PCR. (C) Sequence and design of ASO conjugates and ED₅₀ (mg/kg) in various muscle tissues. Toc = tocopherol, HA = hexylamino, Blue = cEt and black = DNA nucleotides; All ASOs are full PS modified except the underlined nucleotides which are PO. (D) ASO accumulation in the heart, quadriceps and liver as determined by AEX. For Toc-dmpk, the majority of the ASO extracted from tissues was the intact parent conjugate-ASO. All errors are ±std. dev.

Figure 7.

A potential model to rationalize how modulating interactions with plasma proteins by conjugating hydrophobic moieties can enhance ASO potency in muscle tissues. Lipid conjugation enhances affinity for plasma proteins which facilitates transport across the capillary endothelium. The lipid-ASOs partition onto protein acceptors on the cell surface or are internalized as ASO-protein complexes and transported to late endosomal compartments. Cleavage of the linker between the ASO and the lipid by endosomal nucleases can facilitate ASO release from proteins such as albumin and lipoproteins for which PS-ASOs have lower affinity. However, PS-ASOs can stay bound to higher-affinity proteins such as HRG and this may hinder ASO escape from endosomal compartments.