Efficient Synthesis and Biological Evaluation of 5′-GalNAc Conjugated Antisense Oligonucleotides

Conjugation of triantennary <i>N</i>-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. ∼2000) activated as PFP (pentafluorophenyl) esters onto 5′-hexylamino modified antisense oligonucleotides (5′-HA ASOs, mol. wt. ∼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3′-end of an ASO to the 5′-end results in improved potency in cells and animals

Plot showing associations of 1-unit increase in log IL-6, log CRP, and fibrinogen with HR of endpoints on a log scale (after adjusting for randomized treatment, age, gender, LDL cholesterol, HDL cholesterol, triglycerides, BMI, systolic and diastolic blood pressure, current and exsmoking, diabetes, previous CVD, use of antihypertensive therapy, and country).

<p>Plot showing associations of 1-unit increase in log IL-6, log CRP, and fibrinogen with HR of endpoints on a log scale (after adjusting for randomized treatment, age, gender, LDL cholesterol, HDL cholesterol, triglycerides, BMI, systolic and diastolic blood pressure, current and exsmoking, diabetes, previous CVD, use of antihypertensive therapy, and country).</p

Associations of IL-6, CRP, and fibrinogen with risk (HR for 1-unit increase in log IL-6, log CRP, or fibrinogen) of experiencing one of the four categories of events.

<p>Event groupings as defined in the methods. Fatal CVD deaths preceded by nonfatal CVD are excluded. Model A, adjusted for randomized treatment. Model B, adjusted for randomized treatment, age, gender, LDL cholesterol, HDL cholesterol, systolic blood pressure, current smoker, diabetes, previous CVD (CHD, stroke, peripheral arterial disease, stroke, and transient ischaemic attack), use of antihypertensive therapy, and country. Model C, Model B+ adjusted for log triglyceride, BMI, diastolic blood pressure, exsmoker (as well as current smoker).</p

Kaplan-Meier time-to-event plots split by tertiles of IL-6 tertiles for (A) nonfatal CVD (<i>n</i> = 667 events), (B) fatal CVD (<i>n</i> = 189 deaths), (C) fatal other CV (<i>n</i> = 37 deaths), and (D) non-CVD mortality (<i>n</i> = 299 deaths).

<p>Kaplan-Meier time-to-event plots split by tertiles of IL-6 tertiles for (A) nonfatal CVD (<i>n</i> = 667 events), (B) fatal CVD (<i>n</i> = 189 deaths), (C) fatal other CV (<i>n</i> = 37 deaths), and (D) non-CVD mortality (<i>n</i> = 299 deaths).</p

Baseline characteristics by incident primary combined nonfatal and fatal endpoint (<i>p</i>-value versus no event group).

<p>Please note that because of the design structure of the trial—recruiting more participants with hypertension/smokers and diabetes (and women) into the low risk primary prevention group—the significance or nonsignificance of univariate comparisons in this table could be potentially misleading. <i>p</i>-Values for continuous variables are from two-sample <i>t</i>-test and for categorical variables from chi-squared test.</p>a<p>Values are geometric means (SD) calculated from the log-transformed distribution and the (<i>p</i>-value).</p><p>SD, standard deviation; TIA, transient ischemic attack.</p

Comprehensive Structure–Activity Relationship of Triantennary <i>N</i>‑Acetylgalactosamine Conjugated Antisense Oligonucleotides for Targeted Delivery to Hepatocytes

The comprehensive structure–activity relationships of triantennary GalNAc conjugated ASOs for enhancing potency via ASGR mediated delivery to hepatocytes is reported. Seventeen GalNAc clusters were assembled from six distinct scaffolds and attached to ASOs. The resulting ASO conjugates were evaluated in ASGR binding assays, in primary hepatocytes, and in mice. Five structurally distinct GalNAc clusters were chosen for more extensive evaluation using ASOs targeting SRB-1, A1AT, FXI, TTR, and ApoC III mRNAs. GalNAc–ASO conjugates exhibited excellent potencies (ED₅₀ 0.5–2 mg/kg) for reducing the targeted mRNAs and proteins. This work culminated in the identification of a simplified tris-based GalNAc cluster (THA-GN3), which can be efficiently assembled using readily available starting materials and conjugated to ASOs using a solution phase conjugation strategy. GalNAc–ASO conjugates thus represent a viable approach for enhancing potency of ASO drugs in the clinic without adding significant complexity or cost to existing protocols for manufacturing oligonucleotide drugs