(T0930-06-39) Quantitative Characterization of Oligonucleotide Transport Vehicle (OTV) In Vivo Distribution and Pharmacokinetics using Ultra-sensitive, Plate-Based Bioanalytical Methods

Purpose: Crossing the blood brain barrier (BBB) to deliver antisense oligonucleotides (ASOs) requires significant engineering ingenuity. To that end, we have conjugated ASOs directly to our Transport Vehicle platform - termed Oligonucleotide Transport Vehicle (OTV) - that has been engineered to bind the apical domain of human transferrin receptor to enable transport of ASOs across the BBB [1]. Characterization of this novel molecule requires specialized reagents and bioanalytical tools. Herein, we describe the development of plate-based bioanalytical assays to evaluate the pharmacokinetics (PK) of OTV in pre-clinical studies. The bioanalytical toolkit for the PK characterization of OTV consists of three plate-based bioanalytical assays: Total human IgG (huIgG), Total ASO, and Intact OTV.

Methods: Total huIgG concentrations in plasma and tissue homogenates were quantified using a generic MSD (Meso Scale-Discovery)-based sandwich electrochemiluminescence immunoassay (ECLIA). Method qualification data for this generic assay will not be discussed in this work. Quantification of Total ASO (conjugated and liberated) in plasma and tissue homogenates were measured using a method similar to a modified hybridization-based ECLIA [2]. Custom biotinylated-capture and digoxigenin-detection probes were combined with prepared test samples and reference standards. Following hybrid product formation, samples were added to an MSD 96-well streptavidin-coated plate. A secondary SULFO-Tagged sheep anti-digoxygenin antibody was used for detection . 1x MSD Read Buffer T was then added to generate the ECL signal. Quantification of Intact OTV in plasma and tissue homogenates were measured using a method similar to a modified ECLIA [3]. Custom biotinylated capture probe was combined with prepared test samples and relevant standards. The hybridized OTV-probe products were added to an MSD 96-well streptavidin-coated plate. A secondary SULFO-Tagged goat anti-human IgG antibody was used for detection . 1x MSD Read Buffer T was then added to generate the ECL signal.

Results: The total ASO and intact OTV method qualification across different matrices resulted in a dynamic range of 0.03 to 200 nM for both methods (Figure 1A,B). These methods were further assessed for accuracy, precision, sensitivity, selectivity, dilutional linearity and specificity. Accuracy and precision for all quality control (QC) levels in both methods were calculated to be < 25%. In a selectivity assessment, naïve individual lots of matrices were spiked with OTV, and the observed CV% and %Bias met the acceptance criteria (CV% < 25% and %Bias < 25%). Dilutional linearity assessment was performed, and the largest dilution identified for both assays was 1:100,000 with a resulting %CV of < 25% and %Bias of < 25%. Qualification results for both methods are summarized in Table 1 and 2. The specificity of the total ASO method was assessed by comparing molar equivalents of parental ASO to its truncated versions—data indicating high specificity to full-length parent sequence as summarized in Figure 1C. Method qualification data for the huIgG assay is out of scope, since a previously established generic assay was used. The dynamic range of the generic huIgG assay is 33.3 nM – 0.1 nM.

Conclusion: Given the challenges of assessing the three unique components of 1) intact OTV 2) conjugated ASO and 3) total huIgG protein, we have implemented a bioanalytical strategy to assess the biodistribution and pharmacokinetic profile of our OTV. As these molecules are brain penetrant, highly sensitive methods were developed with sub-nanomolar sensitivity in both plasma and brain. In the application of these optimized methods , we were able to capture the full pharmacokinetic profile of our molecule and correlate it exquisitely to target pharmacodynamics and high-resolution imaging of ASO deposition in the brain, highlighting the value of the implemented bioanalytical toolkit.

References: 1. Barker, S. & Thayer, MB., Targeting Transferrin Receptor to Transport Antisense Oligonucleotides Across the Blood-Brain Barrier. BioRxiv. https://doi.org/10.1101/2023.04.25.538145
2. Thayer, MB., POE-Immunoassay: Plate-based oligonucleotide electro-chemiluminescent immunoassay for the quantification of nucleic acids in biological matrices. Scientific reports. 2020.
3. Thayer, MB. & Humphreys SC., Quantification of siRNA-antibody conjugates in biological matrices by triplex-forming oligonucleotide ELISA. Nucleic Acid Ther. 2019.

Acknowledgements: All authors are employees and/or shareholders of Denali Therapeutics, Inc.

Figure 1. Total ASO and Intact OTV methods provide ultra-sensitive and specific quantitation of drug.

Table 1. Total ASO Qualification Results .

Table 2. Intact OTV Qualification Results.