In vivo SELEX strategies to identify potent aptamer-drug conjugates for glioblastoma

PROJECT DESCRIPTION/ABSTRACT Development of effective therapies for glioblastoma (GBM) remains a major challenge despite decades of intensive research. Coupled with intra-tumoral heterogeneity and plasticity, the infiltration of normal brain tissue by GBM cells poses unique therapeutic challenges. Further, the specialized neurovascular unit that forms the blood-brain barrier (BBB) is partially intact in GBM and results in heterogeneous, sub-therapeutic delivery of most cytotoxic chemotherapies to regions of every GBM. We have previously shown that the efficacy of otherwise highly potent antibody-drug conjugates is specifically limited in GBM by poor delivery across the BBB. Like antibodies, single-strand DNA aptamers fold into unique 3-dimensional shapes with epitope binding affinities that rival those of antibodies, and some aptamers also efficiently traverse the BBB. In contrast to the laborious development of antibody-based therapeutics, the integration of solid-support synthesis, PCR amplification, and next-generation sequencing technologies enable massively parallel screening strategies, known as ‘systematic evolution of ligands by exponential enrichment (SELEX)’, to identify individual DNA aptamers with desired physical and biological features through successive rounds of negative and/or positive selection. Based on prior experience with this strategy, we hypothesize that tumor-specific DNA aptamer-drug conjugates (ApDCs) optimized for distribution across the BBB can be efficiently identified using in vivo SELEX with libraries of aptamer-drug conjugates and orthotopic GBM patient-derived xenografts. To address tumor heterogeneity, in vivo selection will be performed across multiple PDXs, and single cell sequencing technology will be leveraged to identify ApDCs that bind to diverse subsets of tumor cells and not normal cells within the brain. The goal of this application is to develop a robust platform for efficient screening of GBM-specific ApDCs. This will be accomplished by addressing three Aims. Aim 1 – R61: Optimize design and sequencing of aptamer-toxin libraries We will optimize strategies for toxin conjugation during library processing through multiple SELEX rounds. Extending our preliminary data demonstrating MMAE toxin stability to PCR thermal cycling, we will optimize toxin-conjugated PCR primers for library preparation and single-cell RNA/aptamer-seq. Aim 2 – R61: Determine optimum training round strategy to identify brain tumor-specific ApDCs We will optimize the time between DNA library injection and tissue collection. We also will evaluate a novel SELEX reward strategy based on selectively capturing aptamers only after cleavage of MMAE from an ApDC. Aim 3 – R33: Apply in vivo SELEX with orthotopic GBM PDXs to train ApDC libraries In vivo SELEX will be performed with an ApDC library across a heterogenous set of orthotopic GBM PDXs to understand the potential for targeting heterogeneous tumor and sparing normal cell populations.