Biology subjects

Krishnamurthy, P. M.

Publications and source records attributed to Krishnamurthy, P. M..

3 recordsLinked to original sources

bioRxiv2021

Quantification of Antisense Oligonucleotides by Splint Ligation and Quantitative Polymerase Chain Reaction

Reliable detection and quantification of antisense oligonucleotides (ASOs) in experimental and clinical specimens is essential to understand the biological function of novel oligonucleotide-based therapeutics. In this study, we describe a method to detect and quantify ASOs in biological samples, whereby the ASO acts as a splint to direct the ligation of complementary probes and quantitative real-time PCR was used to monitor ligation products. Low levels of 2'-O-MOE gapmer ASO in serum, liver, kidney, lung, heart, muscle, and brain tissues can be detected over a 6-log linear range for detection using this method. This method allows quantification of various types of chemically modified ASOs, including PS linkage, 2'-OMe, 2'-O-MOE, locked nucleic acid (LNA), and siRNA. This method does not require probe modifications, and can be performed using standard laboratory equipment; making it a fast, sensitive, and reliable technique that can be widely applied. This detection method may find potential applications in detection of therapeutic oligonucleotides in biological samples.

molecular biology↗

bioRxiv2021

Quantifying and Mitigating Motor Phenotypes Induced by Antisense Oligonucleotides in the Central Nervous System

Antisense oligonucleotides (ASOs) are emerging as a promising class of therapeutics for neurological diseases. When injected directly into the cerebrospinal fluid, ASOs distribute broadly across brain regions and exert long-lasting therapeutic effects. However, many phosphorothioate (PS)-modified gapmer ASOs show transient motor phenotypes when injected into the cerebrospinal fluid, ranging from reduced motor activity to ataxia or acute seizure-like phenotypes. The effect of sugar and phosphate modifications on these phenotypes has not previously been systematically studied. Using a behavioral scoring assay customized to reflect the timing and nature of these effects, we show that both sugar and phosphate modifications influence acute motor phenotypes. Among sugar analogues, PS-DNA induces the strongest motor phenotype while 2-substituted RNA modifications improve the tolerability of PS-ASOs. This helps explain why gapmer ASOs have been more challenging to develop clinically relative to steric blocker ASOs, which have a reduced tendency to induce these effects. Reducing the PS content of gapmer ASOs, which contain a stretch of PS-DNA, improves their toxicity profile, but in some cases also reduces their efficacy or duration of effect. Reducing PS content improved the acute tolerability of ASOs in both mice and sheep. We show that this acute toxicity is not mediated by the major nucleic acid sensing innate immune pathways. Formulating ASOs with calcium ions before injecting into the CNS further improved their tolerability, but through a mechanism at least partially distinct from the reduction of PS content. Overall, our work identifies and quantifies an understudied aspect of oligonucleotide toxicology in the CNS, explores its mechanism, and presents platform-level medicinal chemistry approaches that improve tolerability of this class of compounds.

pharmacology and toxicology↗

bioRxiv2018

5′ Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome Editing

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5'-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

molecular biology↗