# Investigation of the activity of phosphothioate and phosphothioate-LNA-modified oligonucleotides against HIV-1

## TL;DR

This study compares how well phosphothioate and LNA-modified oligonucleotides can block HIV-1 infection in human cells.

## Contribution

It shows that unmodified phosphothioate oligonucleotides are more effective at inhibiting HIV-1 than LNA-modified ones.

## Key findings

- PS oligonucleotides without LNA modifications achieved the lowest IC50 values against HIV-1 integrase.
- LNA-modified oligonucleotides showed reduced antiviral activity compared to unmodified PS oligonucleotides.
- PS-modified oligonucleotides demonstrated better internalization and lower toxicity in cell cultures.

## Abstract

This study investigated the antiretroviral efficacy, toxicity profile, and cellular uptake of phosphothioate (PS) and PS/LNA-modified oligonucleotides within an in vitro HIV infection model. Phosphothioate (PS) oligonucleotides, designed to bind conserved regions of the HIV-1 genome, were modified at the 3’ and/or 5’ ends with LNA nucleotides. The antiviral properties of oligonucleotides against HIV-1 subtype A6 were evaluated using human MT-4 cell cultures. The antiretroviral activity of LNA-oligonucleotides against HIV-1 has been established. Variations in the 50% inhibitory viral reproductive dose (IC50) values among the oligonucleotides were observed, depending upon both the target and the incorporated LNA modification. Optimal IC50 values (90 ± 10 nM) were achieved using a PS oligonucleotide lacking LNA modifications, which targeted the HIV-1 integrase-encoding genomic region. Optimal HIV inhibitory action among LNA constructs was observed in an oligonucleotide with a 5’-end LNA modification targeting the HIV integrase region (IC50 = 1.12 ± 0.03 μM). The introduction of LNA modifications to PS oligonucleotides failed to enhance antiviral activity, as demonstrated by IC50 values revealing significant in vitro HIV-1 inhibitory capacity. The internalization of oligonucleotides demonstrating optimal IC50 values was investigated via flow cytometry and imaging techniques. Antisense oligonucleotides with single PS modification showed better antiretroviral activity with lower IC50 value compared to PS/LNA-modified antisense oligonucleotides. The reason for this difference is the better internalization ability of PS-modified oligonucleotides. The characteristic features included low toxicity (maintaining >92% viable cells after 48 h of culture), high cytoplasmic membrane sorption capacity (approximately 12% FAM + cells after 48 h), high penetration efficiency (approximately 98% FAM + cells showing cytoplasmic signal), and elevated internalization and entropy ratios.