Dual Mechanisms of Action of Self-Delivering, Anti-HIV-1 FANA Oligonucleotides as a Potential New Approach to HIV Therapy

Takahashi et al., Molecular Therapy Nucleic Acids, 2019 tested whether self-delivering antisense oligonucleotides could suppress HIV-1 in the cells the virus infects, and asked how they were working. AUM designed and made the oligonucleotides and co-authored the paper, which was published under the company's former name. Three conserved regions of the viral genome were targeted: the tat and rev region, the U5 region, and the dimerization initiation site in the untranslated region of the genomic RNA, chosen because the first two appear in every spliced and unspliced viral transcript and the third controls how two genome copies pair during assembly.

Delivery came first. A dye-labelled oligonucleotide added to freshly isolated human blood cells, with no carrier and no conjugate, entered 82.9% of them within 4 hours at 100 nM, dose dependently and time dependently, and ended up in the cytoplasm. The same uptake held in a T lymphoblast line. In infected primary cells, treatment at 400 nM cut viral p24 in the supernatant by 69-95% measured 4 days later, with half maximal inhibition at 169.5 nM for the tat and rev design, 176.1 nM for the U5 design, 275.6 nM for the dimerization-site gapmer and 507.7 nM for that gapmer’s sequence made as plain DNA. A single treatment at 3 μM was still working 13 days later, while the DNA comparator faded over the same period.

The mechanism split in two. For the tat and rev design and the U5 design, RNase H1 cleavage products were recovered from treated cells and sequenced, confirming the RNA was cut. The designs against the dimerization site did both, in proportions set by the length of their DNA gap: the nine-base-gap construct, whose half maximal concentration is quoted above, cleaved best, and the fully modified one blocked dimerization best while still cleaving. So one chemistry delivered both a knockdown and a steric block. Neither cytotoxicity nor interferon alpha nor interleukin 6 appeared in cells from multiple donors, against a positive control oligonucleotide that produced more than 500 pg per mL of interferon alpha. The authors also report, without hedging, that the scrambled control of the same chemistry gave about 30% inhibition on its own.