Background:
Gene silencing of the latent virus through the Block and Lock approach can provide a functional cure for HIV-1 by inducing a deep latent state. Our group has found in in-silico studies that a combination of 3 promoter targeted siRNA can cover over 99% of HIV strains. We investigated if these siRNA, delivered together in different microRNA backbones, can be safely and effectively delivered and can promote HIV latency.
Methods:
Individual and multiplexed constructs were designed. The multiplexed constructs included a dual construct on miR30 backbone; two 4-miRshRNA with all siRNA on a miR30 backbone, two 4-miRshRNA constructs with all siRNA on a miR 155 backbone and two constructs with siRNA on various microRNA backbones. 2A self-cleaving peptides or miR 17-92 linkers were used to join individual miRshRNA in each construct. In one instance no linkers were used. These constructs were tested for efficacy by assessing their effect on silencing of HIV virus by measuring HIV core antigen expression by flow cytometry, off-target effects by measuring their effect on interferon stimulating genes and their ability to promote viral latency using various latency reversing agents (LRA). The constructs were compared with a mock construct of miR-30. Statistical significance was determined in GraphPad Prism 10.
Results:
All the multiplexed constructs significantly silenced HIV-BaL or pseudovirus (HIV-NL4.3 iGag delta env mOrange), with four out of six 4-multimiR constructs decreasing HIV-Core Antigen or mOrange expression levels by greater than 90% in TZMBL cells, compared to controls (p<0.0001). The microRNA backbone and linker used affected the expression of various siRNA, with the 2A peptide linked constructs showing greater efficacy. Assessment of interferon stimulating gene expression (RIG-1, OAS-1, IFIT1, Viperin) in transduced TZMBL cells showed no statistical difference between the multiplexed constructs and mock, except for the construct on various loops and no linkers, which was significant for Viperin (p<0.001). Importantly, the constructs blocked virus reactivation by different LRAs (TNFa, TNFa-SAHA, SAHA, Resveratrol, Bryostatin and PMA/Ionomycin) in J-Lat A.72 cells, a HIV-Latency model, to significant levels (p<0.01).
Conclusion
MicroRNA backbones can be used to efficiently and safely deliver siRNA. There is a difference in efficacy and safety between the different backbones, and it is important to screen various miRNA to find an appropriate microRNA backbone for delivery.
Disclosure of Interest Statement:
The project was funded by NHMRC Grant.