Poster Presentation 50 Years Shine-Dalgarno Symposium 2023

Delivery of siRNA inducing transcriptional/epigenetic silencing via Layer-by-Layer particles to the HIV-1 latent reservoir (#133)

Vera Klemm 1 , Ewa Cuba-Wojnilowicz 2 3 , Christina Cortez-Jugo 2 3 , Stuart G Turville 1 4 , Frank Caruso 2 3 , Anthony D Kelleher 1 4 , Chantelle L Ahlenstiel 1 4
  1. Kirby Institute, Sydney, NSW, Australia
  2. Department of Chemical and Biomolecular Engineering, University of Melbourne, Parkville, Victoria, Australia
  3. ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, -, Australia
  4. The UNSW RNA Institute,, Sydney, NSW, Australia

Background:

Delivery of siRNA therapeutics to induce post-transcriptional gene silencing in the cytoplasm and mRNA degradation has been achieved using nanomaterials. However,  nanomaterial delivery of siRNA therapeutics to induce transcriptional gene silencing (TGS) and epigenetic modifications at the promoter in the nucleus is far more challenging, partly due to poor intracellular and nuclear delivery. In the case of the HIV latent reservoir, which is the major barrier to an HIV cure as the reservoir can reactivate when antiretroviral therapy is ceased, the predominate reservoir cell type is resting CD4+ T cells, which do not perform active endocytosis. In this study we investigate Layer-by-Layer particles for delivery of TGS siRNA to HIV latent reservoir cell types, including primary human activated/resting CD4+ T cells and monocyte-derived macrophages.  

 

Methods:

Fluorescently labeled promoter-targeted siPromA or siScrambled control were complexed with multilayered poly(styrene sulfonate) and poly(arginine) particles then incubated with a range of HIV-infected cell types (HeLa T4+, primary activated/resting CD4+ T cells and monocyte-derived macrophages (MDMs). Nuclear delivery of siRNA was assessed at 48h using deconvolution microscopy to measure i) co-localisation of siRNA with NucBlue stained nuclei, ii) arbitrary line profile and iii) 3D cell profile.  Assessment of functional HIV-1 gene silencing by particle-delivered siRNA was measured using Reverse Transcriptase assay and RT-qPCR of gag RNA.

Results:

Image analysis showed delivery of siPromA into the nucleus of all infected cell types assessed.  Significantly decreased levels of Reverse Transcriptase and gag RNA levels demonstrated functional silencing of particle-delivered siPromA in HeLa T4+, primary activated CD4+ T cells and MDMs from three donors, compared to controls.

Conclusion:

This is the first demonstration of particle-delivered siRNA to the nucleus for RNAi via transcription gene silencing and highlights the versatility of nanomaterials for nuclear siRNA delivery to challenging cell types relevant to a range of diseases.