RNA therapeutics are becoming increasingly valuable in clinical applications but their efficient and targeted delivery to the organ and cell type of interest is crucial for clinical translation. Lipid nanoparticles (LNPs) have revolutionised the delivery of mRNA and other small molecules. Tuberculosis (TB) is the single biggest infectious killer worldwide, and current antimicrobial treatment paradigms are increasingly ineffective due to skyrocketing multi-drug resistance interventions need to target alveolar macrophages as these cells represent the major intracellular reservoir for Mycobacterium tuberculosis (Mtb). As proof of concept, we targeted delivery of mRNA coding for mCherry by encapsulating the coding sequence in lipid nanoparticles (LNPs) and we confirmed efficient mRNA transfection into mouse primary bone marrow-derived macrophages (pBMDMs) in vitro. These mRNA-LNPs were then administered to naive mice via intranasal instillation and after 7 h, mCherry expression was detected in the alveoli using IHC. The versatility of this delivery platform allows us to explore novel RNA-based treatment approaches for TB infection.
Using small-molecule IAP antagonists to degrade cellular inhibitors of apoptosis 1 and 2 (cIAP1/cIAP2) to induce apoptosis of Mtb-infected host cells provided proof-of-principle that this host-directed approach can lead to pathogen clearance in vivo. Building on this, we encapsulated cIAP1/2-specific siRNA into LNPs to generate a treatment platform for TB infection. These siRNA-LNPs induced efficient cIAP silencing in mouse pBMDMs, and induction of apoptosis in infected cells.
We combined this approach with another novel RNA-based treatment utilising mRNA that codes for a protective nanobody against a major Mtb virulence factor, ESAT-6. Recombinantly expressed ESAT-6 was used to immunise alpacas and raise ESAT-6-specific nanobodies. Of the resulting nanobody pool, ten ESAT-6 binding nanobodies were selected and their respective mRNA sequences will be encapsulated in LNPs to assess their effect on pathogen virulence in further tests.
With a robust RNA delivery platform suitable for clinical translation, we have a powerful tool at hand to target potent RNA therapeutics directly to the lung. The next step will be to use these LNPs to deliver cIAP1/2-specific siRNA or nanobody-encoding mRNA in vivo and assess their protective potential in a clinically relevant mouse model of TB.