Microbiology & Immunology - Theses
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ItemTargeting the untargetable: Eliminating HIV latency using nanoparticle delivery systems( 2023-04)T cells form an important therapeutic target for the development of immunotherapies or the treatment of T cell-implicated pathologies. Specifically, CD4 T cells are subject to human immunodeficiency virus (HIV) infection. Whilst treatment with antiretroviral therapy (ART) successfully represses the viral load to undetectable levels, HIV treatment is lifelong, posing a medical, social and financial burden to those 38.5 million people currently living with HIV globally. The ability of HIV to establish a reservoir of latently infected cells is the foremost barrier to finding a cure for HIV. One approach towards eliminating HIV latency is the reactivation of viral transcription and subsequent elimination of infected cells through immune-mediated clearance or viral-mediated cytotoxicity. However, this approach so far has suffered from a lack of potency and dose-limiting toxicities due to the use of compounds that affect both host and viral transcription and the inability to specifically target the infected cells. One solution involves the use of nanoparticles for the targeted delivery of existing therapeutics or to advance the development of HIV-specific mRNA-based therapeutics including CRISPR-Cas. However, the generally low rate of endocytosis in CD4+ T cells forms a challenge to efficient nanoparticle-based drug delivery to CD4+ T cells in vitro and in vivo. This thesis describes our efforts towards rationally designing a nanoparticle platform capable of delivering HIV latency-reversing therapeutics to CD4+ T cells with high efficiency. We first established a methodology to improve the assessment of nanoparticle performance in vitro through the generation of absolutely quantitative, comparable data on nanoparticle-cell interactions. We then used this methodology to screen for nanoparticle designs with enhanced uptake kinetics in CD4+ T cells in vitro, using a novel, high-throughput assay to quantify nanoparticle internalization over time. We found that targeting sub-100 nm nanoparticles to T cell surface receptors undergoing rapid receptor cycling can be exploited to actively trigger nanoparticle uptake through receptor-mediated endocytosis and identified CD2 and CD7 as potent candidate receptors for future in vivo T cell targeting. We next aimed to use translate these findings to lipid nanoparticles, a well-established platform for the delivery of nucleic acid-based therapeutics. We investigated whether lipid nanoparticles could be used to deliver a next-generation latency-reversing agent based on CRISPR activation, which specifically targets the HIV proviral genome without affecting host-cell transcription. We identified a novel lipid nanoparticle formulation that can efficiently transfect T cell lines as well as resting CD4+ T cells. We showed that this lipid nanoparticle can co-encapsulate the three RNA components of the CRISPR activation system and induce strong latency reversal in a cell line model for HIV latency. We finally presented preliminary evidence that targeting lipid nanoparticles to rapidly cycling surface receptors increases mRNA delivery, further supporting our findings that targeting receptor-mediated endocytosis could be a viable strategy to increase nanoparticle-mediated drug delivery to traditionally hard-to-transfect T cells. These findings provide a compelling justification for the further assessment of CRISPR activation lipid nanoparticles for the elimination of the latent HIV reservoir, and more broadly contribute to the development of T cell-targeted nanomedicine for HIV and beyond.