Assistant Professor Boston University, United States
Introduction:: Neurodegenerative disease impacts over five million individuals in the US, but current treatments do not halt disease progression1. An emerging contributor to neurodegeneration is the dysfunction of glial cells, which directly support neuronal function and tissue health in the CNS2,3. Microglia, in particular, are essential for maintaining homeostasis in the brain via clearing debris, synaptic pruning, and coordination of immune responses2. Current therapies for neurodegeneration target neurons, however, microglial dysfunction and reactivity often precedes neuronal damage in these disorders3,4. Thus, developing therapies that act selectively on microglia could be an effective way to arrest disease in the early stages. We currently lack effective ways to target microglia for therapy in a cell-specific manner. We hypothesize that targeting microglia for selective therapy delivery can be achieved using non-viral dendrimer nanoparticles (NP) with multivalent presentation of ligands that preferentially engage microglia specific transporters. Here we outline: (1) our use of in silico unsupervised analysis of published glia transcriptomes to identify microglia specific surfaceome targets; (2) a novel dendrimer synthesis method used to prepare NP with multivalent presentation of microglial ligands; and (3) development of an in vitro assay for characterizing ligand specific targeting and cellular uptake mechanisms.
Materials and Methods:: In-silico RNA-Seq analysis used published sequences in Gene Expression Omnibus (GEO) database, analyzed with Galaxy and principal component analysis (PCA). This unbiased screen revealed Slc2a5 as a unique and highly expressed microglia surfaceome gene, which encodes the Glut5 fructose transporter. Expression of Glut5, in healthy mouse brain was evaluated using immunocytochemistry (IHC) methods. To prepare NPs, dendrimers were synthesized using a series of sequential thiol-ene radical and enzymatically catalyzed reactions using pentaerythritol tetrakis (3-mercaptopropinate), trimethylolpropane allyl ether (purchased from Sigma) and ethyl 3-mercaptopropionate (purchased from TCI Chemicals). Dendrimers were functionalized with fructose mono-acrylate (FA) and tertiary amine acrylate (TAA) via 2-(dimethylamino) ethyl acrylate (purchased from TCI Chemicals) and evaluated by NMR and FTIR. To assess Glut5 targeting of NP, we developed an in vitro assay using MCF-7 cells, which controllably expresses Glut5 5,6. Evaluation of Glut5 expression and NP uptake was evaluated using immunocytochemistry (ICC) on fixed cells.
Results, Conclusions, and Discussions:: Evaluation of PCA factor loadings using a cosine similarity cut-off of 0.9 identified 188 microglia specific surfaceome genes, and the list was reduced to the 25 most highly expressed genes based on FPKM value, including Slc2a5, the gene encoding the Glut5 fructose transporter (Fig 1A). Glut5 was detected across the mouse brain but particularly in the striatum where it colocalized with P2yr12 positive microglia along the cell surface (Fig 1B). Generation 1 and Generation 2 dendrimers (Fig 1C) could be prepared on gram scale with accurate control of fructose and amine functionalization. Unique shifts in FTIR spectra correlate with degree of fructose functionalization, allowing the control of functionalization to be assessed. Glut5 expression in cultured MC-7 cells increased 4-fold upon 72 hours exposure to soluble fructose dosed into growth medium (Fig 1D), and preliminary data suggests this effect is consistent at 24 and 48 hours. Work is ongoing to evaluate the formulation of dendrimers into NP and their fructose selective uptake into cells.
In conclusion, we used a bioinformatic analysis to identify Glut5 as a microglia specific target for therapeutic delivery. We confirmed the expression of Glut5 in murine brain with IHC and synthesized dendrimers that permit multivalent presentation of fructose, the ligand for the Glut5 transporter, to permit selective delivery of mRNA therapeutics to microglia. An in vitro assay to induce high expression of Gut5 in MCF-7 cells will enable optimization of NP formulations in future work.
Acknowledgements (Optional): : We thank the Boston University Core Facilities for the use of the NMR, FTIR, and ix83 widefield microscope. This work was supported by funding from Boston University (T.M.O).
References (Optional): : [1] Thorpe, K. E., et al. Emory Univ (2021). [2] Colonna, M. et al. Annu. Rev. Immunol (2017). [3] Kam, T.-I., et al. Neurobiol (2020). [4] Neniskyte, U. & Gross, et al. Nat. Rev. Neurosci (2017). [5] Zamora-León, S. P. et al. Proc. Natl. Acad. Sci (1996). [6] MCF7 - HTB-22 | ATCC.
