Assistant Professor Boston University Boston, Massachusetts, United States
Introduction: Overexpression of key regulatory proteins, such as programmed death ligand one (PDL1), on cancers prevents immune recognition and allows for unregulated cancer growth. Existing immunotherapeutic treatments use monoclonal antibodies that bind to PDL1 which helps with immune recognition, but such therapeutics tend to be large and unstable and target cancers at late stages in their growth cycles. This ultimately leads to poor patient outcomes. Antisense oligonucleotides (ASOs) can be employed to decrease expression of PDL1 on cells and reprogram immune responses against tumor expansion. However, ASOs frequently require continuous high doses to be effective, as they suffer from rapid clearance, fast degradation, and poor cell uptake. To overcome these issues, we have harnessed metal organic framework (MOF) nanomaterials, to deliver ASOs. MOFs are well-defined, highly porous, and biocompatible nanomaterials. Encapsulation of ASOs into a MOF can provide ASOs the stability and protection necessary for intracellular delivery and reduced PDL1 expression, allowing for increased downstream immune recognition of cancers.
Materials and
Methods: ASO sequences were synthesized using solid-phase standard oligonucleotide synthesis, and then encapsulated into zirconium-based nano-sized NU-1000 MOFs. Sequences were chosen for PDL1 specificity, and some contained an additional toll-like receptor 9 (TLR9) agonist region for concurrent immune stimulation. In vitro PDL1 expression was induced in melanoma and triple negative breast cancer cells with interferon gamma (IFNy) prior to incubation with treatments, and PDL1 expression was analyzed via flow cytometry. In vitro PDL1 and co-stimulatory markers expression in immune cells were analyzed via flow cytometry.
Results, Conclusions, and Discussions: We successfully loaded 3 different PDL1-specific ASO sequences into MOFs, averaging ca. 80% encapsulation efficiency. Release of encapsulated ASOs was sustained for up to seven days ex cellulo. MOF encapsulation increased ASO potency in cancer and immune cells, as measured by PDL1 reduction, where treatment led to a three-fold decrease in expression in triple negative breast cancer EMT6 cells and two-fold decrease in melanoma B16F10 cells. In contrast, freely delivered ASO treatments exhibited no reduction in PDL1 expression. To understand the effect of MOF encapsulated ASO delivery in non-cancer cells, PDL1 surface expression on bone marrow-derived dendritic immune cells was measured. Dendritic cells exhibited 25-50% decreases in PDL1 expression and increased co-stimulatory marker expression, ranging from 6 to 12-fold higher than untreated cells. Across all tested cell types, the MOF alone did not induce any significant changes in surface expression, indicating that the role of the MOF in ASO delivery is solely to protect and enable uptake, and that the ASO is key to raising an immune response and reducing tumor evasion. These responses propagate downstream and increase T cell proliferation two-fold at twenty times lower concentrations of ASO when encapsulated into MOFs. Overall, we observe that MOF-mediated delivery of ASOs is capable of increasing anti-tumor immunity, through both enhanced interactions and activation of immune cells and reduced cancer cell evasion and blockade of the immune system. This research highlights how MOFs can be harnessed to bypass ASO limitations. Importantly, as this platform does not require any modifications to the ASO, it can be expanded to other key genes of interest that suffer from overexpression across various cells, providing a path towards improved drug protection and delivery.
