N21 - Strengthening Antisense Oligonucleotide-Mediated Anti-Tumor Immunity Via Metal-Organic Framework Gene Delivery

Introduction: : Overexpression of checkpoint proteins on cancer cells, such as programmed death ligand one (PD-L1), can prevent immune recognition and enable cancer growth. Current monoclonal antibodies that block PD-L1 (e.g., atezolizumab) tend to be large, fragile, and for these reasons unable to penetrate tumors. Moreover, they tend to target cancer at later stages in its growth cycle, which ultimately leads to inconsistent patient outcomes. Antisense oligonucleotides (ASOs) can be employed to decrease expression of PDL1 on cells and reprogram immune responses against tumor expansion. They provide an alternative to decrease PD-L1 expression, but require frequent high dosing due to fast degradation, rapid clearance, and poor cell uptake. Strategies used in the field to overcome these issues have involved utilizing chemical modifications to an ASO, but while these can increase stability and nuclease resistance, extensive modifications can decrease target RNA affinity and negatively affect potency. To overcome these limitations, in this work we have harnessed biocompatible metal-organic framework (MOF) nanoparticles, porous nanomaterials comprising metal nodes and organic linkers, to deliver ASOs effectively to both tumor and immune cells and elevate the resulting antitumor immunity.

Materials and

Methods: : ASO sequences were synthesized using solid-phase standard oligonucleotide synthesis with phosphorothioate backbones, and then encapsulated into zirconium-based 100-200 nm NU-1000 MOFs. Sequences were chosen for PD-L1 specificity, and some contained an additional toll-like receptor 9 (TLR9) agonist region for concurrent immune stimulation. In vitro PD-L1 expression was induced in B16-F10 and EMT6 cancer cells with interferon gamma (IFNy) prior to incubation with treatment. Bone marrow-derived dendritic cells (BMDCs) were differentiated after collection for 5-7 days in 40 ng/mL GM-CSF prior to use. OT1 mice were purchased from Jackson lab and sacrified for splenocyte collection. PD-L1 surface expression, co-stimulatory marker expression on immune cells, T cell proliferation, and Caspase 3 pro-apoptotic marker expression on tumor cells were analyzed via flow cytometry.

Results, Conclusions, and Discussions:: We utilized a zirconium-based nano-sized "NU-1000" MOF, which exhibits a pore diameter of ca. 3.3 nm - large enough to internalize, protect, and deliver large oligonucleotide cargo. Encapsulating ASOs into MOFs specifically can enhance their stability and protection during intracellular delivery, and we observed in this research how this led to reduced PD-L1 expression across multiple tumor types and increased downstream immune recognition. We synthesized three distinct PD-L1-specific ASOs that were cited in past literature to be specific towards targeting the PD-L1 gene. We loaded the three different sequences individually into 100-200 nm NU-1000 MOFs, averaging ~80% encapsulation efficiency and high loading quantities (ca. 9 nmol/mg MOF) regardless of the oligonucleotide sequence. We successfully sustained the release of encapsulated ASOs up to 7 days ex cellulo, which advantageously positions this platform well for reduced and more infrequent dosing compared to current alternatives. MOF encapsulation increased ASO potency and reduced PD-L1 expression ~3-fold and 2-fold in triple negative breast cancer EMT6 and melanoma B16-F10 cells, respectively. Understanding that PD-L1 is expressed on more than just cancer cells but also on healthy immune cells, we evaluated the impact of MOF-delivered ASOs on PD-L1-expressing immune cells. Here, we observed ca. 12-fold increases in dendritic cell co-stimulatory marker expression, and elevated T cell activation and proliferation compared to untreated cells (4-fold and 10-fold, respectively). These propagated responses were not observed by free ASO treatment. Notably, these changes drove a 3-fold increase in B16-F10 caspase-3 expression, a key mediator for apoptosis. Treatment with free ASO or unloaded MOF did not significantly increase the expression of caspase-3 in tumor cells. This research highlights a two-pronged approach for how MOF encapsulation of PD-L1-specific ASOs can impact both tumor and immune cell activity for enhancing propagated anti-tumor immunity. This work also shows how MOFs can be harnessed to bypass ASO limitations without requiring sequence modifications and reducing the necessary required dose. We offer a broadly applicable platform for improved oligonucleotide delivery for various genes of interest across different cancers.

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