Engineering PBAE Nanoparticles for Immunological Reprogramming of Meningioma and Glioblastoma Cancer Cells

Introduction: Poly(beta-amino ester)s (PBAE) nanoparticles are an attractive gene therapy vector due to their low immunogenicity and scalability. Nanoparticles are assembled from positively charged polymers and negatively charged nucleic acids, which can then efficiently deliver genetic cargos into tumor cells for a desired immune response. T cell activation requires three key signals: Signal 1, for recognition of tumor antigens presented on MHC I, Signal 2, for engagement of co-stimulatory molecules, and Signal 3, for secretion of cytokines involved in immune cell recruitment and differentiation. Collectively, this trifecta increases T-cell affinity to tumors and enhance anti-tumor immune responses, without the need to isolate tumor cells and identify tumor antigens ex vivo.
Brain cancers are the fifth most common cancer, with meningioma comprising of 46% of all primary brain tumors and glioblastoma constituting 16.4%. Meningioma affects the membrane enveloping the brain and spinal cord and is the most common non-malignant tumor. Glioblastoma is the most common primary malignant brain tumor. The current standard of care for most meningiomas and glioblastomas is surgical resection, which is highly invasive and requires long recovery times. A nanoparticle gene therapy could a safer and targeted immune response that will eliminate brain tumor cells specifically, eliminating the invasiveness of current treatment options.

Materials and

Methods: This project consisted of three major stages: nanoparticle formulation, tumor reprogramming, and in vivo delivery.
Four PBAE nanoparticle formulations were synthesized and characterized via dynamic light scattering (DLS). To validate gene delivery in vitro, CT-2A (mouse glioblastoma) and IOMM-Lee (human meningioma) cells were transfected with nanoparticles carrying between 300-900 ng of GFP plasmid at 30-90 w/w (weight/weight polymer to DNA ratio). An MTS cell viability assay was conducted to assess toxicity, and GFP transfection was evaluated through flow cytometry. In a following study, IOMM-Lee cells were co-cultured with healthy human astrocytes to understand if there was preferential transfection of cancer cells over healthy cells.
Next, IOMM-Lee cells were transfected with signal 2 genes and IL12 to understand the cytokine expression profiles elicited by these signals. A co-culture with human PBMCs was conducted with 4-1BBL/IL12 delivery to evaluate the IFNγ inflammatory response via ELISA. A more comprehensive ex vivo co-culture of tumor cells with PBMCs after 4-1BBL/IL12 delivery was conducted afterwards to assess tumor cell killing and immune cell activity.
For the in vivo models, three approaches were taken. The first involved implanting IOMM-Lee cells into immunocompromised athymic nude mice brains, followed by delivery of GFP nanoparticles by convection enhanced delivery (CED). The second entailed treatment of CT-2A glioblastoma orthotopically in immunocompetent B6 mice with 4-1BBL/IL12 nanoparticles + anti-PD1. In the final study, we implanted IOMM-Lee cells subcutaneously in the flank of humanized mouse model (MHC double knockout NSG mice humanized with human PBMCs) and treated with 4-1BBL-IL12 nanoparticles + anti-PD1.

Results, Conclusions, and Discussions: PBAE delivery of plasmid GFP demonstrated successful transfection in CT-2A and IOMM-Lee cell lines, across varying dosages and weight/weights. The nanoparticle formulation 4-5-39 demonstrated the highest transfection efficacy across both cell lines and was thus selected for further testing. DLS characterization revelated that the 4-5-39 nanoparticles were ~260 nm in diameter. Upon co-culturing IOMM-Lee cells with healthy human astrocyte cells, we found that the PBAE nanoparticles transfected the cancer cells at a higher rate than the healthy brain cells.
Next, tumor reprogramming was evaluated in the IOMM-Lee cells. From the signal 2 screen, we selected 4-1BBL as the preferred signal 2, as it demonstrated expression levels similar to the reporter gene and had been validated in many previous tumor models before. 4-1BBL was delivered alongside IL12 into IOMM-Lee cells, which were then co-cultured with PBMCs. An IFNγ ELISA demonstrated that the 4-1BBL/IL12 nanoparticles elicited a favorable inflammatory response. This co-culture likewise demonstrated tumor cell killing seven days after nanoparticle delivery, as well as an MHC II shift, indicating tumor programming resulting from 4-1BBL/IL12 delivery.
To validate in vivo gene expression, we performed subcutaneous implantation of IOMM-Lee cells in humanized mice and athymic nude mice flanks, and CT-2A cells into B6 mice. Luciferase nanoparticles were injected intratumorally, and IVIS imaging confirmed robust expression of luciferase 6 hours post-injection. We then implanted 40 immunodeficient mice with IOMM-Lee cells in the brain for the purpose of evaluating orthotopic transfection efficacy. A GFP ELISA was used to measure the concentration of GFP expressed. The expression distribution appeared to be bimodal, with some mice expressing high amounts of GFP while others demonstrated little to no expression.
Two further in vivo survival studies are currently being conducted to evaluate in situ tumor reprogramming. CT-2A cells were implanted orthotopically in B6 immunocompetent mice and treated with 4-1BBL and IL12 PBAE nanoparticles. IOMM-Lee cells were implanted subcutaneously in a humanized immunocompetent mouse model and likewise treated with tumor reprogramming nanoparticles. PBAE NPs can transfect human and rodent meningioma and glioma cells in vitro and in vivo, and local administration of NPs delivering immunostimulatory genes leads to anti-tumor effect.

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