New Jersey Institute of Technology Newark, New Jersey, United States
Introduction: Gene therapy is one of the most promising medical fields which holds the potential to rapidly advance the treatment of difficult ailments such as cancer as well as inherited genetic diseases. However, clinical translation is limited by several drug delivery hurdles including renal clearance, phagocytosis, enzymatic degradation, protein absorption, as well as cellular internalization barriers. Additionally, successful treatments require sustained release of drug payloads to maintain the effective therapeutic level. As such, controlled and sustained release is a significant concern as the localization and kinetics of nucleic acid therapeutics can significantly influence the therapeutic efficacy. There is an unmet need for the development of biodegradable and controlled-release nanoparticle (NP) technologies to further improve the gene therapy efficacy by prolonging the release of nucleic acid drug payload for sustained gene expression or silencing. Herein, we present a 'particle-in-particle' (PNP) nanostructure approach for gene delivery with biodegradable and sustained release properties. Briefly, we have designed a library of lipid-modified PBAEs (termed L-PBAEs) based on the rational hypothesis that the grafting of lipid chains to the PBAE backbone would: 1) facilitate the fusion of the generated nanoparticles with the cell membrane and increasing cellular uptake via endocytosis; 2) increase the nanoparticle mediated gene delivery efficacy and transfection potency, and 3) improve the hydrophobicity of PBAE polymer and the PNPs’ consequent integrity and stability in physiological conditions.
Materials and
Methods: The L-PBAEs were synthesized via Candida antarctica Lipase B (CALB) enzyme-assisted esterification between the hydroxyl groups of PBAE-447 and the carboxylic acid from different commercially available saturated and unsaturated lipid acids. Varying the 8 different lipid acids and 3 different molecular ratios of lipid: PBAE in the PBAE backbone, we synthesized and characterized a library of 24 different cationic L-PBAE polymers. We then prepared a library of gene loaded PNP/L-PBAE NPs, where the PLGA-PEG and L-PBAEs formed to be PNPs through self-assembly and the gene payloads were encapsulated inside L-PBAEs via electrostatic interactions to form L-PBAE/gene nanocomplexes which were embedded within a PLGA-PEG polymeric vector. PNP/C12-PBAE was screened as the top-performing universal transfection formulation for both DNA and mRNA via high-throughput luciferase assay. We screened that the PNP/C12-PBAE NP could effectively transfect fluorescence or luminescence encoded plasmid or mRNA and exhibited very limited cytotoxicity. We further formulated the nanoparticles into a COVID-19 vaccine by encapsulating spike-protein-encoded mRNA and tested whether the formulations could elicit spike-specific antibodies and a Th1-biased T cell immune response in immunized mice.
In addition, we investigated the effect of GSDMBNT mRNA-encapsulated nanoparticles on a-PD1-mediated cancer immunotherapy in immunologically cold tumor models. We locally delivered GSDMBNT mRNA in two immunologically cold tumor models: one that is only moderately sensitive to PD-1 blockade (B16F10 melanoma) and another that is resistant to PD-1 inhibitors (4T1 breast carcinoma). We evaluated whether the pyroptosis-triggered mRNA nanoparticles could enhance cancer immunotherapy.
Results, Conclusions, and Discussions: We demonstrated the excellent stability of PNP for at least 12 months of storage at -20°C after lyophilization, with no loss of transfection efficacy. We further formulated the PNP/C12-PBAE NPs with SARS-CoV-2 spike encoded plasmid DNA and mRNA to develop lipid-modified polymeric nanoparticle-based DNA (DNA-PNP) and mRNA (mRNA-PNP) COVID-19 vaccines. Immunization with mRNA-PNP and DNA-PNP vaccines in BALB/c mice elicited anti-spike antibodies as well as SARS-CoV-2 spike neutralizing antibodies after the priming dose and the antibody titers significantly increased after a booster vaccination in a dose-dependent manner demonstrated by the significant increase of the IgG geometric mean titers (GMTs) compared to the placebo group. Furthermore, the intracellular cytokine staining study detected the secretion of IFN-γ, TNF-α and IL-2, but not IL-4, demonstrating that our PNP system is a universal polymeric nanoparticle-based DNA/mRNA vaccine platform that successfully induces a Th1-biased SARS-CoV-2 spike-specific immune response in vaccinated mice. Additionally, we demonstrate a proof of concept for the design and preclinical trial of local delivery of pyroptosis-triggering mRNA lipid nanoparticles for enhancing ICB therapy in multiple preclinical cold tumor models. Our in vitro data revealed that these GSDMBNT mRNA not only trigger inflammatory pyroptosis but also promote ICD. Animal results further demonstrated that GSDMBNT mRNA reverses the immunosuppressive microenvironment by stimulating the expression of proinflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-18) and promoting the recruitment of CD8+ T cells. Our results illustrated that GSDMBNT mRNA can significantly improve the therapeutic effects of anti-PD-1 immunotherapy and achieve even tumor elimination through revoking antitumor immune responses and remodeling the tumor microenvironment. We also found that this enhanced antitumor immunity locally administered to one tumor stimulates a systemic antitumor response, contributing to cures in the poorly immunogenic tumor model and eliciting control over distant untreated lesions.
Our strategy may provide important steps toward developing a new gene delivery platform with biodegradable and controlled release properties to meet the urgent demand for combating various diseases, including infectious diseases and cancers.
