No abstract

Lipid nanoparticles (LNPs) can be used as delivery vehicles for nucleic acid biotherapeutics. In fact, LNPs are currently being used in the Pfizer/BioNTech and Moderna COVID-19 vaccines. Cationic LNPs composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (chol) LNPs have been classified as one of the most efficient gene delivery systems and are being tested in numerous clinical trials. The objective of this study was to examine the effect of the molar ratio of DOTAP/chol, PEGylation, and lipid to mRNA ratio on mRNA transfection, and explore the applications of DOTAP/chol LNPs in pDNA and oligonucleotide transfection. Here we showed that PEGylation significantly decreased mRNA transfection efficiency of DOTAP/chol LNPs. Among non-PEGylated LNP formulations, 1:3 molar ratio of DOTAP/chol in DOTAP/chol LNPs showed the highest mRNA transfection efficiency. Furthermore, the optimal ratio of DOTAP/chol LNPs to mRNA was tested to be 62.5 µM lipid to 1 μg mRNA. More importantly, these mRNA-loaded nanoparticles were stable for 60 days at 4 °C storage without showing reduction in transfection efficacy. We further found that DOTAP/chol LNPs were able to transfect pDNA and oligonucleotides, demonstrating the ability of these LNPs to transport the cargo into the cell nucleus. The influence of various factors in the formulation of DOTAP/chol cationic LNPs is thus described and will help improve drug delivery of nucleic acid–based vaccines and therapies. Graphical abstract

KRAS is a GTPase involved in the proliferation signaling of several growth factors. The KRAS gene is GC-rich, containing regions with known and putative G-quadruplex (G4) forming regions. Within the middle of the G-rich proximal promoter, stabilization of the physiologically active G4mid structure downregulates transcription of KRAS; the function and formation of other G4s within the gene are unknown. Herein we identify three putative G4-forming sequences (G4FS) within the KRAS gene, explore their G4 formation, and develop oligonucleotides targeting these three regions and the G4mid forming sequence. We tested Polypurine Reverse Hoogsteen hairpins (PPRHs) for their effects on KRAS regulation via enhancing G4 formation or displacing G-rich DNA strands, downregulating KRAS transcription and mediating an anti-proliferative effect. Five PPRH were designed, two against the KRAS promoter G4mid and three others against putative G4FS in the distal promoter, intron 1 and exon 5. PPRH binding was confirmed by gel electrophoresis. The effect on KRAS transcription was examined by luciferase, FRET Melt2, qRT-PCR. Cytotoxicity was evaluated in pancreatic and ovarian cancer cells. PPRHs decreased activity of a luciferase construct driven by the KRAS promoter. PPRH selectively suppressed proliferation in KRAS dependent cancer cells. PPRH demonstrated synergistic activity with a KRAS promoter selective G4-stabilizing compound, NSC 317605, in KRAS-dependent pancreatic cells. PPRHs selectively stabilize G4 formation within the KRAS mid promoter region and represent an innovative approach to both G4-stabilization and to KRAS modulation with potential for development into novel therapeutics.

The oncogene MYC has key roles in transcription, proliferation, deregulating cellular energetics, and more. Modulating the expression or function of the MYC protein is a viable therapeutic goal in an array of cancer types, and potential inhibitors of MYC with high specificity and selectivity are of great interest. In cancer cells addicted to their aberrant MYC function, suppression can lead to apoptosis, with minimal effects on non-addicted, non-oncogenic cells, providing a wide therapeutic window for specific and efficacious anti-tumor treatment. Within the promoter of MYC lies a GC-rich, G-quadruplex (G4)-forming region, wherein G4 formation is capable of mediating transcriptional downregulation of MYC. Such GC-rich regions of DNA are prime targets for regulation with Polypurine Reverse Hoogsteen hairpins (PPRHs). The current study designed and examined PPRHs targeting the G4-forming and four other GC-rich regions of DNA within the promoter or intronic regions. Six total PPRHs were designed, examined in cell-free conditions for target engagement and in cells for transcriptional modulation, and correlating cytotoxic activity in pancreatic, prostate, neuroblastoma, colorectal, ovarian, and breast cancer cells. Two lead PPRHs, one targeting the promoter G4 and one targeting Intron 1, were identified with high potential for further development as an innovative approach to both G4 stabilization and MYC modulation.

Background: Non-Hodgkin's Lymphoma (NHL) is the fifth most common cancer in the United States. Up to 40% of NHL is diffuse large B-cell lymphoma (DLBCL); while only 3% of NHL is classified as mantle cell lymphoma, it is a particularly lethal disease. DLBCL and MCL patients that aberrantly express MYC, which occurs up to 20 and 50% of the time, respectively, have worse survival rates and a lack of sustained clinical response to the standard R-CHOP therapy. We sought to examine both the effect of MYC downregulation on the efficacy of standard of care drugs and enhanced anti-cancer activity with a novel NF-kB targeting steroid, in place of prednisone/prednisolone, and also under conditions of MYC modulation, in NHL cells. Methods and Summary of Findings: The downregulation of MYC was achieved in two distinct manners: with siRNA and with our pioneered MYC G-quadruplex (G4)-stabilizing DNA interference (DNAi) oligonucleotide. This DNAi was optimized for stability, specificity, and in cell activity, from our previously work developing this approach (Hao, et al, NAR 2016). The optimization was done using EMSA, chemical (DMS) footprinting, cell viability assays, qPCR, and microscopy. The lead steroid compound, which is selective for NF-κB signaling inhibition over DNA binding and transcription effects, was more cytotoxic than prednisolone, as determined by cell viability assays in NHL cells. Moreover, its effects on cell growth were additive with doxorubicin, and the overall effect of both drugs on cytotoxicity were enhanced by MYC downregulation. The combination studies were also performed with prednisolone; effects on decreased cell viability were more notable with the novel steroid, as compared to prednisolone. Conclusion: MYC-overexpressing NHL, particularly DLBCL and MCL, respond poorly to the standard R-CHOP therapy. The addition of a MYC-downregulating DNAi, a novel NF-kB targeting steroid, or both, have been noted to enhance the cytotoxic effects of standard drugs. Work is expanding to evaluate the utility of an updated R-CHOP regimen in order to enhance survival for patients with MYC-overexpressing NHL. Citation Format: Alexandra Maria Psaras, Tracy Ann Brooks. Enhancing therapies for MYC-overexpressing Non-Hodgkin's lymphoma: A role for MYC downregulation and new combination therapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 653.