Effective gene therapy of Stargardt disease with PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles.

Stargardt disease (STGD) is the most common form of inherited retinal genetic disorders and is often caused by mutations in ABCA4. Gene therapy has the promise to effectively treat monogenic retinal disorders. However, clinically approved adeno-associated virus (AAV) vectors do not have a loading capacity for large genes, such as ABCA4. Self-assembly nanoparticles composed of (1-aminoethyl)iminobis[N-(oleoylcysteinyl-1-amino-ethyl)propionamide (ECO; a multifunctional pH-sensitive/ionizable amino lipid) and plasmid DNA produce gene transfection comparable with or better than the AAV2 capsid. Stable PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles produce specific and prolonged expression of ABCA4 in the photoreceptors of Abca4 -/- mice and significantly inhibit accumulation of toxic A2E in the eye. Multiple subretinal injections enhance gene expression and therapeutic efficacy with an approximately 69% reduction in A2E accumulation in Abca4 -/- mice after 3 doses. Very mild inflammation was observed after multiple injections of the nanoparticles. PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles are a promising non-viral mediated gene therapy modality for STGD type 1 (STGD1).

Optimization of Synthesis of the Amino Lipid ECO for Effective Delivery of Nucleic Acids.

Nucleic acids are promising for a variety of therapies, such as cancer therapy and the gene therapy of genetic disorders. The therapeutic efficacy of nucleic acids is reliant on the ability of their efficient delivery to the cytosol of the target cells. Amino lipids have been developed to aid in the cytosolic delivery of nucleic acids. This work reports a new and efficient synthetic pathway for the lipid carrier, (1-aminoethyl) iminobis [N-(oleicylcysteinyl-1-amino-ethyl)propionamide] (ECO). The previous synthesis of the ECO was inefficient and presented poor product quality control. A solution-phase synthesis of the ECO was explored, and each intermediate product was characterized with better quality control. The ECO was synthesized with a relatively high yield and high purity. The formulations of the ECO nanoparticles were made with siRNA, miRNA, or plasmid DNA, and characterized. The transfection efficiency of the nanoparticles was evaluated in vitro over a range of N/P ratios. The nanoparticles were consistent in size with previous formulations and had primarily a positive zeta potential. The ECO/siLuc nanoparticles resulted in potent luciferase silencing with minimal cytotoxicity. The ECO/miR-200c nanoparticles mediated the efficient delivery of miR-200c into the target cells. The ECO/pCMV-GFP nanoparticles resulted in substantial GFP expression upon transfection. These results demonstrate that the solution-phase synthetic pathway produced pure ECO for the efficient intracellular delivery of nucleic acids without size limitation.

Efficacy of Targeted ECO/miR-200c Nanoparticles for Modulating Tumor Microenvironment and Treating Triple Negative Breast Cancer as Non-invasively Monitored by MR Molecular Imaging.

PURPOSE: To investigate the effectiveness of targeted ECO/miR-200c in modulating tumor microenvironment and treating triple negative breast cancer (TNBC) using non-invasive magnetic resonance molecular imaging (MRMI) of extradomain B fibronectin (EDB-FN) with a targeted MRI contrast agent. METHODS: MDA-MB-231 and Hs578T TNBC cells were transfected with RGD-PEG-ECO/miR-200c. Invasive and migratory potential was evaluated using transwell, scratch wound, and spheroid formation assays. Athymic nude mice bearing orthotopic MDA-MB-231 and Hs578T xenografts were treated with weekly i.v. injection of RGD-PEG-ECO/miR-200c nanoparticles at 1.0 mg/kg/week RNA for 6 weeks. MRMI of EDB-FN was performed using a targeted contrast agent MT218 [ZD2-N3-Gd(DO3A)] on a 3 T MRS 3000 scanner. T1-weighted images were acquired following intravenous injection of MT218 at dose of 0.1 mmol/kg using a fast spin echo axial sequence with respiratory gating. RESULTS: Systemic administration of RGD-PEG-ECO/miR-200c nanoparticles in mice bearing orthotopic TNBC xenografts significantly suppressed tumor progression without toxic side-effects. MRMI with MT218 revealed that the treatment significantly suppressed tumor proliferation as compared to the control. MRMI also showed that the miR-200c treatment altered tumor microenvironment by reducing EDB-FN expression, as evidenced by decreased contrast enhancement in both MDA-MB-231 and Hs578T tumors. The reduction of EDB-FN was confirmed by immunohistochemistry. CONCLUSIONS: Targeted delivery of miR-200c with RGD-PEG-ECO/miR-200c nanoparticles effectively modulates tumor microenvironment and suppresses TNBC proliferation in animal models. MRMI of tumor EDB-FN expression is effective to non-invasively monitor tumor response and therapeutic efficacy of RGD-PEG-ECO/miR-200c nanoparticles in TNBC.

Formulation and efficacy of ECO/pRHO-ABCA4-SV40 nanoparticles for nonviral gene therapy of Stargardt disease in a mouse model.

It is still a challenge to develop gene replacement therapy for retinal disorders caused by mutations in large genes, such as Stargardt disease (STGD). STGD is caused by mutations in ABCA4 gene. Previously, we have developed an effective non-viral gene therapy using self-assembled nanoparticles of a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid containing rhodopsin promoter (pRHO-ABCA4). In this study, we modified the ABCA4 plasmid with simian virus 40 enhancer (SV40, pRHO-ABCA4-SV40) for enhanced gene expression. We also prepared and assessed the formulations of ECO/pDNA nanoparticles using sucrose or sorbitol as a stablilizer to develop consistent and stable formulations. Results demonstrated that ECO formed stable nanoparticles with pRHO-ABCA4-SV40 in the presence of sucrose, but not with sorbitol. The transfection efficiency in vitro increased significantly after introduction of SV40 enhancer for plasmid pCMV-ABCA4-SV40 with a CMV promoter. Sucrose didn't affect the transfection efficiency, while sorbitol resulted in a fluctuation of the in vitro transfection efficiency. Subretinal gene therapy in Abca4-/- mice using ECO/pRHO-ABCA4 and ECO/pRHO-ABCA4-SV40 nanoparticles induced 36% and 29% reduction in A2E accumulation respectively. Therefore, the ECO/pABCA4 based nanoparticles are promising for non-viral gene therapy for Stargardt disease and can be expended for applications in a variety of visual dystrophies with mutated large genes.

Formulation of Biocompatible Targeted ECO/siRNA Nanoparticles with Long-Term Stability for Clinical Translation of RNAi.

Nanoparticle based siRNA formulations often suffer from aggregation and loss of function during storage. We in this study report a frozen targeted RGD-polyethylene glycol (PEG)-ECO/siß3 nanoparticle formulation with a prolonged shelf life and preserved nanoparticle functionality. The targeted RGD-PEG-ECO/siß3 nanoparticles are formed by step-wised self-assembly of RGD-PEG-maleimide, ECO, and siRNA. The nanoparticles have a diameter of 224.5 ± 9.41 nm and a zeta potential to 45.96 ± 3.67 mV in water and a size of 234.34 ± 3.01 nm and a near neutral zeta potential in saline solution. The addition of sucrose does not affect their size and zeta potential and substantially preserves the integrity and biological activities of frozen and lyophilized formulations of the targeted nanoparticles. The frozen formulation with as low as 5% sucrose retains nanoparticle integrity (90% siRNA encapsulation), size distribution (polydispersity index [PDI] ≤20%), and functionality (at least 75% silencing efficiency) at -80°C for at least 1 year. The frozen RGD-PEG-ECO/siß3 nanoparticle formulation exhibits excellent biocompatibility, with no adverse effects on hemocompatibility and minimal immunogenicity. As RNAi holds the promise in treating the previously untreatable diseases, the frozen nanoparticle formulation with the low sucrose concentration has the potential to be a delivery platform for clinical translation of RNAi therapeutics.

Systemic Delivery of Tumor-Targeting siRNA Nanoparticles against an Oncogenic LncRNA Facilitates Effective Triple-Negative Breast Cancer Therapy.

Long noncoding RNAs (lncRNAs), by virtue of their versatility and multilevel gene regulation, have emerged as attractive pharmacological targets for treating heterogeneous and complex malignancies like triple-negative breast cancer (TNBC). Despite multiple studies on lncRNA functions in tumor pathology, systemic targeting of these "undruggable" macromolecules with conventional approaches remains a challenge. Here, we demonstrate effective TNBC therapy by nanoparticle-mediated RNAi of the oncogenic lncRNA DANCR, which is significantly overexpressed in TNBC. Tumor-targeting RGD-PEG-ECO/siDANCR nanoparticles were formulated via self-assembly of multifunctional amino lipid ECO, cyclic RGD peptide-PEG, and siDANCR for systemic delivery. MDA-MB-231 and BT549 cells treated with the therapeutic RGD-PEG-ECO/siDANCR nanoparticles exhibited 80-90% knockdown in the expression of DANCR for up to 7 days, indicating efficient intracellular siRNA delivery and sustained target silencing. The RGD-PEG-ECO/siDANCR nanoparticles mediated excellent in vitro therapeutic efficacy, reflected by significant reduction in the invasion, migration, survival, tumor spheroid formation, and proliferation of the TNBC cell lines. At the molecular level, functional ablation of DANCR dynamically impacted the oncogenic nexus by downregulating PRC2-mediated H3K27-trimethylation and Wnt/EMT signaling, and altering the phosphorylation profiles of several kinases in the TNBC cells. Furthermore, systemic administration of the RGD-PEG-ECO/siDANCR nanoparticles at a dose of 1 mg/kg siRNA in nude mice bearing TNBC xenografts resulted in robust suppression of TNBC progression with no overt toxic side-effects, underscoring the efficacy and safety of the nanoparticle therapy. These results demonstrate that nanoparticle-mediated modulation of onco-lncRNAs and their molecular targets is a promising approach for developing curative therapies for TNBC and other cancers.