Posterior eye delivery of angiogenesis-inhibiting RNA nanoparticles via subconjunctival injection.

Neovascularization contributes to various posterior eye segment diseases such as age-related macular degeneration and diabetic retinopathy. RNA nanoparticles were demonstrated previously to enter the corneal and retinal cells after subconjunctival injection for ocular delivery. In the present study, antiangiogenic aptamers (anti-vascular endothelial growth factor (VEGF) and anti-angiopoietin-2 (Ang2) aptamers) were conjugated to RNA nanoparticles. The objectives were to investigate the clearance and distribution of these angiogenesis-inhibiting RNA nanoparticles after subconjunctival injection in vivo and their antiangiogenic effects for inhibiting ocular neovascularization in vitro. The results in the whole-body fluorescence imaging study showed that the clearance of RNA nanoparticles was size-dependent with no significant differences between RNA nanoparticles with and without the aptamers except for pRNA-3WJ. The distribution study of RNA nanoparticles by confocal microscopy of the dissected eye tissues in vivo indicated cell internalization of the larger RNA nanoparticles in the retina and retinal pigment epithelium after subconjunctival injection, and the larger nanoparticles with aptamers showed higher levels of cell internalization than those without. In the cell proliferation assay in vitro, RNA nanoparticles with multiple aptamers had higher antiangiogenic effects. With both longer retention time and high antiangiogenic effect, SQR-VEGF-Ang2 could be a promising RNA nanoparticle for posterior eye delivery.

Delivering the next generation of cancer immunotherapies with RNA.

Decades of oncologic clinical use have demonstrated that cancer immunotherapy provides unprecedented therapeutic benefits. Tragically, only a minority of patients respond to existing immunotherapies. RNA lipid nanoparticles have recently emerged as modular tools for immune stimulation. Here, we discuss advancements in RNA-based cancer immunotherapies and opportunities for improvement.

Mesenchymal Stem Cell-Derived Exosomes and Their Potential Agents in Hematological Diseases.

Mesenchymal stem cells (MSCs) are the most exploited stem cells with multilineage differentiation potential and immunomodulatory properties. Numerous lines of findings have reported their successful applications in a multitude of inflammatory conditions and immune disorders. However, it is currently discovered that these effects are mainly mediated in a paracrine manner by MSC-exosomes. Moreover, MSC-exosomes have been implicated in a wide variety of biological responses including immunomodulation, oxidative stress, tumor progression, and tissue regeneration. Meanwhile, they are reported to actively participate in various hematological diseases by the means of transferring different types of exosomal components to the target cells. Therefore, in this review, we briefly discuss the sources and biological features of MSCs and then illustrate the biogenesis and biological processes of MSC-exosomes. Of note, this paper especially highlights the latest research progress of MSC-exosomes in hematological diseases.

Delivering more for less: nanosized, minimal-carrier and pharmacoactive drug delivery systems.

Traditional nanoparticle carriers such as liposomes, micelles, and polymeric vehicles improve drug delivery by protecting, stabilizing, and increasing the circulatory half-life of the encapsulated drugs. However, traditional drug delivery systems frequently suffer from poor drug loading and require an excess of carrier materials. This carrier material excess poses an additional systemic burden through accumulation, if not degradable the need for metabolism, and potential toxicity. To address these shortcomings, minimal-carrier nanoparticle systems and pharmacoactive carrier materials have been developed. Both solutions provide drug delivery systems in which the majority of the nanoparticle is pharmacologically active. While minimal-carrier and pharmacoactive drug delivery systems can improve drug loading, they can also suffer from poor stability. Here, we review minimal-carrier and pharmacoactive delivery systems, discuss ongoing challenges and outline opportunities to translate minimal-carrier and pharmacoactive drug delivery systems into the clinic.

Nanoparticle designs for delivery of nucleic acid therapeutics as brain cancer therapies.

Glioblastoma (GBM) is an aggressive central nervous system cancer with a dismal prognosis. The standard of care involves surgical resection followed by radiotherapy and chemotherapy, but five-year survival is only 5.6% despite these measures. Novel therapeutic approaches, such as immunotherapies, targeted therapies, and gene therapies, have been explored to attempt to extend survival for patients. Nanoparticles have been receiving increasing attention as promising vehicles for non-viral nucleic acid delivery in the context of GBM, though delivery is often limited by low blood-brain barrier permeability, particle instability, and low trafficking to target brain structures and cells. In this review, nanoparticle design considerations and new advances to overcome nucleic acid delivery challenges to treat brain cancer are summarized and discussed.

Radiolabeled RNA Nanoparticles for Highly Specific Targeting and Efficient Tumor Accumulation with Favorable In Vivo Biodistribution.

Therapeutic efficiency and toxicity are two of the three critical factors in molecular therapy and pharmaceutical drug development. Specific tumor targeting and rapid renal excretion contribute to improving efficiency and reducing toxicity. We recently found that RNA nanoparticles display rubber-like properties, enabling them to deliver therapeutics to cancer with high efficiency. Off-target RNA nanoparticles were rapidly cleared by renal excretion, resulting in nontoxicity. However, previous biodistribution studies relied mainly on fluorescent markers, which can cause interference from fluorophore quenching and autofluorescence. Thus, the quantification of biodistribution requires further scrutiny. In this study, radionuclide [3H] markers were used for quantitative pharmacokinetic (PK) studies to elucidate the favorable PK profile of RNA nanoparticles. Approximately 5% of [3H]-RNA nanoparticles accumulated in tumors, in contrast to the 0.7% tumor accumulation reported in the literature for other kinds of nanoparticles. The amount of [3H]-RNA nanoparticles accumulated in tumors was higher than that in the liver, heart, lung, spleen, and brain throughout the entire process after IV injection. [3H]-RNA nanoparticles rapidly reached the tumor vasculature within 30 min and remained in tumors for more than 2 days. Nontargeting [3H]-RNA nanoparticles were found in the urine 30 min after IV injection without degradation and processing, and more than 55% of the IV-injected radiolabeled RNA nanoparticles were cleared from the body within 12 h, while the other 45% includes the radiative counts that cannot be recovered due to whole-body distribution and blood dilution after intravenous injection. The high specificity of tumor targeting, fast renal excretion, and low organ accumulation illustrate the high therapeutic potential of RNA nanoparticles in cancer treatment as efficient cancer-targeting carriers with low toxicity and side effects.

Tumor RNA-loaded nanoliposomes increases the anti-tumor immune response in colorectal cancer.

PURPOSE: Tumor RNA vaccines can activate dendritic cells to generate systemic anti-tumor immune response. However, due to easily degraded of RNA, direct RNA vaccine is less effective. In this study, we optimized the method for preparing PEGylated liposom-polycationic DNA complex (LPD) nanoliposomes, increased encapsulate amount of total RNA derived from CT-26 colorectal cancer cells. Tumor RNA LPD nanoliposomes vaccines improved anti-tumor immune response ability of tumor RNA and can effectively promote anti-tumor therapeutic effect of oxaliplatin. METHODS: Total tumor-derived RNA was extracted from colorectal cancer cells (CT-26 cells), and loaded to our optimized the LPD complex, resulting in the LPD nanoliposomes. We evaluated the characteristics (size, zeta potential, and stability), cytotoxicity, transfection ability, and tumor-growth inhibitory efficacy of LPD nanoliposomes. RESULTS: The improved LPD nanoliposomes exhibited a spherical shape, RNA loading efficiency of 9.07%, the average size of 120.37 ± 2.949 nm and zeta potential was 3.34 ± 0.056 mV. Also, the improved LPD nanoliposomes showed high stability at 4 °C, with a low toxicity and high cell transfection efficacy toward CT-26 colorectal cancer cells. Notably, the improved LPD nanoliposomes showed tumor growth inhibition by activating anti-tumor immune response in CT-26 colorectal cancer bearing mice, with mini side effects toward the normal organs of mice. Furthermore, the effect of the improved LPD nanoliposomes in combination with oxaliplatin can be better than that of oxaliplatin alone. CONCLUSION: The improved LPD nanoliposomes may serve as an effective vaccine to induce antitumor immunity, presenting a new treatment option for colorectal cancer.

A Peptide-Based Small RNA Delivery System to Suppress Tumor Growth by Remodeling the Tumor Microenvironment.

MicroRNAs can regulate a variety of physiological and pathological processes and are increasingly recognized as being involved in regulating the malignant progression of cancer, which is an important direction for the study and treatment of cancer. In addition, the tumor microenvironment has gradually become an important direction of study for combating cancer. Researchers can inhibit tumor growth by remodeling and suppressing an immunosuppressive phenotype in the tumor microenvironment. Therefore, the combination of microRNA delivery and tumor microenvironment remodeling may be a potential research direction. In a previous study, we developed a novel cationic and hydrophilic antimicrobial peptide, DP7, by computer simulation. It was found that cholesterol-modified DP7 (DP7-C) has dual functions as a carrier and an immune adjuvant. In this experiment, we used DP7-C to deliver microRNAs or inhibitors intratumorally, where it played a dual role as a carrier and an immune adjuvant. As a delivery vector, DP7-C has more advantages in terms of transfection efficiency and cytotoxicity than Lipo2000 and PEI25K. Components of the DP7-C/RNA complex can effectively escape endosomes after uptake via caveolin- and clathrin-dependent pathways. As an immune adjuvant, DP7-C can activate dendritic cells and promote macrophage polarization. Moreover, it can transform the immunosuppressive tumor microenvironment into an immune-activated tumor microenvironment, indicating its potential as an anticancer therapy. In conclusion, this study identifies a novel microRNA and inhibitor delivery system that can remodel the tumor microenvironment and introduces an alternative scheme for antitumor treatment.

Strategies for simultaneous and successive delivery of RNA.

Advanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level.

Ultra-thermostable RNA nanoparticles for solubilizing and high-yield loading of paclitaxel for breast cancer therapy.

Paclitaxel is widely used in cancer treatments, but poor water-solubility and toxicity raise serious concerns. Here we report an RNA four-way junction nanoparticle with ultra-thermodynamic stability to solubilize and load paclitaxel for targeted cancer therapy. Each RNA nanoparticle covalently loads twenty-four paclitaxel molecules as a prodrug. The RNA-paclitaxel complex is structurally rigid and stable, demonstrated by the sub-nanometer resolution imaging of cryo-EM. Using RNA nanoparticles as carriers increases the water-solubility of paclitaxel by 32,000-fold. Intravenous injections of RNA-paclitaxel nanoparticles with specific cancer-targeting ligand dramatically inhibit breast cancer growth, with nearly undetectable toxicity and immune responses in mice. No fatalities are observed at a paclitaxel dose equal to the reported LD50. The use of ultra-thermostable RNA nanoparticles to deliver chemical prodrugs addresses issues with RNA unfolding and nanoparticle dissociation after high-density drug loading. This finding provides a stable nano-platform for chemo-drug delivery as well as an efficient method to solubilize hydrophobic drugs.

Evaluation of Newcastle disease virus mediated dendritic cell activation and cross-priming tumor-specific immune responses ex vivo.

We have developed an oncolytic Newcastle disease virus (NDV) that has potent in vitro and in vivo anti-tumor activities and attenuated pathogenicity in chickens. In this ex vivo study using the same recombinant NDV backbone with GFP transgene (NDV-GFP, designated as rNDV), we found that rNDV induces maturation of monocyte-derived immature dendritic cells (iDCs) by both direct and indirect mechanisms, which promote development of antigen-specific T cell responses. Addition of rNDV directly to iDCs culture induced DC maturation, as demonstrated by the increased expression of costimulatory and antigen-presenting molecules as well as the production of type I interferons (IFNs). rNDV infection of the HER-2 positive human breast cancer cell line (SKBR3) resulted in apoptotic cell death, release of proinflammatory cytokines, and danger-associated molecular pattern molecules (DAMPs) including high-mobility group protein B1 (HMGB1) and heat shock protein 70 (HSP70). Addition of rNDV-infected SKBR3 cells to iDC culture resulted in greatly enhanced upregulation of the maturation markers and release of type I IFNs by DCs than rNDV-infected DCs only. When co-cultured with autologous T cells, DCs pre-treated with rNDV-infected SKBR3 cells cross-primed T cells in an antigen-specific manner. Altogether, our data strongly support the potential of oncolytic NDV as efficient therapeutic agent for cancer treatment.

Adenine base editing in an adult mouse model of tyrosinaemia.

In contrast to traditional CRISPR-Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here we show in a mouse model of tyrosinaemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes in the liver, and rescued weight loss in mice. We also generated FAH+ hepatocytes in the liver via lipid-nanoparticle-mediated delivery of a chemically modified sgRNA and an mRNA of a codon-optimized base editor that displayed higher base-editing efficiency than the standard ABEs. Our findings suggest that adenine base editing can be used for the correction of genetic diseases in adult animals.

Core-independent approach for polymer brush-functionalised nanomaterials with a fluorescent tag for RNA delivery.

Here we describe a core-independent approach enabling the grafting of polymer brushes from the surface of nanomaterials and microparticles for oligonucleotide delivery. This method is based on the adsorption of a polyelectrolyte macroinitiator (MI) combined with a fluorescent conjugated polymer for efficient and stable labelling. This allows dense brushes to be generated, with growth kinetics comparable to those observed from mono-functional initiators, for the imaging of nanomaterial cellular localisation and uptake. We also study the impact of brush chemistry on interactions with cell membranes and on transfection efficiency. The method we report offers a unique freedom of design of the core size and shape as well as surface chemistry, whilst enabling tagging, for the study of transfection processes or theragnostic applications.

In vivo delivery of VEGF RNA and protein to increase osteogenesis and intraosseous angiogenesis.

Deficient bone vasculature is a key component in pathological conditions ranging from developmental skeletal abnormalities to impaired bone repair. Vascularisation is dependent upon vascular endothelial growth factor (VEGF), which drives both angiogenesis and osteogenesis. The aim of this study was to examine the efficacy of blood vessel and bone formation following transfection with VEGF RNA or delivery of recombinant human VEGF165 protein (rhVEGF165) across in vitro and in vivo model systems. To quantify blood vessels within bone, an innovative approach was developed using high-resolution X-ray computed tomography (XCT) to generate quantifiable three-dimensional reconstructions. Application of rhVEGF165 enhanced osteogenesis, as evidenced by increased human osteoblast-like MG-63 cell proliferation in vitro and calvarial bone thickness following in vivo administration. In contrast, transfection with VEGF RNA triggered angiogenic effects by promoting VEGF protein secretion from MG-63VEGF165 cells in vitro, which resulted in significantly increased angiogenesis in the chorioallantoic (CAM) assay in ovo. Furthermore, direct transfection of bone with VEGF RNA in vivo increased intraosseous vascular branching. This study demonstrates the importance of continuous supply as opposed to a single high dose of VEGF on angiogenesis and osteogenesis and, illustrates the potential of XCT in delineating in 3D, blood vessel connectivity in bone.

Poly(ß-amino ester)-based gene delivery systems: From discovery to therapeutic applications.

Poly(ß-amino ester)s (PßAE) were firstly synthesized in 1983 but only in 2000 these polymers were used for the first time as gene carrier. Thenceforward, due to their excellent gene delivery properties, PßAE were amply explored to afford very effective non-viral vectors. The promising results obtained both in vitro and in vivo studies involving different areas, from cancer therapy to tissue engineering area have aroused a broad interest of the scientific community for this family of biodegradable cationic polymers. This review is the first comprehensive and critical overview of the use of PßAEs as gene carrier. The rational design of PßAEs is a major step aiming to achieve high transfection efficiencies. Moreover, it has been demonstrated that often very small changes in the structure of these polymers have an impressive impact on the transfection efficiency. A critical discussion on the structure performance relationships is presented as well as the outlook for next developments involving these polymers.

Transplantation studies reveal internuclear transfer of toxic RNA in engrafted muscles of myotonic dystrophy 1 mice.

BACKGROUND: Stem cell transplantation represents a potential therapeutic option for muscular dystrophies (MD). However, to date, most reports have utilized mouse models for recessive types of MD. Here we performed studies to determine whether myotonic dystrophy 1 (DM1), an autosomal dominant type of MD, could benefit from cell transplantation. METHODS: We injected human pluripotent stem (PS) cell-derived myogenic progenitors into the muscles of a novel mouse model combining immunodeficiency and skeletal muscle pathology of DM1 and investigated transplanted mice for engraftment as well as for the presence of RNA foci and alternative splicing pattern. FINDINGS: Engraftment was clearly observed in recipient mice, but unexpectedly, we detected RNA foci in donor-derived engrafted myonuclei. These foci proved to be pathogenic as we observed MBNL1 sequestration and abnormal alternative splicing in donor-derived transcripts. INTERPRETATION: It has been assumed that toxic CUG repeat-containing RNA forms foci in situ in the nucleus in which it is expressed, but these data suggest that CUG repeat-containing RNA may also exit the nucleus and traffic to other nuclei in the syncytial myofiber, where it can exert pathological effects. FUND: This project was supported by funds from the LaBonte/Shawn family and NIH grants R01 AR055299 and AR071439 (R.C.R.P.). R.M-G. was funded by CONACyT-Mexico (#394378).

A Toll-like receptor 3 (TLR3) agonist ARNAX for therapeutic immunotherapy.

Vaccine immunotherapy consisting of tumor antigens combined with an immune-enhancing adjuvant fosters cytotoxic T cell (CTL) proliferation. Clinically, polyI:C has been used as an adjuvant to enhance cancer vaccine protocols. However, according to its long history, polyI:C promotes inflammation that causes cytokine toxicity. Although checkpoint inhibitor immunotherapy has improved the prognoses of patients with progressive cancer, over 75% of patients continue to experience resistance to antibody (Ab) against anti-programmed cell death-protein 1 (PD-1) or its ligand, PD-L1 therapy. In most cases, patients suffer from adverse events resulting from inflammation during anti-PD-1/L1 Ab therapy, which is a serious obstacle to patients' quality of life. We have studied the functional properties of double-stranded (ds)RNA and polyI:C, and developed a nucleic acid adjuvant that barely induces a significant increase in the level of serum inflammatory cytokines in mouse models. This adjuvant, termed ARNAX, consists of DNA-capped dsRNA that specifies the endosomal target for Toll-like receptor 3 (TLR3) in dendritic cells (DCs). We expect that this adjuvant is safe for administration in elderly patients with cancer receiving immunotherapy. Here, we summarize the properties of ARNAX for immunotherapy in mice. We suggest that DC-priming is essential to induce anti-tumor immunity; neither exogenous inflammation nor the administration of tumor antigens is always a prerequisite for DC-mediated CTL proliferation. If our mouse data can be extrapolated to humans, ARNAX and the liberated endogenous tumor antigens may facilitate effect of current therapies on patients with therapy-resistant tumors.

In vitro evolution of enhanced RNA replicons for immunotherapy.

Self-replicating (replicon) RNA is a promising new platform for gene therapy, but applications are still limited by short persistence of expression in most cell types and low levels of transgene expression in vivo. To address these shortcomings, we developed an in vitro evolution strategy and identified six mutations in nonstructural proteins (nsPs) of Venezuelan equine encephalitis (VEE) replicon that promoted subgenome expression in cells. Two mutations in nsP2 and nsP3 enhanced transgene expression, while three mutations in nsP3 regulated this expression. Replicons containing the most effective mutation combinations showed enhanced duration and cargo gene expression in vivo. In comparison to wildtype replicon, mutants expressing IL-2 injected into murine B16F10 melanoma showed 5.5-fold increase in intratumoral IL-2 and 2.1-fold increase in infiltrating CD8 T cells, resulting in significantly slowed tumor growth. Thus, these mutant replicons may be useful for improving RNA therapeutics for vaccination, cancer immunotherapy, and gene therapy.

Pancreatic Cancer Gene Therapy Delivered by Nanoparticles.

Pancreatic cancer is one of the most lethal forms of cancer and has proven to be difficult to treat through conventional methods, including surgery and chemotherapy. Gene therapy serves as a potential novel treatment to interfere with genes that make this cancer so aggressive, but free nucleic acids have low cell uptake due to their negative charge and are unstable in circulation. Nanoparticles can serve as an effective carrier for a wide variety of gene therapies for pancreatic cancer as they can improve the circulation time, decrease the recognition by the immune system, and be functionalized to target specific surface proteins. In this review, we focus on therapeutic strategies using nanoparticles as carriers of small interfering RNA (siRNA), microRNA (miRNA), and gene augmentation (DNA) therapies in the context of pancreatic cancer. Lastly, we discuss the future outlook of nanoparticle-based therapies, including challenges in the clinical setting.

Modular core-shell polymeric nanoparticles mimicking viral structures for vaccination.

Recent advances in the development of protein-based vaccines have expanded the opportunities for preventing and treating both infectious diseases as well as cancer. However, the development of readily and efficient antigen delivery systems capable of stimulating strong cytotoxic T-lymphocyte (CTL) responses remains a challenge. With the attempt to closely mimic the properties of viruses in terms of their size and molecular organization, we constructed RNA (which is a ligand for Toll-like receptor 7 (TLR7) and TLR8) and antigen-loaded nanoparticles resembling the structural organization of viruses. Cationic polymers containing either azide or bicyclo[6.1.0]nonyne (BCN) groups were synthesized as electrostatic glue that binds negatively charged single stranded RNA (PolyU) to form a self-crosslinked polyplex core. An azide-modified model antigen (ovalbumin, OVA) and a BCN-modified mannosylated or galactosylated polymer were sequentially conjugated to the RNA core via disulfide bonds using copper free click chemistry to form the shell of the polyplexes. The generated reducible virus mimicking particles (VMPs) with a diameter of 200 nm and negatively surface charge (-14 mV) were colloidally stable in physiological conditions. The immunogenicity of these VMP vaccines was evaluated both in vitro and in vivo. The surface mannosylated VMPs (VMP-Man) showed 5 times higher cellular uptake by bone marrow derived DCs (BMDCs) compared to galactosylated VMP (VMP-Gal) counterpart. Moreover, VMP-Man efficiently activated DCs and greatly facilitated MHC I Ag presentation in vitro. Vaccination of mice with VMP-Man elicited strong OVA-specific CTL responses as well as humoral immune responses. These results demonstrate that the modular core-shell polymeric nanoparticles described in this paper are superior in inducing strong and durable immune responses compared to adjuvanted protein subunit vaccines and offer therefore a flexible platform for personalized vaccines.