Targeted delivery system using silica nanoparticles coated with chitosan and AS1411 for combination therapy of doxorubicin and antimiR-21.

Herein, a novel targeted delivery system was developed for intracellular co-delivery of doxorubicin (DOX) as a chemotherapeutic drug, antimiR-21 as an oncogenic antagomiR. In this system, DOX was loaded into mesoporous silica nanoparticles (MSNs) and chitosan was applied to cover the surface of MSNs. AS1411 aptamer as targeting nucleolin and antimiR-21 were electrostatically attached onto the surface of the chitosan-coated MSNs and formed the final nanocomplex (AACS nanocomplex). The study of drug release was based on DOX release under pH 7.4 and 5.5. Cellular toxicity and cellular uptake assessments of AACS nanocomplex were carried out in nucleolin positive (C26, MCF-7, and 4T1) and nucleolin negative (CHO) cell lines using MTT assay and flow cytometry analysis, respectively. Also, Anti-tumor efficacy of AACS nanocomplex was evaluated in C26 tumor-bearing mice. Overall, the results show that the combination therapy of DOX and antimiR-21, using AACS nanocomplex, could combat the cancer cell growth rate.

Targeting respiratory diseases using miRNA inhibitor based nanotherapeutics: Current status and future perspectives.

MicroRNAs (miRNAs) play a fundamental role in the developmental and physiological processes that occur in both animals and plants. AntagomiRs are synthetic antagonists of miRNA, which prevent the target mRNA from suppression. Therapeutic approaches that modulate miRNAs have immense potential in the treatment of chronic respiratory disorders. However, the successful delivery of miRNAs/antagomiRs to the lungs remains a major challenge in clinical applications. A range of materials, namely, polymer nanoparticles, lipid nanocapsules and inorganic nanoparticles, has shown promising results for intracellular delivery of miRNA in chronic respiratory disorders. This review discusses the current understanding of miRNA biology, the biological roles of antagomiRs in chronic respiratory disease and the recent advances in the therapeutic utilization of antagomiRs as disease biomarkers. Furthermore our review provides a common platform to debate on the nature of antagomiRs and also addresses the viewpoint on the new generation of delivery systems that target antagomiRs in respiratory diseases.

Design of small molecules targeting RNA structure from sequence.

The design and discovery of small molecule medicines has largely been focused on a small number of druggable protein families. A new paradigm is emerging, however, in which small molecules exert a biological effect by interacting with RNA, both to study human disease biology and provide lead therapeutic modalities. Due to this potential for expanding target pipelines and treating a larger number of human diseases, robust platforms for the rational design and optimization of small molecules interacting with RNAs (SMIRNAs) are in high demand. This review highlights three major pillars in this area. First, the transcriptome-wide identification and validation of structured RNA elements, or motifs, within disease-causing RNAs directly from sequence is presented. Second, we provide an overview of high-throughput screening approaches to identify SMIRNAs as well as discuss the lead identification strategy, Inforna, which decodes the three-dimensional (3D) conformation of RNA motifs with small molecule binding partners, directly from sequence. An emphasis is placed on target validation methods to study the causality between modulating the RNA motif in vitro and the phenotypic outcome in cells. Third, emergent modalities that convert occupancy-driven mode of action SMIRNAs into event-driven small molecule chemical probes, such as RNA cleavers and degraders, are presented. Finally, the future of the small molecule RNA therapeutics field is discussed, as well as hurdles to overcome to develop potent and selective RNA-centric chemical probes.

Yeast Cell wall Particle mediated Nanotube-RNA delivery system loaded with miR365 Antagomir for Post-traumatic Osteoarthritis Therapy via Oral Route.

Post-traumatic osteoarthritis (PTOA) is an acute injury-induced joint inflammation followed by a gradual degradation of articular cartilage. However, there is no FDA-approved Disease-Modifying Osteoarthritis Drug currently. Although gene therapy with microRNA can improve PTOA progression, there is no effective gene delivery vehicle for orally deliver therapeutics due to the harsh environment of the gastrointestinal tract. In this study, we investigated the effect of yeast cell wall particle (YCWP) mediated nanotube-RNA delivery system on PTOA therapy via oral route. Methods: Nontoxic and degradable AAT and miRNA365 antagomir was self-assembled into miR365 antagomir/AAT (NPs). Then NPs-YCWP oral drug delivery system was constructed by using NPs and non-pathogenic YCWP which can be specifically recognized by macrophages. Moreover, surgical destabilization of the medial meniscus induced PTOA mice model was established to evaluate the therapeutic effect of NPs-YCWP via oral route. Results: Compared with control group, NPs showed higher gene inhibition efficiency both in chondrogenic cell line and primary chondrocytes in vitro. Treatment of macrophages with fluorescently labeled NPs-YCWP, the results showed that NPs-YCWP was successfully engulfed by macrophages and participated in the regulation of gene expression in vitro. Under the protection of YCWP, miR365 antagomir/AAT passes through the gastrointestinal tract without degradation after oral administration. After NPs-YCWP therapy, the results of histological staining, gene and protein expression showed that miR365 antagomir/NPs-YCWP improved the symptom of PTOA. Conclusion: Here, we constructed a biodegradable drug delivery system based on non-pathogenic YCWP and nanotubes, which can be used for PTOA therapy via the oral route. It suggests a new gene therapy strategy with YCWP mediated oral nano drug delivery system may serve as a platform for joint degeneration treatment.

Inhibition of miR-155 in MCF-7 breast cancer cell line by gold nanoparticles functionalized with antagomir and AS1411 aptamer.

MicroRNAs are key factors for many biological functions. These regulatory molecules affect various gene networks and involve the subsequent signaling pathways. Therefore, disrupting the expression of these molecules is associated with multiple anomalies in the cells and body. One of the most important related abnormalities is the incidence of cancer. Thus, targeting microRNAs (miRNAs) is an effective approach for cancer gene therapy. Various factors are used for this purpose, including the antagomir nucleotide structure. There are some obstacles in the delivery of nucleotide therapeutics to the target cells, however, the use of nanoparticles could partly overcome these defeciencies. On the other hand, targeted delivery of antagomirs using aptamers, reduces nonspecific effects on nontarget cells. Considering the above, in this study, we designed and fabricated a nanocarrier composed of gold nanoparticles (GNPs), antagomir-155, and nucleolin specific aptamer for breast cancer study and therapy. Here, GNPs were synthesized using citrate reduction and were modified by polyA sequences, AS1411 aptamer, and antagomir-155. Attachment of molecules were confirmed using gel electrophoresis, atomic force microscopy imaging and electrochemical test. The specific entry of modified nanoparticles was investigated by fluorescence microscopy. The efficacy of modified nanoparticles was evaluated using a quantitative polymerase chain reaction (q-PCR) for miR-155 and its target gene. Efficient and specific delivery of AuNP-Apt-anti-miR-155 to target cells was confirmed in comparison with the control cell. The q-PCR analysis showed not only a significant decrease in mir-155 levels but also an elevated TP53INP1 mRNA, direct target of miR-155. The proposed structure inhibits proliferation and stimulates apoptosis by increasing the expression of TP53INP1. Our results suggest that AuNP-Apt-anti-miR-155 could be a promising nano constructor for breast cancer treatment.

The promotion of bone regeneration through CS/GP-CTH/antagomir-133a/b sustained release system.

Few studies reported the application of miRNA in bone regeneration. In this study, the expression of miR133a and miR133b in murine BMSCs was inhibited via antagomiR-133a/b and the osteogenic differentiation in murine BMSCs was evaluated. The RT-PCR, flow cytometry, cell counting kit-8, and annexin V-FITC/PI double staining assays were performed. Double knockdown miR133a and miR133b can promote BMSC osteogenic differentiation. At optimum N/P ration (15:1), the loading efficiency can reach over 90%. CTH-antagomiR-133a/b showed no cytotoxicity to BMSCs and diminished miR133a and miR133b expression in BMSCs. Furthermore, chitosan-based sustained delivery system can facilitate continuous dosing of antagomiR-133a/b, which enhanced calcium deposition and osteogenic specific gene expression in vitro. The new bone formation was enhanced after the sustained delivery system containing CTH-antagomiR-133a/b nanoparticles was used in mouse calvarial bone defect model. Our results demonstrate that CTH nanoparticles could facilitate continuous dosing of antagomiR133a/b, which can promote osteogenic differentiation.

Acid-Triggered Charge-Convertible Graphene-Based All-in-One Nanocomplex for Enhanced Genetic Phototherapy of Triple-Negative Breast Cancer.

Highly efficient and stimulus-responsive nanomedicines for cancer treatment are currently receiving tremendous attention. In this study, an acid-triggered charge-reversible graphene-based all-in-one nanocomplex is appropriately designed by surface modification with multilayer polymers and simultaneous co-transportation of photosensitizer indocyanine green (ICG) and oligonucleotide inhibitor of miR-21 (miR-21i) to achieve highly efficient genetic phototherapy in a controlled manner. The nanocomplex (denoted as GPCP/miR-21i/ICG) effectively protects miR-21i from degradation and exhibits excellent photothermal/photochemical reactive oxygen species (ROS) generation as well as fluorescence imaging ability. The cargoes ICG and miR-21i can significantly be released at acidic pH compared with normal physiological medium and escaped from endosomes/lysosomes due to the acid-triggered charge reversal effect. Typically, the released miR-21i downregulate the endogenous miR-21 and result in the upregulation of the target proteins PTEN and Bax, thus increasing the phototherapeutic efficiency of ICG. High in vivo anticancer efficiency against the MDA-MB-231 triple-negative breast cancer (TNBC) model is obtained due to the combination of genetic regulation of miR-21i and the photokilling effect of ICG. This work highlights the great potential of this smart nanocomplex as an attractive modality of gene-photo combined treatment of cancer, especially for intractable TNBC.

Efficient delivery of anti-miR-210 using Tachyplesin, a cell penetrating peptide, for glioblastoma treatment.

The levels of microRNAs (miRNAs) are altered in various diseases including glioblastoma (GBM) and this alteration may have widespread effects on various hallmarks of cancer cells. MiR210 is overexpressed in GBM and functions as an oncogenic miRNA. Anti-miR210 therapy holds great promise but its efficient delivery remains a major challenge. Our work here explores a novel role of Tachyplesin (Tpl), a cell-penetrating antimicrobial peptide, as a nanocarrier for anti-miR210. Tpl electrostatically interacts with anti-miR210 at 1:25 and 1:50 (anti-miR:Tpl) weight ratios to form a complex and efficiently delivers anti-miR210 inside GBM cells cultured as 2D and 3D spheroid model. Treatment of GBM cells with the complex significantly inhibited miR210 levels (~90%), proliferation, migration and spheroid formation ability and induced apoptosis as evident by increased levels of caspase 3/7 and ROS. GBM cells pre-treated with anti-miR210:Tpl complex were also found to be sensitive to TMZ mediated action. Uptake of the complex in GBM cells induced the levels of miR210 targeted tumor suppressor genes, NeuroD2 and HIF3A. Overall, our work reveals a novel and efficient miRNA delivery ability of Tpl in glioma cells, holding a great promise for treatment of GBM and potentially for other cancers.

Development of Novel antimiRzymes for Targeted Inhibition of miR-21 Expression in Solid Cancer Cells.

MicroRNAs (miRNAs) are short non-coding RNAs that are involved in the regulation of gene expression. Previous reports showed an over-expression of miRNA-21 (miR-21) in various cancer cells, and its up-regulation is closely related to cancer initiation, proliferation and metastasis. In this work, we envisioned the development of novel antimiRzymes (anti-miRNA-DNAzyme) that are capable of selectively targeting and cleaving miR-21 and inhibit its expression in cancer cells using the DNAzyme technique. For this purpose, we have designed different antimiRzyme candidates by systematically targeting different regions of miR-21. Our results demonstrated that RNV541, a potential arm-loop-arm type antimiRzyme, was very efficient (90%) to suppress miR-21 expression in U87MG malignant glioblastoma cell line at 200 nM concentration. In addition, RNV541 also inhibited miR-21 expression (50%) in MDA-MB-231 breast cancer cell line. For targeted delivery, we conjugated RNV541 with a transferrin receptor (TfR) targeting aptamer for TfR-mediated cancer cell delivery. As expected, the developed chimeric structure efficiently delivered the antimiRzyme RNV541 into TfR positive glioblastoma cells. TfR aptamer-RNV541 chimeric construct showed 52% inhibition of miR-21 expression in U87MG glioblastoma cells at 2000 nM concentration, without using any transfection reagents, making it a highly desirable strategy to tackle miR-21 over-expressed malignant cancers. Although these are in vitro based observations, based on our results, we firmly believe that our findings could be beneficial towards the development of targeted cancer therapeutics where conventional therapies face several challenges.

Scaffold-Based Delivery of Nucleic Acid Therapeutics for Enhanced Bone and Cartilage Repair.

Recent advances in tissue engineering have made progress toward the development of biomaterials capable of the delivery of growth factors, such as bone morphogenetic proteins, in order to promote enhanced tissue repair. However, controlling the release of these growth factors on demand and within the desired localized area is a significant challenge and the associated high costs and side effects of uncontrolled delivery have proven increasingly problematic in clinical orthopedics. Gene therapy may be a valuable tool to avoid the limitations of local delivery of growth factors. Following a series of setbacks in the 1990s, the field of gene therapy is now seeing improvements in safety and efficacy resulting in substantial clinical progress and a resurgence in confidence. Biomaterial scaffold-mediated gene therapy provides a template for cell infiltration and tissue formation while promoting transfection of cells to engineer therapeutic proteins in a sustained but ultimately transient fashion. Additionally, scaffold-mediated delivery of RNA-based therapeutics can silence specific genes associated with orthopedic pathological states. This review will provide an overview of the current state-of-the-art in the field of gene-activated scaffolds and their use within orthopedic tissue engineering applications. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1671-1680, 2019.

Comparison of different chemically modified inhibitors of miR-199b in vivo.

MicroRNAs (miRNAs) have recently received great attention for their regulatory roles in diverse cellular processes and for their contribution to several human pathologies. Modulation of miRNAs in vivo provides beneficial therapeutic strategies for the treatment of many diseases, as evidenced by various preclinical studies. However, specific issues regarding the in vivo use of miRNA inhibitors (antimiRs) such as organ-specific delivery, optimal dosing and formulation of the best chemistry to obtain efficient miRNA inhibition remain to be addressed. Here, we aimed at comparing the in vivo efficacy of different chemistry-based antimiR oligonucleotides to inhibit cardiac expression of miR-199b, a highly promising therapeutic target for the treatment of pressure overload-induced cardiac dysfunction. For this purpose, four different designs of oligonucleotides to inhibit miR-199b were initially developed. Systemic administration to wildtype mice on three consecutive days was followed by organ harvesting, seven days after the first injection, in order to quantify the dose-dependent changes in miR-199b expression levels. When comparing the efficiency of each inhibitor at the highest applied dose we observed that the antagomir was the only inhibitor inducing complete inhibition of miR-199b in the heart. LNA reduced expression in the heart by 50 percent while the Zen-AMO and F/MOE chemistries failed to repress miR-199b expression in the heart at any given dose, in vivo. Further optimization was achieved by subjecting the antagomir and LNA nucleotides to additional chemical modifications. Interestingly, antagomir modification by replacing the cholesterol moiety from the 3' to the 5' end of the molecule significantly improved the inhibitory capacity, as reflected by a 75 percent downregulation of miR-199b expression already at a concentration of 5 mg/kg/day. Similar results could be obtained with a LNA-RNA molecule but upon administration of 80 mg/kg/day. These findings show that, from all the chemistries tested by us, an antagomir carrying the cholesterol group at the 5' end was the most efficient inhibitor of miR-199b in the heart, in vivo. Moreover, our data also emphasize the importance of chemistry optimization and best dose range finding to achieve the greatest efficacy in miRNA inhibition in vivo.

Bifunctional Au@Bi2 Se3 Core-Shell Nanoparticle for Synergetic Therapy by SERS-Traceable AntagomiR Delivery and Photothermal Treatment.

For the first time, topological insulator bismuth selenide nanoparticles (Bi2 Se3 NP) are core-shelled with gold (Au@Bi2 Se3 ) i) to represent considerably small-sized (11 nm) plasmonic nanoparticles, enabling accurate bioimaging in the near-infrared region; ii) to substantially improve Bi2 Se3 biocompatibility, iii) water dispersibility, and iv) surface functionalization capability through straightforward gold-thiol interaction. The Au@Bi2 Se3 is subsequently functionalized for v) effective targeting of SH-SY5Y cancer cells, vi) disrupting the endosome/lysosome membrane, vii) traceable delivery of antagomiR-152 and further synergetic oncomiR knockdown and photothermal therapy (PTT). Unprecedentedly, it is observed that the Au shell thickness has a significant impact on evoking the exotic plasmonic features of Bi2 Se3 . The Au@Bi2 Se3 possesses a high photothermal conversion efficiency (35.5%) and a remarkable surface plasmonic effect (both properties are approximately twofold higher than those of 50 nm Au nanoparticles). In contrast to the siRNA/miRNA delivery methods, the antagomiR delivery is based on strand displacement, in which the antagomiR-152 is displaced by oncomiR-152 followed by a surface-enhanced Raman spectroscopy signal drop. This enables both cancer cell diagnosis and in vitro real-time monitoring of the antagomiR release. This selective PTT nanoparticle can also efficiently target solid tumors and undergo in vivo PTT, indicating its potential clinical applications.

Preparation of Anti-miR PNAs for Drug Development and Nanomedicine.

Peptide Nucleic Acids (PNAs) are oligonucleotide mimics that can be used to block the biological action of microRNA, thus affecting gene expression post-transcriptionally. PNAs are obtained with solid-phase peptide synthesis, and can be easily conjugated to other peptides. Conjugation with R8-Peptide or modification of the PNA backbone (at C5 or C2 carbon) with arginine side chains allows efficient cellular uptake. The present protocol describes the synthesis of cationic PNAs that can be used alone as drugs or for efficient co-delivery in suitable inorganic nanocarriers.

Controlled Release of RNAi Molecules by Tunable Supramolecular Hydrogel Carriers.

Local, sustained release and presentation of RNAi therapeutics can be achieved with hydrogel delivery systems. Here we show the development of a supramolecular hydrogel into a local RNAi delivery system. By careful material design, two simple but effective strategies are introduced to obtain controlled release of two classes of RNAi therapeutics, that is, microRNA and antimiR. It was shown that the release of microRNA could be regulated using cholesterol-modification for interaction with the supramolecular hydrogel. Non-modified antimiR release could be controlled via supramolecular introduction of positively charged additive molecules into the supramolecular hydrogel. In this way, either the cholesterol-modification on the drug or the charge introduction into the hydrogel provides handles for controlled RNAi therapy.

Titanium implant functionalized with antimiR-138 delivered cell sheet for enhanced peri-implant bone formation and vascularization.

Patients with compromised bone conditions still suffer the problem of deficient osseointegration during dental implant treatment. Developing mesenchymal stem cell (MSC) sheet functionalized titanium implant with proper inductive cue to promote osteogenesis and angiogenesis coupling shall be a good solution. In the present study, the antimiR-138 delivered MSC sheet is used to functionalize the Ti implant. The cell sheet can well integrate with the Ti implant to form the MSC sheet-implant complex (MSIC). The antimiR-138 delivered MSIC shows greatly improved osteogenesis and angiogenesis coupling both in vitro and in vivo. In vitro, the antimiR-138 delivered MSIC significantly promotes the expression of endogenous osteogenesis and angiogenesis related genes and proteins, alkaline phosphatase activity, extracellular matrix mineralization and collagen secretion compared to the antimiR-control and the nothing delivered control. The in vivo ectopic implantation assay uncovers the robust vascularized bone formation of the antimiR-138 delivered MSIC. The antimiR-138 delivered MSIC with promoted osteogenesis and angiogenesis coupling is anticipated to lead to rigid osseointegration in the compromised bone conditions.

miR-25 Tough Decoy Enhances Cardiac Function in Heart Failure.

MicroRNAs are promising therapeutic targets, because their inhibition has the potential to normalize gene expression in diseased states. Recently, our group found that miR-25 is a key SERCA2a regulating microRNA, and we showed that multiple injections of antagomirs against miR-25 enhance cardiac contractility and function through SERCA2a restoration in a murine heart failure model. However, for clinical application, a more stable suppressor of miR-25 would be desirable. Tough Decoy (TuD) inhibitors are emerging as a highly effective method for microRNA inhibition due to their resistance to endonucleolytic degradation, high miRNA binding affinity, and efficient delivery. We generated a miR-25 TuD inhibitor and subcloned it into a cardiotropic AAV9 vector to evaluate its efficacy. The AAV9 TuD showed selective inhibition of miR-25 in vitro cardiomyoblast culture. In vivo, AAV9-miR-25 TuD delivered to the murine pressure-overload heart failure model selectively decreased expression of miR-25, increased levels of SERCA2a protein, and ameliorated cardiac dysfunction and fibrosis. Our data indicate that miR-25 TuD is an effective long-term suppressor of miR-25 and a promising therapeutic candidate to treat heart failure.

Multi-Functional Peptide-MicroRNA Nanocomplex for Targeted MicroRNA Delivery and Function Imaging.

Targeted delivery of microRNA (miRNA) mimics into specific cells/tissues and real-time monitoring on the biological function of delivered miRNA mimics at molecular level represent two major challenges in the development of miRNA-based therapeutics. Here we report a highly efficient method to address these two challenges simultaneously by using the self-assembled nanocomplex formed by miRNA mimics with a multi-functional peptide conjugate. Using the nanocomplex formed by tumor-suppressive miR-34a and the multi-functional peptide conjugate FA-R9-FPcas3 , we demonstrated the highly efficient and target-selective delivery of miR-34a into HeLa cells and tumors. With the activatable fluorescence probe integrated in the peptide conjugate FA-R9-FPcas3 , the intracellular function of miR-34a delivered by the nanocomplex to upregulate active Caspase-3 was imaged in real-time. The nanocomplex also showed significant therapeutic effects to induce apoptosis in HeLa cells and to suppress tumor growth upon tail vein injection into living mice bearing subcutaneous HeLa tumors.

Combination of Anti-miRNAs Oligonucleotides with Low Amounts of Chemotherapeutic Agents for Pancreatic Cancer Therapy.

Pancreatic ductal adenocarcinoma (PDAC) is the most predominant type of pancreatic cancer and presents one of the highest mortality rates when compared with other carcinomas. The absence of efficient treatment options for PDAC prompted us to investigate whether microRNA inhibition, combined or not with chemotherapeutic agents, would constitute a promising therapeutic approach for this disease. In this chapter, we describe several methods and procedures that can be used to evaluate the potential of new therapeutic strategies involving oligonucleotides against overexpressed microRNAs, in PDAC, either alone or in combination with low amounts of chemotherapeutic drugs.

A multiplex protein-free lateral flow assay for detection of microRNAs based on unmodified molecular beacons.

Lateral flow assays (LFAs) have promising potentials for point-of-care applications. Recently, many LFAs have been reported that are based on hybridization of oligonucleotide strands. Mostly, biotinylated capture DNAs are immobilized on the surface of a nitrocellulose membrane via streptavidin interactions. During the assay, stable colorful complexes get formed that are visible by naked eyes. Here, we present an inexpensive and unique design of LFA that applies unmodified oligonucleotides at capture lines. The presented LFA do not utilize streptavidin or any other affinity protein. We employ structural switch of molecular beacons (MB) in combination with base stacking hybridization (BSH) phenomenon. The unique design of the reported LFA provided high selectivity for target oligonucleotides. We validated potential applications of the system for detection of DNA mimics of two microRNAs in multiplex assays.

RNA Nanoparticle-Based Targeted Therapy for Glioblastoma through Inhibition of Oncogenic miR-21.

Targeted inhibition of oncogenic miRNA-21 has been proposed to treat glioblastoma by rescuing tumor suppressors, PTEN and PDCD4. However, systemic delivery of anti-miR-21 sequences requires a robust and efficient delivery platform to successfully inhibit this druggable target. Three-way-junction (3WJ)-based RNA nanoparticles (RNP), artificially derived from pRNA of bacteriophage phi29 DNA packaging motor, was recently shown to target glioblastoma. Here, we report that multi-valent folate (FA)-conjugated 3WJ RNP constructed to harbor anti-miR-21 LNA sequences (FA-3WJ-LNA-miR21) specifically targeted and delivered anti-miR-21 LNA and knocked down miR-21 expression in glioblastoma cells in vitro and in vivo with favorable biodistribution. Systemically injected FA-3WJ-LNA-miR21 RNP efficiently rescued PTEN and PDCD4, resulting in glioblastoma cell apoptosis and tumor growth regression. Overall survival rate was also significantly improved by FA-3WJ-LNA-miR21 RNP. These results are indicative of the clinical benefit of FA-3WJ RNP-based gene therapy for the successful targeted therapy of developing and even recurring glioblastoma.