Delivery Of miR-34a Research Articles

Developing Folate-Conjugated miR-34a Therapeutic for Prostate Cancer: Challenges and Promises.

Prostate cancer (PCa) remains a common cancer with high mortality in men due to its heterogeneity and the emergence of drug resistance. A critical factor contributing to its lethality is the presence of prostate cancer stem cells (PCSCs), which can self-renew, long-term propagate tumors, and mediate treatment resistance. MicroRNA-34a (miR-34a) has shown promise as an anti-PCSC therapeutic by targeting critical molecules involved in cancer stem cell (CSC) survival and functions. Despite extensive efforts, the development of miR-34a therapeutics still faces challenges, including non-specific delivery and delivery-associated toxicity. One emerging delivery approach is ligand-mediated conjugation, aiming to achieve specific delivery of miR-34a to cancer cells, thereby enhancing efficacy while minimizing toxicity. Folate-conjugated miR-34a (folate-miR-34a) has demonstrated promising anti-tumor efficacy in breast and lung cancers by targeting folate receptor α (FOLR1). Here, we first show that miR-34a, a TP53 transcriptional target, is reduced in PCa that harbors TP53 loss or mutations and that miR-34a mimic, when transfected into PCa cells, downregulated multiple miR-34a targets and inhibited cell growth. When exploring the therapeutic potential of folate-miR-34a, we found that folate-miR-34a exhibited impressive inhibitory effects on breast, ovarian, and cervical cancer cells but showed minimal effects on and targeted delivery to PCa cells due to a lack of appreciable expression of FOLR1 in PCa cells. Folate-miR-34a also did not display any apparent effect on PCa cells expressing prostate-specific membrane antigen (PMSA) despite the reported folate's binding capability to PSMA. These results highlight challenges in the specific delivery of folate-miR-34a to PCa due to a lack of target (receptor) expression. Our study offers novel insights into the challenges and promises within the field and casts light on the development of ligand-conjugated miR-34a therapeutics for PCa.

Combined Photosensitive Gene Therapy Effective Against Triple-Negative Breast Cancer in Mice Model.

Tumor hypoxia and invasion present significant challenges for the efficacy of photodynamic therapy (PDT) in triple-negative breast cancer (TNBC). This study developed a mitochondrial targeting strategy that combined PDT and gene therapy to promote each other and address the challenges. The positively charged amphiphilic material triphenylphosphine-tocopherol polyethylene glycol succinate (TPP-TPGS, TPS) and the photosensitizer chloride e6 (Ce6) formed TPS@Ce6 nanoparticles (NPs) by hydrophobic interaction. They electrostatically condensed microRNA-34a (miR-34a) to form stable TPS@Ce6/miRNA NPs. Firstly, Ce6 disrupted the lysosomal membrane, followed by successful delivery of miR-34a by TPS@Ce6/miRNA NPs. Meanwhile, miR-34a reduced ROS depletion and further enhanced the effectiveness of PDT. Consequently, the mutual promotion between PDT and gene therapy led to enhanced anti-tumor effects. Furthermore, the TPS@Ce6/miRNA NPs promoted apoptosis by down-regulating Caspase-3 and inhibited tumor cell migration and invasion by down-regulating N-Cadherin. In addition, in vitro and in vivo experiments demonstrated that the TPS@Ce6/miRNA NPs achieved excellent anti-tumor effects. These findings highlighted the enhanced anticancer effects and reduced migration of tumor cells through the synergistic effects of PDT and gene therapy. Taken together, the targeted co-delivery of Ce6 and miR-34a will facilitate the application of photodynamic and genic nanomedicine in the treatment of aggressive tumors, particularly TNBC.

Near-Infrared Induced miR-34a Delivery from Nanoparticles in Esophageal Cancer Treatment.

Current nucleic acid delivery methods have not achieved efficient, non-toxic delivery of miRNAs with tumor-specific selectivity. In this study, a new delivery system based on light-inducible gold-silver-gold, core-shell-shell (CSS) nanoparticles is presented. This system delivers small nucleic acid therapeutics with precise spatiotemporal control, demonstrating the potential for achieving tumor-specific selectivity and efficient delivery of miRNA mimics. The light-inducible particles leverage the photothermal heating of metal nanoparticles due to the local surface plasmonic resonance for controlled chemical cleavage and release of the miRNA mimic payload. The CSS morphology and composition result in a plasmonic resonance within the near-infrared (NIR) region of the light spectrum. Through this method, exogenous miR-34a-5p mimics are effectively delivered to human squamous cell carcinoma TE10 cells, leading to apoptosis induction without adverse effects on untransformed keratinocytes in vitro. The CSS nanoparticle delivery system is tested in vivo in Foxn1nu athymic nude mice with bilateral human esophageal TE10 cancer cells xenografts. These experiments reveal that this CSS nanoparticle conjugates, when systemically administered, followed by 850nm light emitting diode irradiation at the tumor site, 6 h post-injection, produce a significant and sustained reduction in tumor volume, exceeding 87% in less than 72 h.

Targeted miR-34a delivery with PD1 displayed bacterial outer membrane vesicles-coated zeolitic imidazolate framework nanoparticles for enhanced tumor therapy

MicroRNA (miRNA) has been widely used as an effective gene drug for tumor therapy, but its chemical instability limited its therapeutic application in vivo. In this research, we fabricate an efficient miRNA nano-delivery system using zeolitic imidazolate framework-8 (ZIF-8) coated with bacterial outer membrane vesicles (OMVs), aimed for cancer treatment. The acid-sensitive ZIF-8 core enables this system to encapsulate miRNA and release them from lysosome quickly and efficiently in the target cells. The OMVs engineered to display programmed death receptor 1 (PD1) on the surface provides a specific tumor-targeting capability. Using a murine breast cancer model, we show that this system has high miRNA delivery efficiency and accurate tumor targeting. Moreover, the miR-34a payloads in carriers can further synergize with immune activation and checkpoint inhibition triggered by OMV-PD1 to enhance tumor therapeutic efficacy. Overall, this biomimetic nano-delivery platform provides a powerful tool for the intracellular delivery of miRNA and has great potential in RNA-based cancer therapeutic applications.

Abstract 3782: Targeting cancer cells using folate conjugated to a fully modified version of miR-34a (FolamiR-34a) to produce enhanced and sustained anti-tumor activity

Abstract Due to the pleiotropic nature of miR-34a, including its ability to downregulate multiple genes that drive resistance to currently used anti-cancer therapies, we hypothesize that the best use of miR-34a in a clinical setting would be in combination with standard-of-care treatments. For in vivo use, such a molecule would necessitate the following: i) a stabilized version of miR-34a that can withstand the harsh environment in circulation and intracellular nucleases, and ii) a robust, specific, and safe delivery vehicle. With regard to stability, we successfully generated the first fully modified version of miR-34a. This new chemically modified molecule is over 400-fold more stable than the previously used partially modified version and induces enhanced and sustained target gene repression. To assess in vivo activity of fully modified miR-34a requires a suitable delivery vehicle. Indeed, while various approaches have been used to restore miR-34a in tumors, clinically relevant mechanisms for delivery of miR-34a have been a challenge due to poor tumor uptake, unfavorable bioavailability, and unwanted toxicity. Previously, we determined that a partially modified version of miR-34a can be delivered specifically and robustly to cancer cells in a targeted, vehicle-free manner through direct conjugation to folate, a ligand of the high-affinity folate receptors. Thus, we conjugated our fully modified miR-34a (FM-miR-34a) to folate generating FM-FolamiR-34a. Following in vivo delivery of FM-FolamiR-34a to mice with breast cancer xenografts we observed enhanced and sustained target gene repression, in comparison to our first-generation partially modified folate-miR-34a conjugate (FolamiR). Critical miR-34a target genes involved in promoting resistance, including MET, AXL, and CD44 were downregulated over 90% for at least 120 hours following a single 1.5 nmol dose of FM-FolamiR-34a. Our final objective is to evaluate FM-FolamiR-34a in combination with standard-of-care agents in vivo. Citation Format: Shreyas Ganesan Iyer, Ahmed M. Abdelaal, Ikjot Singh Sohal, Sudarsan R. Kasireddy, Andrea L. Kasinski. Targeting cancer cells using folate conjugated to a fully modified version of miR-34a (FolamiR-34a) to produce enhanced and sustained anti-tumor activity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3782.

Abstract 2716: Further development of microRNA-34a therapeutics to target castration-resistant prostate cancer

Abstract Prostate cancer (PCa) is heterogeneous and typically presents with multiple distinct cancer foci. Androgen receptor signaling inhibitors (ARSIs) are standard-of-care therapies for advanced PCa and can prolong patient survival by months. Treatment-naïve PCa harbors a population of stem-like cells (AR-/lo) functionally defined as PCa stem cells (PCSCs). During cancer progression or upon therapeutic treatment, non-CSCs may undergo cellular plasticity and be reprogrammed to PCSCs. As a result, PCSCs may become the predominant cell population in advanced (Gleason 9/10) and ARSI-resistant tumors, and the “root cause” for drug resistance. However, current ARSI-based treatment regimens primarily target AR+ CRPC cells or clones but largely ignore AR-/lo cells. microRNA-34a (miR-34a), a tumor-suppressive miRNA dysregulated in PCa, is a potent PCSC inhibitor by targeting cellular processes essential for CSC survival and functions. Here we aim to develop novel miR-34a therapeutics to target PCa cell heterogeneity and CRPC. miR-34a expression negatively correlates with the tumor stage in PCa samples. And miR-34a expression is significantly reduced in p53 mutated tumors. We explored two novel miR-34a delivery platforms. The first is packaging-free ligand-mediated conjugate utilizing 2- [3-(1,3-dicarboxy propyl) ureido] pentanedioic acid (DUPA) as a high-affinity binding ligand for prostate-specific membrane antigen (PSMA), which is upregulated in PCa. The second platform uses cationic lipid nanoparticles to package miR-34a (LNP-miR-34a). We show that during PCa progression and upon treatment, PSMA expression is upregulated and continues to be highly expressed in CRPC and PSMA expression level negatively correlates with overall survival in PCa. Moreover, DUPA-conjugate can specifically target PSMA-expressing PCa both in vitro and in vivo. DUPA-miR-34a significantly induces target gene silencing in vitro. On the other hand, LNP show good encapsulation efficiency for miR-34a, LNP-miR-34a significantly repressed cell proliferation by downregulating critical stemness factors in a dose-dependent manner. Meanwhile, we are pursuing chemical modifications of the miR-34a mimics aiming to improve serum stability and future in vivo applications. Both fully modified miR-34a (FM-miR-34a) and partially-modified-miR-34a (PM-miR-34a) exhibit improved serum stability without compromising the functionality. Our data suggest that DUPA-miR-34a represents a promising novel therapeutic for PCSC-enriched CRPC that continue to express PSMA, whereas LNP-miR-34a could be a systemic therapeutic for CRPC treatment in the combinatorial setting. miR-34a, as a direct target and a major mediator of p53 functions, may represent an effective replacement therapeutic in p53-mutated advanced PCa. Citation Format: Wen (Jess) Li, Xiaozhuo Liu, Ahmed M. Abdelaal, Sudarsan Kasireddy, Yafei Su, Yun Wu, Andrea L. Kasinski, Dean G. Tang. Further development of microRNA-34a therapeutics to target castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2716.

Multi-sensitive functionalized niosomal nanocarriers for controllable gene delivery in vitro and in vivo

MicroRNAs, which can contribute to numerous cellular functions through post-transcriptional silencing, have become well-documented candidates for cancer treatment applications, particularly in chemo-resistant cancers. Herein, several formulations were examined to optimize the essential parameters, and the niosomal formulation consisting of cholesterol:tween-80:DOTAP:PEG with 9:69:15:7 ratio had the best physicochemical parameters including spherical shape, high entrapment efficiency, small diameter (81 ± 0.65 nm), and appropriate positive charge (23 ± 0.64 mV). Here, we aimed to design a system with increased delivery efficiency which was tested by the encapsulation of miR-34a within niosome NPs and assessed the nano-niosomal delivery of miR-34a as a tumor suppressor in MCF-7 human adenocarcinoma cells. The results showed that our novel niosome systems with non-ionic surfactants can successfully eliminate cancer cells by increasing the expression of p53 and reducing the expression of NF-κB. In comparison with the free dispersion of miR-34a, the lysis of a nano-sized delivery system demonstrated a better cytotoxicity effect against cancer cells. Similar results were obtained by performing in vivo test on the 4T1 xenografted Balb/C mouse tumor model and the miR-34a-loaded niosomes displayed a better reduction in tumor size by improving approximately + 13% in tumor inhabitation rate while maintaining the bodyweight close to the first day. Therefore, it is concluded that miR-34a delivery via niosomes has high potential as a tumor suppressor and a reliable procedure for breast cancer treatment.Graphical

MiR34 contributes to spinal muscular atrophy and AAV9-mediated delivery of MiR34a ameliorates the motor deficits in SMA mice

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by the selective loss of spinal motor neurons (MNs) and concomitant muscle weakness. Mutation of SMN1 is known to cause SMA, and restoring SMN protein levels via antisense oligonucleotide treatment is effective for ameliorating symptoms. However, this approach is hindered by exorbitant costs, invasive procedures, and poor treatment responses of some patients. Here, we seek to circumvent these hurdles by identifying reliable biomarkers that could predict treatment efficacy. We uncovered that MiR34 exhibits consistent downregulation during SMA progression in both human and rodent contexts. Importantly, Mir34 family-knockout mice display axon swelling and reduced neuromuscular junction (NMJ) endplates, recapitulating SMA pathology. Introducing MiR34a via scAAV9 improved the motor ability of SMNΔ7 mice, possibly by restoring NMJ endplate size. Finally, we observed a consistent decreasing trend in MiR34 family expression in the cerebrospinal fluid (CSF) of type I SMA patients during the loading phase of nusinersen treatment. Baseline CSF MiR34 levels before nusinersen injection proved predictive of patient motor skills 1 year later. Thus, we propose that MiR34 may serve as a biomarker of SMA since it is associated with the pathology and can help evaluate the therapeutic effects of nusinersen.

Engineering of inhalable nano-in-microparticles for co-delivery of small molecules and miRNAs

In this study, novel Trojan particles were engineered for direct delivery of doxorubicin (DOX) and miR-34a as model drugs to the lungs to raise local drug concentration, decrease pulmonary clearance, increase lung drug deposition, reduce systemic side effects, and overcome multi-drug resistance. For this purpose, targeted polyelectrolyte nanoparticles (tPENs) developed with layer-by-layer polymers (i.e., chitosan, dextran sulfate, and mannose-g-polyethyleneimine) were spray dried into a multiple-excipient (i.e., chitosan, leucine, and mannitol). The resulting nanoparticles were first characterized in terms of size, morphology, in vitro DOX release, cellular internalization, and in vitro cytotoxicity. tPENs showed comparable cellular uptake levels to PENs in A549 cells and no significant cytotoxicity on their metabolic activity. Co-loaded DOX/miR-34a showed a greater cytotoxicity effect than DOX-loaded tPENs and free drugs, which was confirmed by Actin staining. Thereafter, nano-in-microparticles were studied through size, morphology, aerosolization efficiency, residual moisture content, and in vitro DOX release. It was demonstrated that tPENs were successfully incorporated into microspheres with adequate emitted dose and fine particle fraction but low mass median aerodynamic diameter for deposition into the deep lung. The dry powder formulations also demonstrated a sustained DOX release at both pH values of 6.8 and 7.4.

Bone targeted miRNA delivery system for miR-34a with enhanced anti-tumor efficacy to bone-associated metastatic breast cancer

Bone metastatic cancer is the most common occurrence in breast cancer, and the treatment is also facing great challenges. MicroRNA-34a (miRNA-34a) is a promising anti-cancer miRNA for gene therapy to bone metastatic cancer patients. However, the lack of specificity to bone and low accumulation at the site of bone tumor remains the major challenge when used bone-associated tumor. To solve this problem, a bone-targeted vector for delivery of miR-34a to bone metastatic breast cancer was constructed by using the commonly used gene vector branched polyethylenimine 25 k (BPEI 25 k) as the skeleton and linking with alendronate (ALN) moieties for bone targeting group. The constructed gene delivery system PCA/miR-34a can efficiently prevent miR-34a from degradation during blood circulation and enhance the specific bone delivery and distribution. PCA/miR-34a nanoparticles can be uptake into tumor cells through clathrin and caveolae-mediated endocytosis, and directly regulate the expression of oncogenes, thus promoting tumor cell apoptosis and relieving bone tissue erosion. The results of experiments in vitro and in vivo confirmed that the constructed bone-targeted miRNA delivery system PCA/miR-34a can enhance the anti-tumor efficacy in bone metastatic cancer, and provide a potential strategy for gene therapy in bone metastatic cancer.

Abstract 1482: Efficient targeting of prostate cancer using ligand conjugated and chemically modified tumor suppressive miRNA

Abstract MicroRNAs (miRNAs) inhibit the expression of genes through imperfect base pairing with target messenger RNAs (mRNAs), which allows a single miRNA the ability to regulate the expression of multiple genes, potentially acting as a multi-drug cocktail. In principle, tumor-suppressive miRNAs, such as miRNA-34a (miR-34a) are excellent anti-cancer agents; however, due to toxicity associated with currently used delivery vehicles and poor in vivo miRNA stability, the clinical application of miRNAs is thwarted. To overcome these challenges, we engineered two modifications to the miRNA. The first entails conjugating the miRNA to a ligand that promotes delivery specifically to cancer cells while the second involves using a fully chemically modified miRNA (FM-miR) to enhance miRNA stability. The ligand chosen for delivery, 2- [3-(1,3-dicarboxy propyl) ureido] pentanedioic acid (DUPA), is a high-affinity binding partner of the Prostate-Specific Membrane Antigen (PSMA), which is significantly upregulated on prostate cancer cells. We show that DUPA-miR-34a is specifically targeted to tumor cells overexpressing PSMA, is rapidly internalized by the cells, and induces target gene silencing. The binding and uptake of miR-34a by the tumor cells is dependent on conjugating the miRNA to DUPA. In vivo, DUPA-conjugates bind specifically to PSMA expressing prostate tumors with no significant uptake from normal organs or tumors with no PSMA expression. Additionally, Incorporation of the ionophore nigericin in the DUPA ligand enhances miR-34a silencing activity through facilitating its endosomal escape. However, since RNAs are inherently unstable a second modification was engineered to enhance the miRNA stability. Modifying miRNA through the inclusion of phosphorothioate linkages and 2′-fluoro and 2′-O-methyl modified ribose bases, enhances miRNA stability without compromising its activity. Fully modified miR-34a (FM-miR-34a) exhibits enhanced silencing activity of its target genes such as MET, and CD44 in comparison to partially modified miR-34a (PM-miR-34a). In addition, both PM-miR-34a and FM-miR-34a significantly inhibit cell proliferation in vitro, and the silencing of target genes requires the presence of argonaute 2 (AGO2) protein. We plan to identify the targeting differences between FM- and PM-miR-34a and will combine the DUPA ligand with FM-miR-34a to enhance both delivery and activity of miR-34a. Citation Format: Ahmed M. Abdelaal, Sudarshan Kasireddy, Ikjot S. Sohal, Philip S. Low, Andrea L. Kasinski. Efficient targeting of prostate cancer using ligand conjugated and chemically modified tumor suppressive miRNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1482.

Tumor-derived exosomes encapsulating miR-34a promote apoptosis and inhibit migration and tumor progression of colorectal cancer cells under in vitro condition.

MicroRNA (miR)-34a, as a master tumor suppressor in colorectal cancer (CRC), could regulate multiple genes participating in tumor proliferation, invasion, immune evasion, and inflammation-induced progression. Exosomes, as novel nano-carriers, were found to be capable of shuttling crucial mediators to various cells. Since the conventional CRC therapeutics currently are a matter of debate, implication of microRNAs in malignancy remedies have been addressed illustrating promising outlooks. In this study, we aimed to investigate the delivery of miR-34a to CRC cell line CT-26 by encapsulating into tumor-derived exosomes (TEXs), in order to evaluate the anti-proliferative and progressive effects of the novel nano-carrier complex under in vitro condition. Exosomes were purified from the starved CT-26 cells and then enriched by miR-34a using the calcium chloride (Cacl2) modified solution. Following the detection of miR-34a expression in the enriched TEXs, the viability of CT-26 cells treated by multiplicity concentrations of either TEXs or TEX-miR-34a was examined. Moreover, the apoptosis rate of the cells was evaluated, and the migration of CT-26 cells subjected to both TEX-miR-34a and TEX was also measured. Thereafter, the expressions of miR-34a target genes, as IL-6R, STAT3, PD-L1, and VEGF-A, which play roles in tumor progression, were determined in the treated CT-26 cells. The viability of CT-26 cells was harnessed following the treatment with TEX-miR-34a and the apoptosis levels of the cells were also observed to be enhanced dose-dependently. TEX-miR-34a was able to diminish the migration rate of the TEX-miR-34a treated cells and the expressions of IL-6R, STAT3, PD-L1, and VEGF-A were significantly restricted. Moreover, TEXs alone increased the apoptosis rate of tumor cells and repressed the proliferation and migration of these cells which were boosted by enrichment of TEXs with miR-34a. Exosomes isolated from the starved CT-26 cells were found to have a potential to deliver miR-34a into tumor cells properly with high functionality maintenance for miR-34a in case of regulating genes related to tumor progression and TEXs which showed no positive effect favoring cancer cells, presumably act as a favorable adjuvant in the CRC therapy.

Lipoic Acid-Modified Oligoethyleneimine-Mediated miR-34a Delivery to Achieve the Anti-Tumor Efficacy.

MiR-34a, an important tumor suppressor, has been demonstrated to possess great potential in tumor gene therapy. To achieve the upregulation of miR-34a expression level, an oligoethyleneimine (OEI) derivative was constructed and employed as the carrier through the modification with lipoic acid (LA), namely LA-OEI. In contrast to OEI, the derivative LA-OEI exhibited superior transfection efficiency measured by confocal laser scanning microscopy and flow cytometry, owing to rapid cargo release in the disulfide bond-based reduction sensitive pattern. The anti-proliferation and anti-migration effects were tested after the miR-34a transfection to evaluate the anti-tumor response, using human cervical carcinoma cell line HeLa as a model. The delivery of LA-OEI/miR-34a nanoparticles could achieve obvious anti-proliferative effect caused by the induction of cell apoptosis and cell cycle arrest at G1 phase. In addition, it could inhibit the migration of tumor cells via the downregulation of MMP-9 and Notch-1 level. Overall, the LA-OEI-mediated miR-34a delivery was potential to be used as an effective way in the tumor gene therapy.

Smart co-delivery of miR-34a and cytotoxic peptides (LTX-315 and melittin) by chitosan based polyelectrolyte nanocarriers for specific cancer cell death induction

A novel polyelectrolyte nanocarrier was synthesized via layer-by-layer self-assembly of polycationic and polyanionic chains. The nanocarrier is composed of polyglutamate grafted chitosan core, dextran sulfate as a complexing agent, and polyethyleneimine shell decorated with folic acid. This polyelectrolyte complex has unique physicochemical properties so that the core is considered as an efficient carrier for LTX-315 and melittin peptides, and the shell is suitable for delivery of miR-34a. The spherical nanocarriers with an average size of 123 ± 5 nm and a zeta potential of −36 ± 1 mV demonstrated controlled-release of gene and peptides ensured a synergistic effect in establishing multiple cell death pathways on chemoresistance human breast adenocarcinoma cell line, MDA-MB-231. In vitro cell viability assays also revealed no cytotoxicity for the nanocarriers, and an IC50 of 15 μg/mL and 150 μg/mL for melittin and LTX-315, respectively, after 48 h, whereas co-delivery of melittin with miR-34a increased smart death induction by 54%.

Nanocell-mediated delivery of miR-34a counteracts temozolomide resistance in glioblastoma

BackgroundGlioblastoma is the most common primary brain tumor and remains uniformly fatal, highlighting the dire need for developing effective therapeutics. Significant intra- and inter-tumor heterogeneity and inadequate delivery of therapeutics across blood–brain barrier continue to be significant impediments towards developing therapies which can significantly enhance survival. We hypothesize that microRNAs have the potential to serve as effective therapeutics for glioblastoma as they modulate the activity of multiple signaling pathways, and hence can counteract heterogeneity if successfully delivered.MethodsUsing a computational approach, we identified microRNA-34a as a microRNA that maximally reduces the activation status of the three core signaling networks (the receptor tyrosine kinase, p53 and Rb networks) that have been found to be deregulated in most glioblastoma tumors. Glioblastoma cultures were transfected with microRNA-34a or control microRNA to assess biological function and therapeutic potential in vitro. Nanocells were derived from genetically modified bacteria and loaded with microRNA-34a for intravenous administration to orthotopic patient-derived glioblastoma xenografts in mice.ResultsOverexpression of microRNA-34a strongly reduced the activation status of the three core signaling networks. microRNA-34a transfection also inhibited the survival of multiple established glioblastoma cell lines, as well as primary patient-derived xenograft cultures representing the proneural, mesenchymal and classical subtypes. Transfection of microRNA-34a enhanced temozolomide (TMZ) response in in vitro cultures of glioblastoma cells with primary TMZ sensitivity, primary TMZ resistance and acquired TMZ resistance. Mechanistically, microRNA-34a downregulated multiple therapeutic resistance genes which are associated with worse survival in glioblastoma patients and are enriched in specific tumor spatial compartments. Importantly, intravenous administration of nanocells carrying miR-34a and targeted to epidermal growth factor receptor (EGFR) strongly enhanced TMZ sensitivity in an orthotopic patient-derived xenograft mouse model of glioblastoma.ConclusionsTargeted bacterially-derived nanocells are an effective vehicle for the delivery of microRNA-34a to glioblastoma tumors. microRNA-34a inhibits survival and strongly sensitizes a wide range of glioblastoma cell cultures to TMZ, suggesting that combination therapy of TMZ with microRNA-34a loaded nanocells may serve as a novel therapeutic approach for the treatment of glioblastoma tumors.

Study and Preparation of Multifunctional Poly(L-Lysine)@Hyaluronic Acid Nanopolyplexes for the Effective Delivery of Tumor Suppressive MiR-34a into Triple-Negative Breast Cancer Cells.

Non-viral gene delivery using exogenous microRNAs is a potential strategy for fighting cancers with poor prognosis and which lack specific therapies, such as triple-negative breast cancer (TNBC). Herein we report the synthesis of six nontoxic electrostatic polymeric nanocapsules (P1 to P6) for microRNA delivery in TNBC cells. 1H Nuclear Magnetic Resonance (NMR) spectroscopy and Scanning Electron Microscopy (SEM) were used to characterize the nanopolyplexes, synthesized with Poly(L-Lysine) and hyaluronic acid (Ha). Studies on the activity of the ternary HA/PLI/miRNA-34 nanopolyplexes towards TNBC cell line MDA-MB-231 were conducted. The nanopolyplexes mediated intracellular restoration of tumor suppressor miR34a was evaluated by using Western blotting to quantify the expression level of the Bcl-2 protein. The results suggest that the P5, with a ratio PLI/Ha of 0.05, was the most promising for the delivery of miR-34a into TNBC cells; the P5 nanocapsules were able to reduce Bcl-2 expression at a protein level, and had an effect in the overall cell viability after 24 h treatment.

Dual Regulation of miR-34a and Notch Signaling in Triple-Negative Breast Cancer by Antibody/miRNA Nanocarriers

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks expression of the three most common receptors present on other subtypes, leaving it unsusceptible to current targeted or hormonal therapies. In this study, we introduce an alternative treatment strategy for TNBC that exploits its overexpression of Notch1 receptors and its underexpression of the tumor suppressive microRNA (miRNA) miR-34a. Studies have shown that introducing mimics of miR-34a to TNBC cells effectively inhibits cancer growth, but miR-34a cannot be administered in the clinic without a carrier. To enable delivery of miR-34a to TNBC cells, we encapsulated miR-34a mimics in poly(lactic-co-glycolic acid) nanoparticles (NPs) that were functionalized with Notch1 antibodies to produce N1-34a-NPs. In addition to binding Notch1 receptors overexpressed on the surface of TNBC cells, the antibodies in this formulation enable suppression of Notch signaling through signal cascade interference. Herein, we present the results of in vitro experiments that demonstrate N1-34a-NPs can regulate Notch signaling and downstream miR-34a targets in TNBC cells to induce senescence and reduce cell proliferation and migration. These studies demonstrate that NP-mediated co-delivery of miR-34a and Notch1 antibodies is a promising alternative treatment strategy for TNBC, warranting further optimization and in vivo investigation in future studies.

Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours

BackgroundIn this first-in-human, Phase 1 study of a microRNA-based cancer therapy, the recommended Phase 2 dose (RP2D) of MRX34, a liposomal mimic of microRNA-34a (miR-34a), was determined and evaluated in patients with advanced solid tumours.MethodsAdults with various solid tumours refractory to standard treatments were enrolled in 3 + 3 dose-escalation cohorts and, following RP2D determination, expansion cohorts. MRX34, with oral dexamethasone premedication, was given intravenously daily for 5 days in 3-week cycles.ResultsCommon all-cause adverse events observed in 85 patients enrolled included fever (% all grade/G3: 72/4), chills (53/14), fatigue (51/9), back/neck pain (36/5), nausea (36/1) and dyspnoea (25/4). The RP2D was 70 mg/m2 for hepatocellular carcinoma (HCC) and 93 mg/m2 for non-HCC cancers. Pharmacodynamic results showed delivery of miR-34a to tumours, and dose-dependent modulation of target gene expression in white blood cells. Three patients had PRs and 16 had SD lasting ≥4 cycles (median, 19 weeks, range, 11–55).ConclusionMRX34 treatment with dexamethasone premedication demonstrated a manageable toxicity profile in most patients and some clinical activity. Although the trial was closed early due to serious immune-mediated AEs that resulted in four patient deaths, dose-dependent modulation of relevant target genes provides proof-of-concept for miRNA-based cancer therapy.Clinical trial registrationNCT01829971.

Peptide-conjugated liposomes for targeted miR-34a delivery to suppress breast cancer and cancer stem-like population

MicroRNAs perform critical roles in regulation of cancer cells and cancer stem cells (CSCs). CSCs are key contributors of therapy failure; hence, successful tumor therapy requires elimination of both CSCs and tumor cells. MicroRNA-34a (miR-34a) is a well-defined tumor suppressor microRNA which is hypermethylated in breast cancer. Cyclic RGD (cRGD) is a targeting peptide that selectively target integrin receptor-expressing cells. Herein, cRGD-conjugated Polyethylene glycol-modified (PEGylated) liposomes (cRGD-PEG liposomes) encapsulating miR-34a (cRGD-PEG/miR-34a liposomes) was developed for targeted transport of miR-34a into MDA-MB-231 breast cancer cells. A 1.8-fold higher internalization of cRGD-targeted liposomes effectively increased the accumulation of miR-34a in breast carcinoma cells. Notably, cRGD-targeted miR-34a-loaded liposomes showed significant inhibitory outcome on tumor cell growth, migration, and invasion as well as a significant decrease in the percentage of CD44+/CD24−/low cancer stem-like population. The results suggest that cRGD-targeted liposomes may possibly increase the efficiency of therapeutic MicroRNAs in breast carcinoma cells and CSCs.

A High-Throughput Small Molecule Screen Identifies Ouabain as Synergistic with miR-34a in Killing Lung Cancer Cells.

SummaryMicroRNA-34 (miR-34) is one of the major families of tumor suppressor miRNAs often lost in cancers. Delivery of miR-34a mimics to affected tumors as a therapeutic strategy has been tried in pre-clinical studies and in a phase I clinical trial. One approach to increase efficacy and reduce toxicity is to rationally identify drug combinations with small molecules that synergize with miR-34a. In this study we performed a high-throughput screen of a large panel of small molecules with known biological activity and identified ouabain as a candidate small molecule that synergized with miR-34a in killing lung cancer cells. We elucidated autophagy activation as a key mechanism by which miR-34a and ouabain causes increased cytotoxicity in cells. We posit that this combinatorial approach could reduce the active dose of miR-34a needed in vivo to observe tumor shrinkage and potentiate the development of miR-34a combination therapies in the future.