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

BACKGROUND: Glioblastoma 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. METHODS: Using 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. RESULTS: Overexpression 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. CONCLUSIONS: Targeted 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.

Bacterially derived 400 nm particles for encapsulation and cancer cell targeting of chemotherapeutics.

Systemic administration of chemotherapeutic agents results in indiscriminate drug distribution and severe toxicity. Here we report a technology potentially overcoming these shortcomings through encapsulation and cancer cell-specific targeting of chemotherapeutics in bacterially derived 400 nm minicells. We discovered that minicells can be packaged with therapeutically significant concentrations of chemotherapeutics of differing charge, hydrophobicity, and solubility. Targeting of minicells via bispecific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release. This affects highly significant tumor growth inhibition and regression in mouse xenografts and case studies of lymphoma in dogs despite administration of minute amounts of drug and antibody; a factor critical for limiting systemic toxicity that should allow the use of complex regimens of combination chemotherapy.

Phase I/IIa Trial in Advanced Pancreatic Ductal Adenocarcinoma Treated with Cytotoxic Drug-Packaged, EGFR-Targeted Nanocells and Glycolipid-Packaged Nanocells.

PURPOSE: We assessed the safety and efficacy of an EGFR-targeted, super-cytotoxic drug, PNU-159682-packaged nanocells with α-galactosyl ceramide-packaged nanocells (E-EDV-D682/GC) in patients with advanced pancreatic ductal adenocarcinoma (PDAC) who had exhausted all treatment options. PATIENTS AND METHODS: ENG9 was a first-in-man, single-arm, open-label, phase I/IIa, dose-escalation clinical trial. Eligible patients had advanced PDAC, Eastern Cooperative Oncology Group status 0 to 1, and failed all treatments. Primary endpoints were safety and overall survival (OS). RESULTS: Of 25 enrolled patients, seven were withdrawn due to rapidly progressive disease and one patient withdrew consent. All 25 patients were assessed for toxicity, 24 patients were assessed for OS, which was also assessed for 17 patients completing one treatment cycle [evaluable subset (ES)]. Nineteen patients (76.0%) experienced at least one treatment-related adverse event (graded 1 to 2) resolving within hours. There were no safety concerns, dose reductions, patient withdrawal, or treatment-related deaths.Median OS (mOS) was 4.4 months; however, mOS of the 17 ES patients was 6.9 months [208 days; range, 83-591 days; 95.0% confidence interval (CI), 5.6-10.3 months] and mOS of seven patients who did not complete one cycle was 1.8 months (54 days; range, 21-72; 95.0% CI, 1.2-2.2 months). Of the ES, 47.1% achieved stable disease and one partial response. Ten subjects in the ES survived over 6 months, the longest 19.7 months. During treatments, 82.0% of the ES maintained stable weight. CONCLUSIONS: E-EDV-D682/GC provided significant OS, minimal side effects, and weight stabilization in patients with advanced PDAC. Advanced PDAC can be safely treated with super-cytotoxic drugs via EnGeneIC Dream Vectors to overcome multidrug resistance.

Nanocell COVID-19 vaccine triggers a novel immune response pathway producing high-affinity antibodies which neutralize all variants of concern.

Most current anti-viral vaccines elicit a humoral and cellular immune response via the pathway of phagocytic cell mediated viral antigen presentation to B and T cell surface receptors. However, this pathway results in reduced ability to neutralize S-protein Receptor Binding Domains (RBDs) from several Variants of Concern (VOC) and the rapid waning of memory B cell response requiring vaccine reformulation to cover dominant VOC S-proteins and multiple boosters. Here we show for the first time in mice and humans, that a bacterially derived, non-living, nanocell (EDV; EnGeneIC Dream Vector) packaged with plasmid expressed SARS-CoV-2 S-protein and α-galactosyl ceramide adjuvant (EDV-COVID-αGC), stimulates an alternate pathway due to dendritic cells (DC) displaying both S-polypeptides and αGC thereby recruiting and activating iNKT cells with release of IFNγ. This triggers DC activation/maturation, activation of follicular helper T cells (TFH), cognate help to B cells with secretion of a cytokine milieu promoting B cell maturation, somatic hypermutation in germinal centers to result in high affinity antibodies. Surrogate virus neutralization tests show 90-100% neutralization of ancestral and early VOC in mice and human trial volunteers. EDV-COVID-αGC as a third dose booster neutralized Omicron BA. 4/5. Serum and PBMC analyses reveal long lasting S-specific memory B and T cells. In contrast, control EDVs lacking αGC, did not engage the iNKT/DC pathway resulting in antibody responses unable to neutralize all VOCs and had a reduced B cell memory. The vaccine is lyophilized, stored and transported at room temperature with a shelf-life of over a year.

Translational applications of microRNAs in cancer, and therapeutic implications.

The search for targeted novel therapies for cancer is ongoing. MicroRNAs (miRNAs) display a number of characteristics making them an attractive and realisable option. In this review, we explore these applications, ranging from diagnostics, prognostics, disease surveillance, to being a primary therapy or a tool to sensitise patients to treatment modalities such as chemotherapy and radiotherapy. We take a particular perspective towards miRNAs and their impact on rare cancers. Advancement in the delivery of miRNAs, from viral vectors and liposomal delivery to nanoparticle based, has led to a number of pre-clinical and clinical applications for microRNA cancer therapeutics. This is promising, especially in the setting of rare cancers.

Targeted Doxorubicin Delivery to Brain Tumors via Minicells: Proof of Principle Using Dogs with Spontaneously Occurring Tumors as a Model.

BACKGROUND: Cytotoxic chemotherapy can be very effective for the treatment of cancer but toxicity on normal tissues often limits patient tolerance and often causes long-term adverse effects. The objective of this study was to assist in the preclinical development of using modified, non-living bacterially-derived minicells to deliver the potent chemotherapeutic doxorubicin via epidermal growth factor receptor (EGFR) targeting. Specifically, this study sought to evaluate the safety and efficacy of EGFR targeted, doxorubicin loaded minicells (designated EGFRminicellsDox) to deliver doxorubicin to spontaneous brain tumors in 17 companion dogs; a comparative oncology model of human brain cancers. METHODOLOGY/PRINCIPLE FINDINGS: EGFRminicellsDox were administered weekly via intravenous injection to 17 dogs with late-stage brain cancers. Biodistribution was assessed using single-photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). Anti-tumor response was determined using MRI, and blood samples were subject to toxicology (hematology, biochemistry) and inflammatory marker analysis. Targeted, doxorubicin-loaded minicells rapidly localized to the core of brain tumors. Complete resolution or marked tumor regression (>90% reduction in tumor volume) were observed in 23.53% of the cohort, with lasting anti-tumor responses characterized by remission in three dogs for more than two years. The median overall survival was 264 days (range 49 to 973). No adverse clinical, hematological or biochemical effects were observed with repeated administration of EGFRminicellsDox (30 to 98 doses administered in 10 of the 17 dogs). CONCLUSIONS/SIGNIFICANCE: Targeted minicells loaded with doxorubicin were safely administered to dogs with late stage brain cancer and clinical activity was observed. These findings demonstrate the strong potential for clinical applications of targeted, doxorubicin-loaded minicells for the effective treatment of patients with brain cancer. On this basis, we have designed a Phase 1 clinical study of EGFR-targeted, doxorubicin-loaded minicells for effective treatment of human patients with recurrent glioblastoma.

MicroRNA-7 as a tumor suppressor and novel therapeutic for adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) has a poor prognosis with significant unmet clinical need due to late diagnosis, high rates of recurrence/metastasis and poor response to conventional treatment. Replacing tumor suppressor microRNAs (miRNAs) offer a novel therapy, however systemic delivery remains challenging. A number of miRNAs have been described to be under-expressed in ACC however it is not known if they form a part of ACC pathogenesis. Here we report that microRNA-7-5p (miR-7) reduces cell proliferation in vitro and induces G1 cell cycle arrest. Systemic miR-7 administration in a targeted, clinically safe delivery vesicle (EGFREDVTM nanocells) reduces ACC xenograft growth originating from both ACC cell lines and primary ACC cells. Mechanistically, miR-7 targets Raf-1 proto-oncogene serine/threonine kinase (RAF1) and mechanistic target of rapamycin (MTOR). Additionally, miR-7 therapy in vivo leads to inhibition of cyclin dependent kinase 1 (CDK1). In patient ACC samples, CDK1 is overexpressed and miR-7 expression inversely related. In summary, miR-7 inhibits multiple oncogenic pathways and reduces ACC growth when systemically delivered using EDVTM nanoparticles. This data is the first study in ACC investigating the possibility of miRNAs replacement as a novel therapy.

miR-193a-3p is a potential tumor suppressor in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an asbestos-induced cancer with poor prognosis that displays characteristic alterations in microRNA expression. Recently it was reported that the expression of a subset of microRNAs can distinguish between MPM and adenocarcinoma of the lung. However, the functional importance of these changes has yet to be investigated. We compared expression of miR-192, miR-193a-3p and the miR-200 family in normal pleura and MPM tumor specimens and found a statistically significant reduction in the levels of miR-193a-3p (3.1-fold) and miR-192 (2.8-fold) in MPM. Transfection of MPM cells with a miR-193a-3p mimic resulted in inhibition of growth and an induction of apoptosis and necrosis in vitro. The growth inhibitory effects of miR-193a-3p were associated with a decrease in MCL1 expression and were recapitulated by RNAi-mediated MCL1 silencing. Targeted delivery of miR-193a-3p mimic using EDV minicells inhibited MPM xenograft tumour growth, and was associated with increased apoptosis. In conclusion, miR-193a-3p appears to have importance in the biology of MPM and may represent a target for therapeutic intervention.

Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug.

The dose-limiting toxicity of chemotherapeutics, heterogeneity and drug resistance of cancer cells, and difficulties of targeted delivery to tumors all pose daunting challenges to effective cancer therapy. We report that small interfering RNA (siRNA) duplexes readily penetrate intact bacterially derived minicells previously shown to cause tumor stabilization and regression when packaged with chemotherapeutics. When targeted via antibodies to tumor-cell-surface receptors, minicells can specifically and sequentially deliver to tumor xenografts first siRNAs or short hairpin RNA (shRNA)-encoding plasmids to compromise drug resistance by knocking down a multidrug resistance protein. Subsequent administration of targeted minicells containing cytotoxic drugs eliminate formerly drug-resistant tumors. The two waves of treatment, involving minicells loaded with both types of payload, enable complete survival without toxicity in mice with tumor xenografts, while involving several thousandfold less drug, siRNA and antibody than needed for conventional systemic administration of cancer therapies.

Bacterially-derived nanocells for tumor-targeted delivery of chemotherapeutics and cell cycle inhibitors.

Chemotherapeutic drug therapy in cancer is seriously hampered by severe toxicity primarily due to indiscriminate drug distribution and consequent collateral damage to normal cells. Molecularly targeted drugs such as cell cycle inhibitors are being developed to achieve a higher degree of tumor cell specificity and reduce toxic side effects. Unfortunately, relative to the cytotoxics, many of the molecularly targeted drugs are less potent and the target protein is expressed only at certain stages of the cell cycle thus necessitating regimens like continuous infusion therapy to arrest a significant number of tumor cells in a heterogeneous tumor mass. Here we discuss targeted drug delivery nanovectors and a recently reported bacterially-derived 400 nm sized minicell that can be packaged with therapeutically significant concentrations of chemotherapeutic drugs, targeted to tumor cell surface receptors and effect intracellular drug delivery with highly significant anti-tumor effects in vivo. We also report that molecularly targeted drugs can also be packaged in minicells and targeted to tumor cells with highly significant tumor growth-inhibition and regression in mouse xenografts despite administration of minute amounts of drug. This targeted intracellular drug delivery may overcome many of the hurdles associated with the delivery of cytotoxic and molecularly targeted drugs.