RESUMEN
Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.
Asunto(s)
Edición Génica/métodos , Anemia de Células Falciformes/genética , Animales , Reacciones Falso Positivas , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones SCID , Técnicas de Sonda Molecular , Ácidos Nucleicos de Péptidos , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
Noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs), regulate target gene expression and can be used as tools for understanding biological processes and identifying new therapeutic targets. Currently, ncRNA molecules for research and therapeutic use are limited to ncRNA mimics made by chemical synthesis. We have recently established a high-yield and cost-effective method of producing bioengineered or biologic ncRNA agents (BERAs) through bacterial fermentation, which is based on a stable tRNA/pre-miR-34a carrier (~ 180 nt) that accommodates target small RNAs. Nevertheless, it remains a challenge to heterogeneously express longer ncRNAs (e.g., > 260 nt), and it is unknown if single BERA may carry multiple small RNAs. To address this issue, we hypothesized that an additional human pre-miR-34a could be attached to the tRNA/pre-miR-34a scaffold to offer a new tRNA/pre-miR-34a/pre-miR-34a carrier (~ 296 nt) for the accommodation of multiple small RNAs. We thus designed ten different combinatorial BERAs (CO-BERAs) that include different combinations of miRNAs, siRNAs, and antagomirs. Our data showed that all target CO-BERAs were successfully expressed in Escherichia coli at high levels, greater than 40% in total bacterial RNAs. Furthermore, recombinant CO-BERAs were purified to a high degree of homogeneity by fast protein liquid chromatography methods. In addition, CO-BERAs exhibited strong anti-proliferative activities against a variety of human non-small cell lung cancer cell lines. These results support the production of long ncRNA molecules carrying different warhead small RNAs for multi-targeting which may open avenues for developing new biologic RNAs as experimental, diagnostic, and therapeutic tools.
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Antagomirs/biosíntesis , Antagomirs/genética , Bioingeniería/métodos , ARN Largo no Codificante/biosíntesis , ARN Largo no Codificante/genética , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , Cromatografía Liquida , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , ARN Largo no Codificante/aislamiento & purificaciónRESUMEN
Noncoding RNAs (ncRNAs) produced in live cells may better reflect intracellular ncRNAs for research and therapy. Attempts were made to produce biologic ncRNAs, but at low yield or success rate. Here we first report a new ncRNA bioengineering technology using more stable ncRNA carrier (nCAR) containing a pre-miR-34a derivative identified by rational design and experimental validation. This approach offered a remarkable higher level expression (40%-80% of total RNAs) of recombinant ncRNAs in bacteria and gave an 80% success rate (33 of 42 ncRNAs). New FPLC and spin-column based methods were also developed for large- and small-scale purification of milligrams and micrograms of recombinant ncRNAs from half liter and milliliters of bacterial culture, respectively. We then used two bioengineered nCAR/miRNAs to demonstrate the selective release of target miRNAs into human cells, which were revealed to be Dicer dependent (miR-34a-5p) or independent (miR-124a-3p), and subsequent changes of miRNome and transcriptome profiles. miRNA enrichment analyses of altered transcriptome confirmed the specificity of nCAR/miRNAs in target gene regulation. Furthermore, nCAR assembled miR-34a-5p and miR-124-3p were active in suppressing human lung carcinoma cell proliferation through modulation of target gene expression (e.g., cMET and CDK6 for miR-34a-5p; STAT3 and ABCC4 for miR-124-3p). In addition, bioengineered miRNA molecules were effective in controlling metastatic lung xenograft progression, as demonstrated by live animal and ex vivo lung tissue bioluminescent imaging as well as histopathological examination. This novel ncRNA bioengineering platform can be easily adapted to produce various ncRNA molecules, and biologic ncRNAs hold the promise as new cancer therapeutics.
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Perfilación de la Expresión Génica , Ingeniería Genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , MicroARNs/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular/genética , Transformación Celular Neoplásica , Neoplasias Pulmonares/patología , RatonesRESUMEN
The nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor in the regulation of many oxidative enzymes and efflux transporters critical for oxidative stress and cellular defense against xenobiotics. NRF2 is dysregulated in patient osteosarcoma (OS) tissues and correlates with therapeutic outcomes. Nevertheless, research on the NRF2 regulatory pathways and its potential as a therapeutic target is limited to the use of synthetic small interfering RNA (siRNA) carrying extensive artificial modifications. Herein, we report successful high-level expression of recombinant siRNA against NRF2 in Escherichia coli using our newly established noncoding RNA bioengineering technology, which was purified to >99% homogeneity using an anion-exchange fast protein liquid chromatography method. Bioengineered NRF2-siRNA was able to significantly knock down NRF2 mRNA and protein levels in human OS 143B and MG63 cells, and subsequently suppressed the expression of NRF2-regulated oxidative enzymes [heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1] and elevated intracellular levels of reactive oxygen species. In addition, recombinant NRF2-siRNA was effective to sensitize both 143B and MG63 cells to doxorubicin, cisplatin, and sorafenib, which was associated with significant downregulation of NRF2-targeted ATP-binding cassette (ABC) efflux transporters (ABCC3, ABCC4, and ABCG2). These findings support that targeting NRF2 signaling pathways may improve the sensitivity of cancer cells to chemotherapy, and bioengineered siRNA molecules should be added to current tools for related research.
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Antineoplásicos/farmacología , Factor 2 Relacionado con NF-E2/genética , Osteosarcoma/tratamiento farmacológico , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Antineoplásicos/uso terapéutico , Bioingeniería/métodos , Línea Celular Tumoral , Regulación hacia Abajo , Resistencia a Antineoplásicos , Técnicas de Silenciamiento del Gen/métodos , Hemo-Oxigenasa 1/metabolismo , Humanos , Terapia Molecular Dirigida/métodos , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Osteosarcoma/patología , Estrés Oxidativo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/uso terapéutico , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Knowledge of drug absorption, distribution, metabolism, and excretion (ADME) or pharmacokinetics properties is essential for drug development and safe use of medicine. Varied or altered ADME may lead to a loss of efficacy or adverse drug effects. Understanding the causes of variations in drug disposition and response has proven critical for the practice of personalized or precision medicine. The rise of noncoding microRNA (miRNA) pharmacoepigenetics and pharmacoepigenomics has come with accumulating evidence supporting the role of miRNAs in the modulation of ADME gene expression and then drug disposition and response. In this article, we review the advances in miRNA pharmacoepigenetics including the mechanistic actions of miRNAs in the modulation of Phase I and II drug-metabolizing enzymes, efflux and uptake transporters, and xenobiotic receptors or transcription factors after briefly introducing the characteristics of miRNA-mediated posttranscriptional gene regulation. Consequently, miRNAs may have significant influence on drug disposition and response. Therefore, research on miRNA pharmacoepigenetics shall not only improve mechanistic understanding of variations in pharmacotherapy but also provide novel insights into developing more effective therapeutic strategies.
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Epigénesis Genética/genética , Regulación de la Expresión Génica/genética , Inactivación Metabólica/genética , MicroARNs/genética , Preparaciones Farmacéuticas/metabolismo , Procesamiento Postranscripcional del ARN/genética , Animales , Humanos , Factores de Transcripción/genéticaRESUMEN
Development of anticancer treatments based on microRNA (miRNA/miR) such as miR-34a replacement therapy is limited to the use of synthetic RNAs with artificial modifications. Herein, we present a new approach to a high-yield and large-scale biosynthesis, in Escherichia coli using transfer RNA (tRNA) scaffold, of chimeric miR-34a agent, which may act as a prodrug for anticancer therapy. The recombinant tRNA fusion pre-miR-34a (tRNA/mir-34a) was quickly purified to a high degree of homogeneity (>98%) using anion-exchange fast protein liquid chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-34a showed a favorable cellular stability while it was degradable by several ribonucleases. Deep sequencing and quantitative real-time polymerase chain reaction studies revealed that tRNA-carried pre-miR-34a was precisely processed to mature miR-34a within human carcinoma cells, and the same tRNA fragments were produced from tRNA/mir-34a and the control tRNA scaffold (tRNA/MSA). Consequently, tRNA/mir-34a inhibited the proliferation of various types of human carcinoma cells in a dose-dependent manner and to a much greater degree than the control tRNA/MSA, which was mechanistically attributable to the reduction of miR-34a target genes. Furthermore, tRNA/mir-34a significantly suppressed the growth of human non-small-cell lung cancer A549 and hepatocarcinoma HepG2 xenograft tumors in mice, compared with the same dose of tRNA/MSA. In addition, recombinant tRNA/mir-34a had no or minimal effect on blood chemistry and interleukin-6 level in mouse models, suggesting that recombinant RNAs were well tolerated. These findings provoke a conversation on producing biologic miRNAs to perform miRNA actions, and point toward a new direction in developing miRNA-based therapies.
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Antineoplásicos/síntesis química , Bioingeniería/métodos , Supervivencia Celular/efectos de los fármacos , MicroARNs/síntesis química , Profármacos/síntesis química , Animales , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Células Hep G2 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/aislamiento & purificación , MicroARNs/farmacología , Profármacos/aislamiento & purificación , Profármacos/farmacología , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Hematopoietic stem cell transplantation (HSCT) is a curative treatment for patients with many different blood and immune diseases; however, current treatment regimens contain non-specific chemotherapy and/or irradiation conditioning, which carry both short-term and long-term toxicities. The use of such agents may be particularly harmful for patients with Fanconi anemia (FA), who have genetic mutations resulting in deficiencies in DNA repair, leading to increased sensitivity to genotoxic agents. mAb-based conditioning has been proposed as an alternative conditioning strategy for HSCT that minimizes these toxicities by eliminating collateral tissue damage. Given the high need for improved treatments for FA patients, we aimed to evaluate the efficacy of different αCD117 mAb agents and immunosuppression on hematopoietic stem cell (HSC) depletion and explored their ability to safely establish therapeutic donor hematopoiesis post-HSCT in FA disease models. We evaluated the effects of different concentrations of αCD117 mAbs in vitro and in vivo on HSC growth and depletion. To further assess the efficacy of mAb-based conditioning, Fancd2-/- animals were treated with αCD117 mAb and combination agents with αCD47 mAb and antibody-drug-conjugates (ADCs) for syngeneic HSCT. Immunosuppression αCD4 mAb was added to all in vivo experiments due to a slightly mismatched background between the donor grafts and recipients. Immunosuppressant cocktails were also given to Fancd2-/- animals to evaluate the efficacy of mAb-based conditioning in the haploidentical setting. Statistical analyses were done using the unpaired t-test. We found that antagonistic αCD117 mAbs alone do not deplete host HSCs or enhance HSCT effectively in FA mouse models; however, the potency of αCD117 mAbs can be safely augmented through combination with αCD47 mAbs and with ADCs, both of which lead to profound HSC depletion and establishment of long-term donor engraftment post-syngeneic HSCT. This is the first time these approaches have been tested in parallel in any disease setting, with the greatest donor engraftment observed after CD117-ADC conditioning. Interestingly, our data also suggest that HSC-targeted conditioning is not necessary in HSCT for FA, as high donor HSC engraftment was observed with mAb-based immune suppression alone with immunologically matched and mismatched haploidentical grafts. These results demonstrate the safety and efficacy of several different non-genotoxic mAb-based conditioning strategies in the FA setting. In addition, they show that if sufficient immunosuppression is given to obtain initial donor HSC engraftment, turnover of a majority of the hematolymphoid system can result, likely owing to the survival advantage of wild-type HSCs over FA HSCs. Such non-toxic all-mAb-based conditioning strategies could be transformative for FA patients and those with other hematolymphoid diseases.
Asunto(s)
Anemia de Fanconi , Trasplante de Células Madre Hematopoyéticas , Animales , Ratones , Anemia de Fanconi/etiología , Anemia de Fanconi/terapia , Acondicionamiento Pretrasplante/métodos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Inmunosupresores , Terapia de Inmunosupresión/métodos , Anticuerpos MonoclonalesRESUMEN
Hematopoietic stem cells (HSCs) are a rare type of hematopoietic cell that can entirely reconstitute the blood and immune system after transplantation. Allogeneic HSC transplantation (HSCT) is used clinically as a curative therapy for a range of hematolymphoid diseases; however, it remains a high-risk therapy because of its potential side effects, including poor graft function and graft-versus-host disease (GVHD). Ex vivo HSC expansion has been suggested as an approach to improve hematopoietic reconstitution in low-cell dose grafts. Here, we demonstrate that the selectivity of polyvinyl alcohol (PVA)-based mouse HSC cultures can be improved using physioxic culture conditions. Single-cell transcriptomic analysis helped confirm the inhibition of lineage-committed progenitor cells in physioxic cultures. Long-term physioxic expansion also afforded culture-based ex vivo HSC selection from whole bone marrow, spleen, and embryonic tissues. Furthermore, we provide evidence that HSC-selective ex vivo cultures deplete GVHD-causing T cells and that this approach can be combined with genotoxic-free antibody-based conditioning HSCT approaches. Our results offer a simple approach to improve PVA-based HSC cultures and the underlying molecular phenotype, and highlight the potential translational implications of selective HSC expansion systems for allogeneic HSCT.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Animales , Ratones , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/metabolismo , Trasplante Homólogo , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/prevención & control , Enfermedad Injerto contra Huésped/metabolismoRESUMEN
Low dose non-toxic disulfide cross-linked micelle (DCM) encapsulated paclitaxel (PTX) was found to be highly efficacious as a radiosensitizer against oral cancer preclinical model. Intensity-modulated radiation therapy was locally administered for three consecutive days 24 h after intravascular injection of DCM-[PTX] at 5 mg/kg PTX. DCM-[PTX] NPs combined with conventional radiotherapy (2 Gy) resulted in a 1.7-fold improvement in therapeutic efficacy compared to conventional PTX plus radiotherapy. Interestingly, we found that radiotherapy can decrease tight junctions and increase the accumulation of DCM-[PTX] in tumor sites. Stereotactic body radiotherapy (SBRT) given at 6 Gy was used to further investigate the synergistic anti-tumor effect. Tumor tissues were collected to analyze the relationship between the time interval after SBRT and the biodistribution of the nanomaterials. Compared to combination DCM-[PTX] with conventional radiation dose, combination DCM-PTX with SBRT was found to be more efficacious in inhibiting tumor growth.
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Micelas , Neoplasias de la Boca , Línea Celular Tumoral , Disulfuros , Humanos , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/radioterapia , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Distribución TisularRESUMEN
Rationale: Noncoding RNAs (ncRNAs) such as microRNAs (miRs or miRNAs) play important roles in the control of cellular processes through posttranscriptional gene regulation. However, ncRNA research is limited to utilizing RNA agents synthesized in vitro. Recombinant RNAs produced and folded in living cells shall better recapitulate biologic RNAs. Methods: Herein, we developed a novel platform for in vivo fermentation production of humanized recombinant ncRNA molecules, namely hBERAs, carrying payload miRNAs or siRNAs. Target hBERAs were purified by anion exchange FPLC method. Functions of hBERA/miRNAs were investigated in human carcinoma cells and antitumor activities were determined in orthotopic osteosarcoma xenograft spontaneous lung metastasis mouse models. Results: Proper human tRNAs were identified to couple with optimal hsa-pre-miR-34a as new fully-humanized ncRNA carriers to accommodate warhead miRNAs or siRNAs. A group of 30 target hBERAs were all heterogeneously overexpressed (each accounting for >40% of total bacterial RNA), which facilitated large-scale production (8-31 mg of individual hBERAs from 1L bacterial culture). Model hBERA/miR-34a-5p and miR-124-3p were selectively processed to warhead miRNAs in human carcinoma cells to modulate target gene expression, enhance apoptosis and inhibit invasiveness. In addition, bioengineered miR-34a-5p and miR-124-3p agents both reduced orthotopic osteosarcoma xenograft tumor growth and spontaneous pulmonary metastases significantly. Conclusion: This novel ncRNA bioengineering technology and resulting recombinant ncRNAs are unique additions to conventional technologies and tools for basic research and drug development.
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MicroARNs/administración & dosificación , Neoplasias/genética , ARN de Transferencia/biosíntesis , ARN/biosíntesis , Animales , Bioingeniería , Línea Celular Tumoral , Proliferación Celular/genética , Fermentación , Expresión Génica , Terapia Genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Ratones , Terapia Molecular Dirigida , Trasplante de Neoplasias , Neoplasias/terapia , Osteosarcoma/genética , Osteosarcoma/secundario , Osteosarcoma/terapia , Interferencia de ARNRESUMEN
miR-127 is downregulated in breast cancer, where it has been shown to suppress the proliferation, migration, and invasion of breast cancer cells. In triple-negative breast cancer (TNBC), miR-127 downregulation correlates with decreased disease-free and overall patient survival. Tumor suppressor miRNAs may hold therapeutic promise but progress has been limited by several factors, including the lability and high cost of miRNA mimics. Here, we take a novel approach to produce a miR-127 prodrug (miR-127PD), which we demonstrate is processed to mature, functional miR-127-3p in TNBC tumor cells. miR-127PD decreased the viability and motility of TNBC cells, sensitized TNBC cells to chemotherapy, and restricted the TNBC stem cell population. Furthermore, systemic delivery of miR-127PD suppressed tumor growth of MDA-MB-231 and MDA-MB-468 TNBC cells and spontaneous metastasis of MDA-MB-231 cells. In addition, CERK, NANOS1, FOXO6, SOX11, SOX12, FASN, and SUSD2 were identified as novel, functionally important targets of miR-127. In conclusion, our study demonstrates that miR-127 functions as a tumor and metastasis suppressor in TNBC and that delivery of miR-127 may hold promise as a novel therapy. SIGNIFICANCE: Exogenous administration of miR-127, which is functionally activated in target cells, inhibits growth and spontaneous metastasis of triple-negative breast cancer.
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Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/prevención & control , MicroARNs/administración & dosificación , MicroARNs/genética , Profármacos/administración & dosificación , Neoplasias de la Mama Triple Negativas/prevención & control , Animales , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Metástasis Linfática , Ratones , Ratones Endogámicos NOD , Ratones SCID , Pronóstico , Factores de Transcripción SOXC/genética , Factores de Transcripción SOXC/metabolismo , Tasa de Supervivencia , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Drug-metabolizing enzymes, transporters, and nuclear receptors are essential for the absorption, distribution, metabolism, and excretion (ADME) of drugs and xenobiotics. MicroRNAs participate in the regulation of ADME gene expression via imperfect complementary Watson-Crick base pairings with target transcripts. We have previously reported that Cytochrome P450 3A4 (CYP3A4) and ATP-binding cassette sub-family G member 2 (ABCG2) are regulated by miR-27b-3p and miR-328-3p, respectively. Here we employed our newly established RNA bioengineering technology to produce bioengineered RNA agents (BERA), namely BERA/miR-27b-3p and BERA/miR-328-3p, via fermentation. When introduced into human cells, BERA/miR-27b-3p and BERA/miR-328-3p were selectively processed to target miRNAs and thus knock down CYP3A4 and ABCG2 mRNA and their protein levels, respectively, as compared to cells treated with vehicle or control RNA. Consequently, BERA/miR-27b-3p led to a lower midazolam 1'-hydroxylase activity, indicating the reduction of CYP3A4 activity. Likewise, BERA/miR-328-3p treatment elevated the intracellular accumulation of anticancer drug mitoxantrone, a classic substrate of ABCG2, hence sensitized the cells to chemotherapy. The results indicate that biologic miRNA agents made by RNA biotechnology may be applied to research on miRNA functions in the regulation of drug metabolism and disposition that could provide insights into the development of more effective therapies.
RESUMEN
Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related deaths, warranting better therapies. Restoration of tumor-suppressive microRNAs depleted in hepatocellular carcinoma represents a new therapeutic strategy. Herein, we sought to identify a potent microRNA (miRNA) agent that could alleviate HCC tumor burden and improve survival. Among a collection of bioengineered noncoding RNA molecules produced through bacterial fermentation, we identified let-7c agent as the most potent inhibitor of HCC cell viability. Bioengineered let-7c selectively modulated target gene expression (Lin-28 homolog B [LIN28B], AT-rich interactive domain-containing protein 3B [ARID3B], B cell lymphoma-extra large [Bcl-xl], and c-Myc) in HCC cells, and consequently induced apoptosis and inhibited tumorsphere growth. When formulated with liposomal-branched polyethylenimine polyplex, bioengineered let-7c exhibited serum stability up to 24 h. Furthermore, liposomal polyplex-formulated let-7c could effectively reduce tumor burden and progression in orthotopic HCC mouse models, while linear polyethyleneimine-formulated let-7c to a lower degree, as revealed by live animal and ex vivo tissue imaging studies. This was also supported by reduced serum α-fetoprotein and bilirubin levels in let-7c-treated mice. In addition, lipopolyplex-formulated let-7c extended overall survival of HCC tumor-bearing mice and elicited no or minimal immune responses in healthy immunocompetent mice and human peripheral blood mononuclear cells. These results demonstrate that bioengineered let-7c is a promising molecule for advanced HCC therapy, and liposomal polyplex is a superior modality for in vivo RNA delivery.
RESUMEN
Our recent studies have revealed that microRNA-1291 (miR-1291) is downregulated in pancreatic cancer (PC) specimens and restoration of miR-1291 inhibits tumorigenesis of PC cells. This study is to assess the efficacy and underlying mechanism of our bioengineered miR-1291 prodrug monotherapy and combined treatment with chemotherapy. AT-rich interacting domain protein 3B (ARID3B) was verified as a new target for miR-1291, and miR-1291 prodrug was processed to mature miR-1291 in PC cells which surprisingly upregulated ARID3B mRNA and protein levels. Co-administration of miR-1291 with gemcitabine plus nab-paclitaxel (Gem-nP) largely increased the levels of apoptosis, DNA damage and mitotic arrest in PC cells, compared to mono-drug treatment. Consequently, miR-1291 prodrug improved cell sensitivity to Gem-nP. Furthermore, systemic administration of in vivo-jetPEI-formulated miR-1291 prodrug suppressed tumor growth in both PANC-1 xenograft and PC patients derived xenograft (PDX) mouse models to comparable degrees as Gem-nP alone, while combination treatment reduced tumor growth more ubiquitously and to the greatest degrees (70-90%), compared to monotherapy. All treatments were well tolerated in mice. In conclusion, biologic miR-1291 prodrug has therapeutic potential as a monotherapy for PC, and a sensitizing agent to chemotherapy.
Asunto(s)
Albúminas/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de Unión al ADN/metabolismo , Desoxicitidina/análogos & derivados , MicroARNs/farmacología , Paclitaxel/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Profármacos/farmacología , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Chloride intracellular channel 4 (CLIC4) is a tumor suppressor implicated in processes including growth arrest, differentiation, and apoptosis. CLIC4 protein expression is diminished in the tumor parenchyma during progression in squamous cell carcinoma (SCC) and other neoplasms, but the underlying mechanisms have not been identified. Data from The Cancer Genome Atlas suggest this is not driven by genomic alterations. However, screening and functional assays identified miR-142-3p as a regulator of CLIC4. CLIC4 and miR-142-3p expression are inversely correlated in head and neck (HN) SCC and cervical SCC, particularly in advanced stage cancers. In situ localization revealed that stromal immune cells, not tumor cells, are the predominant source of miR-142-3p in HNSCC. Furthermore, HNSCC single-cell expression data demonstrated that CLIC4 is lower in tumor epithelial cells than in stromal fibroblasts and endothelial cells. Tumor-specific downregulation of CLIC4 was confirmed in an SCC xenograft model concurrent with immune cell infiltration and miR-142-3p upregulation. These findings provide the first evidence of CLIC4 regulation by miRNA. Furthermore, the distinct localization of CLIC4 and miR-142-3p within the HNSCC tumor milieu highlight the limitations of bulk tumor analysis and provide critical considerations for both future mechanistic studies and use of miR-142-3p as a HNSCC biomarker.
RESUMEN
Metastasis is the major cause for breast cancer related mortality. The combination of miRNA-based therapy and chemotherapy represents a promising approach against breast cancer lung metastasis. The goal of this study is to develop an improved therapy that co-delivers a novel bioengineered miRNA prodrug (tRNA-mir-34a) and doxorubicin (DOX) via a multifunctional nanomicellar carrier that is based on a conjugate of amphiphilic copolymer POEG-VBC backbone with creatine, a naturally occurring cationic molecule. Co-delivery of DOX leads to more effective processing of tRNA-mir-34a into mature miR-34a and down-regulation of target genes. DOX + tRNA-mir-34a/POEG-PCre exhibits potent synergistic anti-tumor and anti-metastasis activity in vitro and in vivo. Interestingly, the enhanced immune response contributes to the overall antitumor efficacy. POEG-PCre may represent a safe and effective delivery system for an optimal chemo-gene combination therapy.
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Antineoplásicos/uso terapéutico , Bioingeniería , Neoplasias de la Mama/patología , Creatina/química , Sistemas de Liberación de Medicamentos , Neoplasias Pulmonares/secundario , MicroARNs/administración & dosificación , Polímeros/química , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Endocitosis/efectos de los fármacos , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Ratones Endogámicos BALB C , Micelas , Nanopartículas/química , Polímeros/síntesis química , Distribución Tisular/efectos de los fármacosRESUMEN
Recently we have established a novel approach to produce bioengineered noncoding RNA agents (BERAs) in living cells that carry target RNAi molecules (e.g., siRNA and miRNA) and thus act as "prodrugs". Using GFP-siRNA-loaded BERA (BERA/GFP-siRNA) as a model molecule, this study was to define the in vitro and in vivo knockdown efficiency of BERAs delivered by liposome-polyethylenimine nanocomplex (lipopolyplex or LPP). Compared to in vivo-jetPEI® (IVJ-PEI) and polyplex formulations, LPP offered greater protection of BERA/GFP-siRNA against degradation by serum RNases. Particle sizes and zeta potentials of LPP nanocomplex remained stable over 28â¯days when stored at 4⯰C. Furthermore, comparable levels of BERA/GFP-siRNA were delivered by LPP and IVJ-PEI to luciferase/GFP-expressing human SK-Hep1-Luc-GFP or A549-Luc-GFP cells, which were selectively processed into target GFP-siRNA and subsequently knocked down GFP mRNA and protein levels. In addition, LPP-carried BERA/GFP-siRNA was successfully delivered into xenograft tumors and offered more consistent knockdown of tumoral GFP mRNA level in an orthotopic hepatocellular carcinoma (HCC) SK-Hep1-Luc-GFP xenograft mouse model, while IVJ-PEI formulation showed larger variation. These findings demonstrated that lipidation of polyplexes improved serum stability of biologic RNAi molecules, which was efficiently delivered to orthotopic HCC tissues to knock down target gene expression.
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Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Tratamiento con ARN de Interferencia/métodos , Animales , Bioingeniería , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen/métodos , Técnicas de Transferencia de Gen , Humanos , Liposomas , Neoplasias Hepáticas/genética , Luciferasas/genética , Masculino , Ratones , Ratones Desnudos , Nanopartículas/química , Polietileneimina/química , ARN Interferente Pequeño/metabolismo , Ribonucleasas/metabolismo , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Metastasis is a major cause of mortality for cancer patients and remains as the greatest challenge in cancer therapy. Driven by multiple factors, metastasis may not be controlled by the inhibition of single target. This study was aimed at assessing the hypothesis that drugs could be rationally combined to co-target critical DNA, RNA and protein molecules to achieve "saturation attack" against metastasis. Independent actions of the model drugs DNA-intercalating doxorubicin, RNA-interfering miR-34a and protein-inhibiting sorafenib on DNA replication, RNA translation and protein kinase signaling in highly metastatic, human osteosarcoma 143B cells were demonstrated by the increase of γH2A.X foci formation, reduction of c-MET expression and inhibition of Erk1/2 phosphorylation, respectively, and optimal effects were found for triple-drug combination. Consequently, triple-drug treatment showed a strong synergism in suppressing 143B cell proliferation and the greatest effects in reducing cell invasion. Compared to single- and dual-drug treatment, triple-drug therapy suppressed pulmonary metastases and orthotopic osteosarcoma progression to significantly greater degrees in orthotopic osteosarcoma xenograft/spontaneous metastases mouse models, while none showed significant toxicity. In addition, triple-drug therapy improved the overall survival to the greatest extent in experimental metastases mouse models. These findings demonstrate co-targeting of DNA, RNA and protein molecules as a novel therapeutic strategy for the treatment of metastasis.
Asunto(s)
Neoplasias Óseas/patología , Neoplasias Pulmonares/secundario , Terapia Molecular Dirigida , Osteosarcoma/patología , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Terapia Combinada , ADN , Modelos Animales de Enfermedad , Doxorrubicina/farmacología , Sinergismo Farmacológico , Femenino , Humanos , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/terapia , Ratones , MicroARNs/genética , Niacinamida/análogos & derivados , Niacinamida/farmacología , Osteosarcoma/genética , Osteosarcoma/metabolismo , Compuestos de Fenilurea/farmacología , ARN , Sorafenib , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The discovery of functional small noncoding RNAs (ncRNAs), such as microRNAs and small interfering RNAs, in the control of human cellular processes has opened new avenues to develop RNA-based therapies for various diseases including viral infections and cancers. However, studying ncRNA functions and developing RNA-based therapeutics relies on access to large quantities of affordable ncRNA agents. Currently, synthetic RNAs account for the major source of agents for RNA research and development, yet carry artificial modifications on the ribose ring and phosphate backbone in sharp contrast to posttranscriptional modifications present on the nucleobases or unmodified natural RNA molecules produced within cells. Therefore, large efforts have been made in recent years to develop recombinant RNA techniques to cost-effectively produce biological RNA agents that may better capture the structure, function, and safety properties of natural RNAs. In this article, we summarize and compare current in vitro and in vivo methods for the production of RNA agents including chemical synthesis, in vitro transcription, and bioengineering approaches. We highlight the latest recombinant RNA approaches using transfer RNA (tRNA), ribosomal RNA (rRNA), and optimal ncRNA scaffold (OnRS), and discuss the applications of bioengineered ncRNA agents (BERAs) that should facilitate RNA research and development.