RESUMO
To evaluate the status of a 7-month phase 3 study conducted to test the effect of intramuscular injections of VM202 (ENGENSIS), a plasmid DNA encoding human hepatocyte growth factor, into the calf muscles of chronic nonhealing diabetic foot ulcers with concomitant peripheral artery disease. The phase 3 study, originally aimed to recruit 300 subjects, was discontinued because of slow patient recruitment. An unprespecified interim analysis was performed for the 44 subjects enrolled to assess the status and determine the future direction. Statistical analyses were carried out for the Intent-to-Treat (ITT) population and separately for subjects with neuroischemic ulcers, using a t-test and Fisher's exact test. A logistic regression analysis was also conducted. VM202 was safe and potentially should have benefits. In the ITT population (N = 44), there was a positive trend toward closure in the VM202 group from 3 to 6 months but with no statistical significance. Levels of ulcer volume or area were found to be highly skewed between the placebo and VM202 groups. Forty subjects, excluding four outliers in both arms, showed significant wound-closing effects at month 6 (P = .0457). In 23 patients with neuroischemic ulcers, the percentage of subjects reaching complete ulcer closure was significantly higher in the VM202 group at months 3, 4, and 5 (P = .0391, .0391, and .0361). When two outliers were excluded, a significant difference was evident in months 3, 4, 5, and 6 (P = .03 for all points). A potentially clinically meaningful 0.15 increase in Ankle-Brachial Index was observed in the VM202 group at day 210 in the ITT population (P = .0776). Intramuscular injections of VM202 plasmid DNA to calf muscle may have promise in the treatment of chronic neuroischemic diabetic foot ulcers (DFUs). Given the safety profile and potential healing effects, continuing a larger DFU study is warranted with modifications of the current protocol and expansion of enrolling sites.
Assuntos
Diabetes Mellitus , Pé Diabético , Humanos , Pé Diabético/terapia , Pé Diabético/etiologia , DNA , Terapia Genética/efeitos adversos , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/efeitos adversos , Plasmídeos/genética , Plasmídeos/uso terapêutico , Isoformas de Proteínas/genética , Ensaios Clínicos Fase III como Assunto , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Efficient in vivo delivery of a CRISPR/Cas9 plasmid is of paramount importance for effective therapy. Here, we investigated the usability of Salmonella as a plasmid carrier for in vivo therapy against virus-induced cancer using Marek's disease virus (MDV) as a model for study in chickens. A green fluorescent protein-expressing CRISPR/Cas9 plasmid encoding the virulence gene pp38 was constructed against Marek's disease virus. Therapeutic plasmids were transformed into Salmonella carrying lon and sifA gene deletions. The animals in 5 groups were intraperitoneally inoculated with phosphate-buffered saline, vector control, or Salmonella before or after MDV infection, or left uninfected as a naïve control. Therapeutic effectiveness was evaluated by observing disease outcomes and the viral copy number in peripheral blood mononuclear cells. The efficacy of plasmid delivery by Salmonella was 13 ± 1.7% in the spleen and 8.0 ± 1.8% in the liver on the 6th day post-infection. The Salmonella-treated groups showed significant resistance to MDV infection. The maximum effect was observed in the group treated with Salmonella before MDV infection. None of the chickens fully recovered; however, the results suggested that timely delivery of Salmonella could be effective for in vivo CRISPR/Cas9-mediated genetic interference against highly pathogenic MDV. The use of Salmonella in CRISPR systems provides a simpler and more efficient platform for in vivo therapy with CRISPR than the use of conventional in vivo gene delivery methods and warrants further development.
Assuntos
Sistemas CRISPR-Cas , Galinhas , Herpesvirus Galináceo 2/fisiologia , Doença de Marek/prevenção & controle , Plasmídeos/uso terapêutico , Doenças das Aves Domésticas/prevenção & controle , Salmonella/fisiologia , Animais , Feminino , Leucócitos Mononucleares/virologia , Doença de Marek/patologia , Doença de Marek/virologia , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , Salmonella/virologiaRESUMO
Stargardt disease (STGD) is an autosomal recessive retinal disorder caused by a monogenic ABCA4 mutation. Currently, there is no effective therapy to cure Stargardt disease. The replacement of mutated ABCA4 with a functional gene remains an attractive strategy. In this study, we have developed a non-viral gene therapy using nanoparticles self-assembled by a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid. The nanoparticles mediated efficient intracellular gene transduction in wild-type (WT) and Abca4-/- mice. Specific ABCA4 expression in the outer segment of photoreceptors was achieved by incorporating a rhodopsin promoter into the plasmids. The ECO/pRHO-ABCA4 nanoparticles induced substantial and specific ABCA4 expression for at least 8 months, 35% reduction in A2E accumulation on average, and a delayed Stargardt disease progression for at least 6 months in Abca4-/- mice. ECO/plasmid nanoparticles constitute a promising non-viral gene therapy platform for Stargardt disease and other visual dystrophies.
Assuntos
Transportadores de Cassetes de Ligação de ATP/administração & dosagem , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Terapia Genética/métodos , Lipopeptídeos/administração & dosagem , Nanopartículas/química , Rodopsina/administração & dosagem , Doença de Stargardt/terapia , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Lipopeptídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Fotorreceptoras/metabolismo , Plasmídeos/genética , Plasmídeos/uso terapêutico , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Rodopsina/genética , Doença de Stargardt/genética , TransfecçãoRESUMO
The management of men with prostate cancer (PCa) with biochemical recurrence following local definitive therapy remains controversial. Early use of androgen deprivation therapy (ADT) leads to significant side effects. Developing an alternative, clinically effective, and well-tolerated therapy remains an unmet clinical need. INO-5150 is a synthetic DNA therapy that includes plasmids encoding for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA), and INO-9012 is a synthetic DNA plasmid encoding for interleukin-12 (IL-12). This phase 1/2, open-label, multi-center study enrolled men with PCa with rising PSA after surgery and/or radiation therapy. Patients were enrolled into one of four treatment arms: arm A, 2 mg of INO-5150; arm B, 8.5 mg of INO-5150; arm C, 2 mg of INO-5150 + 1 mg of INO-9012; and arm D, 8.5 mg of INO-5150 + 1 mg of INO-9012. Patients received study drug with electroporation on day 0 and on weeks 3, 12, and 24, and they were followed for up to 72 weeks. Sixty-two patients were enrolled. Treatment was well tolerated. 81% (50/62) of patients completed all visits. 85% (53/62) remained progression-free at 72 weeks. PSA doubling time (PSADT) was increased when assessed in patients with day 0 PSADT ≤12 months. Immunogenicity was observed in 76% (47/62) of patients by multiple assessments. Analysis indicated that CD38 and perforin co-positive CD8 T cell frequency correlated with attenuated PSA rise (p = 0.05, n = 50).
Assuntos
Terapia Genética/métodos , Imunidade , Imunoterapia/métodos , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/terapia , Antígeno Prostático Específico/imunologia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/terapia , Linfócitos T Citotóxicos/imunologia , Idoso , Idoso de 80 Anos ou mais , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Seguimentos , Glutamato Carboxipeptidase II/genética , Glutamato Carboxipeptidase II/imunologia , Humanos , Interleucina-12/genética , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/induzido quimicamente , Plasmídeos/genética , Plasmídeos/uso terapêutico , Intervalo Livre de Progressão , Antígeno Prostático Específico/sangue , Antígeno Prostático Específico/genética , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologiaRESUMO
BACKGROUND: Effective methods to deliver therapeutic genes to solid tumors and improve their bioavailability are the main challenges of current medical research on gene therapy. The development of efficient non-viral gene vector with tumor-targeting has very important application value in the field of cancer therapy. Proteolipid integrated with tumor-targeting potential of functional protein and excellent gene delivery performance has shown potential for targeted gene therapy. RESULTS: Herein, we prepared transferrin-modified liposomes (Tf-PL) for the targeted delivery of acetylcholinesterase (AChE) therapeutic gene to liver cancer. We found that the derived Tf-PL/AChE liposomes exhibited much higher transfection efficiency than the commercial product Lipo 2000 and shown premium targeting efficacy to liver cancer SMMC-7721 cells in vitro. In vivo, the Tf-PL/AChE could effectively target liver cancer, and significantly inhibit the growth of liver cancer xenografts grafted in nude mice by subcutaneous administration. CONCLUSIONS: This study proposed a transferrin-modified proteolipid-mediated gene delivery strategy for targeted liver cancer treatment, which has a promising potential for precise personalized cancer therapy.
Assuntos
Acetilcolinesterase/genética , Técnicas de Transferência de Genes , Lipossomos/química , Neoplasias Hepáticas/terapia , Plasmídeos/genética , Transferrina/química , Animais , Linhagem Celular Tumoral , Feminino , Terapia Genética , Humanos , Neoplasias Hepáticas/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , Plasmídeos/administração & dosagem , Plasmídeos/uso terapêutico , TransfecçãoRESUMO
BACKGROUND: The anti-angiogenic fusion protein RBDV-IgG1 Fc (RBDV), which comprises the receptor-binding domain of vascular endothelial growth factor-A (VEGF-A), has shown antitumour effects by reducing angiogenesis in vivo. This study used the cationic lipoplex lipo-PEG-PEI-complex (LPPC) to simultaneously encapsulate both the RBDV targeting protein and the RBDV plasmid (pRBDV) without covalent bonds to assess VEGFR targeting gene therapy in mice with melanoma in vivo. RESULTS: LPPC protected the therapeutic transgene from degradation by DNase, and the LPPC/RBDV complexes could specifically target VEGFR-positive B16-F10 cells both in vitro and in vivo. With or without RBDV protein-targeting direction, the pRBDV-expressing RBDV proteins were expressed and reached a maximal concentration on the 7th day in the sera after transfection in vivo and significantly elicited growth suppression against B16-F10 melanoma but not IgG1 control proteins. In particular, LPPC/pRBDV/RBDV treatment with the targeting molecules dramatically inhibited B16-F10 tumour growth in vivo to provide better therapeutic efficacy than the treatments with gene therapy with IgG1 protein targeting or administration of a protein drug with RBDV. CONCLUSIONS: The simultaneous combination of the LPPC complex with pRBDV gene therapy and RBDV protein targeting might be a potential tool to conveniently administer targeted gene therapy for cancer therapy.
Assuntos
Inibidores da Angiogênese/genética , Terapia Genética/métodos , Lipossomos/química , Melanoma Experimental/terapia , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Células 3T3 , Animais , Linhagem Celular Tumoral , Proliferação de Células , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Masculino , Melanoma Experimental/mortalidade , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/uso terapêutico , Domínios Proteicos/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Taxa de Sobrevida , Transplante Homólogo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Background Ventilator-induced lung injury (VILI) is the most common complication in the mechanical ventilation in clinic. The pathogenesis of VILI has not been well understood. The SRY related High Mobility Group box group-F family member 11(Sox11) is a protein associated with lung development. The focal adhesion kinase(FAK) is a cytoplasmic tyrosine kinase and is regulated by Sox11. The present study, therefore, was undertaken to explore the potential role of Sox11 and FAK in VILI. Methods High volume mechanical ventilation(HMV) was used to establish mouse VILI model under anesthesia. The lung injury was evaluated by analyzing the lung weight, bronchoalveolar lavage fluid, histopathological changes and apoptosis of the lung. The Sox11 and FAK expressions in the lung were investigated by real-time qPCR, western blot and immunohistochemistry analysis. Results HMV induced VILI simultaneously companied with decreased expressions of Sox11 and FAK in alveolar epithelial and interstitial cells either in gene and protein levels. Transfection of Sox11 plasmid significantly upregulated expressions of Sox11 and FAK in gene and protein levels in the lung and particularly effectively alleviated VILI. Furthermore, FAK antagonism by PF562271(FAK antagonist) blocked the alleviating effect of Sox11 plasmid transfection on the VILI. Conclusion The dysregulation in the Sox11 and FAK after HMV play an important role in the pathogenesis of VILI, and facilitating the activity of Sox11and FAK might be an effective target and potential option in the prevention and treatment of VILI in clinic.
Assuntos
Quinase 1 de Adesão Focal/genética , Fatores de Transcrição SOXC/genética , Lesão Pulmonar Induzida por Ventilação Mecânica/genética , Animais , Modelos Animais de Doenças , Regulação para Baixo , Terapia Genética , Masculino , Camundongos Endogâmicos C57BL , Plasmídeos/genética , Plasmídeos/uso terapêutico , Transfecção , Regulação para Cima , Lesão Pulmonar Induzida por Ventilação Mecânica/terapiaRESUMO
Fibrosis contributes to â¼45% of all deaths in industrialized nations, but no direct antifibrotic therapeutic interventions exist to date. Graphene-based nanomaterials exhibit excellent versatility in electronics, and emerging trends exploit their properties for biomedical applications, especially for drug and gene delivery. We designed constructs of graphene nanostars linked to PAMAM-G5 dendrimer for the selective targeting and delivery of a plasmid expressing the collagenase metalloproteinase 9 under the CD11b promoter into inflammatory macrophages in cirrhotic livers. Graphene nanostars preferentially accumulated in inflammatory macrophages M1 in less than 3 h in a manner unaffected by covalent linkage to dendrimers. Dendrimer-graphene nanostars efficiently delivered the plasmid encoding for metalloproteinase 9 into macrophages, allowing the synthesis and secretion of the metalloproteinase to digest adjacent collagen fibers. In turn, metalloproteinase 9 overexpression promoted the macrophage switch from inflammatory M1 to pro-regenerative M2 in 3 days. This targeted gene therapy reduced selectively and locally the presence of collagen fibers in fibrotic tracts where inflammatory macrophages accumulated in cirrhotic mice without affecting the activation state of hepatic stellate cells. Overall, this treatment significantly reduced hepatic injury and improved liver restoration in mice with liver cirrhosis treated for 10 days. Graphene-dendrimer nanostars targeted the macrophage overexpression of metalloproteinase 9, selectively reducing hepatic fibrosis, and might be a good treatment for diseases associated with fibrosis and inflammatory macrophage accumulation.
Assuntos
Dendrímeros/química , Grafite/química , Cirrose Hepática/terapia , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/genética , Nanopartículas/química , Plasmídeos/administração & dosagem , Animais , Técnicas de Transferência de Genes , Terapia Genética , Cirrose Hepática/genética , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/ultraestrutura , Plasmídeos/genética , Plasmídeos/uso terapêutico , Regulação para CimaRESUMO
CRISPR/Cas9 system is a powerful toolbox for gene editing. However, the low delivery efficiency is still a big hurdle impeding its applications. Herein, we report a strategy to deliver Cas9-sgPlk-1 plasmids (CP) by a multifunctional vehicle for tumor therapy. We condensed CPs on TAT peptide-modified Au nanoparticles (AuNPs/CP, ACP) via electrostatic interactions, and coated lipids (DOTAP, DOPE, cholesterol, PEG2000-DSPE) on the ACP to form lipid-encapsulated, AuNPs-condensed CP (LACP). LACP can enter tumor cells and release CP into the cytosol by laser-triggered thermo-effects of the AuNPs; the CP can enter nuclei by TAT guidance, enabling effective knock-outs of target gene (Plk-1) of tumor (melanoma) and inhibition of the tumor both inâ vitro and inâ vivo. This AuNPs-condensed, lipid-encapsulated, and laser-controlled delivery system provides a versatile method for high efficiency CRISPR/Cas9 delivery and targeted gene editing for treatment of a wide spectrum of diseases.
Assuntos
Proteína 9 Associada à CRISPR/genética , Ouro/química , Lipídeos/química , Melanoma Experimental/terapia , Nanopartículas Metálicas/química , Plasmídeos/uso terapêutico , Animais , Apoptose/efeitos da radiação , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Técnicas de Transferência de Genes , Glutationa/química , Humanos , Hipertermia Induzida , Lasers , Melanoma Experimental/patologia , Camundongos , Microscopia Confocal , Fragmentos de Peptídeos/química , Plasmídeos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Guia de Cinetoplastídeos/genética , Ressonância de Plasmônio de Superfície , Quinase 1 Polo-LikeRESUMO
Ultrasound-targeted microbubble destruction (UTMD) is a novel means of tissue-specific gene delivery. This approach systemically infuses transgenes precoupled to gas-filled lipid microbubbles that are burst within the microvasculature of target tissues via an ultrasound signal resulting in release of DNA and transfection of neighboring cells within the tissue. Previous work has shown that adenovirus containing cDNA of UCP-1, injected into the epididymal fat pads in mice, induced localized fat depletion, improving glucose tolerance, and decreasing food intake in obese diabetic mice. Our group recently demonstrated that gene therapy by UTMD achieved beta cell regeneration in streptozotocin (STZ)-treated mice and baboons. We hypothesized that gene therapy with BMP7/PRDM16/PPARGC1A in skeletal muscle (SKM) of obese Zucker diabetic fatty (fa/fa) rats using UTMD technology would produce a brown adipose tissue (BAT) phenotype with UCP-1 overexpression. This study was designed as a proof of concept (POC) project. Obese Zucker rats were administered plasmid cDNA contructs encoding a gene cocktail with BMP7/PRDM16/PPARGC1A incorporated within microbubbles and intravenously delivered into their left thigh. Controls received UTMD with plasmids driving a DsRed reporter gene. An ultrasound transducer was directed to the thigh to disrupt the microbubbles within the microcirculation. Blood samples were drawn at baseline, and after treatment to measure glucose, insulin, and free fatty acids levels. SKM was harvested for immunohistochemistry (IHC). Our IHC results showed a reliable pattern of effective UTMD-based gene delivery in enhancing SKM overexpression of the UCP-1 gene. This clearly indicates that our plasmid DNA construct encoding the gene combination of PRDM16, PPARGC1A, and BMP7 reprogrammed adult SKM tissue into brown adipose cells in vivo. Our pilot established POC showing that the administration of the gene cocktail to SKM in this rat model of genetic obesity using UTMD gene therapy, engineered a BAT phenotype with UCP-1 over-expression. © 2017 IUBMB Life, 69(9):745-755, 2017.
Assuntos
Reprogramação Celular/genética , Diabetes Mellitus Experimental/terapia , Técnicas de Transferência de Genes , Terapia Genética , Obesidade/terapia , Tecido Adiposo Marrom/metabolismo , Animais , Proteína Morfogenética Óssea 7/genética , Diferenciação Celular/genética , Diabetes Mellitus Experimental/genética , Modelos Animais de Doenças , Humanos , Microbolhas/uso terapêutico , Músculo Esquelético/metabolismo , Músculo Esquelético/transplante , Obesidade/genética , Obesidade/fisiopatologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Plasmídeos/genética , Plasmídeos/uso terapêutico , Ratos , Ratos Zucker , Fatores de Transcrição/genéticaRESUMO
PURPOSE: We have developed and evaluated novel peptide-targeted gemini surfactant-based lipoplexes designed for melanoma gene therapy. METHODS: Integrin receptor targeting peptide, cyclic-arginylglycylaspartic acid (cRGD), was either chemically coupled to a gemini surfactant backbone or physically co-formulated with lipoplexes. Several formulations and transfection techniques were developed. Transfection efficiency and cellular toxicity of the lipoplexes were evaluated in an in vitro human melanoma model. Physicochemical properties were examined using dynamic light scattering, zeta-potential, and small-angle X-ray scattering measurements. RESULTS: RGD-modified gemini surfactant based lipoplexes showed significant enhancement in gene transfection activity in A375 cell lines compared to the standard non-targeted formulation, especially when RGD was chemically conjugated to the gemini surfactant (RGD-G). The RGD had no effect on the cell toxicity profile of the lipoplex systems. Targeting specificity was confirmed by using an excess of free RGD and negative control peptide (RAD) and was demonstrated by using normal human epidermal keratinocytes. Physicochemical characterization showed that all nanoparticles were in the optimal size range for cellular uptake and there were no significant differences between RGD-modified and standard lipoplexes. CONCLUSIONS: These findings indicate the potential of RGD-modified gemini surfactant-based lipoplexes for use in melanoma gene therapy as an alternative to conventional chemotherapy.
Assuntos
DNA/administração & dosagem , Lipídeos/química , Melanoma/terapia , Peptídeos Cíclicos/química , Tensoativos/química , Transfecção/métodos , Linhagem Celular , Linhagem Celular Tumoral , DNA/genética , DNA/uso terapêutico , Sistemas de Liberação de Medicamentos , Terapia Genética , Humanos , Integrinas/genética , Integrinas/metabolismo , Queratinócitos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Peptídeos Cíclicos/metabolismo , Plasmídeos/administração & dosagem , Plasmídeos/genética , Plasmídeos/uso terapêutico , Tensoativos/metabolismoRESUMO
Nanoghosts derived from mesenchymal stem cells and retaining their unique surface-associated tumor-targeting capabilities were redesigned as a selective and safe universal nonviral gene-therapy platform. pDNA-loaded nanoghosts efficiently targeted and transfected diverse cancer cells, in vitro and in vivo, in subcutaneous and metastatic orthotopic tumor models, leading to no adverse effects. Nanoghosts loaded with pDNA encoding for a cancer-toxic gene inhibited the growth of metastatic orthotopic lung cancer and subcutaneous prostate cancer models and dramatically prolonged the animals' survival.
Assuntos
DNA/administração & dosagem , Técnicas de Transferência de Genes , Terapia Genética , Neoplasias Pulmonares/terapia , Células-Tronco Mesenquimais , Nanoestruturas , Neoplasias da Próstata/terapia , Animais , Linhagem Celular Tumoral , DNA/genética , DNA/uso terapêutico , Humanos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos C57BL , Nanoestruturas/administração & dosagem , Nanoestruturas/efeitos adversos , Nanoestruturas/ultraestrutura , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Metástase Neoplásica/terapia , Plasmídeos/administração & dosagem , Plasmídeos/genética , Plasmídeos/uso terapêutico , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologiaRESUMO
The increasing incidence of antibiotic-resistant bacterial infections is creating a global public health threat. Because conventional antibiotic drug discovery has failed to keep pace with the rise of resistance, a growing need exists to develop novel antibacterial methodologies. Replication-competent bacteriophages have been utilized in a limited fashion to treat bacterial infections. However, this approach can result in the release of harmful endotoxins, leading to untoward side effects. Here, we engineer bacterial phagemids to express antimicrobial peptides (AMPs) and protein toxins that disrupt intracellular processes, leading to rapid, nonlytic bacterial death. We show that this approach is highly modular, enabling one to readily alter the number and type of AMPs and toxins encoded by the phagemids. Furthermore, we demonstrate the effectiveness of engineered phagemids in an in vivo murine peritonitis infection model. This work shows that targeted, engineered phagemid therapy can serve as a viable, nonantibiotic means to treat bacterial infections, while avoiding the health issues inherent to lytic and replicative bacteriophage use.
Assuntos
Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/genética , Bacteriófagos/genética , Engenharia Genética , Peritonite/terapia , Plasmídeos/uso terapêutico , Toxinas Biológicas/genética , Animais , Escherichia coli/fisiologia , Infecções por Escherichia coli/complicações , Terapia Genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Peritonite/genética , Peritonite/microbiologia , Plasmídeos/genéticaRESUMO
Alzheimer disease involves genetic and non-genetic factors and hence it is rational to be treated with genetic and non-genetic therapeutic agents. Nigella sativa has multiple therapeutic properties including neuroregeneration. Nigella sativa oil (NSO) was encapsulated in PLGA nanoparticles and pDNA was loaded either by adsorption on chitosan-modified particles or encapsulation within PLGA nanoparticles. The particle size and zeta potential of NSO-pDNA-chitosan-PLGA nanoparticles were highly dependent on the medium and exhibited high burst release. Meanwhile, NSO-pDNA-PLGA nanoparticles were more consistent with lower burst release. The fabricated nanoparticles revealed the expected outcomes of both pDNA and NSO. The pDNA transfected N2a cell while the encapsulated NSO promoted neurite outgrowth that is crucial for neuroregeneration. Results from this study suggest that NSO could be added to the gene delivery carrier to enhance treatment benefits for Alzheimer disease.
Assuntos
Doença de Alzheimer/terapia , DNA/administração & dosagem , Portadores de Fármacos/química , Nanopartículas/química , Óleos de Plantas/administração & dosagem , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Animais , Linhagem Celular , DNA/uso terapêutico , Terapia Genética , Humanos , Ácido Láctico/química , Camundongos , Nanopartículas/ultraestrutura , Neurogênese/efeitos dos fármacos , Óleos de Plantas/uso terapêutico , Plasmídeos/administração & dosagem , Plasmídeos/uso terapêutico , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Transfecção/métodosRESUMO
The demand of a plasmid encoding human hepatocyte growth factor gene (pUDK-HGF) in large quantities at high purity and concentration has increased for gene therapy of critical limb ischemia (CLI) in clinical trials. In this article, we produced pUDK-HGF in compliance with current good manufacturing practices at gram scale. The process included a 50-L batch fermentation, continuous alkaline lysis, and integrated three-step chromatography on Sepharose 6 Fast Flow, PlasmidSelect Xtra, and Source 15Q. The production process has been scaled up to yield 4.24 ± 0.41 g of pharmaceutical pUDK-HGF from 1.0 kg bacterial cell paste and the overall yield reached range from 58.37 to 66.70%. The final pUDK-HGF product exhibited high purity with supercoiled percentage of > 95.8% and undetectable residual RNA, contaminated protein, and bacterial endotoxin. The phase I clinical study indicates that intramuscular injection of pUDK-HGF is safe, well tolerated, and may provide symptomatic relief to CLI patients. These results show that our manufacturing process of pUDK-HGF is efficient in producing pharmaceutical-grade plasmid DNA and is safe for clinical applications.
Assuntos
Terapia Genética , Fator de Crescimento de Hepatócito/genética , Isquemia/terapia , Plasmídeos/uso terapêutico , DNA/genética , DNA/uso terapêutico , Desenho de Equipamento , Escherichia coli/genética , Extremidades/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana , Humanos , Microbiologia Industrial/instrumentação , Microbiologia Industrial/métodos , Isquemia/genética , Plasmídeos/genéticaRESUMO
Nanoparticles are becoming promising carriers for gene delivery because of their high capacity in gene loading and low cell cytotoxicity. In this study, a chitosan-based nanoparticle encapsulated within a recombinant pcDNA3.1-dsNKG2D-IL-15 plasmid was generated. The fused dsNKG2D-IL-15 gene fragment consisted of double extracellular domains of NKG2D with IL-15 gene at downstream. The average diameter of the gene nanoparticles ranged from 200 nm to 400 nm, with mean zeta potential value of 53.8 ± 6.56 mV. The nanoparticles which were loaded with the dsNKG2D-IL-15 gene were uptaken by tumor cells with low cytotoxicity. Tumor cells pre-transfected by gene nanopartilces stimulated NK and T cells in vitro. Intramuscular injection of gene nanoparticles suppressed tumor growth and prolonged survival of tumor-bearing mice through activation of NK and CD8(+) T cells. Thus, chitosan-based nanoparticle delivery of dsNKG2D-IL-15 gene vaccine can be potentially used for tumor therapy.
Assuntos
Vacinas Anticâncer/administração & dosagem , Quitosana/química , Interleucina-15/genética , Melanoma/prevenção & controle , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Nanopartículas/química , Vacinas de DNA/administração & dosagem , Animais , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Imunidade , Melanoma/imunologia , Melanoma/patologia , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Plasmídeos/administração & dosagem , Plasmídeos/imunologia , Plasmídeos/uso terapêutico , Transfecção , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêuticoRESUMO
UNLABELLED: Recently GLP-1 was found to have cardioprotective effects independent of those attributable to tight glycemic control. METHODS AND RESULTS: We employed ultrasound targeted microbubble destruction (UTMD) to deliver piggybac transposon plasmids encoding the GLP-1 gene with a nuclear localizing signal to rat hearts with adriamycin cardiomyopathy. After a single UTMD treatment, overexpression of transgenic GLP-1 was found in nuclei of rat heart cells with evidence that transfected cardiac cells had undergone proliferation. UTMD-GLP-1 gene therapy restored LV mass, fractional shortening index, and LV posterior wall diameter to nearly normal. Nuclear overexpression of GLP-1 by inducing phosphorylation of FoxO1-S256 and translocation of FoxO1 from the nucleus to the cytoplasm significantly inactivated FoxO1 and activated the expression of cyclin D1 in nuclei of cardiac muscle cells. Reversal of adriamycin cardiomyopathy appeared to be mediated by dedifferentiation and proliferation of nuclear FoxO1-positive cardiac muscle cells with evidence of embryonic stem cell markers (OCT4, Nanog, SOX2 and c-kit), cardiac early differentiation markers (NKX2.5 and ISL-1) and cellular proliferation markers (BrdU and PHH3) after UTMD with GLP-1 gene therapy. CONCLUSIONS: Intranuclear myocardial delivery of the GLP-1gene can reverse established adriamycin cardiomyopathy by stimulating myocardial regeneration.
Assuntos
Cardiomiopatias/induzido quimicamente , Cardiomiopatias/terapia , Doxorrubicina , Peptídeo 1 Semelhante ao Glucagon/genética , Miocárdio/citologia , Miocárdio/patologia , Plasmídeos/uso terapêutico , Animais , Cardiomiopatias/genética , Cardiomiopatias/patologia , Diferenciação Celular , Proliferação de Células , Ciclina D1/genética , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Transferência de Genes/instrumentação , Terapia Genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Microbolhas , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas do Tecido Nervoso/metabolismo , Plasmídeos/genética , Ratos , Ultrassonografia/instrumentação , Regulação para CimaRESUMO
OBJECTIVE: The purpose of this phase II trial was to evaluate the toxicity and antitumor activity of EGEN-001 in platinum resistant recurrent ovarian cancer. METHODS: Eligible patients had weekly IP infusion of EGEN-001 at a dose of 24mg/m(2). Toxicity and antitumor activity were evaluated using CTCAE and RESIST criteria, respectively. Co-primary endpoints were tumor response and survival without progression (PFS) for at least 6months. Survival without progression before going onto a subsequent therapy (EFS) for at least six months was also considered. RESULTS: A total of 58 EGEN-001 cycles were administered to 20/22 enrolled patients (median 2cycles, range 1-9). The most frequently associated adverse events related specifically to EGEN-001 treatment were grade 1/2 fatigue, fever, chills, abdominal pain, nausea, vomiting, anemia, thrombocytopenia, and leukopenia. Three of 20 EGEN-001 treated patients evaluable for toxicity elected to withdraw from the study motivated in part by grade 1 treatment related toxicities. There were no patients with partial or complete response (0%; 90% CI 0-10.9%). Seven (35%) of 16 patients evaluable for response had stable disease, and 9 (45%) had progressive disease. Six (30%) patients had a PFS of greater than six months, although three had gone off study and onto other therapies before six months. The estimated six-month EFS was 15%. The median PFS and OS were 2.89 and 9.17months, respectively. CONCLUSION: EGEN-001 at the dose and schedule evaluated was associated with some but limited activity and was seemingly less tolerated in platinum resistant recurrent ovarian cancer patients.
Assuntos
Adenocarcinoma/terapia , Neoplasias das Tubas Uterinas/terapia , Terapia Genética/métodos , Interleucina-12/uso terapêutico , Recidiva Local de Neoplasia/terapia , Neoplasias Epiteliais e Glandulares/terapia , Neoplasias Ovarianas/terapia , Neoplasias Peritoneais/terapia , Plasmídeos/uso terapêutico , Adulto , Idoso , Carcinoma Endometrioide/terapia , Carcinoma Epitelial do Ovário , Colesterol/uso terapêutico , Cistadenocarcinoma Seroso/terapia , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Técnicas de Transferência de Genes , Humanos , Infusões Parenterais , Interleucina-12/genética , Pessoa de Meia-Idade , Polietilenoglicóis/uso terapêutico , Polietilenoimina/análogos & derivados , Polietilenoimina/uso terapêutico , Resultado do TratamentoRESUMO
Antimicrobial peptides have been widely recognized as potential candidates for treating tumor, especially for defending against multidrug-resistant cells. Previously, based on the structure of substance P, we have designed a novel class of hybrid antimicrobial peptide NS, which possesses potent antimicrobial activity against a broad spectrum of bacterial pathogens. In this study, we evaluated its cytotoxicity to tumor cells and studied the possible mechanism of action. We showed that NS could efficiently kill tumor cells by rapidly disrupting the tumor cell membrane and inhibiting the DNA synthesis. In addition, we also found that NS could efficiently deliver plasmids into cells and exhibit high transfection efficiency after the introduction of a stearyl moiety to its N-terminus, like many reported cell-penetrating peptides. Taken together, this study revealed the potential multiple functions of NS, providing fundamental support for further therapeutic application as potential antitumor agent.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Plasmídeos/metabolismo , Substância P/análogos & derivados , Neoplasias do Colo do Útero/tratamento farmacológico , Acilação , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Antineoplásicos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/ultraestrutura , Células COS , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Replicação do DNA/efeitos dos fármacos , Feminino , Terapia Genética/métodos , Glioblastoma/metabolismo , Glioblastoma/ultraestrutura , Humanos , Plasmídeos/uso terapêutico , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes/farmacologia , Substância P/genética , Substância P/metabolismo , Substância P/farmacologia , Transfecção/métodos , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/ultraestruturaRESUMO
OBJECTIVE: As angiogenic growth factors can stimulate the development of collateral arteries, a concept called therapeutic angiogenesis, we performed a phase I/IIa open-label clinical trial using intramuscular injection of naked plasmid DNA encoding hepatocyte growth factor (HGF). We reported long-term evaluation of 2 years after HGF gene therapy in 22 patients with severe peripheral arterial disease. METHODS AND RESULTS: Twenty-two patients with peripheral arterial disease or Buerger disease staged by Fontaine IIb (n=7), III (n=4), and IV (n=11) were treated with HGF plasmid, either 2 mg or 4 mg ×2. Increase in ankle-branchial pressure index >0.1 was observed in 11 of 14 patients (79 %) at 2 years after gene therapy and in 11 of the 17 patients (65%) at 2 months. Reduction in rest pain (>2 cm in visual analog scale) was observed in 9 of 9 patients (100%) at 2 years and in 8 of 13 (62%) patients at 2 months. At 2 years, 9 of 10 (90%) ischemic ulcers reduced by >25%, accompanied by a reduction in the size of ulcer. Severe complications and adverse effects caused by gene transfer were not detected in any patient throughout the period up to 2 years. CONCLUSIONS: Overall, the present study demonstrated long-term efficacy of HGF gene therapy up to 2 years. These findings may be cautiously interpreted to indicate that intramuscular injection of naked HGF plasmid is safe, feasible, and can achieve successful improvement of ischemic limbs as sole therapy.