RESUMO
Plants and other organisms, but not insects or vertebrates, express the auxiliary respiratory enzyme alternative oxidase (AOX) that bypasses mitochondrial respiratory complexes III and/or IV when impaired. Persistent expression of AOX from Ciona intestinalis in mammalian models has previously been shown to be effective in alleviating some metabolic stresses produced by respiratory chain inhibition while exacerbating others. This implies that chronic AOX expression may modify or disrupt metabolic signaling processes necessary to orchestrate adaptive remodeling, suggesting that its potential therapeutic use may be confined to acute pathologies, where a single course of treatment would suffice. One possible route for administering AOX transiently is AOX-encoding nucleic acid constructs. Here we demonstrate that AOX-encoding chemically-modified RNA (cmRNA), sequence-optimized for expression in mammalian cells, was able to support AOX expression in immortalized mouse embryonic fibroblasts (iMEFs), human lung carcinoma cells (A549) and primary mouse pulmonary arterial smooth muscle cells (PASMCs). AOX protein was detectable as early as 3 h after transfection, had a half-life of ~4 days and was catalytically active, thus supporting respiration and protecting against respiratory inhibition. Our data demonstrate that AOX-encoding cmRNA optimized for use in mammalian cells represents a viable route to investigate and possibly treat mitochondrial respiratory disorders.
Assuntos
Mitocôndrias , RNA , Animais , Humanos , Camundongos , Fibroblastos/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , RNA/metabolismo , Células A549 , TransfecçãoRESUMO
Extensive research in the past decade has brought mRNA closer to the clinical realization of its therapeutic potential. One common structural feature for all cellular messenger RNAs is a poly(A) tail, which can either be brought in cotranscriptionally via the DNA template (plasmid- or PCR-based) or added to the mRNA in a post-transcriptional enzymatic process. Plasmids containing poly(A) regions recombine in E. coli, resulting in extensive shortening of the poly(A) tail. Using a segmented poly(A) approach, we could significantly reduce recombination of plasmids in E. coli without any negative effect on mRNA half-life and protein expression. This effect was independent of the coding sequence. A segmented poly(A) tail is characterized in that it consists of at least two A-containing elements, each defined as a nucleotide sequence consisting of 40-60 adenosines, separated by a spacer element of different length. Furthermore, reducing the spacer length between the poly(A) segments resulted in higher translation efficiencies compared to homogeneous poly(A) tail and reduced recombination (depending upon the choice of spacer nucleotide). Our results demonstrate the superior potential of segmented poly(A) tails compared to the conventionally used homogeneous poly(A) tails with respect to recombination of the plasmids and the resulting mRNA performance (half-life and translational efficiency).
Assuntos
Engenharia Genética/métodos , Plasmídeos/química , Poli A/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Células A549 , Animais , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Meia-Vida , Humanos , Plasmídeos/metabolismo , Poli A/metabolismo , RNA Mensageiro/metabolismo , Recombinação Genética , TransfecçãoRESUMO
Osteointegration is one of the most important factors for implant success. Several biomolecules have been used as part of drug delivery systems to improve implant integration into the surrounding bone tissue. Chemically modified mRNA (cmRNA) is a new form of therapeutic that has been used to induce bone healing. Combined with biomaterials, cmRNA can be used to develop transcript-activated matrices for local protein production with osteoinductive potential. In this study, we aimed to utilize this technology to create bone morphogenetic protein 2 (BMP2) transcript-activated coatings for titanium (Ti) implants. Therefore, different coating methodologies as well as cmRNA incorporation strategies were evaluated. Three different biocompatible biomaterials were used for the coating of Ti, namely, poly-d,l-lactic acid (PDLLA), fibrin, and fibrinogen. cmRNA-coated Ti disks were assayed for transfection efficiency, cmRNA release, cell viability and proliferation, and osteogenic activity in vitro. We found that cmRNA release was significantly delayed in Ti surfaces previously coated with biomaterials. Consequently, the transfection efficiency was greatly improved. PDLLA coating improved the transfection efficiency in a concentration-dependent manner. Lower PDLLA concentration used for the coating of Ti resulted in higher transfection efficiency. Fibrin and fibrinogen coatings showed even higher transfection efficiencies compared to all PDLLA concentrations. In those disks, not only the expression was up to 24-fold higher but also the peak of maximal expression was delayed from 24 h to 5 days, and the duration of expression was also extended until 7 days post-transfection. For fibrin, higher transfection efficiencies were obtained in the coatings with the lowest thrombin amounts. Accordingly, fibrinogen coatings gave the best results in terms of cmRNA transfection. All biomaterial-coated Ti surfaces showed improved cell viability and proliferation, though this was more noticeable in the fibrinogen-coated disks. The latter was also the only coating to support significant amounts of BMP2 produced by C2C12 cells in vitro. Osteogenesis was confirmed using BMP2 cmRNA fibrinogen-coated Ti disks, and it was dependent of the cmRNA amount present. Alkaline phosphatase (ALP) activity of C2C12 increased when using fibrinogen coatings containing 250 ng of cmRNA or more. Similarly, mineralization was also observed that increased with increasing cmRNA concentration. Overall, our results support fibrinogen as an optimal material to deliver cmRNA from titanium-coated surfaces.
Assuntos
Materiais Revestidos Biocompatíveis/química , Osteogênese/efeitos dos fármacos , Titânio/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Proteína Morfogenética Óssea 2/metabolismo , Osso e Ossos/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fibrinogênio/metabolismo , Camundongos , Células NIH 3T3 , Poliésteres/química , RNA Mensageiro/metabolismo , Propriedades de Superfície/efeitos dos fármacos , Transfecção/métodosRESUMO
Promising improvements in the field of transcript therapeutics have clearly enhanced the potential of mRNA as a new pillar for protein replacement therapies. Synthetic mRNAs are engineered to replace mutated mRNAs and to be immunologically inconspicuous and highly stable while maximizing protein expression. Approaches to deliver mRNA into the cellular cytoplasm safely and efficiently have been further developed so that two mRNA-based approaches replacing vascular endothelial growth factor (VEGF) and cystic fibrosis transmembrane conductance regulator (CFTR) have now made it into clinical trials. These studies bring mRNA therapeutics for protein replacement therapy closer to clinical realization. Herein, we provide an overview of preclinical and clinical developments of mRNA therapeutics for liver diseases.
Assuntos
Sistemas de Liberação de Medicamentos , Hepatopatias/terapia , RNA Mensageiro/genética , RNA Mensageiro/uso terapêutico , Animais , DNA/genética , DNA/uso terapêutico , Terapia de Reposição de Enzimas/métodos , Humanos , Lipídeos/química , Camundongos , Nanopartículas/química , Polímeros/químicaRESUMO
Hypoxia promotes vascularization by stabilization and activation of the hypoxia inducible factor 1α (HIF-1α), which constitutes a target for angiogenic gene therapy. However, gene therapy is hampered by low gene delivery efficiency and non-specific side effects. Here, we developed a gene transfer technique based on magnetic targeting of magnetic nanoparticle-lentivirus (MNP-LV) complexes allowing site-directed gene delivery to individual wounds in the dorsal skin of mice. Using this technique, we were able to control HIF-1α dependent wound healing angiogenesis in vivo via site-specific modulation of the tyrosine phosphatase activity of SHP-2. We thus uncover a novel physiological role of SHP-2 in protecting HIF-1α from proteasomal degradation via a Src kinase dependent mechanism, resulting in HIF-1α DNA-binding and transcriptional activity in vitro and in vivo. Excitingly, using targeting of MNP-LV complexes, we achieved simultaneous expression of constitutively active as well as inactive SHP-2 mutant proteins in separate wounds in vivo and hereby specifically and locally controlled HIF-1α activity as well as the angiogenic wound healing response in vivo. Therefore, magnetically targeted lentiviral induced modulation of SHP-2 activity may be an attractive approach for controlling patho-physiological conditions relying on hypoxic vessel growth at specific sites.
Assuntos
Portadores de Fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Nanopartículas de Magnetita/administração & dosagem , Neovascularização Fisiológica , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Cicatrização/genética , Animais , Linhagem Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Nanopartículas de Magnetita/química , Camundongos , Terapia de Alvo Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteólise , Pele/lesões , Pele/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Recently, chemically modified mRNA (cmRNA) therapeutics have been the subject of extensive application-oriented research in both academia and industry as a safer alternative for gene and recombinant protein therapies. However, the lack of an efficient delivery system hinders widespread application. Here we used â¼100-nm lipoplexes and magnetic lipoplexes that can protect cmRNA from RNases and efficiently deliver it into muscle and fat tissues as well as to the endothelium of the carotid artery. Establishing magnetofection for ex vivo cmRNA delivery for the first time, we suggest this method for potential enhanced and targeted delivery of cmRNA. This study introduces optimal cmRNA complexes with high ex vivo efficiency as good candidates for further in vivo cmRNA delivery.
Assuntos
Lipídeos/química , Magnetismo/métodos , Nanopartículas de Magnetita/química , RNA Mensageiro/administração & dosagem , RNA Mensageiro/química , Transfecção/métodos , Tecido Adiposo/metabolismo , Animais , Células Endoteliais/metabolismo , Lipossomos/química , Camundongos , Músculos/metabolismo , Células NIH 3T3 , RNA Mensageiro/genética , Ovinos , SuínosRESUMO
The development of chemically modified mRNA holds great promise as a new class of biologic therapeutics. However, the intracellular delivery and endosomal escape of mRNA encapsulated in nanoparticles has not been systematically investigated. Here, we synthesized a diverse set of cationic polymers and lipids from a series of oligoalkylamines and subsequently characterized their mRNA delivery capability. Notably, a structure with an alternating alkyl chain length between amines showed the highest transfection efficiency, which was linked to a high buffering capacity in a narrow range of pHâ 6.2 to 6.5. Variation in only one methylene group resulted in enhanced mRNA delivery to both the murine liver as well as porcine lungs after systemic or aerosol administration, respectively. These findings reveal a novel fundamental structure-activity relationship for the delivery of mRNA that is independent of the class of mRNA carrier and define a promising new path of exploration in the field of mRNA therapeutics.
Assuntos
Aminas/química , Lipídeos/química , Polímeros/química , RNA Mensageiro/genética , Animais , Cátions/química , Camundongos , Células NIH 3T3 , Relação Estrutura-Atividade , SuínosRESUMO
PURPOSE: To explore the potential of magnetofection in delivering pDNA to primary mouse embryonic fibroblasts (PMEFs) and porcine fetal fibroblasts (PFFs) and investigate an effect of magnetic cell labeling on transfection efficacy. METHODS: The formulation and a dose of the magnetic vector were optimized. The efficacy of the procedure was quantified by vector internalization, transgene expression and cell iron loading upon specific labeling with Ab-conjugated magnetic beads or non-specific labeling with MNPs. RESULTS: Up to sixty percent of PMEF and PFF cells were transfected at low pDNA doses of 4-16 pg pDNA/cell. Specific labeling of the PMEFs with MNPs, resulted in a 3- and 2-fold increase in pDNA internalization upon magnetofection and lipofection, respectively, that yielded a 2-4-fold increase in percent of transgene-expressing cells. Non-specific cell labeling had no effect on the efficacy of the reporter expression, despite the acquisition of similar magnetic moments per cell. In PFFs, specific magnetic labeling of the cell surface receptors inhibited internalization and transfection efficacy. CONCLUSIONS: Magnetic labeling of cell-surface receptors combined with the application of an inhomogenous magnetic field (nanomagnetic activation) can affect the receptor-mediated internalization of delivery vectors and be used to control nucleic acid delivery to cells.
Assuntos
DNA/administração & dosagem , Portadores de Fármacos/administração & dosagem , Fibroblastos/metabolismo , Campos Magnéticos , Nanopartículas de Magnetita/administração & dosagem , Transfecção/métodos , Animais , Células Cultivadas , DNA/genética , Portadores de Fármacos/química , Nanopartículas de Magnetita/química , Camundongos , Microscopia Eletrônica de Transmissão , Plasmídeos , Cultura Primária de Células , Coloração e Rotulagem , Propriedades de Superfície , SuínosRESUMO
BACKGROUND: Intrauterine growth restriction (IUGR) is intimately linked with postnatal catch-up growth, leading to impaired lung structure and function. However, the impact of catch-up growth induced by early postnatal hyperalimentation (HA) on the lung has not been addressed to date. OBJECTIVE: The aim of this study was to investigate whether prevention of HA subsequent to IUGR protects the lung from 1) deregulation of the transforming growth factor-ß(TGF-ß)/bone morphogenetic protein (BMP) pathway, 2) activation of interleukin (IL)-6 signaling, and 3) profibrotic processes. METHODS: IUGR was induced in Wistar rats by isocaloric protein restriction during gestation by feeding a control (Co) or a low-protein diet with 17% or 8% casein, respectively. On postnatal day 1 (P1), litters from both groups were randomly reduced to 6 pups per dam to induce HA or adjusted to 10 pups and fed with standard diet: Co, Co with HA (Co-HA), IUGR, and IUGR with HA (IUGR-HA). RESULTS: Birth weights in rats after IUGR were lower than in Co rats (P < 0.05). HA during lactation led to accelerated body weight gain from P1 to P23 (Co vs. Co-HA, IUGR vs. IUGR-HA; P < 0.05). At P70, prevention of HA after IUGR protected against the following: 1) activation of both TGF-ß [phosphorylated SMAD (pSMAD) 2; plasminogen activator inhibitor 1 (Pai1)] and BMP signaling [pSMAD1; inhibitor of differentiation (Id1)] compared with Co (P < 0.05) and Co or IUGR (P < 0.05) rats, respectively; 2) greater mRNA expression of interleukin (Il) 6 and Il13 (P < 0.05) as well as activation of signal transducer and activator of transcription 3 (STAT3) signaling (P < 0.05) after IUGR-HA; and 3) greater gene expression of collagen Iα1 and osteopontin (P < 0.05) and increased deposition of bronchial subepithelial connective tissue in IUGR-HA compared with Co and IUGR rats. Moreover, HA had a significant additive effect (P < 0.05) on the increased enhanced pause (indicator of airway resistance) in the IUGR group (P < 0.05) at P70. CONCLUSIONS: This study demonstrates a dual mechanism in IUGR-associated lung disease that is 1) IUGR-dependent and 2) HA-mediated and thereby offers new avenues to develop innovative preventive strategies for perinatal programming of adult lung diseases.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Interleucina-6/metabolismo , Pulmão/crescimento & desenvolvimento , Hipernutrição/prevenção & controle , Fator de Crescimento Transformador beta/metabolismo , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Proteínas Morfogenéticas Ósseas/genética , Dieta com Restrição de Proteínas , Feminino , Retardo do Crescimento Fetal/terapia , Regulação da Expressão Gênica , Interleucina-6/genética , Lactação , Pulmão/patologia , Pneumopatias/prevenção & controle , Masculino , Hipernutrição/patologia , Ratos , Ratos Wistar , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Aumento de Peso/efeitos dos fármacosRESUMO
Research applications and cell therapies involving genetically modified cells require reliable, standardized, and cost-effective methods for cell manipulation. We report a novel nanomagnetic method for integrated cell separation and gene delivery. Gene vectors associated with magnetic nanoparticles are used to transfect/transduce target cells while being passaged and separated through a high gradient magnetic field cell separation column. The integrated method yields excellent target cell purity and recovery. Nonviral and lentiviral magselectofection is efficient and highly specific for the target cell population as demonstrated with a K562/Jurkat T-cell mixture. Both mouse and human enriched hematopoietic stem cell pools were effectively transduced by lentiviral magselectofection, which did not affect the hematopoietic progenitor cell number determined by in vitro colony assays. Highly effective reconstitution of T and B lymphocytes was achieved by magselectofected murine wild-type lineage-negative Sca-1(+) cells transplanted into Il2rg(-/-) mice, stably expressing GFP in erythroid, myeloid, T-, and B-cell lineages. Furthermore, nonviral, lentiviral, and adenoviral magselectofection yielded high transfection/transduction efficiency in human umbilical cord mesenchymal stem cells and was fully compatible with their differentiation potential. Upscaling to a clinically approved automated cell separation device was feasible. Hence, once optimized, validated, and approved, the method may greatly facilitate the generation of genetically engineered cells for cell therapies.
Assuntos
Separação Celular/métodos , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Células-Tronco Hematopoéticas/citologia , Células-Tronco Mesenquimais/citologia , Animais , Antígenos Ly/genética , Vetores Genéticos/química , Células-Tronco Hematopoéticas/metabolismo , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Células Jurkat , Células K562 , Magnetismo , Proteínas de Membrana/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Nanopartículas/química , TransfecçãoRESUMO
BACKGROUND: Intrauterine growth restriction (IUGR) leads to low nephron number and higher incidence of renal disease. We hypothesized that IUGR induces early podocyte alterations based on a dysregulation of Wilms' tumour suppressor gene 1 (WT1), a key player of nephrogenesis and mediator of podocyte integrity. METHODS: IUGR was induced in rats by maternal protein restriction during pregnancy. Kidneys were harvested from male offspring at Days 1 and 70 of life. qRT-PCR, immunohistochemistry and electron microscopy were performed in renal tissue. Albuminuria was assessed by enzyme-linked immunosorbent assay. RESULTS: At Day 70 of life, higher albuminuria and overt alterations of podocyte ultrastructure were detected in IUGR animals in spite of normal blood pressure. Moreover, we found increased glomerular immunoreactivity and expression of desmin, while synaptopodin and nephrin were decreased. Glomerular immunoreactivity and expression of WT1 were increased in IUGR animals at this time point with an altered expressional ratio of WT1 +KTS and -KTS isoforms. These changes of WT1 expression were already present at the time of birth. CONCLUSIONS: IUGR results in early podocyte damage possibly due to a dysregulation of WT1. We suggest that an imbalance of WT1 isoforms to the disadvantage of -KTS affects nephrogenesis in IUGR rats and that persistent dysregulation of WT1 results in a reduced ability to maintain podocyte integrity, rendering IUGR rats more susceptible for renal disease.
Assuntos
Retardo do Crescimento Fetal/patologia , Regulação da Expressão Gênica , Glomérulos Renais/patologia , Néfrons/patologia , Podócitos/patologia , Proteínas WT1/genética , Albuminúria , Animais , Biomarcadores/análise , Determinação da Pressão Arterial , Desmina/genética , Desmina/metabolismo , Feminino , Retardo do Crescimento Fetal/metabolismo , Técnicas Imunoenzimáticas , Glomérulos Renais/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Néfrons/metabolismo , Podócitos/metabolismo , Gravidez , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas WT1/metabolismoRESUMO
Nucleic acids have clear clinical potential for gene therapy. Plasmid DNA (pDNA) was the first nucleic acid to be pursued as a therapeutic molecule. Recently, mRNA came into play as it offers improved safety and affordability. In this study, we investigated the uptake mechanisms and efficiencies of genetic material by cells. We focused on three main variables (1) the nucleic acid (pDNA, or chemically modified mRNA), (2) the delivery vector (Lipofectamine 3000 or 3DFect), and (3) human primary cells (mesenchymal stem cells, dermal fibroblasts, and osteoblasts). In addition, transfections were studied in a 3D environment using electrospun scaffolds. Cellular internalization and intracellular trafficking were assessed by using enhancers or inhibitors of endocytosis and endosomal escape. The polymeric vector TransIT-X2 was included for comparison purposes. While lipoplexes utilized several entry routes, uptake via caveolae served as the main route for gene delivery. pDNA yielded higher expression levels in fast-dividing fibroblasts, whereas, in slow-dividing osteoblasts, cmRNA was responsible for high protein production. In the case of mesenchymal stem cells, which presented an intermediate doubling time, the combination vector/nucleic acid seemed more relevant than the nucleic acid per se. In all cases, protein expression was higher when the cells were seeded on 3D scaffolds.
RESUMO
The threat of spillovers of coronaviruses associated with the severe acute respiratory syndrome (SARS) from animals to humans necessitates vaccines that offer broader protection from sarbecoviruses. By leveraging a viral-genome-informed computational method for selecting immune-optimized and structurally engineered antigens, here we show that a single antigen based on the receptor binding domain of the spike protein of sarbecoviruses elicits broad humoral responses against SARS-CoV-1, SARS-CoV-2, WIV16 and RaTG13 in mice, rabbits and guinea pigs. When administered as a DNA immunogen or by a vector based on a modified vaccinia virus Ankara, the optimized antigen induced vaccine protection from the Delta variant of SARS-CoV-2 in mice genetically engineered to express angiotensin-converting enzyme 2 and primed by a viral-vector vaccine (AZD1222) against SARS-CoV-2. A vaccine formulation incorporating mRNA coding for the optimized antigen further validated its broad immunogenicity. Vaccines that elicit broad immune responses across subgroups of coronaviruses may counteract the threat of zoonotic spillovers of betacoronaviruses.
RESUMO
The application of scaffolds in bone tissue engineering often comes along with side effects such as poor integrity, low regeneration rates of bone tissue with inadequate functionality, and, in case of non-degradable implants, the necessity of a second removal surgery after therapy. In this study, we coated a bioresorbable FDA-approved poly-(ε-caprolactone)-scaffold for bone regeneration with a poly-(D,L-lactide) layer containing copolymer-protected gene vectors to locally provide bone morphogenetic protein-2 (BMP-2). Results show that the presence of such gene vectors did not affect the distribution and attachment of seeded cells on gene-activated surfaces. BMP-2 was released into cell culture supernatants and furthermore detected in homogenised scaffolds. Increased amounts of osteoblastic markers, such as osteocalcin, osteopontin and the activity of alkaline phosphatase, in gene-activated scaffolds in vitro suggest a transdifferentiation of myoblastic C2C12 cells into the osteoblastic phenotype. With this study we present a new technology to bioactivate implant surfaces with non-viral gene vectors. This tool allows the stimulation of tissue regeneration by a local release of therapeutic proteins in vivo.
Assuntos
Proteína Morfogenética Óssea 2 , Transdiferenciação Celular , Mioblastos/citologia , Osteogênese , Engenharia Tecidual/métodos , Fosfatase Alcalina , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Regeneração Óssea/genética , Caproatos/química , Linhagem Celular , Vetores Genéticos , Lactonas/química , Camundongos , Osteocalcina , Osteopontina , Poliésteres/químicaRESUMO
PURPOSE: To target adenoviral vectors to cells of the vasculature and shielding vectors from inactivation by the immune system. METHODS: Complexes of reporter gene expressing adenoviral vectors with positively charged magnetic nanoparticles were formed by electrostatic interaction in presence or absence of additional negatively charged poly(ethylene glycol)-based polymer. Transduction of HUVEC was analyzed in vitro under flow. Protection from inactivation by the immune system was analyzed by pre-incubation of AdV and complexes with neutralizing antibodies and subsequent reporter protein analysis of infected cells. RESULTS: Physical association of AdV with MNP and polymers was demonstrated by radioactive labelling of components and co-sedimentation in a magnetic field. Ad-MNP+/-polymer resulted in efficient transduction of HUVEC, depending on MOI and flow rate in presence of magnetic field, whereas no transduction was observed without complex formation with MNP or in absence of magnetic field. Association with MNP did result in protection from neutralizing antibodies, with slightly increased protection provided by the polymer. CONCLUSIONS: Complex formation of AdV with MNP is a viable means for targeting of vectors to areas of magnetic field gradient. Additional coating with polymer might proof useful in protection from inactivation by the immune system.
Assuntos
Adenoviridae/genética , Células Endoteliais/fisiologia , Magnetismo , Nanopartículas , Transdução Genética/métodos , Adenoviridae/química , Células Endoteliais/química , Células Endoteliais/virologia , Eritrócitos/química , Eritrócitos/metabolismo , Vetores Genéticos/química , Vetores Genéticos/genética , Humanos , Nanopartículas/química , Polietilenoglicóis/química , Eletricidade EstáticaRESUMO
PURPOSE: In the recent years, nanotechnology has boosted the development of potential drug delivery systems and material engineering on nanoscale basis in order to increase drug specificity and reduce side effects. A potential delivery system for immunostimulating agents such as cytosine-phosphate-guanine-oligodeoxynucleotides (CpG-ODN) needs to be developed to maximize the efficacy of immunotherapy against hypersensitivity. In this study, an aerosol formulation of biodegradable, biocompatible and nontoxic gelatin nanoparticle-bound CpG-ODN 2216 was used to treat equine recurrent airway obstruction in a clinical study. METHODS: Bronchoalveolar lavage fluid was obtained from healthy and allergic horses to quantify Th1/Th2 cytokine levels before and after inhalation regimen. Full clinical examinations were performed to evaluate the therapeutic potential of this nebulized gelatin nanoparticle-based CpG formulation. RESULTS: Most remarkable was that regulatory anti-inflammatory and anti-allergic cytokine IL-10 expression was significantly triggered by five consecutive inhalations. Thorough assessment of clinical parameters following nanoparticle treatment indicated a partial remission of the allergic condition. CONCLUSION: Thus this study, for the first time, showed effectiveness of colloidal nanocarrier-mediated immunotherapy in food-producing animals with potential future applicability to other species including humans.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Obstrução das Vias Respiratórias/veterinária , Portadores de Fármacos , Gelatina/química , Doenças dos Cavalos/terapia , Imunoterapia/veterinária , Nanopartículas , Oligodesoxirribonucleotídeos/administração & dosagem , Hipersensibilidade Respiratória/veterinária , Adjuvantes Imunológicos/química , Administração por Inalação , Aerossóis , Obstrução das Vias Respiratórias/imunologia , Obstrução das Vias Respiratórias/terapia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Células Cultivadas , Química Farmacêutica , Citocinas/metabolismo , Esquema de Medicação , Doenças dos Cavalos/imunologia , Cavalos , Nanotecnologia , Nebulizadores e Vaporizadores , Oligodesoxirribonucleotídeos/química , Recidiva , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/terapia , Tecnologia Farmacêutica/métodos , Células Th1/imunologia , Células Th2/imunologia , Receptor Toll-Like 9/agonistasRESUMO
PURPOSE: To optimize silica-iron oxide magnetic nanoparticles with surface phosphonate groups decorated with 25-kD branched polyethylenimine (PEI) for gene delivery. METHODS: Surface composition, charge, colloidal stabilities, associations with adenovirus, magneto-tranduction efficiencies, cell internalizations, in vitro toxicities and MRI relaxivities were tested for the particles decorated with varying amounts of PEI. RESULTS: Moderate PEI-decoration of MNPs results in charge reversal and destabilization. Analysis of space and time resolved concentration changes during centrifugation clearly revealed that at >5% PEI loading flocculation gradually decreases and sufficient stabilization is achieved at >10%. The association with adenovirus occurred efficiently at levels over 5% PEI, resulting in the complexes stable in 50% FCS at a PEI-to-iron w/w ratio of ≥7%; the maximum magneto-transduction efficiency was achieved at 9-12% PEI. Primary silica iron oxide nanoparticles and those with 11.5% PEI demonstrated excellent r(2)* relaxivity values (>600 s(-1)(mM Fe)(-1)) for the free and cell-internalized particles. CONCLUSIONS: Surface decoration of the silica-iron oxide nanoparticles with a PEI-to-iron w/w ratio of 10-12% yields stable aqueous suspensions, allows for efficient viral gene delivery and labeled cell detection by MRI.
Assuntos
Compostos Férricos/química , Técnicas de Transferência de Genes , Vetores Genéticos/química , Magnetismo , Nanopartículas/química , Dióxido de Silício/química , Adenoviridae/genética , Animais , Linhagem Celular , Coloides/química , Estabilidade de Medicamentos , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Imageamento por Ressonância Magnética , Camundongos , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Polietilenoimina/química , Ratos , Propriedades de Superfície , Difração de Raios XRESUMO
Results from large epidemiological studies suggest a clear relation between low birth weight and adverse renal outcome evident as early as during childhood. Such adverse outcomes may include glomerular disease, hypertension, and renal failure and contribute to a phenomenon called fetal programming. Other factors potentially leading to an adverse renal outcome following fetal programming are maternal diabetes mellitus, smoking, salt overload, and use of glucocorticoids during pregnancy. However, clinical data on the latter are scarce. Here, we discuss potential underlying mechanisms of fetal programming, including reduced nephron number via diminished nephrogenesis and other renal (e.g., via the intrarenal renin-angiotensin-aldosterone system) and non-renal (e.g., changes in endothelial function) alterations. It appears likely that the outcomes of fetal programming may be influenced or modified postnatally, for example, by the amount of nutrients given at critical times.
Assuntos
Desenvolvimento Fetal/fisiologia , Nefropatias/etiologia , Rim/anormalidades , Complicações na Gravidez/fisiopatologia , Animais , Feminino , Feto , Humanos , Rim/fisiopatologia , Nefropatias/fisiopatologia , GravidezRESUMO
Site specific vascular gene delivery for therapeutic implications is favorable because of reduction of possible side effects. Yet this technology faces numerous hurdles that result in low transfection rates because of suboptimal delivery. Combining ultrasonic microbubble technology with magnetic nanoparticle enhanced gene transfer could make it possible to use the systemic vasculature as the route of application and to magnetically trap these compounds at the target of interest. In this study we show that magnetic nanoparticle-coated microbubbles bind plasmid DNA and successfully deliver it to endothelial cells in vitro and more importantly transport their cargo through the vascular system and specifically deliver it to the vascular wall in vivo at sites where microbubbles are retained by magnetic force and burst by local ultrasound application. This resulted in a significant enhancement in site specific gene delivery compared with the conventional microbubble technique. Thus, this technology may have promising therapeutic potential. FROM THE CLINICAL EDITOR: This work focuses on combining ultrasonic microbubble technology with magnetic nanoparticle enhanced gene transfer to enable targeted gene delivery via the systemic vasculature and magnetic trapping of these compounds at the target of interest.
Assuntos
Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , Nanopartículas de Magnetita , Microbolhas , Células Endoteliais , Terapia Genética , Humanos , Nanopartículas de Magnetita/administração & dosagem , Nanopartículas de Magnetita/química , Plasmídeos , UltrassomRESUMO
Large segmental osseous defects heal poorly. Recombinant, human bone morphogenetic protein-2 (rhBMP-2) is used clinically to promote bone healing, but it is applied at very high doses that cause adverse side effects and raise costs while providing only incremental benefit. We describe a previously unexplored, alternative approach to bone regeneration using chemically modified messenger RNA (cmRNA). An optimized cmRNA encoding BMP-2 was delivered to critical-sized femoral osteotomies in rats. The cmRNA remained orthotopically localized and generated BMP locally for several days. Defects healed at doses ≥25 µg of BMP-2 cmRNA. By 4 weeks, all animals treated with 50 µg of BMP-2 cmRNA had bridged bone defects without forming the massive callus seen with rhBMP-2. Moreover, such defects recovered normal mechanical strength quicker and initiated bone remodeling faster. cmRNA regenerated bone via endochondral ossification, whereas rhBMP-2 drove intramembranous osteogenesis; cmRNA provides an innovative, safe, and highly translatable technology for bone healing.