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1.
Adv Exp Med Biol ; 1148: 25-54, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482493

RESUMO

Since ERT for several LSDs treatment has emerged at the beginning of the 1980s with Orphan Drug approval, patients' expectancy and life quality have been improved. Most LSDs treatment are based on the replaced of mutated or deficient protein with the natural or recombinant protein.One of the main ERT drawback is the high drug prices. Therefore, different strategies trying to optimize the global ERT biotherapeutic production have been proposed. LVs, a gene delivery tool, can be proposed as an alternative method to generate stable cell lines in manufacturing of recombinant proteins. Since LVs have been used in human gene therapy, clinical trials, safety testing assays and procedures have been developed. Moreover, one of the main advantages of LVs strategy to obtain manufacturing cell line is the short period required as well as the high protein levels achieved.In this chapter, we will focus on LVs as a recombinant protein production platform and we will present a case study that employs LVs to express in a manufacturing cell line, alpha-Galactosidase A (rhαGAL), which is used as ERT for Fabry disease treatment.


Assuntos
Enzimas/biossíntese , Técnicas de Transferência de Genes , Lentivirus , Enzimas/farmacologia , Doença de Fabry/terapia , Vetores Genéticos , Humanos , alfa-Galactosidase/biossíntese , alfa-Galactosidase/farmacologia
2.
Eur J Histochem ; 63(3)2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31455073

RESUMO

RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-transfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have higher viability with respect to Lipofectamine RNAiMAX-administered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.


Assuntos
Técnicas de Transferência de Genes , Lipídeos/química , Polímeros/química , RNA Interferente Pequeno/genética , Transfecção/métodos , Sequência de Bases , Carbocianinas/química , Carbocianinas/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Inativação Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Metabolismo dos Lipídeos , Lipídeos/toxicidade , Lipossomos/química , Lipossomos/metabolismo , Lipossomos/toxicidade , Neoplasias Hepáticas/genética , Polímeros/metabolismo , Polímeros/toxicidade , Interferência de RNA
3.
J Nanobiotechnology ; 17(1): 86, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387581

RESUMO

BACKGROUND: Controllable and multiple DNA release is critical in modern gene-based therapies. Current approaches require complex assistant molecules for combined release. To overcome the restrictions on the materials and environment, a novel and versatile DNA release method using a nano-electromechanical (NEMS) hypersonic resonator of gigahertz (GHz) frequency is developed. RESULTS: The micro-vortexes excited by ultra-high frequency acoustic wave can generate tunable shear stress at solid-liquid interface, thereby disrupting molecular interactions in immobilized multilayered polyelectrolyte thin films and releasing embedded DNA strands in a controlled fashion. Both finite element model analysis and experiment results verify the feasibility of this method. The release rate and released amount are confirmed to be well tuned. Owing to the different forces generated at different depth of the films, release of two types of DNA molecules with different velocities is achieved, which further explores its application in combined gene therapy. CONCLUSIONS: Our research confirmed that this novel platform based on a nano-electromechanical hypersonic resonator works well for controllable single and multi-DNA release. In addition, the unique features of this resonator such as miniaturization and batch manufacturing open its possibility to be developed into a high-throughput, implantable and site targeting DNA release and delivery system.


Assuntos
DNA/química , Nanoestruturas/química , Polieletrólitos/química , Acústica/instrumentação , Fenômenos Biofísicos , Desenho de Equipamento , Análise de Elementos Finitos , Técnicas de Transferência de Genes , Terapia Genética/métodos , Membranas Artificiais , Propriedades de Superfície
4.
Pharm Res ; 36(10): 142, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31376020

RESUMO

BACKGROUND: With the recent approval of the first small interfering RNA (siRNA) therapeutic formulated as nanoparticles, there is increased incentive for establishing the factors of importance for the design of stable solid dosage forms of such complex nanomedicines. METHODS: The aims of this study were: (i) to identify factors of importance for the design of spray-dried siRNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles (LPNs), and (ii) to evaluate their influence on the resulting powders by using a quality-by-design approach. Critical formulation and process parameters were linked to critical quality attributes (CQAs) using design of experiments, and an optimal operating space (OOS) was identified. RESULTS: A series of CQAs were identified based on the quality target product profile. The loading (ratio of LPNs to the total solid content) and the feedstock concentration were determined as critical parameters, which were optimized systematically. Mannitol was chosen as stabilizing excipient due to the low water content of the resulting powders. The loading negatively affected the colloidal stability of the LPNs, whereas feedstock concentration correlated positively with the powder particle size. The optimal mannitol-based solid formulation, defined from the OOS, displayed a loading of 5% (w/w), mass median aerodynamic diameter of 3.3 ± 0.2 µm, yield of 60.6 ± 6.6%, and a size ratio of 1.15 ± 0.03. Dispersed micro-embedded LPNs had preserved physicochemical characteristics as well as in vitro siRNA release profile and gene silencing, as compared to non-spray-dried LPNs. CONCLUSION: The optimal solid dosage forms represent robust formulations suitable for higher scale-up manufacturing.


Assuntos
Dessecação/métodos , Lipídeos/química , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , RNA Interferente Pequeno/química , Administração por Inalação , Animais , Composição de Medicamentos , Excipientes/química , Inativação Gênica , Técnicas de Transferência de Genes , Manitol/química , Camundongos , Nanomedicina , Tamanho da Partícula , Pós , Células RAW 264.7 , RNA Interferente Pequeno/administração & dosagem , Solubilidade , Solventes/química
5.
Int J Nanomedicine ; 14: 5415-5434, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31409996

RESUMO

Over the past two decades, nano-sized biosystems have increasingly been utilized to deliver various pharmaceutical agents to a specific region, organ or tissue for controllable precision therapy. Whether solid nanohydrogel, nanosphere, nanoparticle, nanosheet, micelles and lipoproteins, or "hollow" nanobubble, liposome, nanocapsule, and nanovesicle, all of them can exhibit outstanding loading and releasing capability as a drug vehicle - in particular polymeric nanovesicle, a microscopic hollow sphere that encloses a water core with a thin polymer membrane. Besides excellent stability, toughness and liposome-like compatibility, polymeric nanovesicles offer considerable scope for tailoring properties by changing their chemical structure, block lengths, stimulus-responsiveness and even conjugation with biomolecules. In this review, we summarize the latest advances in stimulus-responsive polymeric nanovesicles for biomedical applications. Different functionalized polymers are in development to construct more complex multiple responsive nanovesicles in delivery systems, medical imaging, biosensors and so on.


Assuntos
Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , Nanopartículas/química , Polímeros/química , Portadores de Fármacos/química , Concentração de Íons de Hidrogênio
7.
J Nanobiotechnology ; 17(1): 85, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31319859

RESUMO

BACKGROUND: Lack of effective tumor-specific delivery systems remains an unmet clinical challenge for successful translation of innovative therapies, such as, therapeutic oligonucleotides. In the past decade, exosomes have been suggested to be ideal drug delivery systems with application in a broad range of pathologies including cancer, due to their organotropic properties. Tumor-derived exosomes, having tumor-homing properties, can efficiently reach cancer cells and therefore behave as carriers for improved drug delivery to the primary tumor and metastases. However, due to their complex composition, and still undefined biological functions, safety concerns arise hampering their translation to the clinics. RESULTS: We propose here the development of exosome-mimetic nanosystems (EMNs) that simulate natural tumor-derived exosomes with respect to their structure and functionality, but with a controlled composition, for the targeted delivery of therapeutic oligonucleotides to lung adenocarcinoma cells (microRNA-145 mimics). Making use of the well-known liposome technology, EMNs can be engineered, loaded with the therapeutic compounds, and tailored with specific proteins (integrin α6ß4) providing them organotropic properties. EMNs show great similarities to natural exosomes with respect to their physicochemical properties, drug loading capacity, and ability to interact with the cancer target cells in vitro and in vivo, but are easier to manufacture, can be produced at high yields, and are safer by definition. CONCLUSIONS: We have designed a multifunctional nanoplatform mimicking exosomes, EMNs, and proved their potential to reach cancer cells with a similar efficient that tumor-derived exosomes but providing important advantages in terms of production methodology and regulations. Additionally, EMNs are highly versatile systems that can be tunable for a broader range of applications.


Assuntos
Antineoplásicos/química , Materiais Biomiméticos/química , Exossomos/química , MicroRNAs/química , Nanocápsulas/química , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Técnicas de Transferência de Genes , Humanos , Integrinas/química , Integrinas/metabolismo , Camundongos , MicroRNAs/administração & dosagem , Propriedades de Superfície
8.
Nat Neurosci ; 22(8): 1345-1356, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285614

RESUMO

Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that a number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo and in the human retina in vitro. We demonstrate applications for recording and stimulation, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.


Assuntos
Dependovirus/genética , Marcação de Genes/métodos , Neuroglia/virologia , Neurônios/virologia , Animais , Técnicas de Transferência de Genes , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Retina/virologia
9.
Chemphyschem ; 20(16): 2110-2121, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31265754

RESUMO

The physicochemical properties and transfection efficacies of two samples of a cationic lipid have been investigated and compared in 2D (monolayers at the air/liquid interface) and 3D (aqueous bulk dispersions) model systems using different techniques. The samples differ only in their chain composition due to the purity of the oleylamine (chain precursor). Lipid 8 (using the oleylamine of technical grade for cost-efficient synthesis) shows lateral phase separation in the Langmuir layers. However, the amount of attached DNA, determined by IRRAS, is for both samples the same. In 3D systems, lipid 8 p forms cubic phases, which disappear after addition of DNA. At physiological temperatures, both lipids (alone and in mixture with cholesterol) assemble to lamellar aggregates and exhibit comparable DNA delivery efficiency. This study demonstrates that non-lamellar structures are not compulsory for high transfection rates. The results legitimate the utilization of oleyl chains of technical grade in the synthesis of cationic transfection lipids.


Assuntos
Aminas/química , DNA/química , Lipídeos/química , Lipossomos/química , Aminas/síntese química , Aminas/normas , Aminas/toxicidade , Animais , Bovinos , Linhagem Celular Tumoral , Colesterol/química , Técnicas de Transferência de Genes/normas , Humanos , Lipídeos/síntese química , Lipídeos/normas , Lipídeos/toxicidade , Lipossomos/normas , Lipossomos/toxicidade , Estrutura Molecular , Transição de Fase , Suínos , Transfecção/normas , Temperatura de Transição
10.
Nat Commun ; 10(1): 2993, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278269

RESUMO

Activated hepatic stellate cell (aHSC)-mediated liver fibrosis is essential to the development of liver metastasis. Here, we discover intra-hepatic scale-up of relaxin (RLN, an anti-fibrotic peptide) in response to fibrosis along with the upregulation of its primary receptor (RXFP1) on aHSCs. The elevated expression of RLN serves as a natural regulator to deactivate aHSCs and resolve liver fibrosis. Therefore, we hypothesize this endogenous liver fibrosis repair mechanism can be leveraged for liver metastasis treatment via enforced RLN expression. To validate the therapeutic potential, we utilize aminoethyl anisamide-conjugated lipid-calcium-phosphate nanoparticles to deliver plasmid DNA encoding RLN. The nanoparticles preferentially target metastatic tumor cells and aHSCs within the metastatic lesion and convert them as an in situ RLN depot. Expressed RLN reverses the stromal microenvironment, which makes it unfavorable for established liver metastasis to grow. In colorectal, pancreatic, and breast cancer liver metastasis models, we confirm the RLN gene therapy results in significant inhibition of metastatic progression and prolongs survival. In addition, enforced RLN expression reactivates intra-metastasis immune milieu. The combination of the RLN gene therapy with PD-L1 blockade immunotherapy further produces a synergistic anti-metastatic efficacy. Collectively, the targeted RLN gene therapy represents a highly efficient, safe, and versatile anti-metastatic modality, and is promising for clinical translation.


Assuntos
Terapia Genética/métodos , Cirrose Hepática Experimental/terapia , Neoplasias Hepáticas/terapia , Hepatopatia Gordurosa não Alcoólica/terapia , Relaxina/genética , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Tetracloreto de Carbono/administração & dosagem , Tetracloreto de Carbono/toxicidade , Linhagem Celular Tumoral/transplante , Progressão da Doença , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/secundário , Camundongos , Nanopartículas/administração & dosagem , Nanopartículas/química , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Plasmídeos/genética , Receptores Acoplados a Proteínas-G/metabolismo , Relaxina/metabolismo , Resultado do Tratamento , Microambiente Tumoral/genética , Regulação para Cima
11.
Int J Nanomedicine ; 14: 4755-4765, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308656

RESUMO

Background: Many techniques and methods have been used clinically to relieve pain from cartilage repair, but the long-term effect is still unsatisfactory. Purpose: The objective of this study was to form an artificial chondroid tissue gene enhanced tissue engineering system to repair cartilage defects via nanosized liposomes. Methods: Cationic nanosized liposomes were prepared and characterized using transmission electron microscope (TEM) and dynamic laser light scattering (DLS). The rat mesenchymal stem cells (rMSCs) were isolated, cultivated, and induced by SRY (Sex-Determining Region Y)-Box 9 (Sox9) via cationic nanosized liposomes. The induced rMSCs were mixed with a thermo-sensitive chitosan hydrogel and subcutaneously injected into the nude mice. Finally, the newly-formed chondroid tissue obtained in the injection parts, and the transparent parts were detected by HE, collagen II, and safranin O. Results: It was found that the presently prepared cationic nanosized liposomes had the diameter of 85.76±3.48 nm and the zeta potential of 15.76±2.1 mV. The isolated rMSCs proliferation was fibroblast-like, with a cultivated confluence of 90% confluence in 5-8 days, and stained positive for CD29 and CD44 while negative for CD34 and CD45. After transfection with cationic nanosized liposomes, we observed changes of cellular morphology and a higher expression of SOX9 compared with control groups, which indicated that rMSCs could differentiate into chondrocyte in vitro. By mixing transfected rMSCs with the thermo-sensitive hydrogel of chitosan in nude mice, chondroid tissue was successfully obtained, demonstrating that rMSCs can differentiate into chondrogenic cells in vivo. Conclusion: This study explored new ways to improve the quality of tissue engineered cartilage, thus accelerating clinical transformation and reducing patient pain.


Assuntos
Condrogênese , Técnicas de Transferência de Genes , Nanopartículas/química , Engenharia Tecidual/métodos , Animais , Cátions , Diferenciação Celular , Forma Celular , Células Cultivadas , Condrogênese/efeitos dos fármacos , Géis , Lipossomos/química , Lipossomos/ultraestrutura , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos Nus , Ratos Sprague-Dawley , Fatores de Transcrição SOX9/metabolismo , Temperatura Ambiente
12.
Adv Mater ; 31(33): e1902575, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31215123

RESUMO

A main challenge to broaden the biomedical application of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9) genome editing technique is the delivery of Cas9 nuclease and single-guide RNA (sgRNA) into the specific cell and organ. An effective and very fast CRISPR/Cas9 genome editing in vitro and in vivo enabled by bioreducible lipid/Cas9 messenger RNA (mRNA) nanoparticle is reported. BAMEA-O16B, a lipid nanoparticle integrated with disulfide bonds, can efficiently deliver Cas9 mRNA and sgRNA into cells while releasing RNA in response to the reductive intracellular environment for genome editing as fast as 24 h post mRNA delivery. It is demonstrated that the simultaneous delivery of Cas9 mRNA and sgRNA using BAMEA-O16B knocks out green fluorescent protein (GFP) expression of human embryonic kidney cells with efficiency up to 90%. Moreover, the intravenous injection of BAMEA-O16B/Cas9 mRNA/sgRNA nanoparticle effectively accumulates in hepatocytes, and knocks down proprotein convertase subtilisin/kexin type 9 level in mouse serum down to 20% of nontreatment. The leading lipid nanoparticle, BAMEA-O16B, represents one of the most efficient CRISPR/Cas9 delivery nanocarriers reported so far, and it can broaden the therapeutic promise of mRNA and CRISPR/Cas9 technique further.


Assuntos
Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Lipídeos/química , Nanopartículas/química , RNA Guia/química , RNA Mensageiro/química , Animais , Transporte Biológico , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes/métodos , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Oxirredução , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , RNA Guia/administração & dosagem , RNA Mensageiro/administração & dosagem
13.
PLoS Genet ; 15(6): e1008226, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199789

RESUMO

Carbonic anhydrase-8 (CA8) is an intracellular protein that functions as an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1) critical to intracellular Ca++ release, synaptic functions and neuronal excitability. We showed previously that murine nociception and analgesic responses are regulated by the expression of this gene in dorsal root ganglion (DRG) associated with a cis-eQTL. In this report, we identify an exon-level cis-eQTL (rs6471859) that regulates human DRG CA8 alternative splicing, producing a truncated 1,697bp transcript (e.g., CA8-204). Our functional genomic studies show the "G" allele at rs6471859 produces a cryptic 3'UTR splice site regulating expression of CA8-204. We developed constructs to study the expression and function of the naturally occurring CA8-204G transcript (G allele at rs6471859), CA8-204C (C allele at rs6471859 reversion mutation) and CA8-201 (full length transcript). CA8-204G transcript expression occurred predominantly in non-neuronal cells (HEK293), while CA8-204C expression was restricted to neuronal derived cells (NBL) in vitro. CA8-204G produced a stable truncated transcript in HEK293 cells that was barely detectable in NBL cells. We also show CA8-204 produces a stable peptide that inhibits pITPR1 and Ca++ release in HEK293 cells. These results imply homozygous G/G individuals at rs6471859, which are common in the general population, produce exclusively CA8-204G that is barely detectable in neuronal cells. CA8 null mutations that greatly impact neuronal functions are associated with severe forms of spinal cerebellar ataxia, and our data suggest G/G homozygotes should display a similar phenotype. To address this question, we show in vivo using AAV8-FLAG-CA8-204G and AAV8-V5-CA8-201 gene transfer delivered via intra-neural sciatic nerve injection (SN), that these viral constructs are able to transduce DRG cells and produce similar analgesic and anti-hyperalgesic responses to inflammatory pain. Immunohistochemistry (IHC) examinations of DRG tissues further show CA8-204G peptide is expressed in advillin expressing neuronal cells, but to a lesser extent compared to glial cells. These findings explain why G/G homozygotes that exclusively produce this truncated functional peptide in DRG evade a severe phenotype. These genomic studies significantly advance the literature regarding structure-function studies on CA8-ITPR1 critical to calcium signaling pathways, synaptic functioning, neuronal excitability and analgesic responses.


Assuntos
Biomarcadores Tumorais/genética , Sinalização do Cálcio/genética , Receptores de Inositol 1,4,5-Trifosfato/genética , Neurônios/metabolismo , Dor/genética , Processamento Alternativo/genética , Animais , Biomarcadores Tumorais/farmacologia , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Camundongos , Mutação/genética , Neurônios/patologia , Especificidade de Órgãos , Dor/patologia , Peptídeos/genética , Peptídeos/farmacologia , Locos de Características Quantitativas/genética , Sítios de Splice de RNA/genética , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo
14.
Parasit Vectors ; 12(1): 303, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31202271

RESUMO

Parasites comprise diverse and complex organisms, which substantially impact human and animal health. Most parasites have complex life-cycles, and by virtue of co-evolution have developed multifaceted, often life-cycle stage-specific relationships with the immune system of their hosts. The complexity in the biology of many parasites often limits our knowledge of parasite-specific immune responses, to in vitro studies only. The relatively recent development of methods to stably manipulate the genetic make-up of many parasites has allowed a better understanding of host-parasite interactions, particularly in vivo. In this regard, the use of transgenic parasites can facilitate the study of immunomodulatory mechanisms under in vivo conditions. Therefore, in this review, we specifically highlighted the current developments in the use of transgenic parasites to unravel the host's immune response to different life-cycle stages of some key parasite species such as Leishmania, Schistosoma, Toxoplasma, Plasmodium and Trypanosome and to some degree, the use of transgenic nematode parasites is also briefly discussed.


Assuntos
Técnicas de Transferência de Genes , Interações Hospedeiro-Parasita/imunologia , Parasitos/genética , Parasitos/imunologia , Animais , Interações Hospedeiro-Parasita/genética , Humanos , Leishmania/genética , Leishmania/imunologia , Estágios do Ciclo de Vida/genética , Estágios do Ciclo de Vida/imunologia , Camundongos , Plasmodium/genética , Plasmodium/imunologia , Toxoplasma/genética , Toxoplasma/imunologia
15.
BMC Genomics ; 20(1): 444, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159719

RESUMO

BACKGROUND: Host genetic backgrounds affect gene functions. The genetic backgrounds of genetically engineered organisms must be identified to confirm their genetic backgrounds identity with those of recipients. Marker-assisted backcrossing (MAB), transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) editing are three commonly used genetic engineering techniques. However, methods for genetic background screening between genetically engineered organisms and corresponding recipients suffer from low efficiency, low accuracy or high cost. RESULTS: Here, we improved our previously reported AmpSeq-SSR method, an amplicon sequencing-based simple sequence repeat (SSR) genotyping method, by selecting SSR loci with high polymorphism among varieties. Ultimately, a set of 396 SSRs was generated and applied to evaluate the genetic backgrounds identity between rice lines developed through MAB, transgenesis, and CRISPR/Cas9 editing and the respective recipient rice. We discovered that the percentage of different SSRs between the MAB-developed rice line and its recipient was as high as 23.5%. In contrast, only 0.8% of SSRs were different between the CRISPR/Cas9-system-mediated rice line and its recipient, while no SSRs showed different genotypes between the transgenic rice line and its recipient. Furthermore, most differential SSRs induced by MAB technology were located in non-coding regions (62.9%), followed by untranslated regions (21.0%) and coding regions (16.1%). Trinucleotide repeats were the most prevalent type of altered SSR. Most importantly, all altered SSRs located in coding regions were trinucleotide repeats. CONCLUSIONS: This method is not only useful for the background evaluation of genetic resources but also expands our understanding of the unintended effects of different genetic engineering techniques. While the work we present focused on rice, this method can be readily extended to other organisms.


Assuntos
Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Repetições de Microssatélites , Oryza/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Proteínas Serina-Treonina Quinases/genética , Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Transferência de Genes , Engenharia Genética , Proteínas de Plantas/antagonistas & inibidores , Polimorfismo Genético , Proteínas Serina-Treonina Quinases/antagonistas & inibidores
16.
J Biomed Nanotechnol ; 15(3): 431-442, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31165690

RESUMO

Human Wnt inhibitory factor-1 (hWIF-1), as an anti-oncogene, holds great promise for non-small-cell lung cancer (NSCLC) therapy. However, the clinical application of hWIF-1 in cancer therapy is limited by elimination and degradation of free hWIF-1 in vivo. Therefore, it is necessary to develop safe and effective gene delivery vectors for hWIF-1 delivery in vivo. In this paper, we synthesized a novel polyethylenimine (PEI) derivative PEI-SP5-2 (PES) based on branched PEI1800 and NSCLC-targeting peptide SP5-2 to deliver hWIF-1 for NSCLC therapy. PES had excellent gene delivery capacity, and the transfection efficiency reached 50.02% ± 4.75% in A549 cell lines when the weight ratio of PES/gene was 100. Besides, the PES/gene particles were monodispersed, and the hydrodynamic diameter and zeta potential were 47.55 nm and 24.9 mV, respectively. In addition, PES/hWIF-1 complexes could inhibit the tumor growth in vitro and in vivo when it was used for non-small-cell lung cancer therapy. We concluded that PES would be promising as a novel gene delivery vector, and PES/hWIF-1 complexes inhibited the tumor growth and showed potential for non-small-cell lung cancer therapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Polietilenoimina , Transfecção
17.
Int J Nanomedicine ; 14: 3557-3569, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31190812

RESUMO

Purpose: Combining siRNA and other chemotherapeutic agents into one nanocarrier can overcome the multidrug resistance (MDR) phenomenon by synergistically MDR relative genes silencing and elevated chemotherapeutic activity. Most of these systems are typically fabricated through complicated procedures, which involves materials preparation, drug loading and modifications. Herein, the purpose of this study is to develop a new and fast co-delivery system of siRNA and doxorubicin for potentially synergistic cancer treatment. Methods: The co-delivery system is constructed conveniently by a stable complex consisting of doxorubicin bound to siRNA via intercalation firstly, followed by interacting with (3-Aminopropyl)triethoxysilane (APTES) electrostatically and Tetraethyl orthosilicate (TEOS) co-condensed, and the characterizations of the resultant nanocarrier are also investigated. Furthermore, this study evaluates the synergistic anti-cancer efficacy in MCF-7/MDR cells after treatment of siRNA and doxorubicin 'two in one' nanocarriers. Results: We establish a new and fast method to craft a co-delivery system of siRNA and doxorubicin with controllable and nearly uniform size, and the entire fabrication process only costs in about 10 minutes. The resultant co-delivery system presents high loading capacities of siRNA and doxorubicin, and the encapsulated doxorubicin plays a pH-responsive control release. Further, biological functionality tests of the synthesized co-delivery nanocarriers show high inhibition of P-gp protein encoded by MDR-1 gene in MCF-7/MDR cells (a variant of human breast cancer cell line with drug resistance) after transfection of these nanocarriers carrying MDR-1 siRNA and doxorubicin simultaneously, which sensitize the MCF-7/MDR cells to doxorubicin, overall leading to improved cell suppression. Conclusion: Collectively, this co-delivery system not only serves as potent therapeutics for synergistic cancer therapy, it also may facilitate the bench-to-bedside translation of combinatorial delivery system as a robust drug nanocarrier by allowing for fabricating a simply and fast nanocarrier for co-delivery of siRNA and doxorubicin with predictable high production rate.


Assuntos
Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , RNA Interferente Pequeno/administração & dosagem , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Inativação Gênica , Humanos , Células MCF-7 , Nanopartículas/administração & dosagem , Nanopartículas/ultraestrutura , RNA Interferente Pequeno/genética , Dióxido de Silício/química
18.
J Biomed Nanotechnol ; 15(7): 1622-1635, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31196364

RESUMO

The construction of a targeted gene delivery system with low cytotoxicity to normal tissues is an urgent need for the clinical treatment of liver cancer. In this study, Antheraea pernyi silk fibroin (ASF) was cationized with low-molecular-weight polyethylenimine (PEI, 1.8 kDa) to synthesize a cationized Antheraea pernyi silk fibroin (CASF). The highly cancer-selective hepatoma targeted peptide, HCBP1 (sequence FQHPSFI), was coupled to the side chains of CASF to synthesize a hepatoma-targeted CASF (CASFP). CASFP relied on the positive charges of CASF could package the pDNA encoded the inhibitor of growth 4 (ING4) and interleukin-24 (IL-24) to form CASFP/pDNA complexes. The results showed that the zeta potential of ASF was reversed from -9.08 ± 0.20 to +11.33 ± 0.38 mV, and its isoelectric point significantly increased from 4.31 to 9.38 after PEI modification. The Fourier transform infrared spectroscopy results and the 1Hydrogen-nuclear magnetic resonance spectra demonstrated that HCBP1 could be coupled to the side chains of CASF under the action of the bifunctional reagent N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). In vitro, human hepatocellular carcinoma HepG2 cells and human normal hepatic L-02 cells were transfected with the CASFP/pDNA complexes. The results of confocal laser scanning microscope analysis and cell viability assays showed that the complexes were able to transfect HepG2 cells and effectively inhibit their proliferation but had no obvious cytotoxicity to L-02 cells. In this study, a new gene delivery system, constructed by using HCBP1-modified CASF and the ING4-IL-24 dual-gene co-expression plasmid, was able to inhibit the proliferation of hepatocellular carcinoma cells but had no obvious cytotoxicity to normal hepatic cells. Therefore, the gene delivery system has the potential for application as a gene therapy in liver cancer.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas de Ciclo Celular , Fibroínas , Técnicas de Transferência de Genes , Proteínas de Homeodomínio , Humanos , Interleucinas , Plasmídeos , Polietilenoimina , Transfecção , Proteínas Supressoras de Tumor
19.
J Biomed Nanotechnol ; 15(8): 1654-1672, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31219018

RESUMO

Stimuli-responsive materials are promising paradigm applied to construct diagnostic and therapeutic intracellular controlled release vectors, while highlighting many challenges and opportunities. In this paper, six α-cyclodextrin-based supramolecular nanovectors were constructed and the efficacy of amine groups, stimuli-responsive profiles and endocytic mechanisms were investigated. The results indicated that the designed supermolecules can compact DNA to form stable complexes and display low cytotoxicity. Among them, PRPEI-2 with suitable PEI amine group exhibited enhanced transfecting performance, high dilution stability, nice serum compatibility, and good acid-responsive profiles to enable endosome escape, significantly higher than commercially available transfecting agent PEI25000, the most effective vector studied to date. The endocytic uptake mechanisms involved in the transfection was mainly through clathrin-mediated pathway, which is closely associated with and can be improved by endosome escape. Moreover, PRPEI-2/DNA polyplex can be effectively expressed in vivo even after 48 h via only single tail-vein injection, and the gene expression and main tissue distribution appeared in the testis, liver, brain and spleen. These excellent characteristics demonstrated that the supramolecular PRPEI-2 represents an excellent prospect as stimuli-responsive nanovectors for gene diagnosis and therapy.


Assuntos
Técnicas de Transferência de Genes , DNA , Endossomos , Polietilenoimina , Transfecção , alfa-Ciclodextrinas
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