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1.
Leuk Lymphoma ; 57(1): 183-92, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-25907616

RESUMO

The poor treatment response of acute myeloid leukemia (AML) overexpressing high-risk oncogenes such as EVI1, demands specific animal models for new treatment evaluations. Evi1 is a common site of activating integrations in murine leukemia virus (MLV)-induced AML and in retroviral and lentiviral gene-modified HCS. Still, a model of overt AML induced by Evi1 has not been generated. Cell lines from MLV-induced AML are growth factor-dependent and non-transplantable. Hence, for the leukemia maintenance in the infected animals, a growth factor source such as chronic immune response has been suggested. We have investigated whether these leukemias are transplantable if provided with growth factors. We show that the Evi1(+)DA-3 cells modified to express an intracellular form of GM-CSF, acquired growth factor independence and transplantability and caused an overt leukemia in syngeneic hosts, without increasing serum GM-CSF levels. We propose this as a general approach for modeling different forms of high-risk human AML using similar cell lines.


Assuntos
Comunicação Autócrina , Proteínas de Ligação a DNA/genética , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proto-Oncogenes/genética , Fatores de Transcrição/genética , Animais , Biomarcadores , Biópsia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Isoenxertos , Leucemia Mieloide Aguda/patologia , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Metástase Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Carga Tumoral
2.
Oncotarget ; 6(31): 30675-703, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26362400

RESUMO

The emergence of genetic engineering at the beginning of the 1970's opened the era of biomedical technologies, which aims to improve human health using genetic manipulation techniques in a clinical context. Gene therapy represents an innovating and appealing strategy for treatment of human diseases, which utilizes vehicles or vectors for delivering therapeutic genes into the patients' body. However, a few past unsuccessful events that negatively marked the beginning of gene therapy resulted in the need for further studies regarding the design and biology of gene therapy vectors, so that this innovating treatment approach can successfully move from bench to bedside. In this paper, we review the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors. At the end of the manuscript, we summarized the main advantages and disadvantages of common gene therapy vectors and we discuss possible future directions for potential therapeutic vectors.


Assuntos
Técnicas de Transferência de Genes , Engenharia Genética/métodos , Terapia Genética/métodos , Vetores Genéticos/genética , Adenoviridae/genética , Dependovirus/genética , Humanos , Lentivirus/genética , Plasmídeos/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Retroviridae/genética , Replicação Viral/genética
3.
Mol Ther Nucleic Acids ; 3: e172, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24983837

RESUMO

Inducible systems for gene expression emerge as a new class of artificial vectors offering temporal and spatial exogenous control of gene expression. However, most inducible systems are less efficient in vivo and lack the target-organ specificity. In the present study, we have developed and optimized an oligonucleotide-based inducible system for the in vivo control of transgenes in the liver. We generated a set of simple, inducible plasmid-vectors based on the addition of four units of liver-specific miR-122 target sites to the 3'untranslated region of the gene of interest. Once the vector was delivered into hepatocytes this modification induced a dramatic reduction of gene expression that could be restored by the infusion of an antagomir for miR-122. The efficiency of the system was tested in vivo, and displayed low background and strong increase in gene expression upon induction. Moreover, gene expression was repeatedly induced even several months after the first induction showing no toxic effect in vivo. By combining tissue-specific control elements with antagomir treatment we generated, optimized and validated a robust inducible system that could be used successfully for in vivo experimental models requiring tight and cyclic control of gene expression.

4.
Pediatr Endocrinol Rev ; 10(4): 473-84, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23957198

RESUMO

The IGF-1R pathway is essential for the initiation and progression of many cancers. In contrast to other receptor tyrosine kinases involved in cancer, it is not frequently mutated or amplified. The classical model of signaling through the IGF-1R centers on ligand initiated kinase activation, allowing binding of adaptor molecules and downstream activation of the MAPK and PI3K pathways. The signaling is terminated through receptor ubiquitination and subsequent degradation. To date, therapies targeting IGF-1R have been designed solely aiming to block phosphorylation mediated signaling by preventing receptor-ligand interaction or by limiting kinase activation. Yet, the classical model is insufficient to explain receptor behavior induced by some IGF-1R inhibitors. This review advocates an updated model of IGF-1R signaling, accommodating the "classical" kinase signaling and the IGF-1R-kinase independent signaling thus providing the theoretical background for receptor downregulation induced by IGF-1R inhibitors. This model should be considered for future design of effective therapies targeting the IGF-1R pathway.


Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Receptor IGF Tipo 1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Criança , Humanos , Neoplasias/patologia , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/fisiologia
5.
Nucleic Acids Res ; 41(5): 3257-73, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23345620

RESUMO

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON-bisLNA-with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson-Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.


Assuntos
DNA Super-Helicoidal/química , Oligonucleotídeos/química , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Soluções Tampão , DNA/química , Clivagem do DNA , Enzimas de Restrição do DNA/química , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Oligonucleotídeos/síntese química , Plasmídeos/química , Temperatura de Transição
6.
Proc Natl Acad Sci U S A ; 109(50): 20620-5, 2012 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-23188799

RESUMO

Owing to its essential role in cancer, insulin-like growth factor type 1 receptor (IGF-1R)-targeted therapy is an exciting approach for cancer treatment. However, when translated into clinical trials, IGF-1R-specific antibodies did not fulfill expectations. Despite promising clinical responses in Ewing's sarcoma (ES) phase I/II trials, phase III trials were discouraging, requiring bedside-to-bench translation and functional reevaluation of the drugs. The anti-IGF-1R antibody figitumumab (CP-751,871; CP) was designed as an antagonist to prevent ligand-receptor interaction but, as with all anti-IGF-1R antibodies, it induces agonist-like receptor down-regulation. We explored this paradox in a panel of ES cell lines and found their sensitivity to CP was unaffected by presence of IGF-1, countering a ligand blocking mechanism. CP induced IGF-1R/ß-arrestin1 association with dual functional outcome: receptor ubiquitination and degradation and decrease in cell viability and ß-arrestin1-dependent ERK signaling activation. Controlled ß-arrestin1 suppression initially enhanced CP resistance. This effect was mitigated on further ß-arrestin1 decrease, due to loss of CP-induced ERK activation. Confirming this, the ERK1/2 inhibitor U0126 increased sensitivity to CP. Combined, these results reveal the mechanism of CP-induced receptor down-regulation and characteristics that functionally qualify a prototypical antagonist as an IGF-1R-biased agonist: ß-arrestin1 recruitment to IGF-1R as the underlying mechanism for ERK signaling activation and receptor down-regulation. We further confirmed the consequences of ß-arrestin1 regulation on cell sensitivity to CP and demonstrated a therapeutic strategy to enhance response. Defining and suppressing such biased signaling represents a practical therapeutic strategy to enhance response to anti-IGF-1R therapies.


Assuntos
Arrestinas/agonistas , Imunoglobulinas Intravenosas/uso terapêutico , Receptor IGF Tipo 1/antagonistas & inibidores , Sarcoma de Ewing/terapia , Anticorpos Monoclonais/uso terapêutico , Arrestinas/antagonistas & inibidores , Arrestinas/genética , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo , Técnicas de Inativação de Genes , Humanos , Sistema de Sinalização das MAP Quinases , RNA Interferente Pequeno/genética , Receptor IGF Tipo 1/imunologia , Receptor IGF Tipo 1/metabolismo , Sarcoma de Ewing/imunologia , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Transdução de Sinais , Ubiquitinação , beta-Arrestinas
7.
Nucleic Acids Res ; 39(9): 3972-87, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21245043

RESUMO

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.


Assuntos
Peptídeos Penetradores de Células/química , Lipopeptídeos/química , Quinolinas/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Animais , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/toxicidade , Células Cultivadas , Endossomos/metabolismo , Humanos , Indicadores e Reagentes , Mediadores da Inflamação/metabolismo , Lipídeos , Lipopeptídeos/metabolismo , Camundongos , Nanopartículas/química , Nanopartículas/toxicidade , Quinolinas/metabolismo
8.
Nucleic Acids Res ; 39(3): 1142-54, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20860997

RESUMO

Zorro-LNA (Zorro) is a newly developed, oligonucleotide (ON)-based, Z-shaped construct with the potential of specific binding to each strand of duplex DNA. The first-generation Zorros are formed by two hybridized LNA/DNA mixmers (2-ON Zorros) and was hypothesized to strand invade. We have now established a method, which conclusively demonstrates that an LNA ON can strand invade into duplex DNA. To make Zorros smaller in size and easier to design, we synthesized 3'-5'-5'-3' single-stranded Zorro-LNA (ssZorro) by using both 3'- and 5'-phosphoramidites. With ssZorro, a significantly greater extent and rate of double-strand invasion (DSI) was obtained than with conventional 2-ON Zorros. Introducing hydrophilic PEG-linkers connecting the two strands did not significantly change the rate or extent of DSI as compared to ssZorro with a nucleotide-based linker, while the longest alkyl-chain linker tested (36 carbons) resulted in a very slow DSI. The shortest alkyl-chain linker (3 carbons) did not reduce the extent of DSI of ssZorro, but significantly decreased the DSI rate. Collectively, ssZorro is smaller in size, easier to design and more efficient than conventional 2-ON Zorro in inducing DSI. Analysis of the chemical composition of the linker suggests that it could be of importance for future therapeutic considerations.


Assuntos
DNA/química , Oligonucleotídeos/química , Inativação Gênica , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Plasmídeos/química
9.
Expert Opin Drug Deliv ; 7(6): 721-35, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20367531

RESUMO

IMPORTANCE OF THE FIELD: Gene therapy is regarded as one of the most promising therapeutic approaches, as it has the potential to treat disorders by correcting malformations at the nucleic acids. AREAS COVERED IN THIS REVIEW: Some of the most recent developments in the process of plasmid DNA vector design and formulation are reviewed with a special focus on: different formulations of nanovectors and a summary of successful cases reported; requirements for systemic administration; and functionalization of the nanocarriers by use of targeting entities. WHAT THE READER WILL GAIN: An understanding of the different physiological barriers and a comprehensive review of the recent strategies used to overcome these obstacles. Particular attention is given to formulations for intravenous administration, colloidal stability properties and different targeting entities used. TAKE HOME MESSAGE: Overall, vector formulation must take into account the administration route and inherent physiological barriers. Critical parameters for the success of pDNA nanovectors are: particles size, colloidal stability of the formulation and interaction between the carrier and plasmid DNA. Highly relevant is the fact that this interaction should be balanced to offer protection to degradation as well as allow dissociation of the therapeutic nucleic acid for obtaining maximal activity.


Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Nanotecnologia
10.
Mol Biotechnol ; 45(2): 171-9, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20238183

RESUMO

Accurate hybridization is dependent on the ratio between sequence-specific and unspecific binding. Dissociation of unspecifically bound, while maintaining specifically hybridized, nucleic acids are key steps to obtain a well-defined complex. We have developed a new method, temperature-assisted, cyclic hybridization (TACH), which increases cognate binding at the expense of unspecific hybridization. The method was used for optimizing binding of peptide nucleic acid (PNA) to supercoiled plasmids and has several advantages over previous methods: (1) it reduces the required amount of bis-PNA by three- to fourfold; (2) it results in less unspecific binding; (3) it extends cooperative hybridization, from 3 bp to 5 bp between two adjacent binding sites; and (4) it decreases the aggregation of bis-PNA. This method might be extended to other forms of hybridizations including the use of additional nucleic acids analogs, such as locked nucleic acid (LNA) and, also, to other areas where PNAs are used such as fluorescence in situ hybridization (FISH), microarrays, or in vivo plasmid delivery.


Assuntos
DNA Super-Helicoidal/química , Hibridização de Ácido Nucleico/métodos , Plasmídeos/química , Análise de Variância , Animais , DNA Super-Helicoidal/metabolismo , Eletroforese em Gel de Ágar , Expressão Gênica , Concentração de Íons de Hidrogênio , Camundongos , Conformação de Ácido Nucleico , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/metabolismo , Plasmídeos/metabolismo , Sensibilidade e Especificidade , Temperatura
11.
Genetica ; 137(1): 47-56, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19488829

RESUMO

In both basic research as well as experimental gene therapy the need to transfer genetic material into a cell is of vital importance. The cellular compartment, which is the target for the genetic material, depends upon application. An siRNA that mediates silencing is preferably delivered to the cytosol while a transgene would need to end up in the nucleus for successful transcription to occur. Furthermore the ability to regulate gene expression has grown substantially since the discovery of RNA interference. In such diverse fields as medical research and agricultural pest control, the capability to alter the genetic output has been a useful tool for pushing the scientific frontiers. This review is focused on nanotechnological approaches to assemble optimised structures of nucleic acid derivatives to facilitate gene delivery as well as promoting down regulation of endogenous genes.


Assuntos
Técnicas de Transferência de Genes , Nanotecnologia/métodos , Animais , Núcleo Celular/genética , Hibridização de Ácido Nucleico , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo
12.
Biomol Eng ; 22(5-6): 185-92, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16144773

RESUMO

Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as "openers" to partially overlapping sites on the opposite DNA strand. The PNA "opener" stabilized the binding of "linear" PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as "openers" to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification.


Assuntos
DNA Super-Helicoidal/química , Terapia Genética , Ácidos Nucleicos Peptídicos/química , Plasmídeos/química , Animais , Humanos , Cinética , Hibridização de Ácido Nucleico
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