RESUMO
Thrombotic microangiopathy (TMA) is an emerging complication of oncologic therapy. Cancer-related causes of renal endothelial cell damage include cytotoxic chemotherapies, radiation given for myeloablation, and direct involvement of renal vasculature by tumor cells. Another class of therapeutic agents that has been implicated in TMA is the vascular endothelial growth factor (VEGF) pathway inhibitors, including the anti-VEGF monoclonal antibody bevacizumab and the VEGF receptor tyrosine kinase inhibitor sunitinib. These TMAs have been termed type II cancer drug-induced TMA and are distinguished from those associated with some cytotoxic chemotherapies (ie, type I) in that they are not dose dependent and patients are more likely to demonstrate some recovery of kidney function. Determination of the cause of TMA in oncologic patients often presents a significant challenge because patients frequently receive multiple chemotherapeutic agents simultaneously and clinicopathologic features often demonstrate substantial overlap, regardless of cause. We present a case of TMA with predominantly chronic features in a 70-year-old patient being treated for adenoid cystic carcinoma of the breast with a single agent, a short interfering RNA targeted against Myc (DCR-MYC).
Assuntos
Proteínas Proto-Oncogênicas c-myc/genética , RNA Interferente Pequeno/efeitos adversos , RNA Interferente Pequeno/genética , Microangiopatias Trombóticas/induzido quimicamente , Microangiopatias Trombóticas/genética , Idoso , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Doença Crônica , Sistemas de Liberação de Medicamentos/efeitos adversos , Sistemas de Liberação de Medicamentos/métodos , Evolução Fatal , Feminino , Marcação de Genes/efeitos adversos , Marcação de Genes/métodos , Humanos , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , Microangiopatias Trombóticas/diagnósticoRESUMO
Conditional gene targeting using Cre recombinase has offered a powerful tool to modify gene function precisely in defined cells/tissues and at specific times. However, in mammalian cells, Cre recombinase can be genotoxic. The importance of including Cre-expressing control mice to avoid misinterpretation and to maximize the validity of the experimental results has been increasingly recognized. While studying the role of podocytes in the pathogenesis of glomerular basement membrane (GBM) thickening, we used Cre recombinase driven by the podocyte-specific podocin promoter (NPHS2-Cre) to generate a conditional knockout. By conventional structural and functional measures (histology by periodic acid-Schiff staining, albuminuria, and plasma creatinine), we did not detect significant differences between NPHS2-Cre transgenic and wild-type control mice. However, surprisingly, the group that expressed Cre transgene alone developed signs of podocyte toxicity, including marked GBM thickening, loss of normal foot process morphology, and reduced Wilms tumor 1 expression. GBM thickening was characterized by altered expression of core structural protein laminin isoform α5ß2γ1. RNA sequencing analysis of extracted glomeruli identified 230 genes that were significant and differentially expressed (applying a q < 0.05-fold change ≥ ±2 cutoff) in NPHS2-Cre mice compared with wild-type control mice. Many biological processes were reflected in the RNA sequencing data, including regulation of the extracellular matrix and pathways related to apoptosis and cell death. This study highlights the importance of including the appropriate controls for potential Cre-mediated toxicity in conditional gene-targeting experiments. Indeed, omitting the Cre transgene control can result in critical errors during interpretation of experimental data.
Assuntos
Marcação de Genes/efeitos adversos , Membrana Basal Glomerular/enzimologia , Integrases/metabolismo , Podócitos/enzimologia , Animais , Regulação da Expressão Gênica , Membrana Basal Glomerular/ultraestrutura , Integrases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Laminina/genética , Laminina/metabolismo , Proteínas de Membrana/genética , Camundongos Transgênicos , Podócitos/ultraestrutura , Regiões Promotoras Genéticas , Fatores de Tempo , Proteínas WT1/genética , Proteínas WT1/metabolismoRESUMO
The CRISPR/Cas9 system provides a revolutionary genome editing tool for all areas of molecular biology. In long non-coding RNA (lncRNA) research, the Cas9 nuclease can delete lncRNA genes or introduce RNA-destabilizing elements into their locus. The nuclease-deficient dCas9 mutant retains its RNA-dependent DNA-binding activity and can modulate gene expression when fused to transcriptional repressor or activator domains. Here, we systematically analyze whether CRISPR approaches are suitable to target lncRNAs. Many lncRNAs are derived from bidirectional promoters or overlap with promoters or bodies of sense or antisense genes. In a genome-wide analysis, we find only 38% of 15929 lncRNA loci are safely amenable to CRISPR applications while almost two-thirds of lncRNA loci are at risk to inadvertently deregulate neighboring genes. CRISPR- but not siPOOL or Antisense Oligo (ASO)-mediated targeting of lncRNAs NOP14-AS1, LOC389641, MNX1-AS1 or HOTAIR also affects their respective neighboring genes. Frequently overlooked, the same restrictions may apply to mRNAs. For example, the tumor suppressor TP53 and its head-to-head neighbor WRAP53 are jointly affected by the same sgRNAs but not siPOOLs. Hence, despite the advantages of CRISPR/Cas9 to modulate expression bidirectionally and in cis, approaches based on ASOs or siPOOLs may be the better choice to target specifically the transcript from complex loci.
Assuntos
Sistemas CRISPR-Cas , RNA Longo não Codificante/genética , Linhagem Celular , Técnicas de Silenciamento de Genes/métodos , Marcação de Genes/efeitos adversos , Marcação de Genes/métodos , Genoma Humano , Células HEK293 , Células HeLa , Humanos , Modelos Genéticos , Regiões Promotoras Genéticas , Interferência de RNARESUMO
BACKGROUND: Gene therapy for inherited serum deficiency disorders has previously been limited by the balance between obtaining adequate expression and causing hepatic toxicity. Our group has previously described modifications of a replication deficient human adenovirus serotype 5 that increase pulmonary vasculature transgene expression. METHODS: In the present study, we use a modified pulmonary targeted adenovirus to express human alpha-1 antitrypsin (A1AT) in C57BL/6 J mice. RESULTS: Using the targeted adenovirus, we were able to achieve similar increases in serum A1AT levels with less liver viral uptake. We also increased pulmonary epithelial lining fluid A1AT levels by more than an order of magnitude compared to that of untargeted adenovirus expressing A1AT in a mouse model. These gains are achieved along with evidence of decreased systemic inflammation and no evidence for increased inflammation within the vector-targeted end organ. CONCLUSIONS: In addition to comprising a step towards clinically viable gene therapy for A1AT, maximization of protein production at the site of action represents a significant technical advancement in the field of systemically delivered pulmonary targeted gene therapy. It also provides an alternative to the previous limitations of hepatic viral transduction and associated toxicities.
Assuntos
Endotélio Vascular/metabolismo , Marcação de Genes/métodos , Terapia Genética/métodos , Pulmão/irrigação sanguínea , Deficiência de alfa 1-Antitripsina/terapia , alfa 1-Antitripsina/genética , Adenovírus Humanos , Animais , Modelos Animais de Doenças , Feminino , Marcação de Genes/efeitos adversos , Técnicas de Transferência de Genes/efeitos adversos , Vetores Genéticos/efeitos adversos , Células HEK293 , Humanos , Inflamação/etiologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transgenes , alfa 1-Antitripsina/sangueRESUMO
Endogenous microRNA (miRNA) expression can be exploited for cell type-specific transgene expression as the addition of miRNA target sequences to transgenic cDNA allows for transgene downregulation specifically in cells expressing the respective miRNAs. Here, we have investigated the potential of miRNA-150 target sequences to specifically suppress gene expression in lymphocytes and thereby prevent transgene-induced lymphotoxicity. Abundance of miRNA-150 expression specifically in differentiated B and T cells was confirmed by quantitative reverse transcriptase PCR. Mono- and bicistronic lentiviral vectors were used to investigate the effect of miRNA-150 target sequences on transgene expression in the lymphohematopoietic system. After in vitro studies demonstrated effective downregulation of transgene expression in murine B220(+) B and CD3(+) T cells, the concept was further verified in a murine transplant model. Again, marked suppression of transgene activity was observed in B220(+) B and CD4(+) or CD8(+) T cells whereas expression in CD11b(+) myeloid cells, lin(-) and lin(-)/Sca1(+) progenitors, or lin(-)/Sca1(+)/c-kit(+) stem cells remained almost unaffected. No toxicity of miRNA-150 targeting in transduced lymphohematopoietic cells was noted. Thus, our results demonstrate the suitability of miRNA-150 targeting to specifically suppress transgene expression in lymphocytes and further support the concept of miRNA targeting for cell type-specific transgene expression in gene therapy approaches.
Assuntos
Linfócitos B/imunologia , Regulação para Baixo , Marcação de Genes , Vetores Genéticos , Hematopoese/genética , MicroRNAs/genética , Linfócitos T/imunologia , Animais , Linhagem Celular , Feminino , Marcação de Genes/efeitos adversos , Masculino , Camundongos , Camundongos Nus , TransgenesRESUMO
One of the most powerful techniques for studying the function of a gene is to disrupt the expression of that gene using genetic engineering strategies such as targeted recombination or viral integration of gene trap cassettes. The tremendous utility of these tools was recognized this year with the awarding of the Nobel Prize in Physiology or Medicine to Capecchi, Evans, and Smithies for their pioneering work in targeted recombination mutagenesis in mammals. Another noteworthy discovery made nearly a decade ago was the identification of a novel class of non-coding genes called microRNAs. MicroRNAs are among the largest known classes of regulatory elements with more than 1000 predicted to exist in the mouse genome. Over 50% of known microRNAs are located within introns of coding genes. Given that currently about half of the genes in mouse have been knocked out, we investigated the possibility that intronic microRNAs may have been coincidentally deleted or disrupted in some of these mouse models. We searched published murine knockout studies and gene trap embryonic stem cell line databases for cases where a microRNA was located within or near the manipulated genomic loci, finding almost 200 cases where microRNA expression may have been disrupted along with another gene. Our results draw attention to the need for careful planning in future knockout studies to minimize the unintentional disruption of microRNAs. These data also raise the possibility that many knockout studies may need to be reexamined to determine if loss of a microRNA contributes to the phenotypic consequences attributed to loss of a protein-encoding gene.
Assuntos
Marcação de Genes/efeitos adversos , MicroRNAs/genética , Animais , Linhagem Celular , Bases de Dados de Ácidos Nucleicos , Expressão Gênica , Íntrons , Camundongos , Camundongos Knockout , FenótipoRESUMO
In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However, there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins. These GE strategies should take into account not only the specificity of the nucleases, but also the fidelity of the DNA donor to carry out their function. The current methods to quantify the fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations. In this work, we have engineered two reporter cell lines (K562_SEWAS84 and K562GWP) that efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS-locus targeting setting. K562_SEWAS84 cells allow the detection of both HDR-and HITI-based donor integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy and specificity of DNA donor-based GE strategies. By using these models, we have found that the specificity of HDR is independent of the delivery method and that the insertion of the target sequence into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that the higher the number of the target sites is, the higher the specificity and efficacy of GE will be.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Recombinação Homóloga , Modelos Genéticos , DNA Recombinante/genética , Marcação de Genes/efeitos adversos , Marcação de Genes/métodos , Genes Reporter , Engenharia Genética , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos , Humanos , Células K562 , Lentivirus/genética , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR), a class of immune-associated sequences in bacteria, have been developed as a powerful tool for editing eukaryotic genomes in diverse cells and organisms in recent years. The CRISPR-Cas9 system can recognize upstream 20 nucleotides (guide sequence) adjacent to the protospacer-adjacent motif site and trigger double-stranded DNA cleavage as well as DNA repair mechanisms, which eventually result in knockout, knockin, or site-specific mutagenesis. However, off-target effect caused by guide sequence misrecognition is the major drawback and restricts its widespread application. In this study, global analysis of specificities of all guide sequences in Arabidopsis thaliana, Oryza sativa (rice), and Glycine max (soybean) were performed. As a result, a simple pipeline and three genome-wide databases were established and shared for the scientific society. For each target site of CRISPR-Cas9, specificity score and off-target number were calculated and evaluated. The mean values of off-target numbers for A. thaliana, rice, and soybean were determined as 27.5, 57.3, and 174.7, respectively. Comparative analysis among these plants suggested that the frequency of off-target effects was correlated to genome size, chromosomal locus, gene density, and guanine-cytosine (GC) content. Our results contributed to the better understanding of CRISPR-Cas9 system in plants and would help to minimize the off-target effect during its applications in the future.
Assuntos
Arabidopsis/genética , Sistemas CRISPR-Cas , Glycine max/genética , Oryza/genética , Biologia Computacional , Edição de Genes/métodos , Marcação de Genes/efeitos adversos , Marcação de Genes/métodos , Genoma de Planta , Melhoramento Vegetal/métodos , Plantas Geneticamente Modificadas/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Gene transfer has long been a compelling yet elusive therapeutic modality. First mainly considered for rare inherited disorders, gene therapy may open treatment opportunities for more challenging and complex diseases such as Alzheimer's or Parkinson's disease. Today, examples of striking clinical proof of concept, the first gene therapy drugs coming onto the market, and the emergence of powerful new molecular tools have broadened the number of avenues to target neurological disorders but have also highlighted safety concerns and technology gaps. The vector of choice for many nervous system targets currently is the adeno-associated viral (AAV) vector due to its desirable safety profile and strong neuronal tropism. In aggregate, the clinical success, the preclinical potential, and the technological innovation have made therapeutic AAV drug development a reality, particularly for nervous system disorders. Here, we discuss the rationale, opportunities, limitations, and progress in clinical AAV gene therapy.
Assuntos
Doença de Alzheimer/terapia , Marcação de Genes/métodos , Terapia Genética/métodos , Doença de Parkinson/terapia , Animais , Dependovirus/genética , Marcação de Genes/efeitos adversos , Terapia Genética/efeitos adversos , HumanosAssuntos
Erupções Acneiformes/induzido quimicamente , Erupções Acneiformes/diagnóstico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Marcação de Genes/efeitos adversos , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Camptotecina/administração & dosagem , Camptotecina/efeitos adversos , Camptotecina/análogos & derivados , Cetuximab , Humanos , Irinotecano , Masculino , Pessoa de Meia-IdadeRESUMO
CRISPR/Cas9 is a recent development in genome editing which is becoming an indispensable element of the genetic toolbox in mice. It provides outstanding possibilities for targeted modification of the genome, and is often extremely efficient. There are currently two main limitations to in ovo genome editing in mice: the first is mosaicism, which is frequent in founder mice. The second is the difficulty to evaluate the advent of off-target mutations, which often imposes to wait for germline transmission to ensure genetic segregation between wanted and unwanted genetic mutations. However rapid progresses are made, suggesting that these difficulties can be overcome in the near future.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Camundongos Transgênicos , Animais , Reparo do DNA por Junção de Extremidades , Modelos Animais de Doenças , Endocrinologistas , Endocrinologia/métodos , Edição de Genes/métodos , Marcação de Genes/efeitos adversos , Marcação de Genes/métodos , Camundongos , Mosaicismo , Oócitos/metabolismo , RNA Guia de Cinetoplastídeos/genética , Reparo de DNA por RecombinaçãoRESUMO
BACKGROUND: Gene delivery in vivo that is tightly focused on the intended target cells is essential to maximize the benefits of gene therapy and to reduce unwanted side-effects. Cell surface markers are immediately available for probing by therapeutic gene vectors and are often used to direct gene transfer with these vectors to specific target cell populations. However, it is not unusual for the choice of available extra-cellular markers to be too scarce to provide a reliable definition of the desired therapeutically relevant set of target cells. Therefore, interrogation of intra-cellular determinants of cell-specificity, such as tissue-specific transcription factors, can be vital in order to provide detailed cell-guiding information to gene vector particles. An important improvement in cell-specific gene delivery can be achieved through auto-buildup in vector homing efficiency using intelligent 'self-focusing' of swarms of vector particles on target cells. Vector self-focusing was previously suggested to rely on the release of diffusible chemo-attractants after a successful target-specific hit by 'scout' vector particles. HYPOTHESIS: I hypothesize that intelligent self-focusing behaviour of swarms of cell-targeted therapeutic gene vectors can be accomplished without the employment of difficult-to-use diffusible chemo-attractants, instead relying on the intra-swarm signalling through cells expressing a non-diffusible extra-cellular receptor for the gene vectors. In the proposed model, cell-guiding information is gathered by the 'scout' gene vector particles, which: (1) attach to a variety of cells via a weakly binding (low affinity) receptor; (2) successfully facilitate gene transfer into these cells; (3) query intra-cellular determinants of cell-specificity with their transgene expression control elements and (4) direct the cell-specific biosynthesis of a vector-encoded strongly binding (high affinity) cell-surface receptor. Free members of the vector swarm loaded with therapeutic cargo are then attracted to and internalized into the intended target cells via the expressed cognate strongly binding extra-cellular receptor, causing escalation of gene transfer into these cells and increasing the copy number of the therapeutic gene expression modules. Such self-focusing swarms of gene vectors can be either homogeneous, with 'scout' and 'therapeutic' members of the swarm being structurally identical, or, alternatively, heterogeneous (split), with 'scout' and 'therapeutic' members of the swarm being structurally specialized. CONCLUSIONS: It is hoped that the proposed self-focusing cell-targeted gene vector swarms with receptor-mediated intra-swarm signalling could be particularly effective in 'top-up' gene delivery scenarios, achieving high-level and sustained expression of therapeutic transgenes that are prone to shut-down through degradation and silencing. Crucially, in contrast to low-precision 'general location' vector guidance by diffusible chemo-attractants, ear-marking non-diffusible receptors can provide high-accuracy targeting of therapeutic vector particles to the specific cell, which has undergone a 'successful cell-specific hit' by a 'scout' vector particle. Opportunities for cell targeting could be expanded, since in the proposed model of self-focusing it could be possible to probe a broad selection of intra-cellular determinants of cell-specificity and not just to rely exclusively on extra-cellular markers of cell-specificity. By employing such self-focusing gene vectors for the improvement of cell-targeted delivery of therapeutic genes, e.g., in cancer therapy or gene addition therapy of recessive genetic diseases, it could be possible to broaden a leeway for the reduction of the vector load and, consequently, to minimize undesired vector cytotoxicity, immune reactions, and the risk of inadvertent genetic modification of germline cells in genetic treatment in vivo.
Assuntos
Marcação de Genes/métodos , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Nanomedicina/métodos , Transdução de Sinais/genética , Transgenes , Animais , Regulação da Expressão Gênica , Marcação de Genes/efeitos adversos , Técnicas de Transferência de Genes/efeitos adversos , Terapia Genética/efeitos adversos , Humanos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Water-soluble chitosan (WSC) was used to increase the stability of chitosan in water and decrease the cytotoxicity induced by acetic acid. Lactobionic acid (LA) bearing galactose group was coupled with WSC for hepatocytes specificity. The composition of galactose in galactosylated chitosan (GC) was determined by NMR spectroscopy. The GC was complexed with plasmid DNA in various GC/DNA (N/P) charge ratios and the resulting complex was characterized by dynamic light scattering, gel retardation, and turbidity to determine the particle sizes, complex formation, and complex stability, respectively. Cytotoxicity and transfection efficiency of GC were also studied in cultured HepG2 human hepatoblastoma cell line and HeLa human cervix epithelial carcinoma cells. The complete GC/DNA complex was formed at the charge ratio of 5 and the GC/DNA complex to DNase I resistance was obtained. Particle sizes decreased with increasing charge ratio of GC to DNA and had a minimum value around 120 nm at the charge ratio of 5. And no significant difference in particle sizes from the charge ratio of 5-20 was found. The suspension of GC/DNA complexes exhibited no significant change in turbidity at the charge ratios of 10, indicating the complete shielding of DNA charge. Cytotoxicity study showed that GC prepared by the water-soluble chitosan had no cytotoxic effects on cells. And transfection efficiency into HepG2, which has asialoglycoprotein receptors (ASGP-R), was higher than that into HeLa without ASGP-R.
Assuntos
Quitina/análogos & derivados , Quitina/química , DNA/administração & dosagem , DNA/química , Portadores de Fármacos/química , Marcação de Genes/métodos , Nanotubos/química , Transfecção/métodos , Absorção , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Quitosana , Portadores de Fármacos/efeitos adversos , Sistemas de Liberação de Medicamentos/efeitos adversos , Sistemas de Liberação de Medicamentos/métodos , Avaliação de Medicamentos , Estabilidade de Medicamentos , Galactose/química , Marcação de Genes/efeitos adversos , Terapia Genética/métodos , Células HeLa , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Nanotubos/efeitos adversos , Nanotubos/ultraestrutura , Tamanho da Partícula , Solubilidade , Água/químicaAssuntos
Ansiedade/genética , Interações Medicamentosas/genética , Tolerância a Medicamentos/genética , Marcação de Genes/efeitos adversos , Camundongos Knockout/genética , Camundongos Knockout/psicologia , Mutação/fisiologia , Animais , Ansiedade/metabolismo , Ansiedade/fisiopatologia , Marcação de Genes/métodos , Camundongos , Camundongos Knockout/metabolismo , Fenótipo , Psicotrópicos/efeitos adversosAssuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/genética , Marcação de Genes , Doenças Genéticas Inatas/terapia , Engenharia Genética , Células Germinativas , Sistemas CRISPR-Cas , Marcação de Genes/efeitos adversos , Marcação de Genes/ética , Marcação de Genes/métodos , Doenças Genéticas Inatas/genética , Engenharia Genética/efeitos adversos , Engenharia Genética/ética , Engenharia Genética/métodos , Mutação em Linhagem Germinativa , HumanosRESUMO
RNA interference (RNAi) can mediate the long- or short-term silencing of gene expression at the DNA, RNA, and/or protein level. Although several triggers of RNAi have been identified, the best characterized of these are small interfering RNAs (siRNAs), which can decrease gene expression through mRNA transcript cleavage, and endogenous microRNAs (miRNAs), which primarily inhibit protein translation. An improved understanding of RNAi has provided new, powerful tools for conducting functional studies in a gene-specific manner. In various applications, RNAi has been used to create model systems, to identify novel molecular targets, to study gene function in a genome-wide fashion, and to create new avenues for clinical therapeutics. Here, we review many of the ongoing applications of RNAi in mammalian and human systems, and discuss how advances in our knowledge of the RNAi machinery have enhanced the use of these technologies.
Assuntos
Mamíferos/genética , Interferência de RNA/fisiologia , Animais , Antivirais/uso terapêutico , Sequência de Bases/fisiologia , Marcação de Genes/efeitos adversos , Marcação de Genes/métodos , Terapia Genética/métodos , Humanos , Sistema Imunitário/fisiologia , Hepatopatias/terapia , Doenças Metabólicas/terapia , MicroRNAs/fisiologia , Modelos Biológicos , Neoplasias/terapia , Doenças do Sistema Nervoso/terapia , Especificidade de Órgãos , RNA Interferente Pequeno/genéticaAssuntos
Terapia Genética/métodos , Terapia Genética/tendências , Doenças Raras/terapia , Animais , Transplante de Células/métodos , Marcação de Genes/efeitos adversos , Marcação de Genes/métodos , Vetores Genéticos/efeitos adversos , Vetores Genéticos/uso terapêutico , Humanos , Doenças Raras/genética , Pesquisa/tendênciasRESUMO
The huge amount of antibiotic substances released in the human environment has probably resulted in an acceleration in the rate of bacterial evolution. It is to note that most interactions between chemotherapeutic agents and microbial populations occur at very low antibiotic concentrations. Thus, natural selection is expected to act on very small increases in the bacterial ability to resist to antibiotic inhibitory effects. On the other hand, there is a wealth of mechanisms to resist to these low antibiotic concentrations. The progressive enrichment in low-level resistant populations favours secondary selections for more specific and effective mechanisms of resistance, particularly in treated patients. These adaptations may have a biological cost in the absence of antibiotics, but frequently compensatory mutations occur, minimizing such genetic burden. In this way, a phenomenon of directional selection takes place, with low possibilities of return to susceptibility. Moreover, low antibiotic concentrations are not only able to select low-level antibiotic resistant variants, but may produce a substantial stress in bacterial populations, that eventually influences the rate of genetic variation and the diversity of adaptive responses. More attention should be devoted to the mechanisms of low-level resistance in microorganisms, as they can serve as stepping stones to develop high level, clinically relevant resistance. These mechanisms should be identified early in the development of drugs in order to adapt the therapeutic strategies (for instance dosage) to minimize the selection of low-level resistant variants, as frequently they emerge by means of concentration-specific selection. At the same time, conventional susceptibility testing should probably be able to detect low-level resistance, and not only clinically-relevant resistance. We should be vigilant of the evolutionary trends of microorganisms; for that a purpose, knowledge of the biology and epidemiology of low-level resistance is becoming a real need.