1,721,274 research outputs found
Biological activity and delivery of peptide nucleic acids (PNA)-DNA chimeras for transcription factor decoy (TFD) pharmacotherapy
No full textPeptide nucleic acids (PNAs) are recently described DNA mimics, in which the sugar-phosphate backbone is replaced by N-(2-aminoethyl)glycine units. These molecules efficiently hybridize with complementary DNA, forming Watson-Crick double helices. In addition, the interest of PNAs and PNA-based analogs is related to the fact that they are resistant to DNases and proteinases. While applications of PNAs as antisense and antigéne molecules in non-viral gene therapy are well documented, their effects as potential transcription factor decoy (TFD) molecules is not demonstrated. PNA/PNA and PNA/DNA duplex are not suitable for TFD. In fact, PNA/PNA duplex does not recognize transcription factors, while, in the case of PNA/DNA hybrids containing nuclear factor binding sites, the interaction with transcription factors is unstable. By sharp contrast, double stranded molecules based on PNA-DNA chimeras exhibit strong TFD activity, display enzymatic stability in serum and cellular extracts and can be delivered to target cells after complexation with liposomes and microspheres. The TFD molecules based on PNA-DNA chimeras can be further engineered by addition of short peptides facilitating cell penetration and nuclear localization. Therefore, these engineered molecules could be of great interest for in vivo experiments on non-viral gene therapy of a variety of diseases, including neoplastic and viral diseases, for which the TFD approach has been already demonstrated as a very useful strategy
The genotype selection in thalassemia: from the basic genetic defect to HbF associated polymorphisms
No full textThe malaria-protective β-globin polymorphisms (causing sickle-cell anemia and β0-thalassaemia) are canonical examples of human adaptation to infectious disease. Accordingly, geographic distribution of β-thalassemia carriers is related with the distribution of malaria. On the other hand, clinical observations show that increased levels of fetal hemoglobin (HbF) can ameliorate the severity of these disorders. High HbF levels are associated with regulation of the the biological activity of several transcription repressors, including MYB, BCL11A, Oct-1, KLF1, LYAR. Accordingly, a second-level of selection of certain genotypes/phenotypes might be associated with high HbF levels, leading to lower mortality, especially in the absence of a optimal clinical management of the β-thalassemia patients. In a recent paper we studied a polymorphism of the Aγ-globin gene in four families with β0-thalassemia (β0-IVSII-1 and β0-IVSI-1) and expressing unusual high HbF levels, congenital or acquired after rejection of bone marrow transplantation (1). This (G→A) polymorphism is located at position +25 of the Aγ-globin genes, a region belonging to a sequence recognized by DNA-binding protein complexes, including LYAR (Ly-1 antibody reactive clone), a zinc-finger transcription factor previously proposed to be involved in down-regulation of the expression of γ-globin genes in erythroid cells. We demonstrated that the (G→A) polymorphism decreases the efficiency of LYAR/DNA interactions. In a more recent study we report the sequencing of Aγ-globin genes performed on genomic DNA isolated from a total of 75 β-thalassemia patients, including 31 β039/β039, 33 β039/β+IVSI-110, 9 β+IVSI-110/β+IVSI-110, one β0IVSI-1/β+IVSI-6 and one β039/β+IVSI-6. The major conclusions of this study are the following: (a) the Aγ(+25 G->A) polymorphism is associated with Gγ-globin-XmnI polymorphism; (b) the Aγ(+25 G->A) and Gγ-globin-XmnI polymorphisms are linked with β039-globin gene, but not with β+IVSI-110-globin gene. The finding that this and the XmnI polymorphisms are linked to the β039-globin gene suggests that this genetic feature has been selected in populations during a time period in which the probability to survive was not associated with the optimal clinical management, but rather to high HbF production
MicroRNA Therapeutics in CF: Targeting CFTR and inflammation networks (MICRORNA-CF)”
No full textIt is clear from numerous reports to date that the involvement of miRNA in the pathology of CF plays a major role. The specific research question here however is a little unclear as the proposal appears to ask two different research questions, firstly, (Tasks 1-4) the aim is to examine pro inflammatory miRNA and their targets with a view to managing inflammatory associated tissue destruction in CF and secondly (Tasks 5-8) is to explore miRNA and CFTR function. The investigators try to link these two aims however the project is very ambitious for a two year period. It could easily be separated into two different proposals. If delivered upon though it will greatly extend the current body of work in this area providing new leads for the management of IL-8 induced inflammation in CF known to potentiate tissue destruction in this condition. The use of PNA’s is a novel approach and may indeed uncover potential therapeutic targets.
The objectives of this proposal MICRORNA-CF are listed as: (a) identification of microRNAs which are either down- or up-regulated in association with P.aeruginosa infection of CF bronchial epithelial cells; (b) identification and validation of the most relevant mRNA targets of the modulated microRNAs; (c) study of changes of the gene expression and secretome profile of CF using PNAs targeting microRNAs; (d) development and biological validation of novel PNA-based molecules interfering with microRNAs involved in inflammation; (e) development of PNA-based antagomiRNAs for CFTR stabilization. Overall it is a really nice project and worthwhile to add to what is currently known about miRNA in the progression of CF and the battle to find new ways to treat this condition but I’m cautious as to the amount of work to be performed in a two year period. Nonetheless the investigators have a proven track record and with their team of collaborators with the experience detailed this may be possible. The use of PNA’s and tasks 5-7 in the experimental plan is very interesting especially the planned testing with CFTR correctors.
Contingency plans should be in place for the possibility that miRNA and their targets identified in immortilised cell lines may not be the same as those observed in primary cells. Transfection of polarised cells can be a difficult process as the cell density required to maintain TEER are very unfavourable for transfection of pre and antimiR’s, this may not be the case for PNA’s however. Again results on polarised cells may not exactly reflect those observed in monolayers yet this is an important part of the experimental plan. If the investigators could gain access to clinical samples it would greatly strengthen their work. I’m not sure about the primary cell model – is this a mix of normal and CF bronchial epithelial cells?
Task 3 (experimental plan) – consideration should also be given to those miRs decreased by PA01 treatment as subsequent increased expression of their targets may be pro-inflammatory
Peptide-nucleic acids (PNAs): A tool for the development of gene expression modifiers
No full textPeptide nucleic acids (PNAs) represent nucleic acid analogues with unique biochemical properties and of great interest for the development of therapeutic agents. The firstly designed and tested PNAs are molecules in which the sugar-phosphate backbone of DNA was replaced with a pseudopeptide chain constituted by N-(2-aminoethyl) glycine monomers. Nucleobases can be linked to this backbone through a carboxymethyl moiety, which allows to maintain a two atom spacer between the backbone and the bases. Since the first reports on PNAs based on N-(2-aminoethyl) glycine backbone, other PNA analogues have been synthesized, with the main purpose of improve biological activities as well as stability and efficient delivery to target cells. Of great interest are chiral PNAs, PNA analogues bearing phosphate groups (PHONA), PNA-DNA and PNA-peptide chimeras, PNA linked to non-peptide vectors. PNAs hybridize to DNA and RNA with high efficiency following the Watson-Crick hybridization rules, forming highly stable PNA/DNA and PNA/RNA duplexes. In addition, homopyrimidine PNAs, as well as PNAs containing a high pyrimidine:purine ratio, are able to bind to DNA or RNA forming highly stable (PNA)(2)-DNA triple helices. Accordingly, therapeutic PNA and PNA analogues could act as antigéne as well as antisense molecules. In addition, recent studies provide evidences for the possible use of PNA-based therapeutic molecules as artificial promoters, as decoy or ribozyme facilitator. Among the therapeutic applications of PNA-based molecules, the most pomising include anti-cancer and anti-viral experimental strategies, but activity of PNAs against bacteria and medically important parasitic organisms have been also reported
Design and synthesis of improved analogs of trimethylangelicin (TMA) for personalized treatment of cystic fibrosis
No full textDesign, synthesis and biological evaluation of a library of TMA analogs were undertaken to identify a lead compound with optimized properties in respect to the parental TMA. Key tasks were to study the different properties of TMA analogs; to test photoreactivity and mutagenic properties; finally to derive structure-activity relationships aimed at rationalizing the structural determinants required to obtain the specific activities. Reserchers carried out comparative analyses of more than 40 newly synthesized TMA analogs and concluded that anti-inflammatory, CFTR potentiator and CFTR corrector activities can be separately obtained in TMA analogues. TMA analogues were identified displaying low or absent antinflammatory effects, but maintaining, and even increasing, the function of CFTR correctors. CFTR correctors were found with no mutagenic effects and without DNA photodamaging activities. Novel TMA analogues are proposed for preclinical studies aimed at the development of protocols for personalized therapy of cystic fibrosis
Identification of dysregulated miRNAs in liquid biopsies from colorectal cancer (CRC) patients: impact in personalized miRNA therapeutics
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A new automated method for isolation of genomic DNA from eukaryotic cells.
No full textIn this study we determine whether the BIOMEK-1000 Laboratory Workstation (Beckman Instruments, Fullerton, CA) can be used to isolate genomic DNA from eukaryotic cells. The results obtained demonstrate that DNA isolated by BIOMEK-1000 is suitable for amplification of genomic sequences by PCR. Our data prove that automated chromatographic DNA isolation employing the BIOMEK-1000 could be used in molecular diagnosis of human pathologies
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