Advanced therapies for the treatment of hemophilia: future perspectives

Monogenic diseases are ideal candidates for treatment by the emerging advanced therapies, which are capable of correcting alterations in protein expression that result from genetic mutation. In hemophilia A and B such alterations affect the activity of coagulation factors VIII and IX, respectively, and are responsible for the development of the disease. Advanced therapies may involve the replacement of a deficient gene by a healthy gene so that it generates a certain functional,structural or transport protein (gene therapy); the incorporation of a full array of healthy genes and proteins through perfusion or transplantation of healthy cells (cell therapy); or tissue transplantation and formation of healthy organs (tissue engineering). For their part, induced pluripotent stem cells have recently been shown to also play a significant role in the fields of cell therapy and tissue engineering.Hemophilia is optimally suited for advanced therapies owing to the fact that, as a monogenic condition, it does not require very high expression levels of a coagulation factor to reach moderate disease status. As a result, significant progress has been possible with respect to these kinds of strategies, especially in the fields of gene therapy (by using viral and non-viral vectors) and cell therapy (by means of several types of target cells). Thus, although still considered a rare disorder, hemophilia is now recognized as a condition amenable to gene therapy, which can be administered in the form of lentiviral and adeno-associated vectors applied to adult stem cells, autologous fibroblasts, platelets and hematopoietic stem cells; by means of non-viral vectors; or through the repair of mutations by chimeric oligonucleotides. In hemophilia, cell therapy approaches have been based mainly on transplantation of healthy cells (adult stem cells or induced pluripotent cell-derived progenitor cells)in order to restore alterations in coagulation factor expression

Induced Pluripotent Stem Cells: Therapeutic Applications in Monogenic and Metabolic Diseases, and Regulatory and Bioethical Considerations

Depto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasTRUEpu

A pharmaceutical model for the molecular evolution of microbial natural products

Abstract Microbes are talented chemists with the ability to generate tremendously complex and diverse natural products which harbor potent biological activities. Natural products are produced using sets of specialized biosynthetic enzymes encoded by secondary metabolism pathways. Here, we present a two-step evolutionary model to explain the diversification of biosynthetic pathways that account for the proliferation of these molecules. We argue that the appearance of natural product families has been a slow and infrequent process. The first step led to the original emergence of bioactive molecules and different classes of natural products. However, much of the chemical diversity observed today has resulted from the endless modification of the ancestral biosynthetic pathways. The second step rapidly modulates the pre-existing biological activities to increase their potency and to adapt to changing environmental conditions. We highlight the importance of enzyme promiscuity in this process, as it facilitates both the incorporation of horizontally transferred genes into secondary metabolic pathways and the functional differentiation of proteins to catalyze novel chemistry. We provide examples where single point mutations or recombination events have been sufficient for new enzymatic activities to emerge. A unique feature in the evolution of microbial secondary metabolism is that gene duplication is not essential but offers opportunities to synthesize more complex metabolites. Microbial natural products are highly important for the pharmaceutical industry due to their unique bioactivities. Therefore, understanding the natural mechanisms leading to the formation of diverse metabolic pathways is vital for future attempts to utilize synthetic biology for the generation of novel molecules.Peer reviewe

Molecular genetics of naringenin biosynthesis, a typical plant secondary metabolite produced by Streptomyces clavuligerus

Background: Some types of flavonoid intermediates seemed to be restricted to plants. Naringenin is a typical plant metabolite, that has never been reported to be produced in prokariotes. Naringenin is formed by the action of a chalcone synthase using as starter 4-coumaroyl-CoA, which in dicotyledonous plants derives from phenylalanine by the action of a phenylalanine ammonia lyase. Results: A compound produced by Streptomyces clavuligerus has been identified by LC-MS and NMR as naringenin and coelutes in HPLC with a naringenin standard. Genome mining of S. clavuligerus revealed the presence of a gene for a chalcone synthase (ncs), side by side to a gene encoding a P450 cytochrome (ncyP) and separated from a gene encoding a Pal/Tal ammonia lyase (tal). Deletion of any of these genes results in naringenin non producer mutants. Complementation with the deleted gene restores naringenin production in the transformants. Furthermore, naringenin production increases in cultures supplemented with phenylalanine or tyrosine. Conclusion: This is the first time that naringenin is reported to be produced naturally in a prokariote. Interestingly three non-clustered genes are involved in naringenin production, which is unusual for secondary metabolites. A tentative pathway for naringenin biosynthesis has been proposedThis work was supported by Grant BIO2012-34723 from the Spanish Ministry of Economy and Competitivity. R. Álvarez-Álvarez received a FPU fellowship from the Spanish Ministry of Education, Culture and Sport

Mycobacterial Phylogenomics: An Enhanced Method for Gene Turnover Analysis Reveals Uneven Levels of Gene Gain and Loss among Species and Gene Families

Species of the genus Mycobacterium differ in several features, from geographic ranges, and degree of pathogenicity, to ecological and host preferences. The recent availability of several fully sequenced genomes for a number of these species enabled the comparative study of the genetic determinants of this wide lifestyle diversity. Here, we applied two complementary phylogenetic-based approaches using information from 19 Mycobacterium genomes to obtain a more comprehensive view of the evolution of this genus. First, we inferred the phylogenetic relationships using two new approaches, one based on a Mycobacterium-specific amino acid substitution matrix and the other on a gene content dissimilarity matrix. Then, we utilized our recently developed gain-and-death stochastic models to study gene turnover dynamics in this genus in a maximum-likelihood framework. We uncovered a scenario that differs markedly from traditional 16S rRNA data and improves upon recent phylogenomic approaches. We also found that the rates of gene gain and death are high and unevenly distributed both across species and across gene families, further supporting the utility of the new models of rate heterogeneity applied in a phylogenetic context. Finally, the functional annotation of the most expanded or contracted gene families revealed that the transposable elements and the fatty acid metabolism-related gene families are the most important drivers of gene content evolution in Mycobacterium

On-resin N-methylation of cyclic peptides for discovery of orally bioavailable scaffolds.

Backbone N-methylation is common among peptide natural products and has a substantial impact on both the physical properties and the conformational states of cyclic peptides. However, the specific impact of N-methylation on passive membrane diffusion in cyclic peptides has not been investigated systematically. Here we report a method for the selective, on-resin N-methylation of cyclic peptides to generate compounds with drug-like membrane permeability and oral bioavailability. The selectivity and degree of N-methylation of the cyclic peptide was dependent on backbone stereochemistry, suggesting that conformation dictates the regiochemistry of the N-methylation reaction. The permeabilities of the N-methyl variants were corroborated by computational studies on a 1,024-member virtual library of N-methyl cyclic peptides. One of the most permeable compounds, a cyclic hexapeptide (molecular mass = 755 Da) with three N-methyl groups, showed an oral bioavailability of 28% in rat

The relationship between target joints and direct resource use in severe haemophilia

Objectives Target joints are a common complication of severe haemophilia. While factor replacement therapy constitutes the majority of costs in haemophilia, the relationship between target joints and non drug-related direct costs (NDDCs) has not been studied. Methods Data on haemophilia patients without inhibitors was drawn from the ‘Cost of Haemophilia across Europe – a Socioeconomic Survey’ (CHESS) study, a cost assessment in severe haemophilia A and B across five European countries (France, Germany, Italy, Spain, and the United Kingdom) in which 139 haemophilia specialists provided demographic and clinical information for 1285 adult patients. NDDCs were calculated using publicly available cost data, including 12-month ambulatory and secondary care activity: haematologist and other specialist consultant consultations, medical tests and examinations, bleed-related hospital admissions, and payments to professional care providers. A generalized linear model was developed to investigate the relationship between NDDCs and target joints (areas of chronic synovitis), adjusted for patient covariates. Results Five hundred and thirteen patients (42% of the sample) had no diagnosed target joints; a total of 1376 target joints (range 1–10) were recorded in the remaining 714 patients. Mean adjusted NDDCs for persons with no target joints were EUR 3134 (standard error (SE) EUR 158); for persons with one or more target joints, mean adjusted NDDCs were EUR 3913 (SE EUR 157; average mean effect EUR 779; p < 0.001). Conclusions Our analysis suggests that the presence of one or more target joints has a significant impact on NDDCs for patients with severe haemophilia, ceteris paribus. Prevention and management of target joints should be an important consideration of managing haemophilia patients

Use of Immortalized Human Hepatocytes to Predict the Magnitude of Clinical Drug-Drug Interactions Caused by CYP3A4 Induction

ABSTRACT: Cytochrome P4503A4 (CYP3A4) is the principal drug-metabolizing enzyme in human liver. Drug-drug interactions (DDIs) caused by induction of CYP3A4 can result in decreased exposure to coadministered drugs, with potential loss of efficacy. Immortalized hepatocytes (Fa2N-4 cells) have been proposed as a tool to identify CYP3A4 inducers. The purpose of the current studies was to characterize the effect of known inducers on CYP3A4 in Fa2N-4 cells, and to determine whether these in vitro data could reliably project the magnitude of DDIs caused by induction. Twenty-four compounds were chosen for these studies, based on previously published data using primary human hepatocytes. Eighteen compounds had been shown to be positive for induction, and six compounds had been shown to be negative for induction. In Cytochrome P4503A4 (CYP3A4) is the major drug-metabolizing enzyme in human liver and is responsible for the clearance of many commonly used drugs, including benzodiazepines, statins, calcium channel blockers, and HIV protease inhibitors. Certain drugs can modulate the level of CYP3A4 activity, thereby causing changes in clearance of coadministered drugs that are CYP3A4 substrates. Levels of CYP3A4 activity can be decreased by inhibition of enzyme activity, or increased by induction of new protein synthesis. Changes in CYP3A4 activity, either through inhibition or induction, can result in potentially serious drug-drug interactions (DDIs). Whereas assays for evaluating inhibition of CYP3A4 are routine and the relationship between in vitro data and in vivo effects relatively well understood Induction of CYP3A4 is thought to occur primarily through transcriptional activation of the gene. The 5Ј-regulatory region of the CYP3A4 gene contains elements that bind various transcription factors that can up-or down-regulate transcription. One such transcription factor is the pregnane X receptor (PXR). PXR is a ligand-activated transcription factor that is activated by a variety of drugs and endogenous compounds to increase transcription of CYP3A4 as well as other drug-metabolizing enzymes and transporter

The interaction of vasoactive substances during exercise modulates platelet aggregation in hypertension and coronary artery disease

<p>Abstract</p> <p>Background</p> <p>Acute vigorous exercise, associated with increased release of plasma catecholamines, transiently increases the risk of primary cardiac arrest. We tested the effect of acute submaximal exercise on vasoactive substances and their combined result on platelet function.</p> <p>Methods</p> <p>Healthy volunteers, hypertensive patients and patients with coronary artery disease (CAD) performed a modified treadmill exercise test. We determined plasma catecholamines, thromboxane A₂, prostacyclin, endothelin-1 and platelet aggregation induced by adenosine diphosphate (ADP) and collagen at rest and during exercise.</p> <p>Results</p> <p>Our results during exercise showed a) platelet activation (increased thromboxane B₂, TXB₂), b) increased prostacyclin release from endothelium and c) decreased platelet aggregation in all groups, significantly more in healthy volunteers than in patients with CAD (with hypertensives lying in between these two groups).</p> <p>Conclusion</p> <p>Despite the pronounced activation of Sympathetic Nervous System (SNS) and increased TXB₂levels during acute exercise platelet aggregation decreases, possibly to counterbalance the prothrombotic state. Since this effect seems to be mediated by the normal endothelium (through prostacyclin and nitric oxide), in conditions characterized by endothelial dysfunction (hypertension, CAD) reduced platelet aggregation is attenuated, thus posing such patients in increased risk for thrombotic complications.</p

Origin and evolution of peptide-modifying dioxygenases and identification of the wybutosine hydroxylase/hydroperoxidase

Unlike classical 2-oxoglutarate and iron-dependent dioxygenases, which include several nucleic acid modifiers, the structurally similar jumonji-related dioxygenase superfamily was only known to catalyze peptide modifications. Using comparative genomics methods, we predict that a family of jumonji-related enzymes catalyzes wybutosine hydroxylation/peroxidation at position 37 of eukaryotic tRNAPhe. Identification of this enzyme raised questions regarding the emergence of protein- and nucleic acid-modifying activities among jumonji-related domains. We addressed these with a natural classification of DSBH domains and reconstructed the precursor of the dioxygenases as a sugar-binding domain. This precursor gave rise to sugar epimerases and metal-binding sugar isomerases. The sugar isomerase active site was exapted for catalysis of oxygenation, with a radiation of these enzymes in bacteria, probably due to impetus from the primary oxygenation event in Earth’s history. 2-Oxoglutarate-dependent versions appear to have further expanded with rise of the tricarboxylic acid cycle. We identify previously under-appreciated aspects of their active site and multiple independent innovations of 2-oxoacid-binding basic residues among these superfamilies. We show that double-stranded β-helix dioxygenases diversified extensively in biosynthesis and modification of halogenated siderophores, antibiotics, peptide secondary metabolites and glycine-rich collagen-like proteins in bacteria. Jumonji-related domains diversified into three distinct lineages in bacterial secondary metabolism systems and these were precursors of the three major clades of eukaryotic enzymes. The specificity of wybutosine hydroxylase/peroxidase probably relates to the structural similarity of the modified moiety to the ancestral amino acid substrate of this superfamily