Optimizing the Design of Oligonucleotides for Homology Directed Gene Targeting

BACKGROUND: Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit. METHODOLOGY/PRINCIPAL FINDINGS: In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA) polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex. CONCLUSION AND SIGNIFICANCE: A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases) in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA

Expanding the clinical spectrum of hereditary fibrosing poikiloderma with tendon contractures, myopathy and pulmonary fibrosis due to <i>FAM111B </i>mutations

BACKGROUND: Hereditary Fibrosing Poikiloderma (HFP) with tendon contractures, myopathy and pulmonary fibrosis (POIKTMP [MIM 615704]) is a very recently described entity of syndromic inherited poikiloderma. Previously by using whole exome sequencing in five families, we identified the causative gene, FAM111B (NM_198947.3), the function of which is still unknown. Our objective in this study was to better define the specific features of POIKTMP through a larger series of patients. METHODS: Clinical and molecular data of two families and eight independent sporadic cases, including six new cases, were collected. RESULTS: Key features consist of: (i) early-onset poikiloderma, hypotrichosis and hypohidrosis; (ii) multiple contractures, in particular triceps surae muscle contractures; (iii) diffuse progressive muscular weakness; (iv) pulmonary fibrosis in adulthood and (v) other features including exocrine pancreatic insufficiency, liver impairment and growth retardation. Muscle magnetic resonance imaging was informative and showed muscle atrophy and fatty infiltration. Histological examination of skeletal muscle revealed extensive fibroadipose tissue infiltration. Microscopy of the skin showed a scleroderma-like aspect with fibrosis and alterations of the elastic network. FAM111B gene analysis identified five different missense variants (two recurrent mutations were found respectively in three and four independent families). All the mutations were predicted to localize in the trypsin-like cysteine/serine peptidase domain of the protein. We suggest gain-of-function or dominant-negative mutations resulting in FAM111B enzymatic activity changes. CONCLUSIONS: HFP with tendon contractures, myopathy and pulmonary fibrosis, is a multisystemic disorder due to autosomal dominant FAM111B mutations. Future functional studies will help in understanding the specific pathological process of this fibrosing disorder

High-resolution CT phenotypes in pulmonary sarcoidosis: a multinational Delphi consensus study

One view of sarcoidosis is that the term covers many different diseases. However, no classification framework exists for the future exploration of pathogenetic pathways, genetic or trigger predilections, patterns of lung function impairment, or treatment separations, or for the development of diagnostic algorithms or relevant outcome measures. We aimed to establish agreement on high-resolution CT (HRCT) phenotypic separations in sarcoidosis to anchor future CT research through a multinational two-round Delphi consensus process. Delphi participants included members of the Fleischner Society and the World Association of Sarcoidosis and other Granulomatous Disorders, as well as members' nominees. 146 individuals (98 chest physicians, 48 thoracic radiologists) from 28 countries took part, 144 of whom completed both Delphi rounds. After rating of 35 Delphi statements on a five-point Likert scale, consensus was achieved for 22 (63%) statements. There was 97% agreement on the existence of distinct HRCT phenotypes, with seven HRCT phenotypes that were categorised by participants as non-fibrotic or likely to be fibrotic. The international consensus reached in this Delphi exercise justifies the formulation of a CT classification as a basis for the possible definition of separate diseases. Further refinement of phenotypes with rapidly achievable CT studies is now needed to underpin the development of a formal classification of sarcoidosis

Effet de certaines séquences de l'ADN sur la recombinaison génétique homologue (mise au point d'un nouvel outil moléculaire pour le ciblage de gène dans les cellules eucaryotes)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

The role of macronutrient balance on appetite, metabolic health and ageing in a mouse model

The fundamental question of what represents a macro-nutritionally balanced diet, and how this influences appetite and maintains health and longevity, remains unanswered. Here, the Geometric Framework, a state-space nutritional modelling method, was used to measure the interactive effects of dietary energy, protein, fat and carbohydrate on food intake, cardiometabolic phenotype and longevity in mice fed one of 25 diets ad libitum. By manipulating both the macronutrient ratios and energy density with the addition of cellulose, these diets addressed both nutrient quality and quantity. Using this 3-D based experimental design, we investigated: 1. the interaction between dietary protein, carbohydrate and fat during long-term dietary manipulation and their effects on food and energy intake, body composition, glucose tolerance and blood pressure in 6 month old mice; 2. the balance of macronutrients and the effect on ageing and age-related cardiometabolic health and survival in 15 month old mice; and 3. the short-term (8 week) high protein feeding vs traditional 40% caloric restriction and the effects on metabolic health in 4 month old mice. Mice have the capacity to regulate individual macronutrients separately from total energy intake. When individual macronutrients were analysed at 4, 6 and 15 months, we found that protein intake was tightly regulated over carbohydrate and fat. Compensatory feeding responses allowed mice to reach this target at the cost of over- or under eating carbohydrate and fat. This is consistent with the protein leverage effect, resulting in increased adiposity and basal blood glucose levels on low % protein diets. Surprisingly, glucose tolerance was markedly better in these mice in comparison to leaner animals on a higher % protein diet. These results remained consistent in 4, 6 and 15 month old mice. Mice fed low-density diets displayed the lowest life expectancy despite consuming 30% less energy, indicating that caloric restriction by dilution did not extend lifespan. Instead, we showed that longevity and latelife cardiometabolic health were optimised with low protein diets, particularly when protein was replaced with carbohydrate to limit compensatory feeding for protein. Replacing protein with fat in the diet resulted in elevated energy intakes due to lack of suppression of compensatory feeding for protein. These consequences are associated with hepatic mammalian target of rapamycin (mTOR) activation and mitochondrial function and, in turn, related to circulating branched-chain amino acids (BCAAs) and glucose. Here, circulating levels of BCAAs were positively correlated with increasing protein to carbohydrate ratio resulting in elevated mTOR activation in the liver. The results suggest that longevity can be extended in ad libitum-fed animals by manipulating the ratio of macronutrients to inhibit mTOR activation. In this thesis, I present the first comprehensive study of the macronutritional phenotype of the mouse by quantifying the interaction of energy and protein, carbohydrate and fat intake during short-term and long-term dietary manipulation and their effects on feeding behaviour, cardiometabolic health and longevity. In translational terms, these data may have important implications for diet management and the development of key features of metabolic syndrome and the extension of a healthy lifespan

PHYSIOPATHOLOGIE DE LA BRONCHIOLITE OBLITERANTE CHEZ LES TRANSPLANTES PULMONAIRES (IMPLICATION DE 4 MEDIATEURS PROFIBROSANTS : TGF-BETA, IGF-1,ET-1 ET PDGF)

PARIS5-BU-Necker : Fermée (751152101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Stimulation of D-loop formation by polypurine/polypyrimidine sequences

Most of the approaches used to correct gene mutations in mammalian cells involve the targeting of short nucleotide molecules to homologous chromosomal sequences and the replacement of resident sequences via homologous recombination and mismatch repair. The limited efficiency and inconsistent reproducibility of these techniques are major constraints to their use in gene therapy. One of the main problems is that it is impossible to obtain reproducible results when the targeted gene loci differ. We investigated the effects of flanking sequences on homologous recombination by means of an in vitro assay of the efficiency of oligonucleotide targeting to its homologous sequence on a large duplex molecule in a reaction catalysed by the Escherichia coli RecA protein. We demonstrated that polypurine·polypyrimidine tracts (PPTs) in duplex DNA strongly stimulate the formation of D-loops with short oligodeoxynucleotides. This result was reproduced with various PPT sequences and oligonucleotides. The stimulatory effect was observed at loci as far as 4000 bp from the PPT. The formation of complexes between the oligonucleotide and the duplex molecule depended on the extent of sequence similarity between the two DNAs and the presence of the RecA protein. The stimulatory effect was inhibited by excess RecA and restored by adding heterologous DNA. We suggest that PPT sequences induce conformational changes in duplex DNA, leading to the aggregation of molecules, facilitating homology searches. We com pared, in vivo, the efficiency of the oligonucleotide-mediated correction of a URA3 chromosomal mutation for sequences with and without a PPT sequence in the vicinity. Consistent with our in vitro results, the efficiency of correction was eight times higher in the presence of the PPT sequence