17 research outputs found
A bioinformatic pipeline for simulating viral integration data
Viral integration is a complex biological process, and it is useful to have a reference integration dataset with known properties to compare experimental data against, or for comparing with the results from computational tools that detect integration. To generate these data, we developed a pipeline for simulating integrations of a viral or vector genome into a host genome. Our method reproduces more complex characteristics of vector and viral integration, including integration of sub-genomic fragments, structural variation of the integrated genomes, and deletions from the host genome at the integration site. Our method [1] takes the form of a snakemake [2] pipeline, consisting of a Python [3] script using the Biopython [4] module that simulates integrations of a viral reference into a host reference. This produces a reference containing integrations, from which sequencing reads are simulated using ART [5]. The IDs of the reads crossing integration junctions are then annotated using another python script to produce the final output, consisting of the simulated reads and a table of the locations of those integrations and the reads crossing each integration junction. To illustrate our method, we provide simulated reads, integration locations, as well as the code required to simulate integrations using any virus and host reference. This simulation method was used to investigate the performance of viral integration tools in our research [6]
Ub-ISAP: a streamlined UNIX pipeline for mining unique viral vector integration sites from next generation sequencing data
Abstract Background The analysis of viral vector genomic integration sites is an important component in assessing the safety and efficiency of patient treatment using gene therapy. Alongside this clinical application, integration site identification is a key step in the genetic mapping of viral elements in mutagenesis screens that aim to elucidate gene function. Results We have developed a UNIX-based vector integration site analysis pipeline (Ub-ISAP) that utilises a UNIX-based workflow for automated integration site identification and annotation of both single and paired-end sequencing reads. Reads that contain viral sequences of interest are selected and aligned to the host genome, and unique integration sites are then classified as transcription start site-proximal, intragenic or intergenic. Conclusion Ub-ISAP provides a reliable and efficient pipeline to generate large datasets for assessing the safety and efficiency of integrating vectors in clinical settings, with broader applications in cancer research. Ub-ISAP is available as an open source software package at https://sourceforge.net/projects/ub-isap/
Isling: A Tool for Detecting Integration of Wild-Type Viruses and Clinical Vectors
Detecting viral and vector integration events is a key step when investigating interactions between viral and host genomes. This is relevant in several fields, including virology, cancer research and gene therapy. For example, investigating integrations of wild-type viruses such as human papillomavirus and hepatitis B virus has proven to be crucial for understanding the role of these integrations in cancer. Furthermore, identifying the extent of vector integration is vital for determining the potential for genotoxicity in gene therapies. To address these questions, we developed isling, the first tool specifically designed for identifying viral integrations in both wild-type and vector from next-generation sequencing data. Isling addresses complexities in integration behaviour including integration of fragmented genomes and integration junctions with ambiguous locations in a host or vector genome, and can also flag possible vector recombinations. We show that isling is up to 1.6-fold faster and up to 170% more accurate than other viral integration tools, and performs well on both simulated and real datasets. Isling is therefore an efficient and application-agnostic tool that will enable a broad range of investigations into viral and vector integration. These include comparisons between integrations of wild-type viruses and gene therapy vectors, as well as assessing the genotoxicity of vectors and understanding the role of viruses in cancer
Probing the closed-loop model of mRNA translation in living cells
<p>The mRNA closed-loop, formed through interactions between the cap structure, poly(A) tail, eIF4E, eIF4G and PAB, features centrally in models of eukaryotic translation initiation, although direct support for its existence <i>in vivo</i> is not well established. Here, we investigated the closed-loop using a combination of mRNP isolation from rapidly cross-linked cells and high-throughput qPCR. Using the interaction between these factors and the opposing ends of mRNAs as a proxy for the closed-loop, we provide evidence that it is prevalent for eIF4E/4G-bound but unexpectedly sparse for PAB1-bound mRNAs, suggesting it primarily occurs during a distinct phase of polysome assembly. We observed mRNA-specific variation in the extent of closed-loop formation, consistent with a role for polysome topology in the control of gene expression.</p
Analysis and comparison of gamma-retroviral vector integration behaviour in human Cd34+Cells transduced under conditions employed in the French and English Scid-X1 clinical trials
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RNA at the epicentre of human development
The engineering of skeletal muscle requires platforms that facilitate the proliferation and maintenance of primary muscle stem cells (myoblasts) and muscle fibre maturation in a manner that reflects native muscle structure. We have been investigating the use of nanostructured conducting polymer surfaces for the orientation and electrical stimulation of cells and tissues and have developed a hybrid conducting polymer and carbon nanotube plaform, suitable for ex vivo muscle growth, differentiation and electrical stimulation. In this study, Carbon nanotube fibres were laid down in parallel on gold coated mylar. Layers of polypyrrole (doped with pTS) were galvinostatically grown over the carbon nanotube fibre mats to create a nanostructured polypyrrole surface. Myoblasts extracted from 8 week old mice were seeded onto polymer platforms and induced to differentiate into myotubes. Analysis of myofibre differentiation and orientation was then performed. In addition, total RNA was harvested from myotubes that had undergone bipolar electrical stimulation on the platforms 8 hours a day, for 3 days. Whole genome arrays (Codelink) were then used to assess gene expression changes in the electrically stimulated myotubes. Platforms were developed to encourage the alignment and differentiation of skeletal muscle stem cells (myoblasts) to reflect the orientation of mature skeletal muscle. Nanostructured platforms were created by orientation of carbon nanotube fibres on a conducting gold mylar surface, over which a layers of conductive polypyrrole were deposited. Human and murine myoblasts could be grown and differentiated on these platforms without the use of cell adhesion molecules. A significant increase in myotube orientation was seen on nanostructured surfaces, i.e. polypyrrole films with an underlying layer of orientated carbon nanotube fibres. This orientation decreased with increasing thickness of the polypyrrole, suggesting a strong influence of the nanostructure on the orientation of myofibres. In addition, a significant number of gene expression changes were detected in myofibres electrically stimulated on the platforms. A number of these genes were associated with muscle differentiation and myoblast fusion, demonstrating that these platforms can be used to influence the differentiation state of skeletal myoblasts through electrical stimulation. These studies demonstrate that novel hybrid platforms can be used to influence skeletal muscle differentiation ex vivo, through electrical stimulation. In addition these platforms can influence muscle fibre orientation in a manner reflecting the in vivo architecture of the parent tissue. Such platforms have application for controlling the regeneration of skeletal muscle in vivo and for the integration of bionic devices designed to facilitate muscle regeneration and function
Modeling correction of severe urea cycle defects in the growing murine liver using a hybrid recombinant adeno-associated virus/piggyBac transposase gene delivery system
Liver-targeted gene therapy based on recombinant adeno-associated viral vectors (rAAV) shows promising therapeutic efficacy in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver, where high rates of hepatocellular proliferation are accompanied by loss of episomal rAAV genomes and subsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an enhanced green fluorescent protein expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks posttreatment compared to traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase-deficient Spf(ash) mouse and the neonatal lethal argininosuccinate synthetase knockout mouse. Finally, transposon integration patterns were analyzed, revealing 127,386 unique integration sites which conformed to previously published piggyBac data. CONCLUSION: Using a hybrid rAAV/piggyBac transposon vector system, we achieved stable therapeutic protection in two urea cycle defect mouse models; a clinically conceivable early application of this technology in the management of severe urea cycle defects could be as a bridging therapy while awaiting liver transplantation; further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration.12 page(s
Genetic diagnosis of Duchenne and Becker muscular dystrophy using multiplex ligation-dependent probe amplification in Rwandan patients.
Duchenne and Becker muscular dystrophies are the most common clinical forms of muscular dystrophies. They are genetically X-linked diseases caused by a mutation in the dystrophin (DMD) gene. A genetic diagnosis was carried out in six Rwandan patients presenting a phenotype of Duchenne and Becker muscular dystrophies and six asymptomatic female carrier relatives using multiplex ligation-dependent probe amplification (MLPA). Our results revealed deletion of the exons 48-51 in one patient, an inherited deletion of the exons 8-21 in two brothers and a de novo deletion of the exons 46-50 in the fourth patient. No copy number variation was found in two patients. Only one female carrier presented exon deletion in the DMD gene. This is the first cohort of genetic analysis in Rwandan patients affected by Duchenne and Becker muscular dystrophies. This report confirmed that MLPA assay can be easily implemented in low-income countries
Coherence analysis discriminates between retroviral integration patterns in CD34+ cells transduced under differing clinical trial conditions
Unequivocal demonstration of the therapeutic utility of γ-retroviral vectors for gene therapy applications targeting the hematopoietic system was accompanied by instances of insertional mutagenesis. These events stimulated the ongoing development of putatively safer integrating vector systems and analysis methods to characterize and compare integration site (IS) biosafety profiles. Continuing advances in next-generation sequencing technologies are driving the generation of ever-more complex IS datasets. Available bioinformatic tools to compare such datasets focus on the association of integration sites (ISs) with selected genomic and epigenetic features, and the choice of these features determines the ability to discriminate between datasets. We describe the scalable application of point-process coherence analysis (CA) to compare patterns produced by vector ISs across genomic intervals, uncoupled from association with genomic features. To explore the utility of CA in the context of an unresolved question, we asked whether the differing transduction conditions used in the initial Paris and London SCID-X1 gene therapy trials result in divergent genome-wide integration profiles. We tested a transduction carried out under each condition, and showed that CA could indeed resolve differences in IS distributions. Existence of these differences was confirmed by the application of established methods to compare integration datasets