Differences in vector-genome processing and illegitimate integration of non-integrating lentiviral vectors

A variety of mutations in lentiviral vector expression systems have been shown to generate a non-integrating phenotype. We studied a novel 12 base-pair U3-long terminal repeats (LTR) integrase (IN) attachment site deletion (U3-LTR att site) mutant and found similar physical titers to the previously reported IN catalytic core mutant IN/D116N. Both mutations led to a greater than two log reduction in vector integration; with IN/D116N providing lower illegitimate integration frequency, whereas the U3-LTR att site mutant provided a higher level of transgene expression. The improved expression of the U3-LTR att site mutant could not be explained solely based on an observed modest increase in integration frequency. In evaluating processing, we noted significant differences in unintegrated vector forms, with the U3-LTR att site mutant leading to a predominance of 1-LTR circles. The mutations also differed in the manner of illegitimate integration. The U3-LTR att site mutant vector demonstrated IN-mediated integration at the intact U5-LTR att site and non-IN-mediated integration at the mutated U3-LTR att site. Finally, we combined a variety of mutations and modifications and assessed transgene expression and integration frequency to show that combining modifications can improve the potential clinical utility of non-integrating lentiviral vectors

Development and Evaluation of Quality Metrics for Bioinformatics Analysis of Viral Insertion Site Data Generated Using High Throughput Sequencing

Integration of viral vectors into a host genome is associated with insertional mutagenesis and subjects in clinical gene therapy trials must be monitored for this adverse event. Several PCR based methods such as ligase-mediated (LM) PCR, linear-amplification-mediated (LAM) PCR and non-restrictive (nr) LAM PCR were developed to identify sites of vector integration. Coupling the power of next-generation sequencing technologies with various PCR approaches will provide a comprehensive and genome-wide profiling of insertion sites and increase throughput. In this bioinformatics study, we aimed to develop and apply quality metrics to viral insertion data obtained using next-generation sequencing. We developed five simple metrics for assessing next-generation sequencing data from different PCR products and showed how the metrics can be used to objectively compare runs performed with the same methodology as well as data generated using different PCR techniques. The results will help researchers troubleshoot complex methodologies, understand the quality of sequencing data, and provide a starting point for developing standardization of vector insertion site data analysis

An investigation into the existence of cyprinid herpesvirus-3 encoded microRNAs

Funder: Dublin City University; Marine Institut

Advancing the Safety of Lentiviral Vector Mediated Gene Therapy

Indiana University-Purdue University Indianapolis (IUPUI)Lentiviral vector mediated gene therapy has made great strides in recent years with several successful clinical trials. However, adverse events encountered with some early trials have highlighted the necessity to improve upon its safety. Improvements can range from early steps in vector production to evaluation of insertion sites post-transduction. We have evaluated an FDA approved DNase for removal of residual plasmid DNA during vector production, developed novel non-integrating lentiviral vectors and employed modified insertion site analysis post-transduction to improve the safety of lentiviral vector mediated gene therapy. To prevent the exposure of gene therapy patients to HIV-1 DNA it is essential to remove residual plasmid DNA during vector production. We evaluated a recombinant human DNase which has been FDA approved for use in patients as an alternative to a bacterially derived DNase. Our results indicate this DNase is an effective alternative with a potentially safer profile for use in patients. The ability of lentiviral vectors to stably integrate their genome into a host cell’s DNA can have negative side-effects due to the risk of insertional mutagenesis. Non-integrating lentiviral vectors have been developed to alleviate this risk in applications where integration is not necessary. However, a low frequency of illegitimate integration persists when using these vectors. We have developed a novel non-integrating vector mutation and evaluated the efficacy of combining it with other mutations for reducing the frequency of illegitimate integration. We demonstrate that combining mutations that inhibit integration can further reduce the frequency of illegitimate integration. Several methodologies have been developed for evaluating the insertion sites of normal integrating lentiviral vectors. Illegitimate integration by non-integrating vectors demonstrates mechanisms which result in insertions and/or deletions at the vector-genome junction. Current methods lack the sensitivity to account for these variables in a high-throughput manner. We have adapted modifications to current methods to improve the capture of these variable insertion sites for analysis. The results of these studies improve the safety of lentiviral vector mediated gene therapy by improving the purity of the vector product, providing a safer vector for non-integrase mediated applications, and allowing more sensitive analysis of insertion sites post-transduction

De novo sequencing and characterization of Picrorhiza kurrooa transcriptome at two temperatures showed major transcriptome adjustments

<p>Abstract</p> <p>Background</p> <p><it>Picrorhiza kurrooa </it>Royle ex Benth. is an endangered plant species of medicinal importance. The medicinal property is attributed to monoterpenoids picroside I and II, which are modulated by temperature. The transcriptome information of this species is limited with the availability of few hundreds of expressed sequence tags (ESTs) in the public databases. In order to gain insight into temperature mediated molecular changes, high throughput <it>de novo </it>transcriptome sequencing and analyses were carried out at 15°C and 25°C, the temperatures known to modulate picrosides content.</p> <p>Results</p> <p>Using paired-end (PE) Illumina sequencing technology, a total of 20,593,412 and 44,229,272 PE reads were obtained after quality filtering for 15°C and 25°C, respectively. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 74,336 assembled transcript sequences were obtained, with an average coverage of 76.6 and average length of 439.5. Guanine-cytosine (GC) content was observed to be 44.6%, while the transcriptome exhibited abundance of trinucleotide simple sequence repeat (SSR; 45.63%) markers.</p> <p>Large scale expression profiling through "read per exon kilobase per million (RPKM)", showed changes in several biological processes and metabolic pathways including <it>cytochrome P450s </it>(<it>CYPs</it>), <it>UDP-glycosyltransferases </it>(<it>UGTs</it>) and those associated with picrosides biosynthesis. RPKM data were validated by reverse transcriptase-polymerase chain reaction using a set of 19 genes, wherein 11 genes behaved in accordance with the two expression methods.</p> <p>Conclusions</p> <p>Study generated transcriptome of <it>P. kurrooa </it>at two different temperatures. Large scale expression profiling through RPKM showed major transcriptome changes in response to temperature reflecting alterations in major biological processes and metabolic pathways, and provided insight of GC content and SSR markers. Analysis also identified putative <it>CYPs </it>and <it>UGTs </it>that could help in discovering the hitherto unknown genes associated with picrosides biosynthesis.</p

Pathogen-host omics analyses of human papillomavirus type 16 sub-lineages in a human epithelial organoid model

Pathogens such as human papillomaviruses (HPVs) have co-evolved with their hosts and form a molecular basis for common diseases. Persistent infection with the “high-risk” HPV type 16 (HPV16) is a potent cause of anogenital and oropharyngeal cancers. Taxonomic HPV16 sub-lineages, based on geographic origin of discovery, are noteworthy due to their variable tumourigenicity. In this dissertation, I present basic research and the resulting biotechnologies we developed, improved, and utilized to study their fascinating pathogen-host relationship with human stratified epithelia. A small number of variations in the E6 gene of HPV16, found in the D2 and D3 sub-lineages, lead to increased tumourigenic risk compared to the prototype A1 sub-lineage. Using an organotypic human epithelial model (or in vitro organoid) we recapitulated the viral life cycle and used “-omics” analyses to assess viral and host molecular differences due to sub-lineage variation. Sub-lineage variants of E6 were associated with host genome instability and viral integration into host DNA. Following these initial findings, I provide perspective on epithelial organoids, namely that the trade-off between model complexity and feasibility should be sensibly considered based on its utility for answering the biological research question at hand. Model applications and improvements are presented, including time-series epithelial stratification measurements, strategies for introducing full-length sub-lineage HPV16 genomes into host keratinocytes, and experiments to study innate immune evasion. These wet-lab works are accompanied by software to aid biologists in analyzing sequencing data. As well, we present current work using The Cancer Genome Atlas to test the association between HPV16 sub-lineage and integration. Overall, this interdisciplinary and interconnected collection has significance for basic researchers, providing insight on how a small number of natural viral variations can lead to increased tumourigenic risk, as well as for experimentalists to gain insight on organoid modelling and novel bioinformatics tools. More broadly, characterizing these molecular interactions between pathogen and host enables us to form a basis for diagnosis, treatment, and ultimately prevention of disease. Future research should aim to closely integrate biological and computational sciences for improving experimental approaches and our ability to make meaningful biological interpretations given the complexity and variability of biological systems

DEVELOPING THERAPEUTIC BIOIMPLANTS FOR HEMOPHILIA A: BIOSAFETY EVALUATION OF TARGETED TRANSGENE INTEGRATION IN PRIMARY HUMAN UMBILICAL CORD-LINING CELLS USING PHIC31 INTEGRASE AND ZINC FINGER NUCLEASES

Ph.DDOCTOR OF PHILOSOPH