Genomic
The FDA Critical Path Initiative has identified toxicogenomics as one of the key opportunities in advancing medical product development. Indeed, transcriptomics (the evaluation of messenger RNA modulation) is one of the most effective tools to elucidate toxic mechanisms and to predict toxicity.
Toxicogenomics is now a standard tool for early detection of safety issues during the discovery phases in pharmaceutical research and development for :
- Biomarker discovery
- Generation of Drug Toxicity signature databases
- Mechanistic toxicology
- Ranking compounds by gene expression profile comparisons
- Early predictive toxicology by comparison with Drug Toxicity signature databases
CIT's services
CIT proposes a complete integrated toxicogenomics solution from in vivo & in vitro studies up to data mining, including:
- Assistance in customized toxicogenomics study design (relevant species selection, choice of appropriate time-points and dose-levels, sequenced organ sampling at necroscopy).
- Large panel of DNA/RNA/uRNA extraction procedures to ensure top level quality DNA/RNA/uRNA from more than 50 tissues, fluids, cells and swabs from rodents and primates.
- Monitoring/detection of any DNA and RNA sequence using real-time quantitative PCR (Applied Biosystems 7900 HT Fast).
- GLP-compliant gene therapy products biodistribution regulatory studies.
- Whole transcriptome profiling (RNA and uRNA) on Affymetrix Genechip® arrays (Whole genome and Gene ST arrays, uRNA chips).
- Clinical genotyping with Affymetrix Human SNP 6.0 arrays.
- Complete data analysis (from preprocessing up to pathway analysis).
- Easy understanding of compound safety and predictive toxicity by data comparison with Drug Toxicity signature databases.
These studies are conducted according to the highest quality standards.
LeadScreen study package
CIT proposes the following short and easy-to-perform toxicogenomics study design for the dissection investigation of toxic mechanism and toxicity prediction, using dose/time combination that produces a maximal impact on gene expression. This design may be customized to your specific objective.
- 3 animals for each sampling occasion.
- 1 control and 2 tests item-treated groups (the fully effective dose and the maximum tolerated dose).
- Minimum of three sampling occasions on a short-time period (usually 1-5 days after treatment, intermediate time-points and/or doses can be added).
- 27 chips (3 tissues : eg.kidney, liver, heart) * 3 groups * 3 animals per group).
- Complete Sponsor-customized data analysis (from preprocessing up to pathway analysis and prediction scoring with DrugMatrix).
- Reporting in 7 days with raw data transferred on a secured FTP server.
Tissues sampling coupled with DNA, RNA and uRNA extraction.
Tissues sampling and nucleic acid extraction processes are GLP compliant with specific procedures and robust quality controls: the key and critical factors for a successful toxicogenomics approach. CIT Genomics team offers expertise and high throughput.
- Dedicated tissue-specific sampling procedures in nuclease-free environment.
- Highly trained and experienced genomics technicians.
- Fast tissue sampling sequence design (from collection to freezing) by giving priority to sensitive or target organs.
- 27 000 RNA extractions performed in the last 36 months.
- DNA/RNA/uRNA extraction dedicated procedures for more than 50 tissues/fluids/swabs/cells.
- Systematic quantification (NanoDrop and Varioskan from Thermo) and quality control (2100 Bioanalyzer from Agilent) on purified nucleic acid prior to further processing.
GLP compliant Real Time qPCR
GLP compliant Real Time qPCR with Applied Biosystems 7900 HT Fast technology and Taqman chemistry, Taqman® Low-Density Array (TLDA) formats available (from 12 to 380 simultaneous gene detection with simplicate to quadruplicate):
- Absolute quantitative simplex/multiplex PCR and RT-PCR including validation of qPCR or RT-qPCR assay (specificity, reproducibility, stability, range linearity, detection limit, inhibition monitoring).
- Quantification of any transcript with a known sequence (up to 380 transcripts on one card).
- Regulatory biodistribution studies for gene therapy products.
- SNP genotyping on clinical samples.
Gene therapy products biodistribution
Regulation biodistribution studies are a pivotal requirement during the preclinical safety evaluation of gene therapy vectors (viral DNA or RNA, plasmid, antisens). They provide essential safety data on the persistence of the DNA or RNA in the organs and its dissemination troughout the tissues and fluids.
CIT provides a complete biodistribution study solution which includes:
- In vivo phase under BSL1/BSL2 conditions.
- Enhanced necropsy procedures in order to avoid organ-to-organ contaminations.
- RNA and DNA extraction from tissues, fluids and swabs (treated vs controls groups extraction rooms).
- Absolute quantitative simplex/multiplex PCR and RT-PCR uncluding validation of qPCR or RT-qPCR assay (specificity, reproducibility, stability, range linearity, detection limit, inhibition monitoring, extraction yields and limits).
- All the study phases are GLP compliant.
Microarrays
Affymetrix platform recognized as Affymetrix Authorized service provider
- Studies on whole transcriptome arrays, including Human Genome U133 Plus 2.0 Mouse Genome 430 2.0, Rat Genome 230 2.0, Canine Genome 2.0, Pig Genome and Bovine, Human Gene ST 1.0, Rat Gene 1.0 ST and Mouse Gene 1.0 ST, uRNA transcriptome using miRNA 2*gain chips.
- Clinical genotyping (SNP clusters and copy number analysis) with Genome_Wide Human SNP 6.0 arrays.
- Highly trained and experimented technicians (over 8000 labelings, hybridizations and scans for the last 12 months).
- Capacity tio process > 300 chips a week.
Bioinformatics & Biostatistics
Versatile data analysis capabilities
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Data Quality Control
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- Image quality control
- Commonly QCmetrics investigations
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Data preprocessing
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- Background adjustment, normalization and summarization
- Different methods available
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Eploratory data analysis
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- Principal component analysis
- Hierarchial clustering
- Correlation matrix
- Boxplots
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Data filtering
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- Based on fold-change and/or p-value cut-offs
- Appropriate statistical tests (Student, paired Student, ANOVA, …)
- Multiple testing adjustments
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Data annotation
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- Based on latest available information from Affymetrix website
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Gene ontology and pathway analysis
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- Molecular function, biological process, cellular component and pathway enrichment from filtered gene lists
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Data reporting
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- Report describing methods used for analysis
- Excel, text, image or html files of generated results
- Customized presentation of results at the request of the Sponsor
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Data transfert
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- Secured web-based workspace eRoom
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Available tools
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- Affymetrix Expression console , R (Bioconductor), SAS
- Easy understanding of compound safety and predictive toxicity by data comparison with DrugMatrix Signatures® database (available end of March after SOT meeting)
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Guidelines
CIT studies comply with the guidance documents, which have been issued by the regulatory agencies.
The FDA guideline entitled "Pharmacogenomics Data Submissions" was released in March 2005 in order to clarify for sponsors the agency current thinking about when pharmacogenomic data must be submitted, or would be welcome on a voluntary basis. To date, several voluntary submissions have been reviewed by the newly formed FDA's Interdisciplinary Pharmacogenomics Review Group.
- The same guideline provides a classification of biomarkers in the context of regulatory submission. This has been expanded to a preclinical safety biomarker validation path prposed by the dedicated "Predictive Safety Testing Consortium" (PSTC), which includes as members, the FDA, the Critical Path Institute and most of major pharmaceutical companies.
- The MAQC (MicroArray Quality Control) study guidance and a FDA preliminary concept paper entitled "Recommendations for the Generation and Submission of Genomic Data, Nov 2006" are aimed to avoid procedural bias in microarray experiments.
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