Whole genome sequencing requires an extremely high amount of sequencing throughput to generate a moderate depth of coverage. The data generated, while comprehensive, does not allow detection of mutations with as much sensitivity as a targeted approach. Exome sequencing is the most cost-effective and efficient solution.
A large portion of relevant mutations occur in the exome. In fact, the exome contains as many as 85% of disease-related mutations. Covering less than 2% of the whole genome, exome sequencing requires only 1/50th of the sequencing throughput to generate the same depth of coverage. This approach provides flexible experimental options:
|gDNA||> 500 ng|
|Fresh frozen tissues||10-30 mg|
We offer two analytically validated NGS-based assays, leveraging targeted approaches to identify, quantify, and monitor viral vector integration events across the genome. Both assays generate information on insertion site location and frequency as well as offer the following unique capabilities:
Target Enrichment Sequencing (TES)
A Hybridization-Capture TES Assay that generates information on insertion site location and frequency, and additionally allows for insertion/transgene integrity study, which is important to resolve mutations or rearrangements.
Quantitative Shearing Linear Amplification Mediated-PCR (qsLAM-PCR)
A Targeted qsLAM-PCR Sequencing Assay that focuses on identifying insertion site location and frequency, offering superior sensitivity on insertion site detection.
Both of our analytically validated NGS-based assays are efficient at generating information about where the viral vector has inserted into the host genome:
Linear Amplification-Mediated-PCR (LAM-PCR) focuses on insertion site location and frequency, offering superior sensitivity on insertion site detection;
Hybrid Capture Targeted Enrichment Sequencing (TES) generates information on insertion site location and frequency, and additionally allows for insertion/transgene integrity study, which is important to resolve mutations or rearrangements.
Lentiviral vectors are efficient gene delivery vehicles suitable for delivering long-term transgene expression in various cell types over time. A drawback of the lentiviral vector include the risk of insertional mutagenesis due to the semi-random integration of genes.
Guidance from the FDA and other regulatory authorities, is that patients treated with cell or gene therapies utilizing viral vectors be monitored for 5 - 15 years for safety. This monitoring includes, but not limited to, vector integration studies of patient samples to ensure stability.
Per the FDA, "LTFU observations are extended assessments that continue some of the scheduled observations of a clinical trial past the active follow-up period and are an integral portion of the study of some investigational GT products. LTFU observations are important to monitor long term safety of GT products. For GT products that present long term risks to subjects, LTFU/surveillance plan(s) should also be put in place post-licensure for monitoring of delayed adverse events (for details we refer you to section VI. of this document)."
Guidelines from the FDA suggest 15 years of long-term follow up after gene therapy treatment with integrating vectors such as lentiviral vectors. Analysis should be performed to determine the site of vector integration if the analysis of a subject’s surrogate cells suggests a predominant clone (e.g., oligoclonal pattern of vector insertions) or monoclonality. In addition, if you detect a predominant integration site, test for persistence by performing another analysis for clonality no more than three months later.
1) Insertion site location, frequency, abundance
2) Integration hotspots & CPG islands
3) Oncogenic gene annotation of Integration Site
4) Longtitudinal profiling (clonal expansion)
5) Vector integrity (TES)
Custom analysis support options are also available upon consultation.