Last month March 2022, the FDA released Draft guidelines for Human Gene Therapy Products Incorporating Human Genome Editing. This guideline is a Draft and as they seek comments and feedback, let’s discuss how exciting this if for those of us in this marketplace, specifically all of us here at KromaTiD! For over 10 years KromaTiD has used our dGH (directional Genome Hybridization) technologies as a service to assist Large Pharma and Gene and Cell Therapy companies to detect and characterize structural variations, chromosomal rearrangements, translocations, and assess the viability of cells, and provide a detailed evaluation and characterization of the effects of these processes, using our directional Genome Hybridization technologies. Furthermore, with additional scrutiny putting it through a rigorous QC as a validation of these methods.
Let’s start with some of the key guidelines outlined in this Draft for Genome Editing using CRISPR:
When describing the manufacturing processes for ex vivo -modified human GE DPs, descriptions of process controls and in-process testing should be included for critical steps that may have significant impact on the efficiency or specificity of editing (e.g., RNP formation step in the case of CRISPR-mediated editing). Acceptance criteria or limits should be provided and justified.
Testing of ex vivo-modified human GE DPs should include evaluation of the following:
- On-target editing efficiency, including characterization of the editing events occurring at the on-target site;
- Off-target editing frequency;
- Chromosomal rearrangements;
- Residual GE components; and
- Total number of genome-edited cells.
We also recommend that the number of edited cells or the frequency of GE be monitored during stability testing of ex vivo-modified human GE DP.
If you would like to read it more in-depth you can download the draft document here:
https://www.fda.gov/media/156894/download
How can KromaTiD help you prepare to meet these Guidelines?
dGH in-Site™ for a Single Cell, Genome-Wide Perspective
KromaTiD’s dGH in-Site provides whole genome tracking inserting DNA cassettes as small as 2kb. Unidirectional dGH probes provide a single cell, genome-wide perspective of cellular engineering outcomes, including tracking of viral and non-viral mediated insert integration (CRISPR/Cas and alternative systems).
- On- and off-target integration metrics
- Detects structural variations in off target CRISPR therapies
- Clean integration data, even from complex or heterogeneous cell populations
- A unique, whole genome, orthogonal method of direct visualization of inserts, without bioinformatic prediction of outcomes
- Multi-channel fluorescence for flexible and multiplex assay design
As an orthogonal analysis to PCR/sequencing techniques, dGH in-Site™ assays enable, through direct visualization, definitive measurement of the presence or absence of inserts. This includes genome-wide distribution and orientation of transgenes or inserted segments. When used in combination with our targeted probes and/or dGH dosimetry paints, dGH in-Site™ assays can provide quantitative, single cell on- vs off-target outcomes of the delivery and editing process and can be used to track the stability of the edited genome in both clonal isolates and non-clonal populations.
directional Genome Hybridization in-Site offers the lowest limit of detection of integrated or genomic DNA targets by fluorescence, dGH in-Site is the most comprehensive tool available for researchers interested in tracking engineering outcomes in a de novo fashion.
dGH SCREEN™ a Single-Cell Rearrangement Event Evaluation and Numbering
KromaTiD’s dGH SCREEN™ is a single cell assay designed to monitor structural variants throughout the genome in an entirely de novo fashion. By utilizing directional Genomic Hybridization technology combined with fluorescence labeling patterns and chromosomal aggregation strategies, dGH SCREEN provides the most comprehensive and high-resolution karyographic analysis available.
Application Benefits of SCREEN™:
- Exchange Events including reciprocal, balanced and allelic translocations
- Orientation Events including inversions, recombinations and sister chromatid exchanges
- Chromosomal Gain & Loss Events including sister chromatid fusions, dicentrics/acentrics, fragmentation/chromothrypsis, polypoidy, aneuploidy, monosomy, polysomy
- Monitoring Cellular Engineering Outcomes: Genome-wide, cell-by-cell and chromosome-by-chromosome assessment of structure, pre- and post modification
- Orthogonal Data for Sequencing: Genome-wide, confirmatory data regarding rearrangements predicted with long read and other NGS analyses
- Structural Integrity QC: A straightforward yardstick by which to measure the relative stability of cell lines, or to screen and compare candidate cell lines, based on total genomic structural variation metrics.
- Variant Discovery: Discover previously unknown mutations by de novo assessment of single cells from patient sub-types
- Genomic Stability Assessment: Track persistence of variants over time, passages, process variable changes, etc.
With dGH SCREEN™, researchers are able to track and localize a wide variety of chromosomal rearrangements within a 10kb limit of detection. In addition to being an unbiased tool for the detection of structural variants throughout the genome SCREEN™ offers High Resolution Analysis of DNA repair activity by monitoring hallmarks of genomic instability, including sister chromatid changes. This analysis produces vast amounts of rearrangement data which can be used to create stability and structural variance profiles for complex and heterogeneous cell populations, making dGH SCREEN one of the most comprehensive and robust karyographic assays available.