CRISPR-Mediated Gene Therapy

What is CRISPR-mediated gene therapy?

CRISPR-mediated gene therapy utilizes CRISPR-Cas (CRISPR-associated) technology to treat genetic disorders by making precise modifications to the DNA within cells. This approach holds promise for correcting disease-causing mutations, inserting therapeutic genes, or regulating gene expression to alleviate symptoms or potentially cure diseases. The Life Sciences companies of Danaher provide a range of solutions crucial for CRISPR-mediated gene therapy development and manufacturing.

CRISPR-Edited Gene Therapy involves several critical steps and technologies:

• Preliminary Screening: Cells from patients are modified using CRISPR-Cas9 with IDT's RUO and cGMP-grade gRNAs, along with Aldevron's cGMP-grade Cas9 nucleases and HDR templates. Flow cytometry aids in selecting desired cell phenotypes, supported by Beckman Coulter Life Sciences' CytoFLEX Flow Cytometer for preclinical labs and the CellMek SPS System for clinical automation. Regulatory compliance is ensured with the Beckman Coulter Life Sciences Navios EX flow cytomete r and certified dry reagents. Initial vector screens utilize the SCIEX ZenoTOF 7600 System for sgRNA evaluation, while Genedata Expressionist and IDBS Polar BioPharma Lifecycle Management software streamline data handling and analysis.
• Isolation & Enrichment: Isolation is achieved with the Beckman Coulter Life Sciences CytoFLEX SRT Benchtop Cell Sorter, followed by cell concentration using the Avanti JXN High-Performance Centrifuge, and viability assessment with the Vi-CELL BLU Cell Viability Analyzer. Experiment and instrument data, including sorted cells for enrichment, are tracked in IDBS Polar BioPharma Lifecycle Management software for comprehensive recordkeeping.
• Gene Modification and Analysis: The chemical transfection process is automated and ensured for consistency using the Beckman Coulter Life Sciences Biomek i-Series Integrated Workstation for delivering gRNA and Cas reagents across different cell lines. Post modification, cell counts and viability are assessed with the Beckman Coulter Life Sciences Vi-CELL BLU Cell Viability Analyzer. More detailed analyses, such as mitotic assessments and fluorescent imaging, can be performed on a Leica Microsystems STELLARIS Confocal Microscope Platform or automated on a Leica Microsystems Mica Microhub. Experiment and instrument data are collected and tracked with IDBS Polar BioPharma Lifecycle Management software.
• Expansion: CRISPR-modified cells are expanded to increase numbers to prepare for downstream characterization. Cell counts and viability during culture expansion are tracked using the Beckman Coulter Life Sciences Vi-CELL BLU Cell Viability Analyzer. Metabolites are characterized using mass spectrometry, with differential mass analyses performed using the SCIEX Triple Quad 7500 System. Mass spectrometry data handling and analyses are streamlined using the Genedata Expressionist software.
• Characterization and Optimization: Assessing CRISPR modification accuracy and identifying off-target effects is crucial, and it is often done via next-generation sequencing (NGS). DNA extraction and NGS library prep are automated with the Beckman Coulter Life Sciences Biomek i-Series Automated Workstation and bead-based nucleic acid extraction (DNA) kits and NGS library cleanup reagents. Genedata Selector software analyzes post-sequencing data for integration efficiency and off-target sites. Evaluating genome editing's impact on cell phenotype and protein expression is essential. Verification of gene-of-interest (GOI) expression and optimization of CRISPR-Cas modifications is facilitated. The SCIEX ZenoTOF 7600 System provides detailed protein and peptide analysis, supported by Genedata Expressionist software for streamlined data processing. Protein expression from CRISPR-modified cells is analyzed using the Beckman Coulter Life Sciences CytoFLEX Flow Cytometer in preclinical labs, with clinical sample preparation automated by the CellMek SPS System. Data analysis employs Beckman Coulter Life Science’s Cytobank and Kaluza C software. Optimizing gRNA sequences and assay readouts requires robust data handling, managed by IDBS Polar BioPharma Lifecycle Management software for regulatory compliance and secure recordkeeping.
• Functional Assessment: CRISPR-modified cells undergo rigorous assessment for functional activity, prioritizing safety and efficacy. Evaluation includes potency and toxicity using readouts such as fluorescent response, ATP assay, and live/dead imaging with a Molecular Devices multi-mode plate reader featuring GxP-compliant software. Functional assays utilize live-cell and time-lapse imaging capabilities provided by Leica Microsystems' STELLARIS Confocal Microscope Platform or Mica Microhub.
• Off-target analysis: The analysis for CRISPR-modified cells employs NGS to ensure that editing has not occurred in unintended genomic regions. NGS libraries are prepared using Beckman Coulter Life Sciences extraction reagents, IDT rhAmpSeq or custom NGS panels, and the Beckman Coulter Life Sciences Biomek i-Series Automated Workstation. Data analysis to characterize off-target effects is conducted using the Genedata Selector software after sequencing.
• Regulatory Interaction: CRISPR-edited gene therapies are subject to internal and external regulatory audits before being transferred to a clinical environment. Data reporting and analysis for patient treatment expansion are ensured to comply with regulatory requirements using IDBS Polar BioPharma Lifecycle Management software, facilitating efficient pre- and post-IND filings.

How does CRISPR technology work in gene therapy?

CRISPR technology employs guide RNA molecules that target specific DNA sequences within the genome. When guided to the target site, the Cas protein (e.g., Cas9) acts as molecular scissors, enabling precise modification of the DNA. This can involve cutting out faulty genes, replacing them with healthy versions, or modifying gene expression levels to treat the underlying causes of genetic diseases.

Through collaborations like with the Innovative Genomics Institute (IGI), Danaher facilitates the development of scalable platforms for CRISPR-mediated gene editing. This partnership enhances the accessibility of advanced CRISPR tools, aiming to tackle rare and underserved genetic diseases effectively.

How does workflow automation benefit the development of CRISPR-mediated gene therapies?

Workflow automation plays a crucial role in advancing CRISPR-mediated gene therapies, particularly for addressing rare and orphan diseases:

• Efficiency: Automation streamlines repetitive tasks such as cell culture, transfection of CRISPR components into cells and analysis of experimental data. This reduces time and labor-intensive processes, accelerating the pace of research and development.
• Consistency and Quality Control: Automated systems ensure consistent experimental conditions and results, minimizing variability and human error. This reliability is essential for reproducibility in research and for meeting stringent regulatory standards required for clinical applications.
• Scalability: Automated workflows can be scaled from small-scale laboratory research to large-scale production for clinical trials and potential commercialization. This scalability facilitates the translation of promising research findings into viable treatments that can benefit larger patient populations.
• Data Management: Integrated automation platforms often include robust data management systems that track experimental parameters, results and compliance with regulatory guidelines. This enhances transparency, reproducibility and traceability throughout the development process, ensuring that gene therapies are safe and effective.

The Life Sciences companies of Danaher provide automation solutions, coupled with their expertise in data management and quality assurance, that empower researchers and developers in the gene therapy field. They provide tools and services that optimize workflow efficiency and support the rigorous demands of CRISPR-mediated therapy development, ultimately aiming to bring transformative treatments to patients worldwide.