CRISPR cell engineering to speed the drug pipeline

CRISPR cell engineering is offering improved disease models and clearer target signals to increase accuracy and speed in the preclinical pipeline.

Over the past few decades, both timelines and the corresponding cost of drug development have escalated, currently requiring an average of 10-15 years and over $2BN to successfully bring a new drug to market^1-3. Lack of efficacy in the intended disease indication is the major cause of failure in clinical phase drug development⁴. Inadequate validation and the high false discovery rate in the preclinical development phase have been noted as a contributing factor to the observed poor target efficacy.

The advent of CRISPR in 2012 brought new methods to the research community to improve the accuracy and speed of the preclinical development phase, and it is increasingly being adopted to accelerate the drug pipeline. CRISPR gene-editing can be used to knock out genes, knock-in new genes for expression, generate single point mutations and with more recent developments of CRISPR activation and interference (CRISPRa/CRISPRi), modulate gene expression to ensure that the most representative models are generated to support drug development.

CRISPR cuts pipeline attrition

The increased speed and accuracy that CRISPR has brought to genetic engineering are transforming the development of more complex disease-relevant cell-based assays to improve predictability for drug therapies. Many of the current preclinical assays lack suitability in being able to address safety issues and evaluate off-target effects ⁵. The ability to more accurately develop disease-relevant models with CRISPR engineering is improving preclinical results to prevent attrition at later stages of development. The typical preclinical process involves several common stages; drug target identification and validation, high-throughput screening, hit validation and lead optimization. CRISPR is being employed at each of these stages to speed workflows and accelerate and validate the pipeline, with the aim to decrease later stage attrition and, ultimately, reduce development costs.

Preclinical pipeline stage	CRISPR edits	Results achieved with CRISPR models
Target identification	Knockout/inhibit a large number of candidates to identify potential drug targets Validate target in knockout or over-expressing model⁶	Identify disease-relevant gene targets Identify druggable targets
High-throughput screening	Full gene knockout for clearer target signals compared to RNAi ⁷ Inducible/ knock-in models to rescue disease phenotypes and confirm target specificity	Understand the functional relationship of drug candidates Confirm target specificity Discriminate between on-target and off-target effects of drug candidates⁵
Hit validation	Multiple gene knockouts for improved disease recapitulation Rapid editing of primary and iPSCs for increased accuracy in disease models ^6,7	Improved accuracy in hit validation for increased success at this stage Determine responsiveness/ resistance of specific disease states to drug candidates ⁷
Lead optimization	Disease-specific knockout, knock-in and variable expression to select lead drug candidate molecules Rapid generation of disease models to speed analysis⁸	ADME characterization^5-7 Safety and toxicity screening for potential drug candidates^5-7

The ability to analyze multiple gene-edits in a single cell line model also offers better disease recapitulation. Using these CRISPR-edited cells in preclinical stage assays is helping to eliminate ineffective compounds and identify the most efficacious molecules earlier in the discovery process ^5-7.

Struggling to get up to speed with CRISPR

The increased accuracy and potential that CRISPR has brought to research offers improved analysis and validation of drug candidates to increase the accuracy of the preclinical phase. Used in this way CRISPR gene-editing is not only cost-effective, it is also cost-saving as it de-risks the drug development process.

Despite the promise and possibilities that CRISPR has brought to drug development, the engineering of CRISPR cell lines or models is not a simple technical process. The full CRISPR workflow requires months of time and resource investment. A single mistake in this process can result in the failure of the entire workflow, with results not realized until the end stages. Studies have shown that researchers typically repeat the entire CRISPR process 3-4 times before achieving the models they need to pursue their projects⁹. With speed to market so vital in the competitive field of drug development, a rapid turnaround of data is needed to support these efforts.

Common pain points when creating CRISPR cell lines

There are currently no universal protocols or guidance for CRISPR cell line development. Consequently, cell engineers spend time painstakingly optimizing each parameter in the protocol for their specific experiment ⁸.

To target the gene region of interest with CRISPR, a guide is used that directs the CRISPR-Cas9 machinery to this section of the DNA for editing. A wide variety of guide formats are available for CRISPR, from single guide RNA (sgRNA) to plasmids and lentivirus and selection of the right format requires knowledge of cell type to be edited and experience with each format to effectively deliver the guide to the cell. Confidence in your editing relies on designing specific guide sequences and being able to accurately predict how the guide will bind to sequences in the genome. Various in silico tools exist to predict the binding of guide sequences, as well as the specificity and efficiency of Cas9 cutting. Additionally, suppliers can help to rapidly manufacture the right guides to support CRISPR scientists.

Cell transfection for CRISPR engineering is difficult with sensitive cell types, such as primary and stem cells, requiring extensive optimization and specialized culture conditions. Even with a deep knowledge of the specific cell type to be edited, the transfection and cell isolation and expansion steps within the CRISPR protocol, often require extensive optimization to be successful.

Following the CRISPR editing of the desired cells, a clonal selection workflow is essential to develop a homogenous gene-edited cell population. This allows full characterization of the gene-edit and simple interpretation of any downstream results working with CRISPR cell models, which is essential to speed the use of CRISPR in drug development. Cell enrichment reduces the number of required cell passages to achieve a clonal population, increasing the health of the cell population in downstream experiments. However, these stages can be time-consuming and technically demanding. Single-cell expansion requires extensive screening of multiple clones to identify the edit of interest, particularly when dealing with CRISPR edits with lower efficiency, like knock-in cell lines. Without fully optimized growth conditions, it can lead to the death of precious cell samples and the loss of all the work.

The technical complexities of CRISPR cell engineering require extensive optimization for success. To enable the necessary number of optimization points across the many different CRISPR protocol steps, the use of automated platforms can be highly beneficial ⁹. However, the requirement for extensive culture facilities and equipment with large lab footprints to develop an effective in-house CRISPR platform, along with the significant time investment and recruitment of appropriately skilled staff, challenges the capacity of many companies.

Working with Abcam for rapid CRISPR solutions

With the increasing use of CRISPR to speed the drug pipeline, outsourcing can provide a more flexible supply of services with improved timelines.

At Abcam, we have a depth of experience delivering the most challenging custom CRISPR-edited cell lines and our robust processes and platform ensure rapid delivery, with standard projects achieved in just 8-12 weeks. Offering end-to-end consultation services, we operate under Broad Institute and Sigma-Aldrich CRISPR Use License Agreements to allow a wide range of edits and to help keep your research on track.

For rapid access to pre-made KO cell lines and lysates explore our catalog.

References

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