PerkinElmer’s Horizon Discovery provides cell engineering tools and services for researchers in drug discovery and development.
Horizon’s portfolio is focused on research reagents; screening and cell line engineering services; bioproduction cell lines; reference standards and base editing.
Horizon’s base editing platform, Pin-point is a gene editing method with the ability to effect multiple single base edits in therapeutically relevant cells without loss of viability while retaining an enhanced safety profile.
Michelle Fraser, business unit manager of base editing at PerkinElmer gives Labiotech the details on this new area of research.
So, what is base editing?
The first generation of CRISPR-Cas applications were groundbreaking, in that for the first time, CRISPR-Cas9 allowed easily programmable, specific targeting of almost any site in the human genome. However, the creation of double stranded DNA breaks (dsDNA), while central to the function of the “bacterial immune system” from which CRISPR was derived, is not ideal for human genome engineering applications, especially in a therapeutic setting.
These dsDNA breaks rely on the natural repair mechanisms of the human cell to re-form intact chromosomes. If more than one gene is targeted at the same time in a cell, there will be more than one cut chromosome. The repair mechanism could generate inaccurate repairs, leading to chromosomal rearrangements such as deletions and translocations. These rearrangements are detrimental to cell health.
The second generation of derived CRISPR tools, such as base editing, do not create dsDNA breaks, but rather harness the targeting power of CRISPR-Cas to more controllably deliver modifications including gene disruption and correction.
In base editing, the Cas enzyme is modified so that it only cuts one of the DNA strands, creating a ‘nick’. The Cas enzyme is targeted to the site that will be edited using a guide RNA, just like the first generation CRISPR platform. Then a deaminase or other effector is introduced to chemically modify the DNA sequence, for example a C>T conversion, on the non-nicked strand. This modified DNA strand is then used as the repair template for the ‘nicked’ strand completing the modification.
How does base editing overcome some of the limitations associated with CRISPR?
The modified Cas enzyme used in base editing only nicks one strand of DNA, so the editing is much more controlled and chromosomal rearrangements are avoided. The intact DNA strand keeps the cut ends in close proximity to each other, restricting the chance for incorrect chromosomal joining.
This allows multiple sites to be edited in a single reaction, making the editing more efficient, i.e. faster and preserving the health of the cells by reducing the number of events needed to achieve the final product. This is particularly important for fragile cells such as iPSCs.
Is there still a place for CRISPR despite the emergence of base editing?
All gene-editing tools have their strengths and weaknesses, and therefore we absolutely believe there is still a place for wild-type CRISPR. For example, if a single-gene knockout is needed, CRISPR offers a quick, simple, efficient and economical solution.
Genome scale libraries of predesigned gRNA for gene knockout are readily available in arrayed and pooled format and are broadly used for functional gene characterization, accelerating target discovery in a wide range of applications. Additionally, not all the base conversions are possible with the current base editors.
Where a base editor is not available for the required modification, or for knock-in requirements, traditional CRISPR/Cas driven HDR remain a valid alternative. Similarly, if a specific size limited delivery system is required, more complex systems may not be suitable. These CRISPR-based technologies along with gene modification technologies such as RNAi, will continue to evolve as new challenges and applications are uncovered, and understanding which tool to deploy and when will be important to understand.
Who is developing base editing technologies?
Base editing was first described in 2016, so it is a very new approach for cell and gene therapy. There are currently only a small number of base editing platforms described in the literature. Besides in-house research systems, the most notable base editing platforms are Dr David Liu’s system that is being used by Beam Technologies and the Pin-point platform by PerkinElmer’s Horizon Discovery.
These two examples use different methods to recruit the deaminase to the site of editing; Beam uses a fusion of the deaminase to the Cas enzyme, while Horizon uses an aptamer fused to the guide RNA and an aptamer binding protein bound to the deaminase. This structural difference can result in different editing characteristics and provides an opportunity to optimize the base editor for the specific therapeutic indication.
For example, by omitting the aptamer from the guide RNA, the Pin-point base editing system allows DNA nicking and insertion of a gene, such as a CAR.
What does the future look like for base editing in the development of cell and gene therapies?
Base editing entered clinical trials in July 2022 with Verve Therapeutics commencing a trial treating patients with familial hypercholesterolemia. This is the first of a pipeline of products in development at many companies, including a significant proportion of the large pharmaceutical companies.
Base editing is a new technology that is quickly finding its way into the clinic, and it represents the future of the use of CRISPR in therapeutics.