This article is from the June 14, 2017 issue of our bi-monthly newsletter Instrument Business Outlook (IBO) published by BioInformatics, LLC. Click here to learn more and sign up for a free trial.
CRISPR gene editing technologies show great promise in accelerating research, development and manufacturing in many applications, such as biomedicine and food production. However, much more research is still needed before CRISPR platforms can be adopted and implemented in broader settings.
For example, Nature Methods recently published a letter in which researchers discussed the issues they came across in their mouse study using CRISPR, namely the frequent off-target effects that were generated. The letter states that “[m]ore work may be needed to increase the fidelity of CRISPR/Cas9 with regard to off-target mutation generation before the CRISPR platform can be used without risk, especially in the clinical setting.”
Companies have been working to come up with new methods and technologies that can solve common issues with CRISPR and open up the technology to more applications. Horizon Discovery and MilliporeSigma both released innovative CRISPR platforms last month that aim to optimize CRISPR platforms and address some of the specific concerns that arise with gene editing.
Horizon Discovery’s Helitron Transposon-based Technology
UK-based Horizon Discovery announced in May that it acquired the rights to use a Helitron transposon-based technology platform to build upon its existing gene editing capabilities. The technology platform was co-invented by Tilmann Buerckstuemmer, PhD, head of Innovation at Horizon Discovery.
Transposons are pieces of DNA that are able to “jump” from site to site within a genome. They play an important role in the removal and integration of genomic sequences, as their inherent capabilities support cell functions and aid in the creation of new genes. Helitron transposons are transposons that “capture and mobilize gene fragments in eukaryotes,” according to a research paper published in Nature Communications in March 2016.
“We are particularly excited about using this transposon for cell or gene therapy applications because it is cheap to manufacture and likely to deliver cargo at high efficiency.”
According to the company, Horizon’s platform enables extremely efficient DNA delivery into a genome, making it a highly useful tool in applications such as cell engineering and therapy, as well as gene therapy. The Helitron-based platform enables a unique method of transferring and delivering DNA. While the majority of transposon-based platforms utilize a “cut and paste” mechanism to transfer DNA, Horizon’s platform uses a “copy and paste” method, allowing users to integrate many copies of a single DNA sequence that are then incorporated into the genome. This makes the platform unique in its sequence and “mechanism of action,” according to Dr. Buerckstuemmer.
This novel “copy and paste” mechanism enables the platform to be used for a variety of applications. “Horizon has full freedom to operate in all areas including cell products, services and therapeutics,” he stated. “The copy and paste mechanism could be superior where particularly high levels of gene expression are needed and where the random integration of the transposon represents an acceptable safety risk.”
Examples are the manufacturing of drugs, which can be costly and challenging, as well as manufacturing standards for cancer cells. The Helitron-based platform can be used as a more efficient and cost effective alternative. “The generation of cell lines that express high levels of biologicals (e.g. therapeutic antibodies) can potentially be obtained much quicker and at lower cost,” explained Dr. Buerckstuemmer. “Another application relates to the manufacturing of reference standards for genomic amplifications such as Her2 that will be used to bench mark diagnostic tests for breast cancer.”
While useful for many applications, the platform shows much promise in optimizing the manufacturing of cell and gene therapies due to its unique mechanism. “We are particularly excited about using this transposon for cell or gene therapy applications because it is cheap to manufacture and likely to deliver cargo at high efficiency [through its copy and paste function],” stated Dr. Buerckstuemmer. “Such characteristics are desirable, for instance, for approaches that re-wire T cells in cancer ([such as] CAR-T cell therapy).”
The technology is meant to complement existing CRISPR technologies and is part of a broader offering that Horizon is in the process of developing and in-licensing, according to Dr. Buerckstuemmer. “With this tool kit at hand, Horizon will be able to broaden its scope and utilize the right tool at the right time—depending on the specific application,” he said.
While Horizon’s Helitron transposon–based “cut and paste” mechanism boosts efficiency in gene editing by allowing for greater DNA transfer and delivery in CRISPR platforms, MilliporeSigma’s recently developed technique increases efficiency by providing greater target specificity.
MilliporeSigma’s Proximal CRISPR Technique
In MilliporeSigma’s research paper on proximal CRISPR (proxy-CRISPR), published in Nature Communications in April, various CRISPR-Cas systems are discussed, including the hypothesis that type II-B FnCas9 from Francisella novicida has the “ability to access target sites in certain mammalian chromatin contexts.” FnCas9, as used in the proxy-CRISPR technique, specifically helps with common CRISPR challenges, such as off-target effects, by requiring two CRISPR DNA binding events for cutting, making the proxy-CRISPR method much more target specific. “The requirement for two binding events doubles the size of the DNA target sequence, making it exponentially more unique,” said Martha S. Rook, PhD, head of Gene Editing & Novel Modalities at MilliporeSigma.
The “two binding events” that Dr. Rook referred to relates to the proxy-CRISPR method, which requires the use of catalytically dead SpCas9 to bind to the DNA. By using two dead SpCas9, which are able to penetrate the chromatin in human cells, the FnCas9’s nuclease activity is restored in the cell. If the FnCas9 was used on its own, its activity would be inhibited due to its inability to target the chromatin structures. This makes the proxy-CRISPR method much more efficient and specific than traditional CRISPR methods.
The Nature research paper also indicates that the same genomic sites in various genes can be “selectively modified” with proxy-CRISPR through using selected proximal dCas9-binding sites to distinguish the identical genomic sites. This enables the proxy-CRISPR method to be extremely useful in increasing the specificity in diseases for which genome editing can be quite complicated, such as blood diseases. “It is well known in the genome editing community that the genes involved in blood disease share high homology, meaning the DNA sequences are the same across many blood genes,” explained Dr. Rook. “This creates challenges when gene therapy researchers want to target only one hemoglobin gene.”
“The requirement for two binding events doubles the size of the DNA target sequence, making it exponentially more unique.”
Citing the research in the Nature paper, Dr. Rook pointed out how proxy-CRISPR can target a specific site in a specific area. “Proxy-CRISPR, using dead-SpCas9 and active CjCas9, can be used to cut a site in only one location, Hemoglobin Subunit Beta (HBB), even when that exact DNA sequence also exists in Hemoglobin Subunit Delta (HBD),” she said. “[In this example,] there is some DNA sequence surrounding the CjCas9 target site, which is different between HBB and HBD, and dead-SpCas9 was targeted to only the HBB allele, allowing CjCas9 to cut only the HBB allele.” This example illustrates how proxy-CRISPR can “improve specificity in challenging genome editing scenarios,” she said.
Deciding what CRISPR-Cas system to use is largely driven by what the DNA sequence of a specific gene is. “Different diseases have different DNA sequence requirements,” stated Dr. Rook. According to Dr. Rook, knowing what these DNA sequences require can only be predicted by examining the DNA sequences that are thought to play a major role in disease phenotypes, as opposed to solely examining the phenotypic nature of the disease itself. “Simply put, the greater the variety of DNA sequences you can target with CRISPR, the greater the number of diseases you can address,” explained Dr. Rook. “Depending on the priority of efficacy vs specificity, the appropriate CRISPR combination can then be employed.”
She added, “The great thing about proxy-CRISPR is that it increases the number of CRISPR systems that can be used, and thus the number of applicable DNA sequences and diseases.” The proxy-CRISPR method is expected to open new gene editing opportunities for MilliporeSigma’s customers, as well as the company’s business partners and internal cell engineering R&D teams, according to Dr. Rook.