Special Focus on CRISPR: Regarding lipids and CRISPR

On one side, the gene-editing technology tackles lipids; on the other, it uses them for transportation; plus additional news of CRISPR research and technology and a bonus guest commentary

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Special Focus on CRISPR: Regarding lipids and CRISPR
On one side, the gene-editing technology tackles lipids; on the other, it uses them for transportation; plus additional news of CRISPR research and technology and a bonus guest commentary
Lipids have always been a classic “two sides of the same coin” biological feature, if for no other reason than the whole HDL (“good” cholesterol) vs. LDL (“bad” cholesterol) issue. In much the same way, in leading off our CRISPR special focus section this issue, we thought we might visit two sides of lipids in terms of CRISPR/Cas9 gene editing: one side being lipid control, and the other one being lipids as a form of therapeutic delivery.
And to quickly recap for those not fully in the know about clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9), the CRISPR/Cas9 system is based on an antiviral defense mechanism in bacteria in which the Cas9 enzyme recognizes the viral DNA sequences of previous infections and cuts up invading DNA during re-infection. Researchers have engineered the CRISPR/Cas9 system to not only locate and cut specific sequences of DNA, but to also turn on or off the expression of targeted genes without making permanent changes to the DNA coding sequence.
Silencing a high-cholesterol gene
In late April, biomedical engineers at Duke University reported that they had used a CRISPR/Cas9 genetic engineering technique to turn off a gene that regulates cholesterol levels in adult mice, leading to reduced blood cholesterol levels and gene repression lasting for six months after a single treatment. The study appeared online in Nature Communications on April 26 under the title “RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors.”
According to Duke, this marks the first time researchers have delivered CRISPR/Cas9 repressors for targeted therapeutic gene silencing in adult animal models. Certainly, this CRISPR/Cas9 repressor technique has emerged as a robust tool for disrupting gene regulation in cell culture models, but there have been challenges in adapting it for delivery to adult animals in applications such as gene therapy.
In their most recent study, Dr. Charles Gersbach, the Rooney Family Associate Professor of Biomedical Engineering at Duke, and members of his laboratory developed an approach to efficiently package and deliver the CRISPR/Cas9 repressor system to mice. They tested their delivery system by silencing Pcsk9, a gene that regulates cholesterol levels. While several drugs have been developed to treat high cholesterol and cardiovascular disease by blocking the activity of Pcsk9, this new approach would prevent Pcsk9 from being made.
“We previously used these same types of tools to turn genes on and off in cultured cells, and we wanted to see if we could also deliver them to animal models with an approach that is relevant for gene therapy,” Gersbach said. “We wanted to change the genes in a way that would have a therapeutic outcome, and Pcsk9 is a useful proof of concept given its role regulating cholesterol levels, which in turn affect health issues like heart disease.”
To test the targeted Pcsk9 repressor in an adult animal, the team opted to use adeno-associated viral (AAV) vectors—small viruses that have been engineered to target a variety of tissue types in human gene therapy clinical trials. Due to the vector’s small cargo limit, the team couldn’t use the common Cas9 enzyme from Streptococcus pyogenes. Instead, they opted to use a smaller Cas9 from Staphylococcus aureus. They also deactivated the DNA-cutting function of Cas9, creating a “dead” version of the enzyme, dCas9, that binds to but does not cut the targeted DNA sequence.
The dCas9 can be combined with a KRAB protein that silences gene expression, creating a CRISPR/Cas9 repressor that blocks transcription, reduces chromatin accessibility and silences gene expression without altering the underlying DNA sequence. Using an AAV vector to deliver CRISPR/Cas9-based repressors to the mouse liver, the researchers reduced Pcsk9 and cholesterol levels in treated mice.
While the experiment was successful, the researchers also observed the release of liver enzymes into the blood only in treatments that included Cas9. While these liver enzyme levels remained below a critical threshold and normalized over time, their elevated levels indicated that the therapy potentially caused immune responses in the liver, where the virus and Cas9 enzyme accumulate. That raises questions about the efficacy of multiple injections.
“One of the interesting things we found looked like an immune response against the Cas9 protein,” said Pratiksha Thakore, the Ph.D. student who led the work in Gersbach’s lab. “Following injection, we saw that levels of our target gene, Pcsk9, were reduced, but we also observed increases in expression of many immune cell genes, which indicates that immune cells were infiltrating the liver after we delivered Cas9 to the mice. Gaining a better understanding of this immune response and how to modulate it will be important for using Cas9 technologies for therapies.”
This might be due to the fact that the Cas9 enzyme is derived from bacteria; as such, the immune system might see it as an “invader” and move to attack it. In addition, researchers worry that potential patients for CRISPR/Cas9 therapies may already be primed to resist such therapies because the Cas9 enzymes most commonly used in research come from common bacteria to which humans are routinely exposed.
“The field is just starting to look at this, and it’s clear that immune response is an important issue,” said Gersbach. “Although we did see an immune response in the mice when we administered Cas9, the levels of liver enzymes in the serum seemed to mitigate over time without any intervention, and the effect of Pcsk9 repression was sustained regardless.”
“There are still lots of things for us to explore with this approach,” added Thakore. “CRISPR/Cas9 tools have worked so well in cell culture models that it’s exciting to apply them more in vivo, especially when we’re examining important therapeutic targets and using delivery vehicles that would be relevant to treating human diseases.”
Driving CRISPR with a lipid vehicle
Meanwhile, in news from earlier this year, back in February, Intellia Therapeutics Inc. announced that Cell Reports had published its manuscript, “A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing.”
According to the company, the lipid nanoparticle (LNP) delivery of Cas9 mRNA and sgRNA resulted in 97-percent reduction in mouse transthyretin (TTR) protein levels in the liver, and the reduction was sustained for at least 12 months. The publication also documented that CRISPR/Cas9 components were undetectable in mice within three days after administration of Intellia’s LNP delivery system. Researchers further demonstrated that Intellia’s LNP technology is a similarly robust and effective delivery method for CRISPR/Cas9-mediated knockdown in rats, a higher rodent species.
“These data show that our proprietary lipid nanoparticle technology achieves significant and enduring editing of the TTR gene through a single dose,” said Dr. David Morrissey, senior vice president of platform and delivery technology at Intellia. “Our lipid nanoparticle system is a transient expression system that enables CRISPR/Cas9 to make the intended gene edit and then clear from the cells. Minimizing the duration of CRISPR/Cas9 components in cells is desirable, as that may reduce the potential for safety issues associated with the continued presence of those components. The LNPs also allow us to re-dose, if needed, to attain the desired target effect. This paper details the most effective systemic delivery of CRISPR/Cas9 components reported to date, further supporting our IND-enabling activities this year and future potential treatments for liver-based genetic diseases.”
The data included in this publication build on earlier findings initially released last year at the Le Stadium Conference on Messenger RNA Therapeutics, and later presented at the 20th Annual Meeting of the American Society of Gene and Cell Therapy and the 13th Annual Meeting of the Oligonucleotide Therapeutics Society.

Guest commentary: A CRISPR view of gene editing
By Leala Thomas of Bio-Rad Laboratories
The power to edit a gene is the power to change its function. From generating novel cell lines and better animal models for the discovery and preclinical phases of therapeutic research to creating a therapeutic itself, CRISPR gene editing is emerging as the leading technique that allows rapid advances to happen. Downstream applications are numerous and changing the way we approach treating and curing disease.
Gene-editing techniques
The earliest forms of gene editing were based on homologous recombination—the ability to exchange nucleotide sequences between similar pieces of DNA. These methods only worked in a small subset of model systems where recombination frequency was high enough to achieve results. Even in those systems, the process was laborious, requiring stringent and time-consuming selection protocols. The field of genome editing was propelled forward with the discovery that the frequency of homologous recombination could be increased by using an endonuclease to generate a double-stranded DNA break. Over the past 20 years, this discovery has led to the development of a range of genome-editing technologies—meganucleases, zinc finger nucleases (ZFNs), TALENs and CRISPR/Cas9—that have expedited basic research and become incorporated into clinical trials.
CRISPR/Cas9 is the simplest gene-editing technique developed to date and has truly revolutionized the field of gene editing. Because it uses RNA rather than a protein to target nuclease activity, CRISPR/Cas9 can be retargeted by simply synthesizing or ordering a new guide RNA (gRNA). The RNA guide also makes this technique amenable to high-order multiplexing; large-scale screens using hundreds or even thousands of gRNAs in a single experiment have been performed. Complex animal models that once took years to generate are now completed in just a few months.
CRISPR also has the potential to address the failure rate that many promising drug candidates currently experience when transitioning from preclinical animal testing to early-stage testing in human clinical trials. Unlike traditional gene-editing techniques that were limited to a small number of species, CRISPR has been used successfully in a wide range of species, resulting in a vast expansion of available model systems tailored to the study of specific diseases which could provide a more human-like animal model.
Gene therapy
Thousands of diseases have an underlying genetic basis, some in the form of a single-gene mutation, such as cystic fibrosis and hemophilia, while others are more complex, like cancer. The field has made the largest strides in monogenic diseases of the blood such as sickle cell disease, followed by diseases where it is possible to genetically modify the liver (currently treated by enzyme replacement therapy) and finally cancer. The first ZFN gene-editing therapy to move to clinical trials was for HIV. Others followed as potential treatments for hemoglobinopathies, such as β-thalassemia and sickle cell disease.
Some therapies in clinical trials appear very promising, because they can offer a cure for the disease and also save on healthcare costs. For example, gene editing is being used to introduce a corrective gene into the DNA of liver cells from patients with mucopolysaccharidosis II (MPS II), enabling the liver to produce the missing enzymes. In two additional clinical trials, hemophilia B and MPS I are being targeted with similar therapies that impact the enzymes involved in those diseases.
In August 2017, the U.S. Food and Drug Administration (FDA) approved the first gene therapy in the United States, a drug called Kymriah from Novartis for B cell acute lymphoblastic leukemia. This CAR-T cell therapy removes T cells from the patient’s blood and introduces a gene that expresses the chimeric antigen receptor (CAR) protein that, once reinfused, directs T cells to kill leukemia cells in the blood.
Cost and pricing models
Gene-editing therapies are usually accompanied with a hefty price tag often based on an outcomes-based pricing model. This model creates an almost money-back guarantee for payers, in that if the treatment works, the payer trades a lifetime of treatments for the disease and its complications for a single upfront cost.
When these costs are weighed, many expect the one-time cost for gene-editing therapies to pale in comparison to current estimates for lifetime treatment, assuming the disease is not terminal at a young age. Each proposed system is not without its issues. What if a patient fails to return to the doctor to test for remission? What if the patient switches insurance companies? As more gene therapies reach the market and patients demand them, industry stakeholders must find solutions to allow for widespread access.
Overall, the best gene therapies will be those that provide cures in one-time doses and actually reduce the lifetime patient healthcare costs related to the disease. If a pharmaceutical company can make that case, it is expected that payers will cover the cost of its gene therapy treatment.
Gene-editing workflow
Of significant importance to researchers doing genome editing are the time, cost and efficiency of the methods used. The following gene-editing workflow proposes tips for streamlining the process.
Step 1: Transfection
A common first step in a CRISPR gene-editing workflow is to identify the best method for delivering the CRISPR/Cas9 system into the cells of interest: animal or human, tumor-derived or wild type. Transfer efficiency and subsequent cell viability are very important when considering which method to use. The standard approach to transfection, which involves injecting cells with separate CRISPR elements, is costly and inefficient. However, injecting pre-assembled CAS9 protein/ RNA complexes can increase success in challenging cell types. Electroporation and lipid transfection are the two primary methods used; each lab will need to test to find the preferred method for their cell type.
Step 2: Enrichment and single-cell isolation
After transfection, a researcher then selects for edited cells to increase the efficiency of the workflow. The standard CRISPR workflow takes two-and-a-half months and a series of steps to isolate the cells of interest. Because not all of the cells will be transfected, inclusion of a reporting protein, such as GFP, may be used as part of the gene-editing strategy. This fluorescent tag can be used to enrich for only those cells that have GFP, as a surrogate for those cells which carry the edit, using flow cytometry. Integrating a cell sorter into this workflow can potentially reduce the timeline by about 30 days, which could reduce the unnecessary costs associated with reagents and consumables. Enrichment also reduces the number of passages cells undergo, ensuring healthy cells for downstream assays.
Step 3: Confirmation of edits
After enrichment, the presence of the desired edits is verified using a variety of methods. Sequencing of the edited gene may be performed to identify the location of the edits. In some cases, droplet digital PCR (ddPCR) can be used to detect HDR and NHEJ edits. Based on the results, an appropriate number of single cell clones are then harvested. The ddPCR method provides precise and sensitive detection and quantification of edits at or below 0.5 percent frequency. In addition, standard surveyor tests may be utilized, such as high-resolution melt assays, which provide a low-cost option for quickly screening mutations.
Confirmation of an edit may also be made at the protein level, whereby protein expression is monitored either using western blotting or immunohistochemistry. For example, some edits may disrupt a gene sequence leaving a modified or truncated protein. This analysis will allows the researcher not only to see changes in expression of the entity gene but also any off-target effects or other genes.
Step 4: Downstream analysis
Downstream analysis can take the form of genetic, phenotypic, cellular or proteomic analyses. Lead target validation and discovery scientists can benefit from multiplex assays that enable measurement of several genes, proteins or cellular markers simultaneously, ensuring the edit is serving its intended purpose in the cell.
Next steps in gene editing
The possibilities are endless. Gene-editing applications are expanding rapidly as tools become cheaper and easier to use. As access increases, demand will also increase. This technology allows researchers to build any cell that they need by adding missing genes or removing genes that cause toxicity. Gene editing could provide hope for millions of people suffering from diseases with insufficient treatment options or for those without current treatment options.
Leala Thomas is a marketing development manager at Bio-Rad Laboratories. In her current position, she focuses on campaign development for the drug discovery and development group, a role which includes outbound marketing and global training. Please visit here for more information on genome editing. Please visit here for more information on genome editing.

Positive data on CRISPR CAR-T
ZUG, Switzerland & CAMBRIDGE, Mass.—Mid-April saw CRISPR Therapeutics, a biopharmaceutical company focused on creating gene-based medicines for serious diseases, announce the presentation of new data from the company’s allogeneic chimeric antigen receptor T cell (CAR-T) program at the American Association for Cancer Research (AACR) Annual Meeting 2018.
According to the company, the data presented “demonstrate the generation of CAR-T cells targeted to BCMA and CD70 through CRISPR/Cas9 gene editing that have high editing rates, consistent expression and selective and potent cell killing,” adding that the findings confirm and expand on work already completed on CTX101, the company’s lead allogeneic CAR-T cell therapy in development for CD19+ malignancies.
“In the studies presented today, we used multiplexed CRISPR gene editing to modify healthy donor T cells to make CAR-T cells that selectively and potently target the tumor antigen of choice,” said Dr. Tony Ho, head of research and development at CRISPR Therapeutics. “These data provide further evidence that CRISPR/Cas9 can play a major role in enabling the creation of next-generation CAR-T cell therapies that may work for a broader population of patients, including those with solid tumors.”
New RNA CRISPR tool normalizes tau splicing
LA JOLLA, Calif.—Dr. Patrick Hsu and colleagues at the Salk Institute recently identified a family of bacterial RNA-guided, RNA-selective nucleases. As they are considerably smaller than known Cas nucleases, they are said to efficiently and specifically knocked down mRNAs in human cells. Putting the technique to a unique test, Hsu and colleagues used the ribonucleases to manipulate mRNA splicing, too—the scientists reversed aberrant tau mRNA splicing in neurons derived from a patient with frontotemporal dementia, foreshadowing future therapeutic possibilities.
As an extra bonus, Salk noted that “the petite ribonucleases are easier to package into viral vectors and introduce into mammalian cells.” Hsu’s work appeared in Cell on March 15. A second group, led by David Cheng and David Scott from Arbor Biotechnologies in Cambridge, Mass., independently discovered the same enzyme family and published their work the same day in Molecular Cell.
While much attention has focused on using CRISPR/Cas editing of DNA to treat disease, Hsu sees advantages to targeting RNA. DNA editing permanently alters the genome, and introduces the risk of DNA damage. Changes to DNA tend to be all or nothing; that is good for ablating or replacing a gene wholesale, but not for fine-tuning protein or isoform expression, he has noted. In contrast, targeting dynamic changes in RNA allows for reversible tweaking of both. In addition, an estimated 15 percent of genetic diseases stem from RNA mis-splicing, and would be more amenable to RNA- than DNA-targeted editing approaches.
New CRISPR system discovered via discovery platform
CAMBRIDGE, Mass.—Arbor Biotechnologies, an early-stage life-sciences company, recently published its first findings of a new member of the CRISPR/Cas13 enzyme family—Cas13d, arising from Arbor’s discovery platform.  
In a paper published in the journal Molecular Cell, Arbor reported a new enzyme, Cas13d, that is significantly smaller than other members of the CRISPR-Cas13 family and has exciting implications for applications such as RNA manipulation and highly sensitive diagnostics. The discovery was made utilizing Arbor’s proprietary platform, which enables the high-throughput discovery and identification of enzymes that provide new protein functionalities and catalytic activities.
“Arbor’s revolutionary platform accelerates the rate of discovery and characterization of new biomolecules by orders of magnitude,” said Arbor founder David Scott. Added Arbor founder Winston Yan: “We are now at the cusp of being able to convert sequence data into a catalog of protein functions. The possibilities are limitless.”
The discovery of CRISPR-Cas13d represents the first significant milestone for Arbor's discovery pipeline and, the company says, illustrates the benefit of scaling protein characterization.
The market and potential for CRISPR/Cas9 gene editing
Kalorama Information shares insights about CRISPR’s present and future in a new report
CRISPR/Cas9 is a revolutionary approach that enables rapid, economical model generation through precise genome editing. Kalorama notes that in 2018, more than 30 players participate in this market, from large multinational corporations to small, niche life-sciences companies. Key players include Agilent, MilliporeSigma, GE Healthcare Dharmacon, GeneScript, Horizon Discovery Group, OriGene, ThermoFisher Scientific, Transposagen and ToolGen.
These companies develop and commercialize various products and services for each of the major gene-editing technologies. As Kalorama points out, the nature of CRISPR’s complex technology requires partnerships between the developers of the technology and the healthcare concerns with distribution strength and a broader product base. These developments are outlined in the report “The Market and Potential for CRISPR/Cas9 Gene Editing,” and highlights are provided below.
Recent lawsuit and industry risk analysis
Currently, one of the more significant factors in commercializing CRISPR/Cas9 as a viable therapeutic tool is the current legal battles surrounding the technology. The issue started when both the University of California, Berkeley and the Broad Institute of MIT both filed patents related to this technology. While there's been some resolution, many questions about intellectual property rights remain.
Recent market developments
  • The most important funding source that fuels a market for research instrumentation is the National Institutes of Health. Overall though, grants for CRISPR in 2015 represented $267 million, but expanded to more than $1 billion by 2017.
  • CRISPR/Cas9-based technologies have strongly increased genome engineering efficiencies in bacteria. This has enabled more rapid metabolic engineering of both the model host Escherichia coli and non-model organisms like Clostridia and Streptomycetes, opening new possibilities to use these organisms as improved cell factories. The discovery of novel Cas9-like systems from diverse microbial environments will extend the repertoire of applications and broaden the range of organisms in which it can be used to create novel production hosts.
  • In December 2017, Vertex Pharmaceuticals and CRISPR Therapeutics announced that the companies will co-develop and co-commercialize CTX001, an investigational gene-editing treatment.
  • GE Healthcare Dharmacon now offers an arrayed synthetic form of CRISPR/Cas9 that allows many different assays (e.g., enzymatic, endpoint, secreted factors) to be performed without resorting to antibiotic selection, long time points or cell splitting.
  • Near the beginning of last year, the FDA proposed draft guidance suggesting modifications intentionally introduced into animal genomes should be regulated in a manner comparable to new drugs, meaning developers would have to show efficacy, animal and human safety and safety for the environment.
  • In late April 2017, researchers from the Broad Institute of MIT and Harvard, other institutes and departments at MIT and Harvard, the Howard Hughes Medical Institute and the National Center for Biotechnology Information published an article in Science titled “Nucleic acid detection with CRISPR-Cas13a/C2c2.”  In this article, they describe a CRISPR-based diagnostic which they called Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK). This platform combines the Cas13a enzyme with isothermal amplification. The authors used this platform to detect the Zika and Dengue viruses, pathogenic bacteria, cell-free tumor DNA and other genetic targets.

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