State of Research

CRISPR: Growing in Popularity, But Problems Remain

The CRISPR gene-editing technology is taking the scientific world by storm, but researchers are still uncovering the platform’s potential and pitfalls.

The “Democratization” of Gene Editing
The idea of modifying human genomes using homology directed repair (HDR) has been around for decades. HDR is a widely-used repair mechanism to fix double-strand breaks in the cell’s DNA. By supplying an exogenous, homologous piece of DNA to the cell and increasing the probability of HDR occurring, changes in the DNA sequence can be introduced to the targeted area.

Some of the first gene editing platforms taking advantage of HDR used engineered endonucleases such as zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). Both ZFNs and TALENs required a custom protein to target a specific DNA sequence, making them pricey and very difficult to engineer. The more recently developed CRISPR/Cas9 platform works differently: instead of using a protein, the Cas enzyme uses a small guide RNA to locate the targeted DNA, which is then cut. CRISPR is more low tech and “user-friendly” than other platforms, as users are no longer required to do the onerous chore of protein engineering. Now, the ability to modify genomes can be done without extensive training or expensive equipment. CRISPR can be used for knocking out genes, creating reporter or selection genes, or modifying disease-specific mutations—by practically anyone with basic knowledge of molecular biology.

The CRISPR Revolution
Because of its relative simplicity and accessibility, it is no wonder that life scientists from all over the world are eager to incorporate CRISPR into their research. One of the most remarkable things about CRISPR technology is how quickly its popularity has allowed the platform to evolve. The discovery revealing that CRISPR can be used for RNA-programmable genome editing was first published in 2012 (1). Since then, about 2,000 manuscripts have been published including this technology and millions of dollars have been invested into CRISPR-related research and start-up companies. Scientists have developed a myriad of applications for CRISPR, which can be used in practically any organism under the sun. The popularity and progress of gene editing promises revolutionary advancements in virtually every scientific field: from eradicating disease-carrying mosquitoes to creating hypoallergenic eggs and even curing genetic diseases in humans.

There is no doubt that CRISPR has enormous potential – the widespread interest and rapid progress are evidence of that. The main reason CRISPR has been so widely adapted is because development and customization is way less labor-intensive and time-consuming than previous methods. But material preparation is a miniscule part of the CRISPR platform. Contrary to popular belief, just because CRISPR is easier to use than other methods of gene editing, it is not “easy.”

Hitting the Bull’s-Eye
To understand the limitations of this gene editing technology, it’s important to understand more about how CRISPR works. The CRISPR/Cas9 system functions by inducing double-strand breaks at a specific target and allowing the host DNA repair system to fix the site of interest. Cells have two major repair pathways to fix these types of breaks, Non-Homologous End Joining (NHEJ) and homology directed repair (HDR). NHEJ is faster and more active than HDR and does not require a repair template, so NHEJ is the principle means by which CRISPR/Cas9-induced breaks are repaired. If the editing goal is to induce insertions or deletions through NHEJ to cut out a part of a gene, then using the CRISPR platform is less complicated. But directing CRISPR to correct a gene with exogenous DNA does not work very well, as the rate of HDR occurring at the site of interest is often very low, sometimes less than 1%. For precise genome-editing through CRISPR, it is essential to have HDR while minimizing damaging NHEJ events.

Increasing the rate of HDR is not trivial. Recent research has shown that the conditions for the two repair pathways vary based on the cell type, location of the gene, and the nuclease used (2). Besides, optimizing CRISPR to better control HDR versus NHEJ events is only half the battle. One of the greatest challenges in using CRISPR is to be able to quickly and accurately detect different genome-editing events. Many scientists measure changes by sequencing. A more sophisticated method is to use droplet digital PCR (ddPCR) (3), but many labs have limited accessibility to ddPCR systems. After screening multiple clones and detecting the desired genetic edit comes what is usually the most laborious and time-consuming step: pure clonal isolation. Because each individual cell is affected by CRISPR independently, isogenic cell lines must be established.

Improving CRISPR
CRISPR has become the gold standard for many scientists now that the potential to perform gene editing has become so universal. Top journals expect isogenic lines for characterizing genes and mutations, and many researchers feel pressured to include such experiments to stay competitive in grant proposals. Moreover, a federal biosafety committee has recently approved the first study in patients using this genome-editing technology. To keep up with the pace of this rapidly moving field, significant improvements in CRISPR technology need to be made.

First, a better grasp of the basic principles behind CRISPR is likely to lead to improvements in targeting and efficiency. For example, understanding how the cell type, locus, and genomic landscape affect the targeting and cutting of Cas9 could lead to higher efficiencies of HDR. Similarly, engineering Cas9 and the guide RNA to maximize on-target activity could also accelerate the technology’s success. Screening the ability of the guide RNA to target the intended sequence can be performed in vitro by assessing the Cas9-mediated cuts on a PCR-amplified fragment of DNA; this method often gives a good indication of what to expect in cells. Another hindrance to CRISPR technology is that currently, all methods to detect genome changes require destruction of the cells of interest. Development of an assay to detect chromosomal changes in live cells – reminiscent of live-cell RNA detection – would immensely improve the processes of clonal screening and isolation.

Besides the technical challenges that come with maximizing on-target edits is that Cas9 frequently has off-target effects, producing insertions or deletions at unintended sites. Online algorithms can predict where some of these cuts are likely to occur, but currently, there is no efficient method to identify all possible off-target sites (4). To complicate things further, no two human genomes are identical. Due to genetic variation, predicting off-target effects based on reference genomes remains a challenge. Because so much of the hype around CRISPR surrounds the potential to treat human diseases, it is imperative to make sure that CRISPR does not introduce detrimental changes elsewhere in the genome before therapeutic use in humans. Standard screening methods and biological assays need to be established to robustly assess potential damage done to other sites in the genome and to measure its impact on cell function and mutagenesis.

Despite its shortcomings, the hub of activity surrounding CRISPR has been astounding. And with the force of thousands of scientists working on this technology, the possibilities for the future of CRISPR are boundless.

 


 

References

  1. Jinek, M., et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816-21.
  2.  Miyaoka, Y., et al., Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci Rep, 2016. 6: p. 23549.
  3.  Hindson, B.J., et al., High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem, 2011. 83(22): p. 8604-10.
  4.  Stella, S. and G. Montoya, The genome editing revolution: A CRISPR-Cas TALE off-target story. Bioessays, 2016. 38 Suppl 1: p. S4-s13.

Generation of Human Stem Cells under Good Manufacturing Practice: Facility Update

cGMP Facility on Nancy Ridge Dr.

Allele’s New cGMP Facility on Nancy Ridge Dr.

Last year Allele dedicated a new building space for cleanroom operations to provide a cell banking service for personalized medicine. This facility will be the center of current Good Manufacturing Practice (cGMP) production of human induced pluripotent stem cells (iPSCs) using Allele’s proprietary synthetic mRNA platform. Over the past three months, progress to get the facility up and running has been substantial. Our facility includes four main modules: the reception area and doctors’ offices, a Fibroblast Isolation and Maintenance room, a Reprogramming and iPSC Maintenance room, and a Quality Control room. Air handling, which is a major component of the environmental control system, has been installed and validated. Equipment such as biosafety cabinets, incubators, and refrigerators have been installed and qualified, as well as equipment for performing essential quality control steps. To standardize personnel-related steps of cGMP processing, we have prepared rigorous SOPs and have extensively trained individual manufacturing operators. Overall, we are enthusiastic about the facility’s progress and are committed to delivering the best possible service as the industry leader in iPSC banking.

ScientistHood

iPSC roomCells

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cGMP Compliance: What Does It Mean for Your Cell Lines?

As the promise for cell-based therapy grows, the interest in making clinically relevant cell lines has skyrocketed for industrial and academic researchers alike. For translation into human therapies, cell-based products must be made following current Good Manufacturing Practice (cGMP). Many groups have already claimed to generate cell lines that are “cGMP-compliant,” “cGMP-ready,” or “certifiable under cGMP.” But what does it take to be truly cGMP-compliant, and what practices can you introduce in your lab to comply with cGMP standards?

A common misconception in the United States is that a facility is granted a ‘cGMP license’ from the government to manufacture cGMP-grade products. Rather, the Food and Drug Administration (FDA) evaluates the manufacturing process for each product to determine if it is compliant with cGMP standards. The primary concern when it comes to deriving cell-based products for therapies is making sure that the product is derived in a safe and reproducible manner. To ensure maximum quality assurance, researchers should

• choose reliable, xenogeneic-free raw materials,
• establish and monitor a clean environment,
• qualify all equipment and software,
• remove variation in laboratory procedures by creating detailed Standard Operating Procedures (SOPs) and by providing rigid process validation at each step.

Nevertheless, even establishing robust quality assurance does not imply that the process is scalable for commercial production. In the world of biologics, “the product is the process.” A requisite step to ensure a smooth transition to cGMP practice is to ensure that the process of manufacturing is not altered due to changes in production scale. For example, depending on the therapy, millions or billions of cells may be required for a single patient. Therefore, it is in the best interest of the researchers to develop a scalable method at the beginning to avoid revamping the entire process (e.g., changing from adherent cells to suspension). Along these lines, the quality control (QC) requirements of cell-based products should be carefully considered and not have to include difficult-to-assay tests. For example, some cell lines have been qualified as cGMP-compliant upon conversion from research-grade conditions to cGMP quality standards. Rigorous tests were performed on the converted lines to ensure that the cells were free of contamination. Even though strict measures were carried out to ensure cGMP compliancy, deriving cell lines in this manner makes scalability and reproducibility a challenge. Ideally, the entire process of deriving cell products for clinical use should be performed under cGMP conditions: from the acquisition of human tissue to the manufacturing, testing, and storage of derivative cell products.

Another important consideration when instituting cGMP-compliance is documentation. Each process must be described with rigorous SOPs, the training of individual manufacturing operators must be well-documented, and the entire established process must be validated and well noted. Failure to document—in the eyes of the FDA—is often equated with failure to perform the underlying activity. It is equally important to remain ‘current.’ The FDA expects manufacturing processes to stay up-to-date with current regulations, even as policies change.

For an academic lab, closely aligning with cGMP standards can ensure that the resulting cell lines are comparable to other truly cGMP-produced products used during clinical trials. It is in the best interest of academic researchers to establish rigorous SOPs and use qualified reagents and equipment, even if it is not possible to carry out all steps in a certified cleanroom. Whenever possible, it is advisable to acquire truly cGMP cell lines from appropriate sources for preclinical projects; if prohibited by costs or other reasons, it is recommended to use a protocol that is as close to cGMP as possible.

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Press Release: Allele Biotechnology & Pharmaceuticals Closes Purchase of cGMP Facility for Production of Clinical-Grade Cells for Cell Therapy Applications

SAN DIEGO–(BUSINESS WIRE)–Allele Biotechnology & Pharmaceuticals, Inc. (“Allele”), a leader in the development of specialized cells for pharmaceutical drug discovery and regenerative medicine, today announced that it has closed the purchase of a new facility intended for its cGMP (current good manufacturing practices) production of clinical-grade cells for cell therapy applications.

The 18,000 square-foot facility, located near the main headquarters of Allele in San Diego, California, will be the center of production of human induced pluripotent stem cells (hiPSCs) using Allele’s proprietary synthetic mRNA platform, a technology that generates hiPSCs with neither the random integration of foreign DNA nor the use of whole virus or virus-based elements, drawbacks that are common to other technologies for making hiPSCs. Such “footprint-free” cells will be produced by Allele for industrial and academic partnerships, as well as Allele’s own efforts in the area of cellular therapeutics.

hiPSCs, as cells that have the potential to become any cell in the human body, hold great promise for therapies that can alleviate or cure human disease. Towards this end, Allele has recently made a number of advances regarding the differentiation of hiPSCs towards cells of specific lineages, such as neural progenitor cells, neurons, astrocytes, mesenchymal stem cells, cardiomyocytes, skeletal muscle cells, hepatocytes, and adipocytes, including brown fat cells. These cells would also be produced in the cGMP facility when intended for specific therapies.

“This dedicated facility will help us to realize a number of our visions in bringing the benefits of pluripotent stem cells to society,” said Jiwu Wang, Ph.D., President and CEO of Allele. “The first step in helping people in need with all the stem cell technologies developed in labs is to clear a path to move them from bench to bedside, which requires high-quality, controlled production that can be monitored by the FDA. Together with our licensees, drug development partners, investors, and individuals who would like to participate in banking hiPSCs for research and therapy, we anticipate even faster pace in our business development in this area.”

Contacts

Allele Biotechnology & Pharmaceuticals, Inc.
Jiwu Wang, 858-587-6645
info@allelebiotech.com

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The NIH is one step closer to be short of $260 million from its 2011 budget

The National Institutes of Health (NIH) 2011 budget will be cut by $260 million in the budget that the House has just passed based on the last minute pact reached last Friday to avoid federal government shut down.

The NIH’s 2011 budget will be $30.7 billion, down 0.8% from its 2010 budget of $30.9 billion, according to news releases that can be found from various sources. Previously, the President proposed a $32.1 billion budget for the NIH and the House of Representatives allocated $29.4 billion to the agency. President Obama asked for a $1 billion increase for the NIH in 2012, which will be in new debate to start immediately. Chances are the 2012 budget for the agency will be less than what the administration wanted.

Combined with “the cliff effect” from the ending of the stimulus money the NIH has epically managed since 2009 to fund extra research projects, the negative growth of the NIH budget could mean less academic positions and tighter lab budgets ahead. Cutting-edge technology and cost effectiveness will be the key for survival of the fittest in the biomedical research jungle.

Promotion of the week: Save 10% on any purchase of feeder cells. Email brianahasey@allelebiotech.com with offer code : FDST11

New Product of the week: Damage-free cloning kit for difficult cloning projects—get recombined plasmids or failed ligation? Your DNA is damaged by purification bugger and/or UV, ask us how to deal with it oligo@allelebiotech.com

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Thursday, April 14th, 2011 NIH Budget and You, State of Research 1 Comment