Cas-CLOVER achieves targeted knock-ins in induced pluripotent stem cell lines

Hera Admin on November 17, 2022

As cell line development bioprocessing turns from traditional random plasmid integration to targeted knock-ins for maximizing yield and stability, it is important to choose the right gene editing tools. The RNA-guided Cas9 nuclease from Streptococcus pyogenes (SpCas9), which is the most commonly used enzyme in genome-editing applications, can yield significant levels of off-target activity, with reported off-target cutting as high as 13%. Multiplex editing with SpCas9 for cell line engineering can also lead to high rates of translocations, up to 4% for individual translocations or rearrangements and up to 5% cumulatively.1

A recent study published in Molecular Therapy Nucleic Acids saw the authors at Poseida Therapeutics deploy the dual RNA-guided dimeric endonuclease Cas-CLOVER, as an alternative to CRISPR/Cas9. Hera and Poseida both use Cas-CLOVER technology in different fields of use. Let’s take a closer look at the targeted knock-in data in this study with Cas-CLOVER.

Comparison of knockout and knock-in success in iPSCs between Cas-CLOVER and Cas9

In this study, the team determined HDR (homology-directed repair)-mediated knock-in potential using Cas-CLOVER by examining reporter cassette delivery at several loci (GAPDH, B2M, and HBB) in induced Pluripotent Stem Cells (iPSCs).

Figure 1: Schematic showing targeting strategy.

1 Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched allogeneic CAR-T cells. Madison et al.Molecular Therapy – Nucleic Acids. August 8, 2022.

At most loci, Cas-CLOVER’s indel – as well as knock-in – efficiency is equal to or greater than that of Cas9, as demonstrated in the below example for B2M.

Figure 2: Quantification of indel (left) and HDR rates (right) at the B2M locus.

At HBB, knockin efficiency was higher using Cas-CLOVER, but the difference was probably not due to differential efficiency of target cleavage, as gauged by Cas-CLOVER’s lower indel generation (see image below). Rather, the higher efficiency was likely a result of the larger deletions, with overhangs, that are characteristic of Cas-CLOVER. Cas9 indels generally result in small 1-3 bp blunt-ended cuts.

Figure 2: Quantification of indel (left) and HDR rates (right) at the HBB locus. Flow for HDR measurement at the HBB locus was performed 14 days post-nucleofection to ensure episomal GFP expression from the donor plasmid had decreased below the detection limit.

After Cas-CLOVER editing, potential off-target sites were queried for the presence of indel mutations. Indel frequency at off-target sites did not occur at a statistically significant level above background. The team concluded that, within primary T cells from multiple donors, the Cas-CLOVER platform performs as well as any other targeted nuclease, but with much lower off-target activity. Notably, Cas-CLOVER yields efficient multiplexed gene editing.

Targeted knock-in capability is becoming essential for cell line development

Induced pluripotent stem cells (iPSCs) are being used more and more in therapeutic bioprocessing and in synthetic biology. Moreover, the level of targeting that the Poseida team was able to achieve with Cas-CLOVER is relevant for any cell line development professional – including those engineering custom cells for preclinical research purposes.

Learn more about Hera’s Cas-CLOVER and piggyBac technology

To learn more about Cas-CLOVER, piggyBac, and how Hera can help accelerate your gene editing research with our toolkit, click here. We have everything for your gene editing needs, including flexible front or back-loaded service terms and licensing options. Access our technology as quickly as possible by shopping online today.

References

  1. Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched allogeneic CAR-T cells. Madison et al.Molecular Therapy – Nucleic Acids. August 8, 2022.

Rapid Creation of Bioluminescent Cell Lines for Orthotopic and Metastatic Rodent Oncology Studies

Hera Admin on February 25, 2022

The piggyBac (PB) transposon system delivers highly stable transgenes without gene silencing for over 40 passages, making PB superior to viral methods for creating reporter cell lines. The PB transposon is a mobile genetic element that is integrated into the host genome via the PB transposase “cut and paste” mechanism. To achieve stable genomic integration, The PB transposon is co-transfected with a PB transposase protein or expression vector. The transposase inserts the gene cargo and ITRs into the host genome at TTAA sites (Figure 1).

how-piggybac-works

Figure 1: The piggyBac transposon system

Advantages to piggyBac in Bioluminescent Cell Line Creation

Hera BioLabs has used PB to generate numerous tumorigenic cell lines with robust luciferase expression. With previously validated cell lines, it takes Hera as few as 4 weeks to generate stable luciferase(+) cells for downstream use. These cell lines serve as an invaluable resource for monitoring tumor xenograft growth in either subcutaneous or orthotopic anatomical locations. In the subcutaneous space, in vivo luminescent imaging allows for assessment of primary tumor growth using the AMI HT from Spectral Imaging Instruments Imaging. For example, Hera recently made H358 non-small cell lung cancer cells with stable luciferase expression for subcutaneous inoculation in our SRG OncoRats and tracked tumor growth with luminescent imaging (Figure 2). Importantly, high luciferase expression confers adequate luminescent intensity for visualization of deep orthotopic tumors and metastases.

bioluminescent-rats

Figure 2: In vivo bioluminescent imaging using the AMI HT (Spectral Instruments Imaging). SRG OncoRats were given subcutaneous inoculations with 4 x 106 NCI-H358-Luciferase cells and tumors grown for 5 weeks.

If your efficacy testing requires in vivo imaging or tracking metastases, contact us for studies in luciferase-expressing cell lines. Use one of our previously engineered lines or let us express luciferase in your cell line!

Learn more by contacting us today!

Gene Editing With piggyBac: Creating A Curative Treatment For Beta-Thalassemia

Jack Crawford on October 21, 2020

Genetic Engineering Can Soon Lead To Lifelong Cures For Diseases That Were Previously Incurable – And ß-thalassemia Is The Perfect Example.

Through novel therapeutic methods like cell therapy, scientists have found a way to cure a disease that used to only be manageable. Previous gene editing methods used to treat ß-thalassemia were limited and nonspecific; however, advances like piggyBac and CRISPR-Cas9 based gene editing tools are opening doors for better patient outcomes.

What Is ß-Thalassemia?

Beta-thalassemia is a heritable genetic disease caused by a mutation in the hemoglobin beta (HBB) gene. Inheriting a mutated HBB gene leads to a lower production of hemoglobin.
Decreased hemoglobin is a problem because red blood cells, which carry hemoglobin, are used to transport oxygen throughout the body, so patients diagnosed with ß-thalassemia are often anemic. Patients with severe symptoms typically require invasive and frequent treatments with frequent blood transfusions and iron chelation therapy.

Previously, one patient diagnosed with ß-thalassemia had been treated successfully by having lentiviral delivery of a normal HBB gene into his hematopoietic stem and progenitor cells (HSPC). While successful for this patient, there is a high risk of off-target integration that makes lentiviral delivery an undesirable treatment option.

Viral vector directed gene therapies can be harmful, usually through insertion into multiple sites of the host genome. Problems in immune response could occur, and there remains a possibility of insertional mutations.

Steps To A Cure Through Next Generation Gene Editing

An improved ß-thalassemia treatment would correct the underlying mutation of the HBB gene. Correction of the HBB gene would allow for normal production of hemoglobin and ultimately better oxygen transport.

Using fibroblasts collected from the same patient, it is possible to generate induced pluripotent stem cells (iPSCs), which are then modified to carry the corrected form of the HBB gene. These cells could then be introduced back into the patient as HSPCs, curing the patient.

The bacteria derived RNA-directed Cas9 nuclease, also known as CRISPR-Cas9, can perform double stranded breaks in DNA in a directed manner. This makes room in the genome for insertion of desired genes. After the break is performed, the CRISPR-Cas9 technology can be combined with piggyBac in iPSCs to correct the HBB gene mutation.

How Does piggyBac Work?

The piggyBac system is a gene editing technology that can insert and remove genetic cargo from cells. This is done through transposase activity, allowing for the target gene to be cloned into a transposon vector and inserted into the region of interest.

PiggyBac has been shown to have little to no unintentional downstream effects, and is able to insert and remove genetic material without leaving behind any kind of “footprint” – meaning only the desired gene is inserted. Alternatively, the Cre/loxP system, another tool to insert/remove genes, leaves behind 34 bp that are foreign to the cell.

In the case of ß-thalassemia, piggyBac is able to remove the selection cassette and all foreign sequences, leaving behind only the corrected HBB gene and restoring normal hemoglobin activity.

Gene Editing Tools That Can Be Used With piggyBac

To use piggyBac to its full potential, it can be used in combination with gene editing tools that cause double stranded breaks in DNA. These tools are all nuclease based, meaning the breaks are caused by an enzyme.
There are three comparable DNA cleaving methods that target specific sites within DNA, which are:

  • Zinc finger nucleases (ZFN)
  • Transcription activator-like effector nucleases (TALENs),
  • Cas9 nucleases

Of these, Cas9 is the most reliable, readily available, and easiest to synthesize.

ZFNs have been known to be both expensive and difficult when it comes to their design, making them difficult to acquire for research.

TALENs are inefficient and difficult to validate, because many pairs are needed to ensure their cutting efficiencies.

Cas9 nucleases are efficient when used in systems such as CRISPR-Cas9, but off-target effects can be a limiting factor. However, Hera has built on the CRISPR-Cas9 platform to create the precise genome editing tool Cas-CLOVER which functions with no observable off-target effects.

Learn More About piggyBac, Cas-CLOVER And Hera BioLabs

Here at Hera, we use our proprietary gene editing system, Cas-CLOVER, which expands on the CRISPR-Cas9 platform for more accurate gene editing. Unlike CRISPR-Cas9, which is directed by one guide RNA, Cas-CLOVER has a dimeric guide system with one guide RNA and one Cas9 protein on each side of the target site. Because of its dimeric nature, Cas-CLOVER is more accurate than CRISPR-Cas9 and results in no observable off-target effects.
Moreover, Cas-CLOVER falls under a different set of patents than CRISPR-Cas9 which allows for hassle-free licensing agreements. Hera provides access to the Cas-CLOVER technology for drug discovery and early development.

Hera also provides licenses to piggyBac, which can be combined with Cas-CLOVER for scarless gene editing.
Whether you’re performing basic research or developing curative treatments like those for ß-thalassemia, Hera is ready to work with you to push your science forward.

To learn more about Hera how Hera utilizes the piggyBac transposase system click here. If you’d like to discuss licensing and forming a partnership reach out to the Hera team today by clicking here.

References

Xie, F., Ye, L., Chang, J. C., Beyer, A. I., Wang, J., Muench, M. O., & Kan, Y. W. (2014). Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome research, 24(9), 1526–1533. https://doi.org/10.1101/gr.173427.114

Advancements In Stem Cell-Based Therapies For Malignant Brain Tumors

Jack Crawford on October 26, 2016

Despite many great developments in the overall treatment of cancers, glioblastoma (GBM) patients still have a pretty dire prognosis, with an estimated 5% five year survival rate and a median survival of approximately 15 months from diagnosis. One of the biggest challenges associated with the treatment of malignant brain tumors, including GBM, is how to effectively deliver drugs to the site without causing serious adverse side effects – especially since any treatment therapy needs to cross the blood-brain barrier without losing significant potency, be able to diffuse throughout the brain and specifically target cancer cells rather than indiscriminately impacting all cells, including healthy cells.

Researchers have known for some time now that mesenchymal stem cells (MSCs), particularly human adipose-derived mesenchymal stem cells (hAMSCs), have great potential as brain tumor-targeting carriers of drug therapies. Until recently, it was widely accepted that the most effective way of engineering hAMSCs for drug delivery was through the use of viral vectors; unfortunately, large scale preclinical trials of such virus delivered gene therapy have raised significant questions about the risk of immunotoxicity as well as the potential activation of latent viruses, inflammatory responses or systemic autoimmunity.

A team from Johns Hopkins University School of Medicine, however, has recently published a study in Biomaterials that demonstrated the successful use of non-viral nanobiotechnology to deliver safe and effective gene and cell therapies in the treatment of GBM. By using synthetic nanoparticles (NPs), they were able to engineer hAMSCs to produce BMP4, a growth factor known to decrease tumor growth, which allowed for successful migration past various biological barriers and ultimately delivered a “therapeutic payload”. Using a cancer xenograft rat model, Mangravati et all were able to provide in vivo evidence that “NP-engineered hAMSCs administered locally and systemically in a rodent glioma model retain their intrinsic tumor-homing efficiency by migrating towards the brain and penetrating the tumor, and hAMSCs engineered to secrete BMP4 significantly increased survival” in brain tumor initiating cell-bearing rats without producing the debilitating side effects associated with viral vectors. This gives great hope to the future of drug and stem-cell based therapies for a wide array of human diseases, including, but not limited to, malignant brain cancer such as glioblastoma.