NEW YORK As CRISPR researchers develop new and better ways to edit the genome while leaving as few unintended consequences as possible behind, a team led by scientists at the University of California, San Francisco and MIT has developed a method that would create completely reversible gene edits.
In a recent study in Cell, UCSF's Luke Gilbert, MIT's Jonathan Weissman, and their colleagues described their method, called CRISPRoff a programmable epigenome editor consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. This transient CRISPRoff expression initiates highly specific repression of genes and DNA methylation that's maintained through cell division and differentiation of stem cells to neurons. In their experiments, they found that the epigenome editing was highly specific, with minimal off-target editing.
In order to reverse this effect, the researchers then engineered a switch they called CRISPRon, through which they used Cas9-mediated gene editing to inactivate DNMT1 the main DNA methylation maintenance enzyme in mammalian cells in cells where they had previously silenced specific genes. Post-DNMT1 knockout, 60 percent to 80 percent of cells demonstrated reactivated gene expression. Similarly, treatment of cells with a small-molecule inhibitor of DNMT1 showed reactivated expression of genes that had previously been silenced, demonstrating that depletion of DNA methylation was sufficient to reverse CRISPRoff-mediated gene silencing.
"If you want to fix a pathogenic mutation, then CRISPR is really enabling. But we felt that for many applications, you may not want to permanently mutate the genome," Gilbert said, explaining the method's genesis. "So, we were searching for ways to turn gene expression off or on, without manipulating the sequence of the genome and just manipulating the transcripts that are produced by a cell."
Medically, he noted, there could be many applications where patients might feel more comfortable with using genome editing if they know that their genes won't be permanently changed, in part based on the concept that gene therapy has already been in use for a variety of applications for more than a decade.
Gilbert further noted that while he, Weissman, and many other CRISPR researchers have been working with tools such as CRISPR interference (CRISPRi) that can downregulate gene expression rather than turning it off entirely, these tools are more awkward to work with from a therapeutic standpoint. While a regular CRISPR-Cas system uses a Cas nuclease to latch onto a gene a mutate it in some fashion to turn it off, CRISPRi uses deactivated Cas9, resulting in RNA-directed transcriptional control of the target region. In other words, it functions "almost like normal transcription factors within a cell, where you constitutively express proteins to target the gene, and then that turns the gene on or off," Gilbert explained. "One of the advantages of Cas9 is you can express it briefly and it'll make a change to the genome that's permanent, and carry it out for a long time. We were looking for ways to basically leverage the strengths of Cas9's permanence and durability, but also leverage this epigenetic editing feature of not having to permanently mutate a gene."
The dead Cas9 works as a programmable DNA binding element rather than as a programmable nuclease, Weissman added.
In terms of therapeutic applications for human beings, the technology has a lot of possible uses, the researchers believe. Before there was an Ebola vaccine, for example, they were working on CRISPRoff as a way to confer programable immunity for anyone who might be affected by the disease.
"If you have a virus where you know the receptor, you could use CRISPRoff to turn gene expression off," Gilbert said. For Ebola that receptor is a protein called NPC1. "We know if you turn NPC1 off in the liver, you're immune to Ebola. But you don't want to permanently mutate NPC1 because you cause cholesterol processing defects and lysosomal storage disorder phenotypes," he added. The idea they had, therefore, was to deliver CRISPRoff to the liver of healthcare workers traveling to Ebola hotspots so that they'd be completely immune to the disease while working with patients.
"And when they left the Ebola hotspot, to avoid detrimental effects of mutating or permanently silencing NPC1, then you could redeliver CRISPRon to restore gene expression and therefore not have any detrimental phenotypes from permanently losing that gene function," Gilbert added.
He further noted that the technology could even be used to modulate pain response. If someone were planning to have surgery, or recovering from an injury, CRISPRoff could be administered to shut down pain receptors for a short time. Once the patient recovered, the pain receptors could be turned back on. It would also help people avoid opioid pain killers.
Another example, according to Weissman, would be in the area of oncology. Cancer studies often reveal the presence of genes or gene mutations that lead to resistance to chemotherapy or radiotherapy. CRISPR is now being considered as a possible addition to some late-stage cancer patients' therapies as a way to knock out resistance genes and reawaken therapeutic response.
In May 2019, Christiana Care's Health System's Gene Editing Institute was preparing to file an investigational new drug application with the US Food and Drug Administration for a clinical trial protocol that would use CRISPR genome editing to improve the efficacy of chemotherapy for KRAS-positive non-small-cell lung cancer (NSCLC) patients. The protocol involved using CRISPR-Cas9 gene editing to knock down NRF2 in order to render patients more sensitive to chemotherapeutic agents.
Under a scenario using CRISPRoff, that gene's expression may only be off for the time it takes to administer the necessary cancer treatment. "You can imagine turning on or off genes in your intestine or in your blood stem cells," Weissman said. "The cells are more sensitive to radiation. But then after you have the radiotherapy, [the cells could return] to normal states so you don't have to worry about the long-term consequences of turning off the gene."
Weissman noted there may be some instances where CRISPRon isn't needed to turn gene expression back on. While conducting their experiments, the researchers noted that the gene silencing in certain loci would decay over a period of days or weeks, depending on the cell cycle turnover rate.
"If that can be tuned, we can now come in [with] one type of treatment and over the period of, say, weeks or months, it would naturally restore and you don't have to come in with the second," Weissman said.
That rate of decay would depend on the tissue in question and the dynamics of tissue turnover "will dictate how long these program changes last," Gilbert added. "In post-mitotic cells like muscle or neuron, these methyl marks in non-replicating cells may last for years and years. So, it depends on the cell type."
There are still many elements to CRISPRoff that have to be worked out and refined before it can be used in the clinic. As with any CRISPR system meant to be used as a therapy, delivery into the right cells at the right time is currently the principal challenge, Gilbert said. The researchers are also working on making the CRISPRoff complex smaller, and capable of targeting more than one loci at once, Weissman added.
But there's already been clear interest in commercializing the technology, he said. Both he and Gilbert, as well as a few other researchers who authored the paper, have already filed for patents on CRISPRoff and CRISPRon.
Indeed, Weissman said, the technology could have applications in cell therapy, and could even aid in the development of so-called off-the-shelf allogeneic CAR-T cells. The current procedure for making CAR Ts is expensive and time-consuming because it involves harvesting an individual's cells, engineering them, and re-administering them as a treatment. As of now, allogeneic CAR Ts could cause life-threatening graft-versus-host disease, and could be rejected by the host immune system.
Using CRISPRoff, however, Weissman envisions being able to edit allogeneic CAR Ts in ways that would camouflage them from an individual's immune system, while also adding safety controls that would allow a physician to turn the CAR Ts off, if needed. "It could make it a much more accessible treatment," he said. "You could have a safer and more universal cell therapy, and you can then do much more complicated engineering because you only have to do it once for many patients, as opposed to trying to do this complicated engineering in a bespoke way for each patient."
Overall, he added, what the study really shows is that cutting DNA and then repairing it is quite difficult. And while researchers have gotten better at avoiding off-target effects, so-called on-target off-targets unintended consequences of on-targets editing such as DNA damage response, large indels, and even chromothripsis, can still do damage to the genome.
"So, when you don't have to do that, therapeutically, there are lot of advantages," Weissman said. "Things like base editor and prime editor are examples of this, and we see CRISPRoff as a complement to this, which allows you to do epigenome editing from beginning to end, and to do it in a clean and controlled way."
- 5 potential benefits of exosome treatment - AZ Big Media - June 8th, 2021
- Orion and the Blood Service launch collaboration to develop new CAR T-cell cancer therapy - Yahoo Finance Australia - June 8th, 2021
- Improvements in the Treatment of Patients With MCL Over the Past Decade - Oncology Learning Network - June 8th, 2021
- Novo Nordisk partners with Heartseed on heart failure cell therapy - PMLiVE - June 8th, 2021
- Citius Pharmaceuticals to be Added to Russell 2000 Index - BioSpace - June 8th, 2021
- IN8bio Completes Treatment of First Cohort in Phase 1 Clinical Trial with Gamma Delta T-Cell Therapy in Patients with Newly Diagnosed Glioblastoma... - June 8th, 2021
- Regenerative medicine: moving next-gen treatments from lab to clinic - Pharmaceutical Technology - June 8th, 2021
- Global $30+ Billion Cell Therapy Bioprocessing Market to 2028: Market Opportunities in Automated Procedures to Produce Cell Therapies - PRNewswire - June 8th, 2021
- Global Stem Cell & Regenerative Medicine Market Report Forecast to 2027 3M Group Novartis AG Integra Gene Therapies The Manomet Current - The... - June 8th, 2021
- Novel CAR-T Cell Therapy Produces Early and Deep Responses in Certain Patients with Multiple Myeloma - Curetoday.com - June 8th, 2021
- BioRestorative Therapies to Present at the Emerging Growth Conference on June 9, 2021 - StreetInsider.com - June 8th, 2021
- Innovent Biologics and IASO Biotherapeutics to Present Updated Data from its Anti-BCMA CAR-T Therapy in Relapsed/Refractory Multiple Myeloma at... - June 8th, 2021
- High Rate of Response Noted in MCL and CLL With Cirmtuzumab Plus Ibrutinib - Cancer Network - June 8th, 2021
- Dr. Ghosh on the Role of Off-the-Shelf CAR T-Cell Therapy in Myeloma June - OncLive - June 8th, 2021
- Triplet Combo of Polatuzumab Vedotin, Rituximab and Lenalidomide Safe, Effective to Treat Relapsed/Refractory DLBCL - Cancer Network - June 8th, 2021
- Income, Education Affect Treatment and Survival in MDS - DocWire News - June 8th, 2021
- Polatuzumab With Rituximab and Lenalidomide Shown Safe and Effective for Relapsed/Refractory DLBCL - Targeted Oncology - June 8th, 2021
- Early Promise Observed With Lisaftoclax in Relapsed/Refractory CLL and Other Hematologic Malignancies - Targeted Oncology - June 8th, 2021
- Mustang Bio to Host Key Opinion Leader Webinar on MB-106 CD20-Targeted CAR T for - GlobeNewswire - June 8th, 2021
- FDA Lifts Hold on bluebird bio's Sickle Cell and Beta-Thalassemia Gene Therapies - BioSpace - June 8th, 2021
- Abecma Continues to Improve Survival in Heavily Pretreated Patients With Multiple Myeloma - Curetoday.com - June 8th, 2021
- HSCT-Sparing, Chemotherapy-Free Treatment of Ph+ ALL With Ponatinib/Blinatumomab Leads to 100% CR Rate - Cancer Network - June 8th, 2021
- Fate Therapeutics Highlights Positive Interim Data from its Phase 1 Study of FT516 in Combination with - GlobeNewswire - June 8th, 2021
- Action Taken by Governor Phil Scott on Legislation - June 7, 2021 - Office of Governor Phil Scott - June 8th, 2021
- Cancer research: New advances and innovations - Medical News Today - June 8th, 2021
- Naturally Treat & Heal The Cause of Pain with Regenerative Therapies - The Mountaineer - May 15th, 2021
- CAR T-Cell Therapy UCARTCS1A Shows Early Activity in Relapsed/Refractory Myeloma - OncLive - May 15th, 2021
- Rinri Therapeutics Secures Innovate UK Funding Grant for 3.2m Project to Advance its Novel Cell-Based Therapy to Restore Hearing Loss - PRNewswire - May 15th, 2021
- With $52 Million Series A, Appia Bio Anticipates a Bright Future in Cell Therapy - BioSpace - May 15th, 2021
- Genenta Phase I/II Glioblastoma Data at ASGCT Show Temferon Delivered Tumor-Focused Interferon ExpressionData presented at the 2021 American Society... - May 15th, 2021
- Safety and Efficacy of Consolidation Therapy with Ipilimumab Plus Nivolumab after Autologous Stem Cell Transplantation - DocWire News - May 15th, 2021
- Fascination with stem cell sensation leads to Fulbright scholarship - Monash University - May 15th, 2021
- Omeros' Narsoplimab Pivotal Trial Data to Be Shared as an Oral Presentation at the European Hematology Association Congress - Business Wire - May 15th, 2021
- Munshi Explains Staging, Prognosis, and Treatment for a Patient With Acute Graft-vs-Host-Disease - Targeted Oncology - May 15th, 2021
- KD-PACE Salvage Therapy for Aggressive Relapsed Refractory Multiple Myeloma, Plasma Cell Leukemia and Extramedullary Myeloma - DocWire News - May 15th, 2021
- Icy Microneedle Patch Delivers Cell Therapy, Then Melts - Freethink - May 15th, 2021
- FDA Responds to Creative Medical Technology Holdings Regarding Its ImmCelz IND for Stroke Treatment - BioSpace - May 15th, 2021
- Global Stem Cell and Platelet Rich Plasma (PRP) Alopecia Therapies Market 2021 Type and End-use Industry 2026 Orange County Hair Restoration Center,... - May 15th, 2021
- Allogeneic Mesenchymal Stem Cell Segment Is Expected To Lead In the Global Rheumatoid Arthritis Stem Cell Therapy Market over the Forecast Period,... - May 15th, 2021
- Advanced Therapy Medicinal Products Market Size Worth $21.2 Billion By 2028: Grand View Research, Inc. - PRNewswire - May 15th, 2021
- New connection between metabolism and red blood cell development - Harvard Gazette - May 15th, 2021
- Heart attack recovery aided by injecting heart muscle cells that overexpress cyclin D2 - The Mix - May 15th, 2021
- Vertex and CRISPR Therapeutics to Present New Clinical Data - GlobeNewswire - May 15th, 2021
- Rocket Pharmaceuticals Presents Positive Clinical Data from Fanconi Anemia, Leukocyte Adhesion Deficiency-I, and Pyruvate Kinase Deficiency Programs... - May 15th, 2021
- Stem Cell Therapy Global Market Report 2021: COVID-19 Growth And Change To 2030 - Yahoo Finance - May 3rd, 2021
- Stem cell therapy for COPD: Cost, effectiveness, and more - Medical News Today - May 3rd, 2021
- Thomas Smeenk on Hemostemix's autologous stem cell therapy technology and why some call it 'the fountain of youth' - InvestorIntel - May 3rd, 2021
- Mesoblast says its stem cell treatment saved lives of severely ill COVID patients - Stockhead - May 3rd, 2021
- Antiviral T cells protected, viable as off-the-shelf treatment for painful complication stem cells - Microbioz India - May 3rd, 2021
- Investigating CAR T-Cell Therapy for Use in Different Disease Types - Targeted Oncology - May 3rd, 2021
- Should scientists be allowed to grow human embryos in a dish beyond 14 days? Is it scientifically important or morally wrong? - USA TODAY - May 3rd, 2021
- Hope, hype and exploitation the wild history of stem cell science - ABC News - May 3rd, 2021
- Brave New World is being reinvented with synthetic embryosand the right reasons - SYFY WIRE - May 3rd, 2021
- Stem Cell Therapy Market Trends Evaluation 2021 By Leading Players Updates, Consumer-Demand, Key Strategies, Consumption, Industry Development, Market... - May 3rd, 2021
- STEM NOLA Teams with Obatala Science & CellSpring to Expose Students to Careers and Medical Research at Biotech Startups - PRNewswire - May 3rd, 2021
- Breakthrough gene therapies in sight | UNC-Chapel Hill - UNC Chapell Hill - May 3rd, 2021
- FDA's Abecma Approval a 'Great Blessing' for Patients with Multiple Myeloma - Curetoday.com - May 3rd, 2021
- Enhanced inhibition of tumor growth using TRAIL-overexpressing adipose-derived stem cells in combination with the chemotherapeutic agent CPT-11 in... - May 3rd, 2021
- Global Stem Cell Therapy Market 2020-2027 Trends, industry vertical, along with the geography, delivering valuable insights | Reportspedia Clark... - May 3rd, 2021
- Rheumatoid Arthritis Stem Cell Therapy Market Report 2021 by Global Key Players, Types, Applications, Countries, Size, Forecast to 2027 Clark County... - May 3rd, 2021
- Cell Therapy Market To Expand At An expeditious Growth Rate From 2021-2027 | Thermo Fisher Scientific, Nanofiber Solutions, Advanced Biomatrix,... - May 3rd, 2021
- FTC continues to crack down on companies peddling fake COVID treatments and cures - HamletHub - May 3rd, 2021
- ThermoGenesis : The History of Cell and Gene Therapy - marketscreener.com - April 18th, 2021
- Russell Health Honored in Global Business Leaders Magazine's '20 Leading Companies of the Year 2021' - PRNewswire - April 18th, 2021
- Durable B-ALL Control With Allogeneic Transplant After CAR T-Cell Therapy - Cancer Therapy Advisor - April 18th, 2021
- CRISPR lauds easy scale-out of cell therapy - BioProcess Insider - BioProcess Insider - April 18th, 2021
- NeoProgen, Inc. Receives First Granted Patent for Method to Treat Cardiac Conditions using Neonatal Heart-derived Medicinal Signaling Cells (nMSCs)... - April 18th, 2021
- CRISPR gene therapy for sickle cell disease approved by the FDA - BioNews - April 18th, 2021
- Man's heart healed by stem cell therapy and love of an old flame - Leeds Live - April 18th, 2021
- Autologous Stem Cell Based Therapies Market Professional Report 2021 Witness Robust Expansion by 2026 SoccerNurds - SoccerNurds - April 18th, 2021
- Stem Cell Therapy Market Analysis 2021: Size, Share, Sales, Growth, Revenue, Type, Application & Forecast To 2027 SoccerNurds - SoccerNurds - April 18th, 2021
- Mastitis therapy explored | AG | kmaland.com - KMAland - April 18th, 2021
- CAR T-Cell Therapy Enters the Arena in Multiple Myeloma With Ongoing Research Ahead - OncLive - April 18th, 2021
- Leukemia Cutis: Symptoms and Treatment - Healthline - April 18th, 2021
- Stem Cell Therapy Market is Set to Witness Impressive Growth | Top Players Osiris Therapeutics, Inc. (US), MEDIPOST Co., Ltd. KSU | The Sentinel... - April 18th, 2021
- Global Stem Cell Therapy Market 2020 SWOT Analysis, Growth Rate, and Market Share By Type, Application and Global Forecast 2027 - Los Hijos de la... - April 18th, 2021
- Stem Cell Therapy Market Potential Growth, Size, Share, Demand and Analysis of Key Players Research Forecasts to 2025 - The Courier - March 8th, 2021
- Animal Stem Cell Therapy Market Size, Industry Analysis, Growth Factors, Trends, and Regional Forecast to 2027 - The Courier - March 8th, 2021
- Pericardial Injection Effective, Less Invasive Way to Get Regenerative Therapies to Heart - NC State News - March 8th, 2021
- Biomarkers Help Predict the Role of Chemotherapy in Biologic Aging - OncLive - March 8th, 2021