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Posts Tagged ‘CRISPR-Cas9’

Novartis uses a ‘dimmer switch’ medication to fine-tune gene therapy candidates

Reporter: Amandeep Kaur, BSc., MSc.

Using viral vectors, lipid nanoparticles, and other technologies, significant progress has been achieved in refining the delivery of gene treatments. However, modifications to the cargo itself are still needed to increase safety and efficacy by better controlling gene expression.

To that end, researchers at Children’s Hospital of Philadelphia (CHOP) have created a “dimmer switch” system that employs Novartis’ investigational Huntington’s disease medicine branaplam (LMI070) as a regulator to fine-tune the quantity of proteins generated from a gene therapy.

According to a new study published in Nature, the Xon system altered quantities of erythropoietin—which is used to treat anaemia associated with chronic renal disease—delivered to mice using viral vectors. The method has previously been licenced by Novartis, the maker of the Zolgensma gene therapy for spinal muscular atrophy.

The Xon system depends on a process known as “alternative splicing,” in which RNA is spliced to include or exclude specific exons of a gene, allowing the gene to code for multiple proteins. The team used branaplam, a small-molecule RNA-splicing modulator, for this platform. The medication was created to improve SMN2 gene splicing in order to cure spinal muscular atrophy. Novartis shifted its research to try the medication against Huntington’s disease after a trial failure.

A gene therapy’s payload remains dormant until oral branaplam is given, according to Xon. The medicine activates the expression of the therapy’s functional gene by causing it to splice in the desired way. Scientists from CHOP and the Novartis Institutes for BioMedical Research put the dimmer switch to the exam in an Epo gene therapy carried through adeno-associated viral vectors. The usage of branaplam increased mice Epo levels in the blood and hematocrit levels (the proportion of red blood cells to whole blood) by 60% to 70%, according to the researchers. The researchers fed the rodents branaplam again as their hematocrit decreased to baseline levels. The therapy reinduced Epo to levels similar to those seen in the initial studies, according to the researchers.

The researchers also demonstrated that the Xon system could be used to regulate progranulin expression, which is utilised to treat PGRN-deficient frontotemporal dementia and neuronal ceroid lipofuscinosis. The scientists emphasised that gene therapy requires a small treatment window to be both safe and effective.

In a statement, Beverly Davidson, Ph.D., the study’s senior author, said, “The dose of a medicine can define how high you want expression to be, and then the system can automatically ‘dim down’ at a pace corresponding to the half-life of the protein.”

“We may imagine scenarios in which a medication is used only once, such as to control the expression of foreign proteins required for gene editing, or only on a limited basis. Because the splicing modulators we examined are administered orally, compliance to control protein expression from viral vectors including Xon-based cassettes should be high.”

In gene-modifying medicines, scientists have tried a variety of approaches to alter gene expression. For example, methyl groups were utilised as a switch to turn on or off expression of genes in the gene-editing system CRISPR by a team of researchers from the Massachusetts Institute of Technology and the University of California, San Francisco.

Auxolytic, a biotech company founded by Stanford University academics, has described how knocking down a gene called UMPS could render T-cell therapies ineffective by depriving T cells of the nutrition uridine. Xon could also be tailored to work with cancer CAR-T cell therapy, according to the CHOP-Novartis researchers. The dimmer switch could help prevent cell depletion by halting CAR expression, according to the researchers. According to the researchers, such a tuneable switch could help CRISPR-based treatments by providing “a short burst” of production of CRISPR effector proteins to prevent undesirable off-target editing.

Source: https://www.fiercebiotech.com/research/novartis-fine-tunes-gene-therapy-a-huntington-s-disease-candidate-as-a-dimmer-switch?mkt_tok=Mjk0LU1RRi0wNTYAAAF-q1ives09mmSQhXDd_jhF0M11KBMt0K23Iru3ZMcZFf-vcFQwMMCxTOiWM-jHaEvtyGOM_ds_Cw6NuB9B0fr79a3Opgh32TjXaB-snz54d2xU_fw

Other Related Articles published in this Open Access Online Scientific Journal include the following:

Gene Therapy could be a Boon to Alzheimer’s disease (AD): A first-in-human clinical trial proposed

Reporter: Dr. Premalata Pati, Ph.D., Postdoc

https://pharmaceuticalintelligence.com/2021/03/22/gene-therapy-could-be-a-boon-to-alzheimers-disease-ad-a-first-in-human-clinical-trial-proposed/

Top Industrialization Challenges of Gene Therapy Manufacturing

Guest Authors: Dr. Mark Szczypka and Clive Glover

https://pharmaceuticalintelligence.com/2021/03/29/top-industrialization-challenges-of-gene-therapy-manufacturing/

Dysregulation of ncRNAs in association with Neurodegenerative Disorders

Curator: Amandeep Kaur

https://pharmaceuticalintelligence.com/2021/01/11/dysregulation-of-ncrnas-in-association-with-neurodegenerative-disorders/

Cancer treatment using CRISPR-based Genome Editing System 

Reporter: Irina Robu, PhD

https://pharmaceuticalintelligence.com/2021/01/09/59906/

CRISPR-Cas9 and the Power of Butterfly Gene Editing

Reporter: Madison Davis

https://pharmaceuticalintelligence.com/2020/08/23/crispr-cas9-and-the-power-of-butterfly-gene-editing/

Gene Editing for Exon 51: Why CRISPR Snipping might be better than Exon Skipping for DMD

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2016/01/23/gene-editing-for-exon-51-why-crispr-snipping-might-be-better-than-exon-skipping-for-dmd/

Gene Editing: The Role of Oligonucleotide Chips

Curators: Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2016/01/07/gene-editing-the-role-of-oligonucleotide-chips/

Cause of Alzheimer’s Discovered: protein SIRT6 role in DNA repair process – low levels enable DNA damage accumulation

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2017/06/15/cause-of-alzheimers-discovered-protein-sirt6-role-in-dna-repair-process-low-levels-enable-dna-damage-accumulation/

Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting

Author & Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2015/08/30/delineating-a-role-for-crispr-cas9-in-pharmaceutical-targeting/

Brain Science

Larry H Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2015/11/03/brain-science/

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Cancer treatment using CRISPR-based Genome Editing System 

Reporter: Irina Robu, PhD

CRISPR, stands for “clusters of regularly interspaced short palindromic repeats” is one of the biggest accomplishments in science of this decade and it is the simplest tool for altering DNA sequences and modifying gene functions. The technology is adapted form the natural defense mechanism of bacteria. Bacteria uses CRISPR-derived RNA and different Cas proteins to foil attacks by viruses and foreign bodies.

Scientists in the laboratory of Prof. Dan Peer, VP for R&D and Head of the Laboratory of Precision Nanomedicine at the Shmunis School of Biomedicine and Cancer Research at TAU  have shown that CRISPR/Cas9 system is efficient in treating metastatic cancer. They developed a novel lipid nanoparticle-based delivery system that targets cancer cells and ends them by genetic manipulation, called CRISPR-LNPs, which were published in published in November 2020 in Science Advances.

Professor Peer and his team of scientists chose two of the deadliest cancers: glioblastoma and metastatic ovarian cancer to prove that CRISPR genome editing system can be used to treat cancer effectively in a living animal. Since, glioblastoma is the most aggressive type of brain cancer with a life expectancy of 15 months after diagnosis, the researchers showed that the single treatment with CRISPR-LNPs doubled the average life expectancy of mice with glioblastoma tumors.  At the same time, ovarian cancer is the most lethal cancer of female reproductive system and many patients are usually diagnosed at the advance stage of the disease. Treatment with CRISPR-LNPs in a metastatic ovarian cancer mice model increased their overall survival rate by 80%.

Despite CRISPR genome editing technology being capable of identifying and altering  any genetic segment, clinical implementation is still in its infancy because the inability to accurately deliver the CRISPR to the target cells.  In order to solve the issue, Professor Peer developed a delivery system that targets the DNA responsible for the cancer cells.

By demonstrating that the technology can treat two aggressive cancers, researchers open the technology to numerous new possibilities for treating other types of cancer. They intend to go on to experiments with blood cancers which are very interesting genetically.

SOURCE

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The Nobel Prize in Chemistry 2020: Emmanuelle Charpentier & Jennifer A. Doudna

Reporters: Stephen J. Williams, Ph.D. and Aviva Lev-Ari, PhD, RN

The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry 2020 to

Emmanuelle Charpentier
Max Planck Unit for the Science of Pathogens, Berlin, Germany

Jennifer A. Doudna
University of California, Berkeley, USA

“for the development of a method for genome editing”

Genetic scissors: a tool for rewriting the code of life

Emmanuelle Charpentier and Jennifer A. Doudna have discovered one of gene technology’s sharpest tools: the CRISPR/Cas9 genetic scissors. Using these, researchers can change the DNA of animals, plants and microorganisms with extremely high precision. This technology has had a revolutionary impact on the life sciences, is contributing to new cancer therapies and may make the dream of curing inherited diseases come true.

Researchers need to modify genes in cells if they are to find out about life’s inner workings. This used to be time-consuming, difficult and sometimes impossible work. Using the CRISPR/Cas9 genetic scissors, it is now possible to change the code of life over the course of a few weeks.

“There is enormous power in this genetic tool, which affects us all. It has not only revolutionised basic science, but also resulted in innovative crops and will lead to ground-breaking new medical treatments,” says Claes Gustafsson, chair of the Nobel Committee for Chemistry.

As so often in science, the discovery of these genetic scissors was unexpected. During Emmanuelle Charpentier’s studies of Streptococcus pyogenes, one of the bacteria that cause the most harm to humanity, she discovered a previously unknown molecule, tracrRNA. Her work showed that tracrRNA is part of bacteria’s ancient immune system, CRISPR/Cas, that disarms viruses by cleaving their DNA.

Charpentier published her discovery in 2011. The same year, she initiated a collaboration with Jennifer Doudna, an experienced biochemist with vast knowledge of RNA. Together, they succeeded in recreating the bacteria’s genetic scissors in a test tube and simplifying the scissors’ molecular components so they were easier to use.

In an epoch-making experiment, they then reprogrammed the genetic scissors. In their natural form, the scissors recognise DNA from viruses, but Charpentier and Doudna proved that they could be controlled so that they can cut any DNA molecule at a predetermined site. Where the DNA is cut it is then easy to rewrite the code of life.

Since Charpentier and Doudna discovered the CRISPR/Cas9 genetic scissors in 2012 their use has exploded. This tool has contributed to many important discoveries in basic research, and plant researchers have been able to develop crops that withstand mould, pests and drought. In medicine, clinical trials of new cancer therapies are underway, and the dream of being able to cure inherited diseases is about to come true. These genetic scissors have taken the life sciences into a new epoch and, in many ways, are bringing the greatest benefit to humankind.

Illustrations

The illustrations are free to use for non-commercial purposes. Attribute ”© Johan Jarnestad/The Royal Swedish Academy of Sciences”

Illustration: Using the genetic scissors (pdf)
Illustration: Streptococcus’ natural immune system against viruses:CRISPR/Cas9 pdf)
Illustration: CRISPR/Cas9 genetic scissors (pdf)

Read more about this year’s prize

Popular information: Genetic scissors: a tool for rewriting the code of life (pdf)
Scientific Background: A tool for genome editing (pdf)

Emmanuelle Charpentier, born 1968 in Juvisy-sur-Orge, France. Ph.D. 1995 from Institut Pasteur, Paris, France. Director of the Max Planck Unit for the Science of Pathogens, Berlin, Germany.

Jennifer A. Doudna, born 1964 in Washington, D.C, USA. Ph.D. 1989 from Harvard Medical School, Boston, USA. Professor at the University of California, Berkeley, USA and Investigator, Howard Hughes Medical Institute.

 

Other Articles on the Nobel Prize in this Open Access Journal Include:

2020 Nobel Prize for Physiology and Medicine for Hepatitis C Discovery goes to British scientist Michael Houghton and US researchers Harvey Alter and Charles Rice

CONTAGIOUS – About Viruses, Pandemics and Nobel Prizes at the Nobel Prize Museum, Stockholm, Sweden 

AACR Congratulates Dr. William G. Kaelin Jr., Sir Peter J. Ratcliffe, and Dr. Gregg L. Semenza on 2019 Nobel Prize in Physiology or Medicine

2018 Nobel Prize in Physiology or Medicine for contributions to Cancer Immunotherapy to James P. Allison, Ph.D., of the University of Texas, M.D. Anderson Cancer Center, Houston, Texas. Dr. Allison shares the prize with Tasuku Honjo, M.D., Ph.D., of Kyoto University Institute, Japan

2017 Nobel prize in chemistry given to Jacques Dubochet, Joachim Frank, and Richard Henderson  for developing cryo-electron microscopy

2016 Nobel Prize in Chemistry awarded for development of molecular machines, the world’s smallest mechanical devices, the winners: Jean-Pierre Sauvage, J. Fraser Stoddart and Bernard L. Feringa

Correspondence on Leadership in Genomics and other Gene Curations: Dr. Williams with Dr. Lev-Ari

Programming life: An interview with Jennifer Doudna by Michael Chui, a partner of the McKinsey Global Institute

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Human gene editing continues to hold a major fascination within a biomedical and biopharmaceutical industries. It’s extraordinary potential is now being realized but important questions as to who will be the beneficiaries of such breakthrough technologies remained to be answered. The session will discuss whether gene editing technologies can alleviate some of the most challenging unmet medical needs. We will discuss how research advances often never reach minority communities and how diverse patient populations will gain access to such breakthrough technologies. It is widely recognize that there are patient voids in the population and we will explore how community health centers might fill this void to ensure that state-of-the-art technologies can reach the forgotten patient groups . We also will touch ethical questions surrounding germline editing and how such research and development could impact the community at large.

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New CRISPR Approach Transforms Skin Cells into Pluripotent Stem Cells

Reporter: Irina Robu, PhD

Dr. Timo Otonkoski, University of Helsinki and Dr.Juha Kere, King’s College London succeeded on reprograming skin cells into pluripotent stem cells by activating cell’s own genes using gene editing technology, CRISPR-Cas9-based gene activation (CRISPRa) that can be used to activate genes. The method uses a blunt version of Cas9 ‘gene scissors’ that does not cut DNA and can consequently be used to activate gene expression without mutating the genome. Previously, reprogramming was only possible by artificially introducing the critical transformation genes known as Yamanaka Factors into skin cells where they are normally inactive.

According to a study that is published in Nature Communication, called Human Pluripotent Reprogramming with CRISPR activators which show that CRISPRa is an attractive tool for cellular reprogramming applications due to its high multiplex capacity and direct alignment of endogenous loci. In the article, it is presented that reprogramming of primary human dermal fibroblasts to induced pluripotent stem cells with CRISPRa, the aimed at endogenous cells. The data shows that human body cells can only be reprogrammed into iPS cells with CRISPRa, and the findings reveal the involvement of EEA motif-associated mechanisms in cellular reprogramming.

The discovery also advocates that it might be likely to improve many other reprogramming tasks by addressing genetic elements that are typical of the intended target cell type. According to Jere Weltner, PhD student working on the project “the technology can find practical application in biobanking and many other applications of tissue technology.

SOURCE

https://www.sciencedaily.com/releases/2018/07/180706091723.htm

 

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

The CRISPR-Cas9 system has proven to be a powerful tool for genome editing allowing for the precise modification of specific DNA sequences within a cell. Many efforts are currently underway to use the CRISPR-Cas9 system for the therapeutic correction of human genetic diseases. CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells.

 

CRISPR–Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR–Cas9.

 

Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells. Using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), an average insertion or deletion (indel) efficiency greater than 80% was achieved. This high efficiency of insertion or deletion generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs.

 

The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. These results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. As hPSCs can acquire P53 mutations, cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.

 

CRISPR-based editing of T cells to treat cancer, as scientists at the University of Pennsylvania are studying in a clinical trial, should also not have a p53 problem. Nor should any therapy developed with CRISPR base editing, which does not make the double-stranded breaks that trigger p53. But, there are pre-existing humoral and cell-mediated adaptive immune responses to Cas9 in humans, a factor which must be taken into account as the CRISPR-Cas9 system moves forward into clinical trials.

 

References:

 

https://techonomy.com/2018/06/new-cancer-concerns-shake-crispr-prognosis/

 

https://www.statnews.com/2018/06/11/crispr-hurdle-edited-cells-might-cause-cancer/

 

https://www.biorxiv.org/content/early/2017/07/26/168443

 

https://www.nature.com/articles/s41591-018-0049-z.epdf?referrer_access_token=s92jDP_yPBmDmi-USafzK9RgN0jAjWel9jnR3ZoTv0MRjuB3dEnTctGtoy16n3DDbmISsvbln9SCISHVDd73tdQRNS7LB8qBlX1vpbLE0nK_CwKThDGcf344KR6RAm9k3wZiwyu-Kb1f2Dl7pArs5yYSiSLSdgeH7gst7lOBEh9qIc6kDpsytWLHqX_tyggu&tracking_referrer=www.statnews.com

 

https://www.nature.com/articles/s41591-018-0050-6.epdf?referrer_access_token=2KJ0L-tmvjtQdzqlkVXWVNRgN0jAjWel9jnR3ZoTv0Phq6GCpDlJx7lIwhCzBRjHJv0mv4zO0wzJJCeuxJjzoUWLeemH8T4I3i61ftUBkYkETi6qnweELRYMj4v0kLk7naHF-ujuz4WUf75mXsIRJ3HH0kQGq1TNYg7tk3kamoelcgGp4M7UTiTmG8j0oog_&tracking_referrer=www.statnews.com

 

https://www.biorxiv.org/content/early/2018/01/05/243345

 

https://www.nature.com/articles/nmeth.4293.epdf

 

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Translational Gene Editing – June 16-17, 2016 in Boston, MA

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Learn More | Sponsorship & Exhibit Details | Register by April 29 & SAVE up to $200!

IMPROVING CRISPR FOR BETTER FUNCTIONAL SCREENING

Optimized sgRNA Libraries for Genetic Screens with CRISPR-Cas9
John Doench, Ph.D., Associate Director, Genetic Perturbation Platform, Broad Institute of Harvard and MIT

Optimizing CRISPR for Pooled Genome-Wide Functional Genetic Screens
Paul Diehl, Ph.D., Director, Business Development, Cellecta, Inc.

CRISPR-Cas9 Whole Genome Screening: Going Where No Screen Has Gone Before
Ralph Garippa, Ph.D., Director, RNAi Core Facility, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center

Cross-Species Synthetic Lethal Screens and Applications to Drug Discovery
Norbert Perrimon, Ph.D., Professor, Department of Genetics, Harvard Medical School and Investigator, Howard Hughes Medical Institute

Interactive Breakout Discussion Groups with Continental Breakfast
This session features various discussion groups that are led by a moderator/s who ensures focused conversations around the key issues listed. Attendees choose to join a specific group and the small, informal setting facilitates sharing of ideas and active networking. Continental breakfast is available for all participants.

Topic: CRISPR/Cas9 System for In vivo Drug Discovery
Moderator: Danilo Maddalo, Ph.D., Lab Head, ONC Pharmacology, Novartis Institutes for BioMedical Research

  • Impact of CRISPR/Cas9 system on in vivo mouse models
  • Application of the CRISPR/Cas9 system in in vivo screens
  • Technical limitations/safety issues

Topic: Getting Past CRISPR Pain Points
Moderators: John Doench, Ph.D., Associate Director, Genetic Perturbation Platform, Broad Institute of Harvard and MITStephanie Mohr, Ph.D., Lecturer, Genetics & Director of the Drosophila RNAi Screening Center, Harvard Medical School

  • Challenges and solutions for CRISPR gRNA design
  • Methods for detecting engineered changes

Topic: Cellular Delivery of CRISPR/Cas9
Moderator: Daniel E Bauer M.D., Ph.D., Assistant Professor of Pediatrics, Harvard Medical School and Staff Physician in Pediatric Hematology/Oncology, Boston Children’s Hospital and Dana-Farber Cancer Institute, Principal Faculty, Harvard Stem Cell Institute

GENE EDITING FOR SCREENING DISEASE PATHWAYS AND DRUG TARGETS

Scouring the Non-Coding Genome by Saturating Edits
Daniel E. Bauer, M.D., Ph.D., Assistant Professor of Pediatrics, Harvard Medical School and Staff Physician in Pediatric Hematology/Oncology, Boston Children’s Hospital and Dana-Farber Cancer Institute, Principal Faculty, Harvard Stem Cell Institute

Parallel shRNA and CRISPR/Cas9 Screens Reveal Biology of Stress Pathways and Identify Novel Drug Targets
Michael Bassik, Ph.D., Assistant Professor, Department of Genetics, Stanford University

BUILDING THE CRISPR TOOLBOX

Beyond Cas9: Discovering Single Effector CRISPR Tools
Jonathan Gootenberg, Member, Laboratories of Dr. Aviv Regev and Dr. Feng Zhang, Department of Systems Biology, Harvard Medical School, and Broad Institute of Harvard and MIT

CRISPR-Cas9 Genome Editing Improves Sub-Cellular Localization Studies
Netanya Y. Spencer, M.D., Ph.D., Research Fellow in Medicine, Joslin Diabetes Center, Harvard Medical School

TECHNOLOGY PANEL: Trends in CRISPR Technologies
Panelists to be Announced

This panel will bring together 2-3 technical experts from leading technology and service companies to discuss trends and improvements in CRISPR libraries, reagents and platforms that users can expect to see in the near future. (Opportunities Available for Sponsoring Panelists)

APPLICATIONS OF CRISPR FOR DRUG DISCOVERY

Use of CRISPR and Other Genomic Technologies to Advance Drug Discovery
Namjin Chung, Ph.D., Head, Functional Genomics Platform, Discovery Research, AbbVie, Inc.

Application of Genome Editing Tools to Model Human Genetics Findings in Drug Discovery
Myung Shin, Ph.D., Senior Principal Scientist, Genetics and Pharmacogenomics, Merck & Co. Inc.

In vivo Application of the CRISPR/Cas9 Technology for Translational Research
Danilo Maddalo, Ph.D., Lab Head, ONC Pharmacology, Novartis Institutes for BioMedical Research

DEVELOPING TOOLS FOR BETTER TRANSLATION

Improving CRISPR-Cas9 Precision through Tethered DNA-Binding Domains
Scot A. Wolfe, Ph.D., Associate Professor, Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School

Nucleic Acid Delivery Systems for RNA Therapy and Gene Editing
Daniel G. Anderson, Ph.D., Professor, Department of Chemical Engineering, Institute for Medical Engineering & Science, Harvard-MIT Division of Health Sciences & Technology and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology

Translating CRISPR/Cas9 into Novel Medicines
Alexandra Glucksmann, Ph.D., COO, Editas Medicine

2nd Annual Translational Gene Editing: Exploiting CRISPR/Cas9 for Building Tools for Drug Discovery & Development: June 16, 2016, Boston, MA, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

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Rice University researches develop new CRISPR-CAS9 strategy to reduce off-target gene editing effects, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

New strategies, tools offered for genome editing

 Reported from Science Daily at  https://www.sciencedaily.com/

Bioengineer Gang Bao and team explore CRISPR-Cas9 alternatives

Date:
February 8, 2016
Source:
Rice University
Summary:
Bioengineers have studied alternative CRISPR-Cas9 systems for precision genome editing, with a focus on improving its accuracy and limiting ‘off-target’ errors.
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FULL STORY

A Cas9 protein (light blue) with guide RNA (purple) and DNA (red) shows a DNA bulge, marking a sequence that would be considered off-target for CRISPR-Cas9 editing. The Rice University lab of bioengineer Gang Bao has developed Web-based tools to search for such off-targets.
Credit: Bao Lab/Rice University

Rice University bioengineers have found new techniques for precision genome editing that are more accurate and have fewer off-target errors.

The new strategies are shared in three papers in an upcoming special issue of the Nature journal Molecular Therapy on improving the revolutionary genome-editing technique called CRISPR-Cas9.

Bioengineering Professor Gang Bao and his colleagues present ideas for maximizing on-target gene editing with biological catalysts capable of cutting DNA called “engineered nucleases.” Several such systems have been studied for years, but for the past three, the promise of cut-and-paste editing via CRISPR-Cas9 has captured the attention of scientists worldwide.

CRISPR-Cas9, a naturally occurring defense system in bacteria, allows researchers to design a short sequence of RNA called “guide RNA” that targets a specific section of genetic code (DNA) in a cell. An associated Cas9 protein then cuts the section, disrupts it or replaces it with the desired code.

That’s how bacteria use CRISPR-Cas9 to immunize themselves from disease. Exposure to an invader causes the bacteria to adapt by adding the invader’s genetic signature to a CRISPR database. The bacteria then recognize future enemies and destroy them with an appropriate Cas9 protein.

About three years ago researchers discovered that bacterial CRISPR-Cas9 could be modified to edit DNA in human cells by, for instance, replacing mutant sequences with normal, or “wild-type,” sequences in much the same way a bacterium banks an invader’s DNA signature. The technique is seen as having great potential for disease modeling and treatment, synthetic biology and molecular pathway dissection.

But CRISPR-Cas9 is still vulnerable to snipping the wrong sequences — called “off-targets” — in addition to the right ones. In therapeutic applications, Bao said, off-target cutting by CRISPR-Cas9 could cause many detrimental effects, including cancer.

Bao, who moved to Rice’s BioScience Research Collaborative (BRC) in 2015 with a grant from the Cancer Prevention and Research Institute of Texas, is studying ways to refine CRISPR-Cas9, which he described as “nanoscissors for editing genes.”

One of his goals is to treat the hereditary disease sickle cell anemia, which he hopes CRISPR-Cas9 will eventually cure. But first the therapy must become much better at avoiding off-targets that can cause unwanted side effects.

In two of the papers, the researchers study different orthologs: Cas9 proteins from species with the same ancestors as the Streptococcus pyogenes (Spy)bacterium commonly used in CRISPR/Cas9.

“Our approach in these papers is to explore the possibility of using different Cas9 orthologs,” Bao said. “There are many possibilities.”

In the first paper, Bao and his group used experiments on mammalian cells to characterize a CRISPR-Cas9 system from the Neisseria meningitides (Nme) bacterium. It differs from Spy in a way that bioengineers can use to reduce the risk of off-target edits, he said.

That difference lies primarily in a sequence of code that is not part of the target, but close by. Known as a protospacer-adjacent motif (PAM), it’s a marker for target DNA sequences and necessary for Cas9 protein binding. InSpyCas9 editing, the PAM sequence is generally three nucleotides long. For Nme, the required PAM sequence is significantly longer — eight nucleotides. While Nme may find fewer targets, those targets are more likely to be the correct ones, according to the researchers. That, they argue, may make it a safer alternative for gene editing.

The second paper, a collaboration with colleagues at the University of Freiburg, Germany, addresses highly specific human-gene editing using yet another bacteria’s immune system. For this study, Cas9 proteins from Spy were replaced with Streptococcus thermophiles (Sth) proteins that also recognize longer PAMs. Tests carried out in human cells found Sth proteins with more stringent PAM requirements were significantly better than SpyCas9 proteins at avoiding off-targets.

Bao and company also looked at the effect of bulges in DNA and RNA that can influence targeting. Bulges appear when a sequence is one nucleotide longer or one nucleotide shorter than the expected DNA sequence targeted by guide RNA.

“We found that even with DNA or RNA bulges, the Cas9 protein can still cut,” he said. “That’s a unique contribution. Nobody saw that would be the case, but we demonstrated it. Consequently, we’ve developed a Web-based tool to search for three cases of potential off-target sites that contain base mismatches, RNA bulges and DNA bulges.”

Bao noted the Nme and Sth Cas9 proteins, unlike Spy, are small enough to be packaged within an adeno-associated virus for delivery to and treatment of specific cells in an animal. “That’s another advantage, and why we want to go on to explore these two systems,” he said.

The third paper is a review of current CRISPR-Cas9 techniques that focuses on genome-editing tools available for target selection, experimental methods and validation. Bao and his team also lay out a list of challenges yet to be solved to eliminate off-target effects.

He said there is a path forward, represented in part by his investigation of two new bacterial systems as well as the fact that CRISPR-Cas9 is a much easier technique to implement in the lab than other genome-editing systems such as TALEN and zinc finger nuclease.

Bao said that unlike those older genome-editing techniques, CRISPR-Cas9 is straightforward enough for students to learn and use in a short time.

Bao hopes to establish his lab as a focal point for genome editing in the Texas Medical Center. To that end, he brought the TMC genome-editing community together for a well-attended workshop at the BRC last December.

“We had a lot of good discussions,” he said. “One thing I would like to stimulate is the formation of a consortium among the many labs in TMC using CRISPR. They have needs to design CRISPR systems for different applications, but there are a lot of common issues. If we work together, it will be easier to address them.”


Story Source:

The above post is reprinted from materials provided by Rice University.Note: Materials may be edited for content and length.


Journal References:

  1. Ciaran M. Lee, Thomas J. Cradick, Gang Bao. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells. Molecular Therapy, 2016; DOI:10.1038/mt.2016.8
  2. Maximilian Müller, Ciaran M Lee, Giedrius Gasiunas, Timothy H Davis, Thomas J Cradick, Virginijus Siksnys, Gang Bao, Toni Cathomen, Claudio Mussolino. Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome. Molecular Therapy, 2015; DOI: 10.1038/mt.2015.218
  3. Ciaran M. Lee, Thomas J. Cradick, Eli J Fine, Gang Bao. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing. Molecular Therapy, 2016; DOI: 10.1038/mt.2016.1

Cite This Page:

Rice University. “New strategies, tools offered for genome editing: Bioengineer Gang Bao and team explore CRISPR-Cas9 alternatives.” ScienceDaily. ScienceDaily, 8 February 2016. <www.sciencedaily.com/releases/2016/02/160208135449.htm>.

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From GEN News Highlights

Reposted from GEN News

Nov 18, 2015
2.2.8

2.2.8   RNA-Based Drugs Turn CRISPR/Cas9 On and Off, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

RNA-Based Drugs Turn CRISPR/Cas9 On and Off

  • This image depicts a conventional CRISPR-Cas9 system. The Cas9 enzyme acts like a wrench, and specific RNA guides act as different socket heads. Conventional CRISPR-Cas9 systems act continuously, raising the risk of off-target effects. But CRISPR-Cas9 systems that incorporate specially engineered RNAs could act transiently, potentially reducing unwanted changes. [Ernesto del Aguila III, NHGRI]

    By removing parts of the CRISPR/Cas9 gene-editing system, and replacing them with specially engineered molecules, researchers at the University of California, San Diego (UCSD) and Isis Pharmaceutical hope to limit the CRISPR/Cas9 system’s propensity for off-target effects. The researchers say that CRISPR/Cas9 needn’t remain continuously active. Instead, it could be transiently activated and deactivated. Such on/off control could prevent residual gene-editing activity that might go awry. Also, such control could be exploited for therapeutic purposes.

    The key, report the scientists, is the introduction of RNA-based drugs that can replace the guide RNA that usually serves to guide the Cas9 enzyme to a particular DNA sequence. When Cas9 is guided by a synthetic RNA-based drug, its cutting action can be suspended whenever the RNA-based drug is cleared. The Cas9’s cutting action can be stopped even more quickly if a second, chemically modified RNA drug is added, provided that it is engineered to direct inactivation of the gene encoding the Cas9 enzyme.

    Details about temporarily activated CRISPR/Cas9 systems appeared November 16 in the Proceedings of the National Academy of Sciences, in a paper entitled, “Synthetic CRISPR RNA-Cas9–guided genome editing in human cells.” The paper’s senior author, the USCD’s Don Cleveland, Ph.D., noted that the RNA-based drugs described in the study “provide many advantages over the current CRISPR/Cas9 system,” such as increased editing efficiency and potential selectivity.

    “Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells,” wrote the authors of the PNAS paper. “Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA–RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA.”

    Not only did the synthetic RNA functionally replace the natural crRNA, it produced enhanced cleavage activity at a target DNA site with apparently reduced off-target cleavage. These findings, Dr. Cleveland explained, could provide a platform for multiple therapeutic applications, especially for nervous system diseases, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 activity. “In addition,” he said, “[these designer RNAs] can be synthesized efficiently, on an industrial scale and in a commercially feasible manner today.”

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Use of CRISPR/CAS9 to Edit Genome of Pigs: Recominetics announces $10M Funding Round, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

Gene-editing startup raising $10M to expand staff
Nov 25, 2015

Reporter: Stephen J. Williams,Ph.D.

From the Mineapolis/St. Paul Journal

source from: http://www.bizjournals.com/twincities/news/2015/11/25/gene-editing-startup-raising-10m-to-expand-staff.html 

Katharine Grayson
Staff reporter
Minneapolis / St. Paul Business Journal

Recombinetics Inc. is seeking $10 million in funding as it ramps up sales of its genetically tweaked animals.
The St. Paul-based biotech company’s recent round has already brought in about about $2.8 million from friends and family, said Chief Operating Officer Kyle Dawley. Company officials hope to close out the round within the next two months and add about 10 employees to its staff of 25.

 

 

Recombinetics edits pigs' genes for biomedical research purposes

Recombinetics edits pigs’ genes for biomedical research purposes. Photo source: Simone Van Den Berg

Recombinetics uses gene-editing technology to tweak animals for the agribusiness and biomedical markets. It’s biomedical business centers around pigs, which the company modifies for research purposes. That side of the company’s business already generates revenue, Dawley said, though he declined to reveal sales figures.

The company focuses on pigs, touting them as better research subjects than mice when it comes to testing medical devices and drugs for use in humans.

“Pigs are — size-wise and genetically — a lot more like humans than rats and mice,” Dawley said.

One of Recombinetics’ long-term goals is grow human organs inside pigs.

The company aims to modify livestock for food consumption as well. One of its projects calls for creating hornless cattle by taking a gene from one breed and putting into another.

Recombinetics expects food ventures may get a boost from the Food and Drug Administration’s recent approval of a genetically engineered salmon called “AquAdvantage.” The fish grows faster than traditional salmon thanks to the introduction of trout genes.

Recombinetics has raised $15 million since its founding.

Katharine Grayson covers med tech, clean tech, technology, health care and venture capital.

 

See also Surrogen, Inc., which produces transgenic pigs for purpose of large animal models of disease.

Other posts on this Open Access Journal where I have discussed the utility of the minipig as a large animal model of disease include:

The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research

The SCID Pig II: Researchers Develop Another SCID Pig, And Another Great Model For Cancer Research

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