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In a first, researchers from the Oregon Health and Science University along with colleagues in California, China and South Korea repaired a mutation in human embryos by using a gene-editing tool called CRISPR-Cas9.

The mutation seen in the MYBPC3 gene causes a common heart condition called hypertrophic cardiomyopathy, which is marked by thickening of the heart muscle.

The mutation is seen in about one in 500 people and can lead to sudden death later in life. It is an inherited cardiac disease and the presence of even one copy of the gene can cause symptoms, which usually manifest as heart failure. Correcting the mutation in the embryo ensures that the child is born healthy and the defective gene is not passed on to future generations. There is currently no cure for the condition.

CRISPR-Cas9 is a system used by bacterial cells to recognise and destroy viral DNA as a form of adaptive immunity. Using components of the CRISPR system, researchers can remove, add or alter specific DNA sequences in the genome of higher organisms.

The gene editing tool has two components a single-guide RNA (sgRNA) that contains a sequence that can bind to DNA, and the Cas9 enzyme which acts as a molecular scissor that can cleave DNA. The genetic sequence of the sgRNA matches the target sequence of the DNA that has to be edited. In order to selectively edit a desired sequence in DNA, the sgRNA is designed to find and bind to the target.

Upon finding its target, the Cas9 enzyme swings into an active form that cuts both strands of the target DNA. One of the two main DNA-repair pathways in the cell then gets activated to repair the double-stranded breaks. While one of the repair mechanisms result in changes to the DNA sequence, the other is more suitable for introducing specific sequences to enable tailored repair. In theory, the guide RNA will only bind to the target sequence and no other regions of the genome.

But the CRISPR-Cas9 system can also recognise and cleave different regions of the genome than the one that was intended to be edited. These off-target changes are very likely to take place when the gene-editing tool binds to DNA sequences that are very similar to the target one. Though many studies have found few unwanted changes suggesting that the tool is probably safe, researchers are working on safer alternatives.

Along with sperm from a man with hypertrophic cardiomyopathy, the gene-editing tool was also introduced into eggs from 12 healthy women before fertilisation. In normal conditions, a piece of DNA with the correct sequence serves as a template for the repair to work, although the efficiency can be significantly low. Instead of the repair template that was provided by the researchers, the cells used the healthy copy of the DNA from the egg as a template. This came as a big surprise.

Normally, if sperm from a father with one mutant copy of the gene is fertilized in vitro with normal eggs, 50% of the embryos would inherit the condition. When the gene-editing tool was used, 42 out of the 58 embryos did not carry the mutation. The remaining 16 embryos had unwanted additions or deletions of DNA.

Thus the probability of inheriting the healthy gene increased from 50 to 72.4%. There was no off-target snipping of the DNA. According to Nature, the edited embryos developed similarly to the control embryos, with 50% reaching an early stage of development (blastocyst). This indicates that editing does not block development.

Clinical trials are under way in China and in the U.S. to use this tool for treating cancer. In May this year, it was shown in mice that it is possible to shut down HIV-1 replication and even eliminate the virus from infected cells. In agriculture, a new breed of crops that are gene-edited will become commercially available in a few years. In February this year, the National Academy of Sciences (NAS) and the National Academy of Medicine said scientific advances make gene editing in human reproductive cells a realistic possibility that deserves serious consideration.

R. Prasad

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The lowdown on genome editing - NATIONAL - The Hindu - The Hindu

The use of genome-editing techniques in medical therapies has proved to be a promising development in the treatment of certain diseases, such as cancer, HIV and rare diseases, by genetically altering specific types of cells. Compared to other techniques used to insert, delete or replace DNA in the genome of an organism, CRISPR/Cas9 is much quicker, easier to use and less costly, may be more precise in its application, and can also be used to edit multiple genes simultaneously. The technique therefore has the potential to be a true game-changer in medicine with profound beneficial effects on human health. However, the enthusiasm for the opportunities of this promising technology should be accompanied by adequate regulatory oversight to guarantee the safety of products and applications that use this technology.

The first clinical trial using CRISPR-edited immune cells began in patients with lung cancer in China in 2016. Earlier this year, FDAs Recombinant DNA Advisory Committee did not find any objections to the first clinical protocol to use CRISPR/Cas9-mediated gene editing, and the first US clinical trial is expected to start shortly. Several more clinical trials have since been approved and started in China, including one which proposes to perform gene editing in vivo i.e. directly within the body of live patients (as opposed to ex vivo, e.g. using cells extracted from donors).

Most recently, in August 2017, a team of US-based scientists at Oregon Health and Science University published a paper describing the successful use of CRISPR/Cas9 to fix a disease-causing DNA error in dozens of early-stage human embryos, which, according to biologist Shoukrat Mitalipov, brings us much closer to clinical applications. Clinical use of this work would mean actually implanting some of these embryos with the goal of children being born that possess genes which have been artificially edited using CRISPR technology and would be capable of passing those edited genes to their offspring. These developments are exciting for patients and their loved ones, but in equal measure represent a challenge to existing regulatory structures and society at large. At any rate, with the pace of development in the CRISPR field around the world, clinical trials involving CRISPR in the EU may not be far away.

Regulators in the EU and abroad will need to stay abreast of this new (r)evolution in genome-editing technologies. In this respect, different groups established within the European Commission, including the European Group on Ethics in Science and New Technologies, have emphasised the great potential [of the CRISPR/Cas9 genome-editing technology] due to its many advantages to previous methods and acknowledged that the CRISPR/Cas9 system challenges the international regulatory landscape for the modification of human cells in the near to medium term.

While no specific regulatory guidance has been issued to date, the European Medicines Agency (EMA) has started to lay the groundwork for the regulatory implications to come, by launching a public consultation on the revision of its Guideline on medicinal products containing genetically modified cells on 20 July 2017. The EMA specifically recognises that the current 2012 guideline focuses on genetic modifications by traditional methods (based on the use of vectors carrying recombinant nucleic acids), but that the introduction of the CRISPR/Cas9 system has rapidly increased the use of genome-editing technologies to genetically modify cells ex vivo for clinical applications, and aims to take these aspects into consideration in its revised draft guideline, which is expected by March 2018.

Regulation of gene-edited products in the EU

1.EU-wide classification and authorisation of Advanced Therapy Medicinal Products

Currently, in the EU, new medicinal products based on genes (gene therapy), cells (cell therapy) and tissues (tissue engineering) also known as advanced therapy medicinal products or ATMP are regulated by the ATMP Regulation (Regulation (EC) No. 1394/2007 on advanced therapy medicinal products). The ATMP Regulation is a lex specialis supplementing the provisions of Directive 2001/83/EC and Regulation (EC) No 726/2004. It regulates ATMPs which are intended to be placed on the market in [EU] Member States and either prepared industrially or manufactured by a method involving an industrial process.

Like all other modern biotechnology medicinal products, ATMPs are regulated at EU level and are subject to the centralised marketing authorisation procedure. In addition to the general regulatory requirements that apply to all medicinal products, given their complexity, ATMPs are subject to specific technical requirements, including the type and amount of quality pre-clinical and clinical data necessary to demonstrate the quality, safety and efficacy of the product and obtain a marketing authorisation. To facilitate the development of these products and help pharmaceutical companies prepare for marketing authorisation applications, the EMA has adopted a raft of scientific guidelines. Whether the existing regulatory framework and guidelines will be fit for purpose for genome-editing applications remains to be seen. Some clarifications and modifications seem unavoidable as is reflected by the EMAs on-going revision of its overarching guideline on medicinal products containing genetically modified cells in light of the CRISPR/Cas9 advances.

The ATMP Regulation distinguishes three types of ATMPs, two of which are of interest when considering CRISPR products and applications: (i) gene therapy medicinal products (GTMPs) and (ii) somatic cell therapy medicinal products (sCTMPs).

Pursuant to Directive 2001/83/EC (Annex I, Part IV, Section 2.1), a GTMP corresponds to a biological medicinal product with the following characteristics:

a)it contains an active substance which contains or consists of a recombinant nucleic acid used in or administered to human beings with a view to regulating, repairing, replacing, adding or deleting a genetic sequence; and

b)its therapeutic, prophylactic or diagnostic effect relates directly to the recombinant nucleic acid sequence it contains, or to the product of genetic expression of this sequence.

In contrast, the Directive (Annex I, Part IV, Section 2.2) defines an sCTMP as a biological medicinal product that:

a)contains or consists of cells or tissues that have been subject to substantial manipulation so that biological characteristics, physiological functions or structural properties relevant for the intended clinical use have been altered, or of cells or tissues that are not intended to be used for the same essential function(s) in the recipient and the donor; and

b)is presented as having properties for, or is used in or administered to human beings with a view to treating, preventing or diagnosing a disease through the pharmacological, immunological or metabolic action of its cells or tissues.

Depending on the primary mode of action of a therapy, CRISPR-modified cells that are used in therapy could likely be categorised as either GTMPs or sCTMPs (though given the early stages of CRISPR-related clinical trials, this has not yet been confirmed by regulatory authorities to date). For example, where the primary use of genome-edited haematopoietic stem cells (HSCs) is immune reconstitution and the genetic modification is for the secondary purpose of limiting risk of graft versus host disease, the therapy is likely to be classified as an sCTMP. This is because HSCs themselves can reconstitute a patients immune system without any genetic modification. In contrast, where the primary mode of action is a direct result of the genetic modification, it is likely to be classified as a GTMP. For example, where a gene is inserted into T-cells, resulting in a receptor being expressed on the cell surface designed to recognise and attack target cells (such as cancer cells), this is likely to be considered a GTMP because the T-cells alone, without this genetic modification, would not provide any therapeutic effect. Notwithstanding the above, the ATMP Regulation requires that a product meeting the definition of both GTMP and sCTMP be classified as a GTMP.

If an applicant is unsure whether a product is an ATMP, it can request a recommendation from the EMAs specialised Committee for Advanced Therapies (CAT), which must respond within 60 days (after consultation with the European Commission). Non-confidential summaries of these recommendations are publicly available. The CAT provides advice on whether a product falls within the definition of an ATMP, formulates draft opinions on the quality, safety and efficacy of ATMPs for final approval by the Committee for Medicinal Products for Human Use (CHMP), and advises the latter on any data generated in the development of ATMPs. The CAT has previously evaluated cell therapies involving genetically modified cells and the evaluation of CRISPR-modified medicinal products is likely to be analogous (see for example Autologous anti-BCMA CAR T-cells which were classified as a gene therapy medicinal product). Companies interested in the development and marketing of CRISPR edited medicinal products should consider monitoring CAT recommendations, reports and publications in order to better understand how CRISPR products will be classified and regulated in the context of the ATMP Regulation.

2.National approvals by competent authorities and ethics committees of clinical trials with ATMPs

ATMPs must go through clinical trials in the same way as any other medicine. Clinical trials are approved on a national basis, by the national competent regulatory authorities after the provision of the opinion of an ethics committee, in accordance with the harmonised procedures and principles established by the Clinical Trials Directive (Directive 2001/20/EC). However, the application procedure will be streamlined significantly as applications will be submitted through a single EU portal and undergo a (partly) harmonised assessment (by all Member States involved) once the Clinical Trials Regulation (EU) No 536/2014 will become applicable (at the earliest, October 2018 according to the timeframe drawn up by the EMA but most likely later).

Given the complexity of ATMPs (and the corresponding clinical trial dossiers), specific written authorisation is required, and the timelines for approval of clinical trials with these products are often longer than for regular medicinal products. Currently, the time period for the national competent authority to consider a request for authorisation of a clinical trial which in principle may not exceed 60 days may be extended by 30 days in the case of GTMPs and sCTMPs. This maximum period of 90 days may be extended by a further 90 days in the event of consultation of a group or a committee in accordance with the regulations and procedures of the Member States concerned. Under the new Clinical Trials Regulation, review timelines will remain lengthier in the case of clinical trials involving an ATMP.

Ethical aspects remain the responsibility of individual EU Member States and (even under the new Clinical Trial Regulation) local ethics committees need to give their opinion before a clinical trial can be authorised. It is clear that the debate around trials involving products created by CRISPR techniques (or therapies involving the direct in vivo use of CRISPR in patients) is likely to be complex given the numerous ethical issues (such as the fear of designer babies and other eugenic applications) raised by this technology. Currently, for ATMPs, ethics committees can extend the time period to give their reasoned opinion on a clinical trial with these products from 60 to 90 days (which may be further extended by 90 days in the event of consultation of a specific committee), and despite the intention of the Clinical Trials Regulation to shorten the timelines for clinical trial approvals in the EU these extended timelines are likely to be indispensible for CRISPR related trials.

However, importantly, to date, it is clearly established in EU law that gene therapy trials resulting in modifications to the subjects germ line genetic identity are prohibited (and this prohibition is maintained under the EU Clinical Trials Regulation). Therefore, one of the most controversial applications of CRISPR (which was the subject of the August 2017 Mitalipov paper) the editing of genes at a germ-line level (in egg cells, sperm cells and embryos) so that the edited gene is inheritable by future generations is unlikely to be permitted in the EU for the foreseeable future.

3.Other regulations to consider

Additional legislation supporting the ATMP regulation will have to be considered as well. For example, where tissues and cells are used as starting materials, the donation, procurement and testing of the cells are covered by the Tissues and Cells Directive (Directive 2004/23/EC). Other relevant legislation includes Directive 2005/28/EC that lays down detailed guidelines for good clinical practice (GCP) and the requirements for authorisation of the manufacturing or importation of ATMPs.

This is an exciting time for research and development wherein the use of CRISPR in medicine may lead to rapid and significant progress for human health. The regulation of the developments triggered by this new technology is important to ensure that, on the one hand, appropriate quality and safety standards are adhered to, by way of evaluating and mitigating potential risks and, on the other, a clear and certain regulatory environment is created to encourage researchers to explore fully the potential of this technology within the ethical bounds society deems appropriate. It appears that the regulation of gene and cell therapies under the EU ATMP Regulation possibly with some regulatory modifications and the adoption of adequate scientific guidelines - could also govern revolutionary gene-editing techniques such as CRISPR/Cas9, and is therefore the legal instrument to watch as CRISPR continues to conquer the world of medicine.

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August 2017 Regulating CRISPR genome editing in humans: where do we go from here? - JD Supra (press release)

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New York, NY When doctors cant find a diagnosis for a patients disease, they turn to genetic detectives. Equipped with genomic sequencing technologies available for less than 10 years, these sleuths now routinely search through a patients DNA looking for mutations responsible for mysterious diseases.

Despite many successes, the search still comes back empty more often than not. In fact, disease-causing mutations are found in only about one in three to four patients suspected of having a strongly genetic condition.

A big reason why most investigations turn up empty-handed is the dark genome. Only two percent of the human genome is well understood by scientists. This small fraction contains the 20,000 genes that encode instructions for making the cells proteins. The remaining 98 percentthe dark genomeis largely a mystery. Although its known that the dark, non-coding genome regulates genesturning them on and off, for examplethe details remain obscure.

As a consequence, sequencing data from the entire genome is currently considered almost uninterpretable, saysDavid Goldstein, PhD, the John E. Borne Professor of Medical and Surgical Research and director of the Institute for Genomic Medicine at Columbia University Medical Center, and todays genetic detectives restrict their search for disease-causing mutations to the sliver of genome that contains protein-coding genes.

Related Article:UVA Finds Way to Dramatically Speed Research into Cancer, Genetic Diseases

To help locate pathogenic mutations in the vast non-coding genome, Dr. Goldstein and his colleagues Ayal Gussow and Andrew Allen have developed a new technique called Orion. Orion is designed to flag regions of the non-coding genome that are likely to contain disease-causing genetic changes by identifying parts of the genome that are under selection in the human population.

We anticipate that researchers will immediately start using Orion to help them find pathogenic mutations in patients in which previous sequencing efforts were negative, says Dr. Goldstein. Details about the method were published online Aug. 10 in PLoS One.

Orion was developed by comparing the entire genomes of 1,662 people and identifying stretches of DNA that vary little from person to person. Because these regions are intolerant to change, they are most likely doing something important, says Dr. Goldstein, lead author of the paper.

That means a mutation in an intolerant region is more likely to cause disease than a mutation in a tolerant (read: less important) region. This prediction was confirmed when the researchers mapped the locations of previously identified non-coding mutations: More mutations fell within Orions intolerant regions.

Previous methods to explore the non-coding genome focused on areas of the non-coding genome that have been retained in multiple species over evolutionary time, suggesting they, too, have an important function. However, this approach is not able to identify regions of the genome that have taken on important new functions in humans.

Orion isnt yet a finished product, Dr. Goldstein says. As more genomes are sequenced, the resolution of Orions regions will improve dramatically.

At that point, we are optimistic that Orion will constitute one helpful tool in the effort to identify variants throughout the genome that influence the risk of both rare and common diseases, says Dr. Goldstein.

Thestudyis titled Orion: Detecting Regions of the Human Non-Coding Genome that are Intolerant to Variation Using Population Genetics. Authors are Ayal Gussow (Duke University, Durham, NC, and Columbia University Medical Center, New York, NY), Brett Copeland (CUMC), Ryan Dhindsa (CUMC), Quanli Wang (CUMC), Slave Petrovski (CUMC and University of Melbourne, Victoria, Australia), William Majoros (Duke), Andrew Allen (Duke), and David Goldstein (CUMC).

The study was supported by the National Institutes of Health (1U01MH105670, 1UM1HG00901,F31NS092362,RC2NS070344;U01NS077303;U01NS053998,RC2MH089915,K01MH098126,R01MH097971,U01HG007672, andUM1AI100645); Biogen Inc.; SAIC Fredrick Inc.; the Joseph and Kathleen Bryan Alzheimers Disease Research Center; the Duke Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery; the Bill and Melinda Gates Foundation; the Ellison Medical Foundation; and the Murdock Study Community Registry and Biorepository.

David Goldstein is a founder of and holds equity in Pairnomix and Praxis and receives support from Janssen, Gilead, Biogen, AstraZeneca, and UCB. The authors declare no other conflicts of interest.

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New Technique Searches 'Dark Genome' for Disease Mutations - Lab Manager Magazine