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1st Patients To Get CRISPR Gene-Editing Treatment Continue To Thrive – NPR https://www.npr.org/sections/health-shots/2020/12/15/944184405/1st-patients-to-get-crispr-gene-editing-treatment-continue-to-thrive
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Victoria Gray (second from left) with children Jamarius Wash, Jadasia Wash and Jaden Wash. Now that the gene-editing treatment has eased Gray’s pain, she has been able be more active in her kids’ lives and looks forward to the future. “This is really a life-changer for me,” she says.

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<p>The last thing a lot of people want to do these days is get on a plane. But even a pandemic would not stop Victoria Gray. She jumped at the chance to head to the airport this summer.</p> <p>”It was one of those things I was waiting to get a chance to do,” says Gray.</p><p>She had never flown before because she was born with <a href=”https://www.cdc.gov/ncbddd/sicklecell/facts.html”>sickle cell disease</a>. She feared the altitude change might trigger one of the worst complications of the devastating genetic disease — a sudden attack of excruciating pain.</p> <p>But Gray is the first person in the United States to be successfully<strong> </strong>treated for a genetic disorder with the help of CRISPR, a revolutionary gene-editing technique that makes it much <a href=”https://www.npr.org/series/773368439/the-crispr-revolution”>easier to make very precise changes in DNA</a>.</p>

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<p>About a year after getting the treatment, it was working so well that Gray felt comfortable flying for the first time. She went to Washington, D.C., to visit her husband, who has been away for months on deployment with the National Guard.</p> <p>”It was exciting. I had a window. And I got to look out the window and see the clouds and everything,” says Gray, 35, of Forest, Miss.</p> <p>Gray wore a mask the whole time to protect herself against the coronavirus, kept her distance from other people at the airport, and arrived happily in Washington, D.C., even though she’s afraid of heights.</p> <aside id=”ad-backstage-wrap” aria-label=”advertisement”>

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</aside> <p>”I didn’t hyperventilate like I thought I would,” Gray says, laughing as she recounts the adventure in an interview with NPR.</p> <p>NPR has had exclusive access to <a href=”https://www.npr.org/sections/health-shots/2019/12/25/784395525/a-young-mississippi-womans-journey-through-a-pioneering-gene-editing-experiment”>follow Gray through her experience</a> since she underwent the landmark treatment on July 2, 2019. Since <a href=”https://www.npr.org/sections/health-shots/2020/06/23/877543610/a-year-in-1st-patient-to-get-gene-editing-for-sickle-cell-disease-is-thriving”>the last time NPR checked in with Gray</a> in June, she has continued to improve. Researchers have become increasingly confident that the approach is safe, working for her and will continue to work. Moreover, they are becoming far more encouraged that her case is far from a fluke.</p> <p>At a recent meeting of the American Society for Hematology, researchers <a href=”https://ash.confex.com/ash/2020/webprogram/Paper139575.html”>reported the latest results from the first 10 patients</a> treated via the technique in a research study, including Gray, two other sickle cell patients and seven patients with a related blood disorder, <a href=”https://medlineplus.gov/genetics/condition/beta-thalassemia/#:~:text=Beta%20thalassemia%20is%20a%20blood,many%20parts%20of%20the%20body.”>beta thalassemia</a>. The patients now have been followed for between three and 18 months.</p> <p>All the patients appear to have responded well. The only side effects have been from the intense chemotherapy they’ve had to undergo before getting the billions of edited cells infused into their bodies.</p> <p>The <em>New England Journal of Medicine</em> <a href=”https://www.nejm.org/doi/full/10.1056/NEJMoa2031054″>published online</a> this month the first peer-reviewed research paperfrom the study, focusing on Gray and the first beta thalassemia patient who was treated.</p> <p>”I’m very excited to see these results,” says <a href=”https://doudnalab.org/”>Jennifer Doudna of the University of California, Berkeley</a>, who shared the Nobel Prize this year for her role in the development of CRISPR. “Patients appear to be cured of their disease, which is simply remarkable.”</p> <p>Another nine patients have also been treated, according to <a href=”http://www.crisprtx.com/”>CRISPR Therapeutics</a> in Cambridge, Mass., and <a href=”https://www.vrtx.com/”>Vertex Pharmaceuticals</a> in Boston, two companies sponsoring the research. Those individuals haven’t been followed long enough to report any results, officials say.</p> <p>But the results from the first 10 patients “represent an important scientific and medical milestone,” says <a href=”https://www.vrtx.com/about-us/leadership/david-altshuler-md-phd/”>Dr. David Altshuler,</a> Vertex’s chief scientific officer.</p> <p>The treatment boosted levels of a protein in the study subjects’ blood known as fetal hemoglobin. The scientists believe that protein is compensating for defective adult hemoglobin that their bodies produce because of a genetic defect they were born with. Hemoglobin is necessary for red blood cells to carry oxygen.</p> <p>Analyses of samples of bone marrow cells from Gray six months after getting the treatment, then again six months later, showed the gene-edited cells had persisted the full year — a promising indication that the approach has permanently altered her DNA and could last a lifetime.</p> <p>”This gives us great confidence that this can be a one-time therapy that can be a cure for life,” says <a href=”http://www.crisprtx.com/about-us/leadership/dr.-samarth-kulkarni”>Samarth Kulkarni</a>, the CEO of CRISPR Therapeutics.</p> <p>Gray and the two other sickle cell patients haven’t had any complications from their disease since getting the treatment, including any pain attacks or hospitalizations. Gray has also been able to wean off the powerful pain medications she’d needed most of her life.</p> <p>Prior to the treatment, Gray experienced an average of seven such episodes every year. Similarly, the beta thalassemia patients haven’t needed the regular blood transfusions that had been required to keep them alive.</p> <p>”It is a big deal because we we able to prove that we can edit human cells and we can infuse them safely into patients and it totally changed their life,” says <a href=”https://tristarchildrensspecialists.com/physicians/profile/Dr-Haydar-A-Frangoul-MD”>Dr. Haydar Frangoul</a> at the Sarah Cannon Research Institute in Nashville. Frangoul is Gray’s doctor and is helping run the study.</p> <p>For the treatment, doctors remove stem cells from the patients’ bone marrow and use CRISPR to edit a gene in the cells, activating the production of fetal hemoglobin. That protein is produced by fetuses in the womb but usually shuts off shortly after birth.</p> <p>The patients then undergo a grueling round of chemotherapy to destroy most of their bone marrow to make room for the gene-edited cells, billions of which are then infused into their bodies.</p> <p>”It is opening the door for us to show that this therapy can not only be used in sickle cell and thalassemia but potentially can be used in other disorders,” Frangoul says.</p> <p>Doctors have already started trying to use CRISPR to treat cancer and to <a href=”https://www.npr.org/sections/health-shots/2020/03/04/811461486/in-a-1st-scientists-use-revolutionary-gene-editing-tool-to-edit-inside-a-patient”>restore vision to people blinded</a> by a genetic disease. They hope to try it for many other diseases as well, including heart disease and AIDS.</p> <p>The researchers stress that they will have to follow Gray and many other patients for a lot longer to be sure the treatment is safe and that it keeps working. But they are optimistic it will.</p> <p>Gray hopes so too.</p> <p>”It’s amazing,” she says. “It’s better than I could have imagined. I feel like I can do what I want now.”</p><p>The last year hasn’t always been easy for Gray, though. Like millions of other Americans, she has been sheltering at home with three of her children, worrying about keeping them safe and helping them learn from home much of the time.</p> <p>”I’m trying to do the things I need to do while watch them at the same time to make sure they’re doing the things they need to do,” Gray says. “It’s been a tough task.”</p><p>But she has been able do other things she never got to do before, such as watch her oldest son’s football games and see her daughter cheerleading.</p> <p>”This is really a life-changer for me,” she says. “It’s magnificent.”</p> <p>She’s now looking forward to going back to school herself, learning to swim, traveling more when the pandemic finally ends, and watching her children grow up without them worrying about their mother dying.</p> <p>”I want to see them graduate high school and be able to take them to move into dorms in college. And I want to be there for their weddings — just everything that the normal people get to do in life. I want to be able to do those things with my kids,” she says. “I can look forward now to having grandkids one day — being a grandmama.”</p>
<p><strong><a href=”https://blockads.fivefilters.org”></a></strong> <a href=”https://blockads.fivefilters.org/acceptable.html”>(Why?)</a></p> Tue, 15 Dec 2020 10:02:00 +0000 Rob Stein

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Using CRISPR, new technique makes it easy to map genetic networks: Nucleotide barcodes attached to guide RNAs uniquely identify regulatory genes – Science Daily https://www.sciencedaily.com/releases/2020/12/201210145755.htm
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<div><img src=”https://www.sciencedaily.com/images/scidaily-icon.png” class=”ff-og-image-inserted”></div><p id=”first”>CRISPR-Cas9 makes it easy to knock out or tweak a single gene to determine its effect on an organism or cell, or even another gene. But what if you could perform several thousand experiments at once, using CRISPR to tweak every gene in the genome individually and quickly see the impact of each?</p><div id=”text” readability=”206″>
<p>A team of University of California, Berkeley, scientists has developed an easy way to do just that, allowing anyone to profile a cell, including human cells, and rapidly determine all the DNA sequences in the genome that regulate the expression of a specific gene.</p>
<p>While the technique will mostly benefit basic researchers who are interested in tracking the cascade of genetic activity — the genetic network — that impacts a gene they’re interested in, it will also help researchers quickly find the regulatory sequences that control disease genes and possibly find new targets for drugs.</p>
<p>”A disease where you might want to use this approach is cancer, where we know certain genes that those cancer cells express, and need to express, in order to survive and grow,” said Nicholas Ingolia, UC Berkeley associate professor of molecular and cell biology. “What this tool would let you do is ask the question: What are the upstream genes, what are the regulators that are controlling those genes that we know about?”</p>
<p>Those controllers may be easier to target therapeutically in order to shut down the cancer cells.</p>
<p>The new technique simplifies something that has been difficult to do until now: backtrack along genetic pathways in a cell to find these ultimate controllers.</p>

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<p>”We have a lot of good ways of working forward,” he said. “This is a nice way of working backward, figuring out what is upstream of something. I think it has a lot of potential uses in disease research.”</p>
<p>”I sometimes use the analogy that when we walk into a dark room and flip a light switch, we can see what light gets switched on. That light is like a gene, and we can tell, when we flip the switch, what genes it turns on. We are already very good at that,” he added. “What this lets us do is work backward. If we have a light we care about, we want to find out what are the switches that control it. This gives us a way to do that.”</p>
<p>Ryan Muller, a graduate student in the Ingolia lab, and colleagues Lucas Ferguson and Zuriah Meacham, along with Ingolia, will publish the details of their technique online on Dec. 10 in the journal <em>Science</em>.</p>
<p><strong>Barcoding the genome</strong></p>
<p>Since the advent of CRISPR-Cas9 gene-editing eight years ago, researchers who want to determine the function of a specific gene have been able to precisely target it with the Cas9 protein and knock it out. Guided by a piece of guide RNA complementary to the DNA in the gene, the Cas9 protein binds to the gene and cuts or, as with CRISPR interference (CRISPRi), inhibits it.</p>

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<p>In the crudest type of assay, the cell or organism either lives or dies. However, it’s possible to look for more subtle effects of the knockout, such as whether a specific gene is turned on or off, or how much it’s turned up or down.</p>
<p>Today, that requires adding a reporter gene — often one that codes for a green fluorescent protein — attached to an identical copy of the promoter that initiates expression of the gene you’re interested in. Since each gene’s unique promoter determines when that gene is expressed, if the Cas9 knockout affects expression of your gene of interest, it will also affect expression of the reporter, making the culture glow green under fluorescent light.</p>
<p>Nevertheless, with 6,000 total genes in yeast — and 20,000 total genes in humans — it’s a big undertaking to tweak each gene and discover the effect on a fluorescent reporter.</p>
<p>”CRISPR makes it easy to comprehensively survey all the genes in the genome and perturb them, but then the big question is, How do you read out the effects of each of those perturbations?” he said.</p>
<p>This new technique, which Ingolia calls CRISPR interference with barcoded expression reporter sequencing, or CiBER-seq, solves that problem, allowing these experiments to be done simultaneously by pooling tens of thousands of CRISPR experiments. The technique does away with the fluorescence and employs deep sequencing to directly measure the increased or decreased activity of genes in the pool. Deep sequencing uses high-throughput, long-read next generation sequencing technology to sequence and essentially count all the genes expressed in the pooled samples.</p>
<p>”In one pooled CiBER-seq experiment, in one day, we can find all the upstream regulators for several different target genes, whereas, if you were to use a fluorescence-based technique, each of those targets would take you multiple days of measurement time,” Ingolia said.</p>
<p>CRISPRing each gene in an organism in parallel is straightforward, thanks to companies that sell ready-made, single guide RNAs to use with the Cas9 protein. Researchers can order sgRNAs for every gene in the genome, and for each gene, a dozen different sgRNAs — most genes are strings of thousands of nucleotides, while sgRNAs are about 20 nucleotides long.</p>
<p>The team’s key innovation was to link each sgRNA with a unique, random nucleotide sequence — essentially, a barcode — connected to a promoter that will only transcribe the barcode if the gene of interest is also switched on. Each barcode reports on the effect of one sgRNA, individually targeting one gene out of a complex pool of thousands of sgRNAs. Deep sequencing tells you the relative abundances of every barcode in the sample — for yeast, some 60,000 — allowing you to quickly assess which of the 6,000 genes in yeast has an effect on the promoter and, thus, expression of the gene of interest. For human cells, a researcher might insert more than 200,000 different guide RNAs, targeting each gene multiple times.</p>
<p>”This is really the heart of what we were able to do differently: the idea that you have a big library of different guide RNAs, each of which is going to perturb a different gene, but it has the same query promoter on it — the response you are studying. That query promoter transcribes the random barcode that we link to each guide,” he said. “If there is a response you care about, you poke each different gene in the genome and see how the response changes.”</p>
<p>If you get one barcode that is 10 times more abundant than any of the others, for example, that tells you that that query promoter is switched on 10 times more strongly in that cell. In practice, Ingolia attached about four different barcodes to each guide RNA, as a quadruple check on the results.</p>
<p>”By looking more directly at a gene expression response, we can pick up on a lot of subtlety to the physiology itself, what is going on inside the cell,” he said.</p>
<p>In the newly reported experiments, the team queried five separate genes in yeast, including genes involved in metabolism, cell division and the cell’s response to stress. While it may be possible to study up to 100 genes simultaneously when CRISPRing the entire genome, he suspects that, for convenience, researchers would limit themselves to four or five at once.</p>
<p>The work was funded by the National Institutes of Health (DP2 CA195768, R01 GM130996).</p>
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CRISPR gene therapy shows promise against blood diseases – Nature.com https://www.nature.com/articles/d41586-020-03476-x
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</figure><p>In 1949, biochemist Linus Pauling declared<sup><a href=”https://www.nature.com/articles/d41586-020-03476-x#ref-CR1″ data-track=”click” data-action=”anchor-link” data-track-label=”go to reference” data-track-category=”references”>1</a></sup> <a href=”https://science.sciencemag.org/content/110/2865/543″ data-track=”click” data-label=”https://science.sciencemag.org/content/110/2865/543″ data-track-category=”body text link”>sickle-cell anaemia the first “molecular disease”</a> after discovering that the condition is caused by a flaw in the body’s oxygen-carrying protein, haemoglobin. Now, more than 70 years later, cutting-edge genetic techniques could provide a molecular treatment.</p><p>In <i>The New England Journal of Medicine</i><sup><a href=”https://www.nature.com/articles/d41586-020-03476-x#ref-CR2″ data-track=”click” data-action=”anchor-link” data-track-label=”go to reference” data-track-category=”references”>2</a></sup><sup>,</sup><sup><a href=”https://www.nature.com/articles/d41586-020-03476-x#ref-CR3″ data-track=”click” data-action=”anchor-link” data-track-label=”go to reference” data-track-category=”references”>3</a></sup>, separate research teams report promising results from trials of two pioneering gene therapies that target the root cause of sickle-cell anaemia. Both aim to boost the production of an alternative form of haemoglobin, called fetal haemoglobin. One study does so using <a href=”https://www.nature.com/articles/d41586-020-02765-9″ data-track=”click” data-label=”https://www.nature.com/articles/d41586-020-02765-9″ data-track-category=”body text link”>CRISPR–Cas9 genome editing</a>. And, because it is the first published account of using the gene-editing system to treat a heritable disease, it provides an important proof of concept for that technology.</p><p>The other approach shuttles in the code for an RNA that alters expression of the fetal haemoglobin gene. Both treatments relieved participants of the debilitating episodes known as pain crises that come with sickle-cell disease.</p><p>“To have something like these two techniques is a great opportunity,” says Renee Garner, a paediatrician at the Louisiana State University School of Medicine in New Orleans. “It would just open the doors of hope for these patients.”</p><p>Both clinical trials have enrolled only a handful of participants, and it is too soon to say how long the effects will last — the first participant in the RNA study was treated nearly two-and-a-half years ago. The CRISPR–Cas9 approach is also being used to treat people with severe forms of a related genetic disorder called β-thalassaemia, and those participants have not required the blood transfusions usually needed to manage the disease.</p><p>“It is very promising,” says Marina Cavazzana, a gene therapy researcher at Necker Children’s Hospital in Paris. “We need new technologies and more than one product in the market to face the huge problem of sickle cell.”</p><p>Sickle-cell disease and β-thalassaemia are two of the most common genetic disorders attributable to mutations in a single gene. Both conditions affect the production of β-globin, a component of haemoglobin. People with severe β-thalassaemia have anaemia; in sickle-cell anaemia, the blood cells become deformed, clump together and can clog blood vessels, sometimes starving tissues of oxygen and causing pain episodes. Each year, 60,000 people are diagnosed worldwide with a severe form of β-thalassaemia, and 300,000 are diagnosed with sickle-cell disease.</p><p>Both diseases can be cured by a bone-marrow transplant, although most people with the conditions cannot find a suitably matched donor. But in recent years, a variety of experimental gene-therapy approaches have burst onto the scene. Last year, the European Union approved a gene therapy called Zynteglo to treat β-thalassaemia. That approach uses a virus to shuttle a functioning copy of the β-globin gene into blood-producing stem cells. Bluebird Bio, a biotechnology company in Cambridge, Massachusetts, is conducting clinical trials of a similar approach in people with sickle-cell disease as well.</p><p>The CRISPR and RNA approaches take a different tack. They seek to boost expression of a form of haemoglobin that is normally produced in the fetus and then switched off shortly after birth. Researchers had hypothesized that turning this fetal haemoglobin back on could compensate for the disabled β-globin produced by people with sickle cell anaemia or β-thalassaemia.</p><p>Both studies suggest that this is the case. In one, a team that includes researchers from two Massachusetts companies — Vertex Pharmaceuticals in Boston and CRISPR Therapeutics in Cambridge — used CRISPR–Cas9 to alter a region of a gene called <i>BCL11A</i>, which is required to switch off production of fetal haemoglobin. By disabling this gene, the team hoped to turn fetal-haemoglobin production back on in adult red blood cells.</p><p>The other study’s team — led by haematologist David Williams at Boston Children’s Hospital and researchers from Bluebird Bio — used a snippet of RNA that switches off expression of the <i>BCL11A</i> gene in red blood cells.</p><p>The CRISPR–Cas9 publication reports data from two participants, one with β-thalassaemia and one with sickle-cell disease, but the trial has now treated a total of 19 people, says David Altshuler, chief scientific officer at Vertex. Williams’ publication, meanwhile, reports data from six participants with sickle-cell disease, and his trial has since treated three more.</p><p>So far, the participants with β-thalassaemia have not needed blood transfusions, and participants with sickle-cell disease have not reported pain crises since the treatment. Side effects from the therapies — which included infection and abdominal pain — were temporary and linked to the treatments needed to prepare the bone marrow for the procedure.</p><p>In both cases, blood stem cells are removed from the marrow, then modified and reinfused into the patients. But before the cells are reintroduced, the participant is treated with drugs to ablate the remaining blood stem cells. This treatment can be difficulty and risky, and leaves the participant at risk of infection until the marrow recuperates; it can also damage fertility. Researchers are now hunting for gentler ways to <a href=”https://www.nature.com/articles/d41586-019-03601-5″ data-track=”click” data-label=”https://www.nature.com/articles/d41586-019-03601-5″ data-track-category=”body text link”>prepare the bone marrow for such infusions</a>.</p><p>Until the therapies are made safer, such approaches will probably be restricted only to people with severe disease that does not respond to treatment using other drugs, says haematologist David Rees at King’s College Hospital, London. “Scientifically, these studies are quite exciting,” he says. “But it’s hard to see this being a mainstream treatment in the long term.”</p>
<p><strong><a href=”https://blockads.fivefilters.org”></a></strong> <a href=”https://blockads.fivefilters.org/acceptable.html”>(Why?)</a></p> Tue, 08 Dec 2020 08:00:00 +0000 Heidi Ledford
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