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More Progress Toward Gene Editing for Kids with Muscular Dystrophy

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Normal and treated muscles with DMD
Caption: Muscles of untreated mouse model of Duchenne muscular dystrophy (left) compared to muscles of similar mice one year after gene-editing treatment (right). Dystrophin production (green) is restored in treated animals, despite therapy-related immune response to the Cas9 editing enzyme (dark spots in white inset). Credit: Charles Gersbach, Duke University, Durham, NC

Thanks to CRISPR and other gene editing technologies, hopes have never been greater for treating or even curing Duchenne muscular dystrophy (DMD) and many other rare, genetic diseases that once seemed tragically out of reach. The latest encouraging news comes from a study in which a single infusion of a CRISPR editing system produced lasting benefits in a mouse model of DMD.

There currently is no way to cure DMD, an ultimately fatal disease that mainly affects boys. Caused by mutations in a gene that codes for a critical protein called dystrophin, DMD progressively weakens the skeletal and heart muscles. People with DMD are usually in wheelchairs by the age of 10, with most dying before the age of 30.

The exquisite targeting ability of CRISPR/Cas9 editing systems rely on a sequence-specific guide RNA to direct a scissor-like, bacterial enzyme (Cas9) to just the right spot in the genome, where it can be used to cut out, replace, or repair disease-causing mutations. In previous studies in mice and dogs, researchers directly infused CRISPR systems directly into the animals bodies. This “in vivo” approach to gene editing successfully restored production of functional dystrophin proteins, strengthening animals’ muscles within weeks of treatment.

But an important question remained: would CRISPR’s benefits persist over the long term? The answer in a mouse model of DMD appears to be “yes,” according to findings published recently in Nature Medicine by Charles Gersbach, Duke University, Durham, NC, and his colleagues [1]. Specifically, the NIH-funded team found that after mice with DMD received one infusion of a specially designed CRISPR/Cas9 system, the abnormal gene was edited in a way that restored dystrophin production in skeletal and heart muscles for more than a year. What’s more, lasting improvements were seen in the structure of the animals’ muscles throughout the same time period.

As exciting as these results may be, much more research is needed to explore both the safety and the efficacy of in vivo gene editing before it can be tried in humans with DMD. For instance, the researchers found that older mice that received the editing system developed an immune response to the bacterially-derived Cas9 protein. However, this response didn’t prevent the CRISPR/Cas9 system from doing its job or appear to cause any adverse effects. Interestingly, younger animals didn’t show such a response.

It’s worth noting that the immune systems of mice and people often respond quite differently. But the findings do highlight some possible challenges of such treatments, as well as approaches to reduce possible side effects. For instance, the latest findings suggest CRISPR/Cas9 treatment might best be done early in life, before an infant’s immune system is fully developed. Also, if it’s necessary to deliver CRISPR/Cas9 to older individuals, it may be beneficial to suppress the immune system temporarily.

Another concern about CRISPR technology is the potential for damaging, “off-target” edits to other parts of the genome. In the new work, the Duke team found that its CRISPR system made very few “off-target” edits. However, the system did make a surprising number of complex edits to the targeted dystrophin gene, including integration of the viral vector used to deliver Cas9. While those editing “errors” might reduce the efficacy of treatment, researchers said they didn’t appear to affect the health of the mice studied.

It’s important to emphasize that this gene editing research aimed at curing DMD is being done in non-reproductive (somatic) cells, primarily muscle tissue. The NIH does not support the use of gene editing technologies in human embryos or human reproductive (germline) cells, which would change the genetic makeup of future offspring.

As such, the Duke researchers’ CRISPR/Cas9 system is designed to work optimally in a range of muscle and muscle-progenitor cells. Still, they were able to detect editing of the dystrophin-producing gene in the liver, kidney, brain, and other tissues. Importantly, there was no evidence of edits in the germline cells of the mice. The researchers note that their CRISPR system can be reconfigured to limit gene editing to mature muscle cells, although that may reduce the treatment’s efficacy.

It’s truly encouraging to see that CRISPR gene editing may confer lasting benefits in an animal model of DMD, but a great many questions remain before trying this new approach in kids with DMD. But that time is coming—so let’s boldly go forth and get answers to those questions on behalf of all who are affected by this heartbreaking disease.

Reference:

[1] Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nelson CE, Wu Y, Gemberling MP, Oliver ML, Waller MA, Bohning JD, Robinson-Hamm JN, Bulaklak K, Castellanos Rivera RM, Collier JH, Asokan A, Gersbach CA. Nat Med. 2019 Feb 18.

Links:

Muscular Dystrophy Information Page (National Institute of Neurological Disorders and Stroke/NIH)

Gersbach Lab (Duke University, Durham, NC)

Somatic Cell Genome Editing (Common Fund/NIH)

NIH Support: National Institute of Arthritis and Musculoskeletal and Skin Diseases; National Institute of Biomedical Imaging and Bioengineering


Gene Editing in Dogs Boosts Hope for Kids with Muscular Dystrophy

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Dystrophin before and after treatment

Caption: A CRISPR/cas9 gene editing-based treatment restored production of dystrophin proteins (green) in the diaphragm muscles of dogs with Duchenne muscular dystrophy.
Credit: UT Southwestern

CRISPR and other gene editing tools hold great promise for curing a wide range of devastating conditions caused by misspellings in DNA. Among the many looking to gene editing with hope are kids with Duchenne muscular dystrophy (DMD), an uncommon and tragically fatal genetic disease in which their muscles—including skeletal muscles, the heart, and the main muscle used for breathing—gradually become too weak to function. Such hopes were recently buoyed by a new study that showed infusion of the CRISPR/Cas9 gene editing system could halt disease progression in a dog model of DMD.

As seen in the micrographs above, NIH-funded researchers were able to use the CRISPR/Cas9 editing system to restore production of a critical protein, called dystrophin, by up to 92 percent in the muscle tissue of affected dogs. While more study is needed before clinical trials could begin in humans, this is very exciting news, especially when one considers that boosting dystrophin levels by as little as 15 percent may be enough to provide significant benefit for kids with DMD.


Cool Videos: Myotonic Dystrophy

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Myotonic Dystrophy Video screenshot

Today, I’d like to share a video that tells the inspirational story of two young Massachusetts Institute of Technology (MIT) researchers who are taking aim at a genetic disease that has touched both of their lives. Called myotonic dystrophy (DM), the disease is the most common form of muscular dystrophy in adults and causes a wide variety of health problems—including muscle wasting and weakness, irregular heartbeats, and profound fatigue.

If you’d like a few more details before or after watching these scientists’ video, here’s their description of their work:  “Eric Wang started his lab at MIT in 2013 through receiving an NIH Early Independence Award. Learn about the path that led him to study myotonic dystrophy, a disease that affects his family. Eric’s team of researchers includes Ona McConnell, an avid field hockey goalie who is affected by myotonic dystrophy herself. Determined to make a difference, Eric and Ona hope to inspire others in their efforts to better understand and treat this disease.”

Links:


Engineering a Better Way to Deliver Therapeutic Genes to Muscles

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Green adenovirus delivers therapeutic genes to muscles which glow green

Amid all the progress toward ending the COVID-19 pandemic, it’s worth remembering that researchers here and around the world continue to make important advances in tackling many other serious health conditions. As an inspiring NIH-supported example, I’d like to share an advance on the use of gene therapy for treating genetic diseases that progressively degenerate muscle, such as Duchenne muscular dystrophy (DMD).

As published recently in the journal Cell, researchers have developed a promising approach to deliver therapeutic genes and gene editing tools to muscle more efficiently, thus requiring lower doses [1]. In animal studies, the new approach has targeted muscle far more effectively than existing strategies. It offers an exciting way forward to reduce unwanted side effects from off-target delivery, which has hampered the development of gene therapy for many conditions.

In boys born with DMD (it’s an X-linked disease and therefore affects males), skeletal and heart muscles progressively weaken due to mutations in a gene encoding a critical muscle protein called dystrophin. By age 10, most boys require a wheelchair. Sadly, their life expectancy remains less than 30 years.

The hope is gene therapies will one day treat or even cure DMD and allow people with the disease to live longer, high-quality lives. Unfortunately, the benign adeno-associated viruses (AAVs) traditionally used to deliver the healthy intact dystrophin gene into cells mostly end up in the liver—not in muscles. It’s also the case for gene therapy of many other muscle-wasting genetic diseases.

The heavy dose of viral vector to the liver is not without concern. Recently and tragically, there have been deaths in a high-dose AAV gene therapy trial for X-linked myotubular myopathy (XLMTM), a different disorder of skeletal muscle in which there may already be underlying liver disease, potentially increasing susceptibility to toxicity.

To correct this concerning routing error, researchers led by Mohammadsharif Tabebordbar in the lab of Pardis Sabeti, Broad Institute of MIT and Harvard and Harvard University, Cambridge, MA, have now assembled an optimized collection of AAVs. They have been refined to be about 10 times better at reaching muscle fibers than those now used in laboratory studies and clinical trials. In fact, researchers call them myotube AAVs, or MyoAAVs.

MyoAAVs can deliver therapeutic genes to muscle at much lower doses—up to 250 times lower than what’s needed with traditional AAVs. While this approach hasn’t yet been tried in people, animal studies show that MyoAAVs also largely avoid the liver, raising the prospect for more effective gene therapies without the risk of liver damage and other serious side effects.

In the Cell paper, the researchers demonstrate how they generated MyoAAVs, starting out with the commonly used AAV9. Their goal was to modify the outer protein shell, or capsid, to create an AAV that would be much better at specifically targeting muscle. To do so, they turned to their capsid engineering platform known as, appropriately enough, DELIVER. It’s short for Directed Evolution of AAV capsids Leveraging In Vivo Expression of transgene RNA.

Here’s how DELIVER works. The researchers generate millions of different AAV capsids by adding random strings of amino acids to the portion of the AAV9 capsid that binds to cells. They inject those modified AAVs into mice and then sequence the RNA from cells in muscle tissue throughout the body. The researchers want to identify AAVs that not only enter muscle cells but that also successfully deliver therapeutic genes into the nucleus to compensate for the damaged version of the gene.

This search delivered not just one AAV—it produced several related ones, all bearing a unique surface structure that enabled them specifically to target muscle cells. Then, in collaboration with Amy Wagers, Harvard University, Cambridge, MA, the team tested their MyoAAV toolset in animal studies.

The first cargo, however, wasn’t a gene. It was the gene-editing system CRISPR-Cas9. The team found the MyoAAVs correctly delivered the gene-editing system to muscle cells and also repaired dysfunctional copies of the dystrophin gene better than the CRISPR cargo carried by conventional AAVs. Importantly, the muscles of MyoAAV-treated animals also showed greater strength and function.

Next, the researchers teamed up with Alan Beggs, Boston Children’s Hospital, and found that MyoAAV was effective in treating mouse models of XLMTM. This is the very condition mentioned above, in which very high dose gene therapy with a current AAV vector has led to tragic outcomes. XLMTM mice normally die in 10 weeks. But, after receiving MyoAAV carrying a corrective gene, all six mice had a normal lifespan. By comparison, mice treated in the same way with traditional AAV lived only up to 21 weeks of age. What’s more, the researchers used MyoAAV at a dose 100 times lower than that currently used in clinical trials.

While further study is needed before this approach can be tested in people, MyoAAV was also used to successfully introduce therapeutic genes into human cells in the lab. This suggests that the early success in animals might hold up in people. The approach also has promise for developing AAVs with potential for targeting other organs, thereby possibly providing treatment for a wide range of genetic conditions.

The new findings are the result of a decade of work from Tabebordbar, the study’s first author. His tireless work is also personal. His father has a rare genetic muscle disease that has put him in a wheelchair. With this latest advance, the hope is that the next generation of promising gene therapies might soon make its way to the clinic to help Tabebordbar’s father and so many other people.

Reference:

[1] Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Tabebordbar M, Lagerborg KA, Stanton A, King EM, Ye S, Tellez L, Krunnfusz A, Tavakoli S, Widrick JJ, Messemer KA, Troiano EC, Moghadaszadeh B, Peacker BL, Leacock KA, Horwitz N, Beggs AH, Wagers AJ, Sabeti PC. Cell. 2021 Sep 4:S0092-8674(21)01002-3.

Links:

Muscular Dystrophy Information Page (National Institute of Neurological Disorders and Stroke/NIH)

X-linked myotubular myopathy (Genetic and Rare Diseases Information Center/National Center for Advancing Translational Sciences/NIH)

Somatic Cell Genome Editing (Common Fund/NIH)

Mohammadsharif Tabebordbar (Broad Institute of MIT and Harvard and Harvard University, Cambridge, MA)

Sabeti Lab (Broad Institute of MIT and Harvard and Harvard University)

NIH Support: Eunice Kennedy Shriver National Institute of Child Health and Human Development; Common Fund


CRISPR-Based Anti-Viral Therapy Could One Day Foil the Flu—and COVID-19

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Artistic rendering of CRISPR Cas13a as scissors

CRISPR gene-editing technology has tremendous potential for making non-heritable DNA changes that can treat or even cure a wide range of devastating disorders, from HIV to muscular dystrophy Now, a recent animal study shows that another CRISPR system—targeting viral RNA instead of human DNA—could work as an inhaled anti-viral therapeutic that can be preprogrammed to seek out and foil potentially almost any flu strain and many other respiratory viruses, including SARS-CoV-2, the coronavirus that causes COVID-19.

How can that be? Other CRISPR gene-editing systems rely on a sequence-specific guide RNA to direct a scissor-like, bacterial enzyme (Cas9) to just the right spot in the genome to cut out, replace, or repair disease-causing mutations. This new anti-viral CRISPR system also relies on guide RNA. But the guide instead directs a different bacterial enzyme, called Cas13a, to the right spot in the viral genome to bind and cleave viral RNA and stop viruses from replicating in lung cells.

The findings, recently published in the journal Nature Biotechnology [1], come from the lab of Philip Santangelo, Georgia Institute of Technology and Emory University, Atlanta. Earlier studies by other groups had shown the potential of Cas13 for degrading the RNA of influenza viruses in a lab dish [2,3]. In this latest work, Santangelo and colleagues turned to mice and hamsters to see whether this enzyme could actually work in the lung tissue of a living animal.

What’s interesting is how Santangelo’s team did it. Rather than delivering the Cas13a protein itself to the lungs, the CRISPR system works by supplying a messenger RNA (mRNA) with the instructions to make the anti-viral Cas13a protein. This is the same idea as the Pfizer and Moderna mRNA-based COVID-19 vaccines, which temporarily direct your muscle cells to produce viral spike proteins that launch an immune response. In this case, the lung cells translate the Cas13a mRNA to produce the protein. Directed by the guide RNA that was also delivered to the same cells, Cas13a degrades the viral RNA and stops the infection. Because mRNA doesn’t enter the cell’s nucleus, it doesn’t interact with DNA and raise potential concerns about causing unwanted genetic changes.

The researchers designed guide RNAs that were specific to a shared, highly conserved portion of influenza viruses involved in replicating their genome and infecting other cells. They also designed another set directed to key portions of SARS-CoV-2.

Next, they delivered the Cas13a mRNA and guides straight to the lungs of animals using an adapted nebulizer, just like those used to deliver medicines to the lungs of people. In mice with influenza, Cas13a degraded influenza RNA in the lungs and the animals recovered without any apparent side effects. In SARS-CoV-2-infected hamsters, the same approach limited the virus’s ability to replicate in cells as the animals COVID-19-like symptoms improved.

The findings are the first to show that mRNA can be used to express the Cas13a protein in living lung tissue, not just in cells in a dish. It’s also the first to show that the bacterial Cas13a protein is effective at slowing or stopping replication of SARS-CoV-2. The latter raises hope that this CRISPR system could be quickly adapted to fight any future novel coronaviruses that develop the ability to infect humans.

The researchers report that this approach has potential to work against the vast majority—99 percent—of the flu strains that have circulated around the world over the last century. It also should be equally effective against the new and more contagious variants of SARS-CoV-2 now circulating around the globe. While more study is needed to understand the safety of such an anti-viral approach before trying it in humans, what’s clear is basic research advances like this one hold great potential for helping us to fight life-threatening respiratory viruses of the past, present, and future.

References:

[1] Treatment of influenza and SARS-CoV-2 infections via mRNA-encoded Cas13a in rodents. Blanchard EL, Vanover D, Bawage SS, Tiwari PM, Rotolo L, Beyersdorf J, Peck HE, Bruno NC, Hincapie R, Michel F, Murray J, Sadhwani H, Vanderheyden B, Finn MG, Brinton MA, Lafontaine ER, Hogan RJ, Zurla C, Santangelo PJ. Nat Biotechnol. 2021 Feb 3. [Published online ahead of print.]

[2] Programmable inhibition and detection of RNA viruses using Cas13. Freije CA, Myhrvold C, Boehm CK, Lin AE, Welch NL, Carter A, Metsky HC, Luo CY, Abudayyeh OO, Gootenberg JS, Yozwiak NL, Zhang F, Sabeti PC. Mol Cell. 2019 Dec 5;76(5):826-837.e11.

[3] Development of CRISPR as an antiviral strategy to combat SARS-CoV-2 and influenza. Abbott TR, Dhamdhere G, Liu Y, Lin X, Goudy L, Zeng L, Chemparathy A, Chmura S, Heaton NS, Debs R, Pande T, Endy D, La Russa MF, Lewis DB, Qi LS. Cell. 2020 May 14;181(4):865-876.e12.

Links:

COVID-19 Research (NIH)

Influenza (National Institute of Allergy and Infectious Diseases/NIH)

Santangelo Lab (Georgia Institute of Technology, Atlanta)


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