Posts Tagged ‘health’

Liam Shore is a third-year researcher at the University of Liverpool, in the Department of Philosophy. His research interests fall within the domain of ethics, notably on the ethics of digital and biotechnologies.

The Making of a Philosopher

Im a philosopher, but I havent always been one, so how does someone become a philosopher? And more fundamentally, why would anyone want to become one?

As a rare vocation, youd be forgiven for supposing that philosophers are an extinct species who once roamed the Athenian plazas during early antiquity, gesticulating poignantly and wearing togas. Well, happily they do exist today, sans the togas, largely unnoticed, behind the scenes on ethics boards, or engaging in fundamental first-principles critiques of.well.everything.

A question arises: if philosophers critique everything, how do they develop knowledge to criticise specialist areas? This becomes particularly poignant in an applied ethics context. My own personal journey, from Technologist to Philosopher, shows that one practically needs to be educated in two disciplines to become a bona fide philosopher.

When deciding what subjects to study, and what career to pursue, I was torn between multiple strong interests. In third place, Technology; in second place, Medicine; and in first place, Philosophy. In my case, I took the reverse path toward becoming a philosopher. Namely, I studied technology, worked in the biological sciences industry, and returned to academia with domain-specific expertise to enter into the philosophy sub-field of ethics. The beauty of philosophy for me, and the reason why I personally had the desire to pursue becoming a philosopher, is that philosophy, being able to critique everything, can powerfully converge disparate interests. It is this quality that made philosophy my first love, and so my PhD journey began, delving into the ethics of radical life extension.

Understanding Rejuvenation Biotechnologies

Recently, breakthroughs in rejuvenation biotechnologies, particularly those of the Strategies for Engineered Negligible Senescence (SENS) variant, have garnered little attention, and yet constitute steps towards a paradigm-altering event. SENS therapies, like maintaining classic cars to prolong their lifespan, seeks to do the same for our bodies as we age. SENS suggests that ageing is caused by the accumulation of cellular and molecular damage throughout the body over time, and advocates posit that by repairing or reversing this damage, it is possible to rejuvenate tissues and organs, thereby extending a persons healthy lifespan. Ultimately, by seeking to tackle age-related diseases at their root, via interventions such as stem cell therapies, the aim is to bring age-related diseases fully under comprehensive medical control. Overall, the eventual aim of SENS is to combine a panel of these therapies to combat all preceding causes of age-related diseases, and consequently, tackle ageing itself!

Although this sounds futuristic, there are therapies in various stages of development, with the furthest along being in clinical trials. Advocates claim that these therapies could, in due course, function well enough to rejuvenate a persons body to a youthful state. In effect, this is a process that, amongst other things, removes damage and replaces cells, enabling the body to regain a healthy condition. The outcome of extending good health is that it prolongs life, as it postpones the onset of age-related diseases until higher chronological ages. Accordingly, if someone repeatedly receives these therapies throughout life, this could constitute a potentially radical life-extending situation, as periods of poor health may be postponed repeatedly, allowing one to maintain optimal physiological functioning for longer, thereby delaying death itself!

A Case for Philosophical Inquiry The SENS approach to rejuvenation biotechnologies represents a bold vision for extending healthy lifespans and combating age-related diseases. However, realising this vision requires careful consideration of the ethical implications of extending human lifespan, making the SENS approach a question for philosophical research.

The most common ethical concerns for life-extending technologies are Health Equity i.e. fairness in health opportunities for all; Longevity/Population Dynamics i.e. understanding how long people live & how populations change; Environmental Impacts i.e. the effects of human activities on nature and Informed Consent/Autonomy i.e. respecting peoples right to make their own decisions.

Nevertheless, although important, these concerns dont engage with how this technology impacts what we find meaningful at the profoundest level as human beings. However, my research incorporates all the aforementioned ethical concerns and delves deeper into the realms of identity, purpose, and meaning in life, primarily through an existentialist lens.

Existentialism, as a philosophical theory, concerns itself with questions of: the nature of individual existence, authenticity of self, human freedom, and the search for purpose/meaning in life. It is via this prism that Im currently defining a taxonomy of values supported by radical life extension advocates, with this taxonomy categorising virtues like fairness, compassion, and autonomy, providing a structured framework for ethical analysis. In addition, Im exploring how a SENS-induced radically extended life may impact what we value. And next, I plan to explore whether the consequences of SENS therapies could result in mental ageing, in essence a feeling of listlessness, a sense of ennui, or a notion of world-weariness.

Overall, I hope that my research will deliver original insights to help us work towards a future where radically extended healthspans are possible, while fully prioritising and ensuring human well-being.

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Becoming an Expert: Exploring the Ethics of Radical Life Extension - News - University of Liverpool - News

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Genetically engineered B cells could produce super-antibodies to HIV

Researchers at Washington University School of Medicine in St. Louis have received a $6.2 million grant from the National Institutes of Health (NIH) to develop a gene therapy that would modify the immune systems B cells to spur them to produce broadly neutralizing antibodies against HIV. In theory, such an approach could control or eliminate the infection without need for ongoing antiretroviral therapy. Shown is the engineered adenovirus designed to deliver HIV superantibody genes into B cells.

HIV infections can be controlled with medication, but such therapy must continue throughout patients lives because no strategy exists to eliminate the virus from the body or control the infection without ongoing treatment.

With the aim of developing such a strategy, researchers at Washington University School of Medicine in St. Louis have received a $6.2 million grant from the National Institutes of Health (NIH) to develop a gene therapy that would modify the immune systems B cells to spur them to produce broadly neutralizing antibodies against HIV. In theory, such an approach could control or eliminate the infection without need for ongoing antiretroviral therapy.

Permanent ways to control or eliminate HIV infection remain elusive, and their development is a major goal of the field, said David T. Curiel, MD, PhD, the Distinguished Professor of Radiation Oncology. The idea of modifying B cells which naturally produce antibodies to ensure that they manufacture specific antibodies that are broadly effective at targeting HIV is an exciting strategy. We have brought together a great team with expertise in HIV, gene therapy, and animal models of infection to work toward this goal.

Curiels co-principal investigators are Michael R. Farzan, PhD, of Harvard Medical School and Boston Childrens Hospital, and Mauricio de Aguiar Martins, PhD, of the University of Florida.

Over the decades since HIV appeared, researchers have learned that about 1% of people with the virus are able to produce what might be considered superantibodies against the virus. Such individuals known as elite neutralizers can produce antibodies against multiple strains of HIV.

Some people naturally have antibodies that can bind and destroy or deactivate very diverse strains of HIV, and we now have the ability to build those types of antibodies in the lab, said Paul Boucher, a doctoral student in Curiels lab. But just giving other patients these superantibodies is not an ideal solution, because these proteins would stay in the body only temporarily. Instead, our approach is to genetically modify the cells responsible for making antibodies the immune systems B cells so they can always produce superantibodies against HIV whenever they may need to.

Such engineered B cells could create, in theory, a state of permanent vaccination against the virus. Even if such a gene therapy doesnt fully clear HIV from the body, the strategy could allow the amount of virus in the body to be controlled, keeping it at a minimal level and creating a functional cure, according to the researchers.

The strategy involves modifying a different type of virus, called adenovirus. When used in gene therapy, such viruses are genetically disabled so they cant cause disease. The researchers then could engineer the adenovirus to carry the gene responsible for manufacturing broadly neutralizing antibodies to HIV. In the same viral vector, they also could include genes responsible for manufacturing the CRISPR/Cas9 gene editing proteins. In this way, the gene therapy delivery vehicle would carry into the body both the antibody gene that will be edited into the B cell genome and the genes to build the molecular tools to carry out that editing.

Using a three-part targeting strategy, the researchers would design the adenovirus to deliver its genetic payload only to B cells, avoiding other cell types. They have developed ways to modify the virus so that it is targeted directly to a protein that is expressed on the surface of B cells and no other cell types. The researchers can further restrict the targeting by using genetic methods to ensure that the CRISPR/Cas9 proteins can only be manufactured when their genes are delivered into B cells. Finally, they have developed strategies to modify the adenovirus in a way that stops its natural tendency to accumulate in the liver.

This strategy to modify B cells is distinct from another adenoviral gene therapy approach to HIV treatment that is currently in clinical trials led by principal investigator Rachel M. Presti, MD, PhD, a professor of medicine in the Division of Infectious Diseases at Washington University School of Medicine. HIV is difficult to eliminate from the body because the virus integrates its genome into the DNA of the infected individuals T cells. The strategy currently in clinical trials is focused on using precise targeting of the CRISPR/Cas9 gene editing proteins to excise the virus from the genomes of all of a patients infected T cells. This strategy is being tested in a first-in-human, phase 1 clinical trial to determine its safety and preliminary efficacy at various doses.

Curiel said engineered B cells are ripe for developing new therapies to treat a wide variety of diseases. In November, a genetically engineered B cell therapy was administered to a patient for the first time at the University of Minnesota Medical Center. In that case, the therapy was designed to treat mucopolysaccharidosis type 1, a life-threatening condition in which the body lacks an enzyme necessary to break down large sugar molecules inside cells.

Gene therapy with engineered B cells is an exciting new area of research, Curiel said. We look forward to combining our expertise in adenovirus gene therapy, HIV infection and preclinical models of disease to realize our plan for developing an HIV therapy that we hope can permanently control the infection.

This work is supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH), grant number 1R01-AI174270-01A1. This content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

About Washington University School of Medicine

WashU Medicine is a global leader in academic medicine, including biomedical research, patient care and educational programs with 2,900 faculty. Its National Institutes of Health (NIH) research funding portfolio is the second largest among U.S. medical schools and has grown 56% in the last seven years. Together with institutional investment, WashU Medicine commits well over $1 billion annually to basic and clinical research innovation and training. Its faculty practice is consistently within the top five in the country, with more than 1,900 faculty physicians practicing at 130 locations and who are also the medical staffs of Barnes-Jewish and St. Louis Childrens hospitals of BJC HealthCare. WashU Medicine has a storied history in MD/PhD training, recently dedicated $100 million to scholarships and curriculum renewal for its medical students, and is home to top-notch training programs in every medical subspecialty as well as physical therapy, occupational therapy, and audiology and communications sciences.

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$6.2 million to help develop gene therapy for HIV Washington University School of Medicine in St. Louis - Washington University School of Medicine in...

Freezing your body after death with the hope of coming back to life one day sounds like something out of a science fiction movie.

Southern Cryonics, the first body-freezing facility in the Southern Hemisphere, tries to turn this idea into reality.

Were sort of in a race against time, says Southern Cryonics director, Peter Tsolakides, to Neos Kosmos.

Cryonics, coming from the Greek word kros for icy cold, involves the preservation of legally declared dead bodies at extremely low temperatures for potential future revival.

The facility in Holbrook, New South Wales, uses this practice, with the expectation that one day, advancements in medical technology and science will restore patients to health and in the young body.

But the timeframe of this future remains uncertain.

Tsolakides says once you preserve a body, you can keep it (stored) for thousands of years, but the chance of coming back depends on when you freeze it.

A matter of life after death

He says, currently 50 people, are willing to take the risk for a chance at life after death, and the number is growing.

This group consists of 35 investors each contributing $50,000 to $70,000, and 15 subscribers or customers who have paid $150,000 through life insurance.

Tsolakides says there are no guarantees, despite how great the dream of being brought back from the dead might be for some people.

Most of them know something about it (cryonics), but they also look at it and say, look, theres no guarantees, but theres a chance.

And that chance versus being buried in the ground or cremated is a much higher chance coming back.

He estimates the chance of a well-preserved body being revived in 200 years to be around 20%.

He says although its hard to predict what the world will be like in be like in 1,000 years, bodies might be revived when technological advancements have found the key to immortality.

In the real world, nobody will be dying, and most diseases will be cured. So, we will know how to prevent death in a sense, and the next step is to bring back those who have already died, but in a good condition.

Tsolakides says that while they dont know how to bring a person back to life, current developments give you inklings, of what the future is going to be like.

He says progress has to start somewhere, and right now billions are invested in medical research aimed at disease cures.

This includes groups working on brain revival, organ regeneration, cloning, and advancements in artificial intelligence and nanotechnology.

How does cryonics work?

When a person is declared legally dead at the hospital, a cooling process begins.

Chemicals are used to stabilise the body, lowering it to about ice temperature.

Once taken to the funeral home, the body is further cooled and infused with an antifreeze substance until it reaches about -80C.

Next, it goes to the cryonics facility, gradually cooled to -180C and preserved below that temperature, in a large vacuum flask container filled with liquid nitrogen.

Southern Cryonics Greek-Australian director says, theres a brief window of a few hours after legal death, where no deterioration occurs to the body.

Once preserved in liquid nitrogen, it can be stored for thousands of years due to almost no chemical or biological activity at that temperature.

Its a race against time to keep the temperature going down, he says.

But is it possible to freeze a human brain to revive it later?

If you catch them (bodies) under our optimal time, very little damage is occurring to the brain, but that doesnt mean that 200 years from now, that damage cant be repaired, Tsolakides says.

The facility can currently hold up to 40 patients, with each container fitting 4 bodies, but can expand to hold up to six or seven hundred patients if necessary.

The birth and evolution of Southern Cryonics

Tsolakides got interested in cryonics from a young age.

When he came back to Australia around 2012 after working overseas, he saw there were only cryonics facilities in the US and Russia.

He connected with like-minded people and talked about building one in Australia.

We started getting what we call founding members, says Tsolakides, each contributing $50,000 to kickstart the project, eventually totalling 35 members of a non-profit organisation.

We started the facility and that was how it sort of developed.

He says, they chose Holbrook, a small town with about 1,500 people, for a few reasons.

Land there wasnt expensive, and it was halfway between Melbourne and Sydney, making it accessible to over half of Australias population.

Holbrooks nearby Albury airport is crucial for quick patient transportation, and the support from the local council made the decision easier.

Another advantage is its proximity to liquid nitrogen suppliers along the Hume Highway, crucial for the facility.

Holbrooks low history of natural disasters made it a safe choice after a thorough analysis of several years.

The legalities

Tsolakides says Southern Cryonics got all the official approvals from the NSW Department of Health and the local council, to operate as a cemetery but uses a recognised funeral home for mortuary work.

The government groups that we work with helped us a lot. It wasnt like we got resistance or anything like that.

A good idea but not for everyone

Tsolakides was born in Israel to Greek parents.

His mother was from the Greek island of Syros, and his father from Athens.

They briefly lived in Greece before moving to Australia in 1955 when Tsolakides was five years old.

He has a degree in Chemistry and later pursued one in Business Administration.

Throughout his career, he worked primarily in marketing for an oil company.

He grew up and lived in Melbourne for many years before moving to Sydney, a place he now calls home.

His passion in cryonics sparked at about18 after reading Robert Ettingers book, The Prospect of Immortality.

At that age, he didnt worry much about death.

He assumed this will be everywhere, by the time he got old, but soon realised that very few people worldwide were interested in it.

He says that while some are intrigued by cryonics, most view it as a good idea but not for themselves.

Even the US organisation have about five to six thousand members only, with 400 or 500 people suspended, and theyve been going for 50 years.

But that didnt stop him for pursuing his curiosity around cryonics.

Keeping an eye on scientific developments

Tsolakides is determined to improve their techniques and increase success chances, despite challenges or doubts about cryonics.

He says Southern Cryonics along with overseas organisations is monitoring the best way to store a body, leaving the revival work to other scientists.

Its (suspending the body) physically possible to do it now, he says, but of course, you can always improve the processes.

While cryonics remains a controversial field and the chances of revival seem low now, it is yet to be seen whether future technology will ever be able to bring the dead back to life.

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Is it possible to come back from the dead? Australia's first body-freezing facility explores the boundaries of mortality - Neos Kosmos

Researchers have shown that dangerous cysts, which form over time in polycystic kidney disease (PKD), can be prevented by a single normal copy of a defective gene. This means the potential exists that scientists could one day tailor a gene therapy to treat the disease. They also discovered that a type of drug, known as a glycoside, can sidestep the effects of the defective gene in PKD. The discoveries could set the stage for new therapeutic approaches to treating PKD, which affects millions worldwide. The study, partially funded by the National Institutes of Health (NIH), is published in Cell Stem Cell.

Scientists used gene editing and 3-D human cell models known as organoids to study the genetics of PKD, which is a life-threatening, inherited kidney disorder in which a gene defect causes microscopic tubes in the kidneys to expand like water balloons, forming cysts over decades. The cysts can crowd out healthy tissue, leading to kidney function problems and kidney failure. Most people with PKD are born with one healthy gene copy and one defective gene copy in their cells.

Human PKD has been so difficult to study because cysts take years and decades to form. This new platform finally gives us a model to study the genetics of the disease and hopefully start to provide answers to the millions affected by this disease."

Benjamin Freedman, Ph.D., senior study authorat the University of Washington, Seattle

To better understand the genetic reasons cysts form in PKD, Freedman and his colleagues sought to determine if 3-D human mini-kidney organoids with one normal gene copy and one defective copy would form cysts. They grew organoids, which can mimic features of an organ's structure and function, from induced pluripotent stem cells, which can become any kind of cell in the body.

To generate organoids containing clinically relevant mutations, the researchers used a gene editing technique called base editing to create mutations in certain locations on the PKD1 and PKD2 genes in human stem cells. They focused on four types of mutations in these genes that are known to cause PKD by disrupting the production of polycystin protein. Disruptions in two types of the protein polycystin-1 and polycystin-2 are associated with the most severe forms of PKD.

They then compared cells with two gene copy mutations in organoids to cells with only one gene copy mutation. In some cases, they also used gene editing to correct mutations in one of the two gene copies to see how this affected cyst formation. They found organoids with two defective gene copies always produced cysts and those that carried one good gene copy and one bad copy did not form cysts.

"We didn't know if having a gene mutation in only one gene copy is enough to cause PKD, or if a second factor, such as another mutation or acute kidney injury was necessary," Freedman said. "It's unclear what such a trigger would look like, and until now, we haven't had a good experimental model for human PKD."

According to Freedman, the cells with one healthy gene copy make only half the normal amount of polycystin-1 or polycystin-2, but that was sufficient to prevent cysts from developing. He added that the results suggest the need for a second trigger and that preventing that second hit might be able to prevent the disease.

The organoid models also provided the first opportunity to study the effectiveness of a class of drugs known as eukaryotic ribosomal selective glycoside on PKD cyst formation.

"These compounds will only work on single base pair mutations, which are commonly seen in PKD patients," explained Freedman. "They wouldn't be expected to work on any mouse models and didn't work in our previous organoid models of PKD. We needed to create that type of mutation in an experimental model to test the drugs."

Freedman's team found that the drugs could restore the ability of genes to make polycystin, increasing the levels of polycystin-1 to 50% and preventing cysts from forming. Even after cysts had formed, adding the drugs slowed their growth.

Freedman suggested that a next step would be to test existing glycoside drugs in patients. Researchers also could explore the use of gene therapy as a treatment for PKD.

The research was supported by NIH's Nation Center for Advancing Translational Sciences, National Institute of Diabetes and Digestive and Kidney Diseases, and National Institute of General Medical Sciences through awards R01DK117914, UH3TR002158, UH3TR003288, U01DK127553, U01AI176460, U2CTR004867, UC2DK126006, P30DK089507, R21DK128638, and R35GM142902; an Eloxx Pharmaceuticals Award; the Lara Nowak-Macklin Research Fund; and a Washington Research Foundation fellowship.

Source:

Journal reference:

Vishy, C. E.,et al.(2024) Genetics of cystogenesis in base-edited human organoids reveal therapeutic strategies for polycystic kidney disease. Cell Stem Cell. doi.org/10.1016/j.stem.2024.03.005.

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Gene therapy and glycoside drugs offer new hope for polycystic kidney disease treatment - News-Medical.Net

Menopause is a natural part of aging that marks the end of the female reproductive years but many people dont know what to expect until theyre in the midst of it. Did you know, for example, that you could experience symptoms up to a decade before menopause actually begins?

Menopause specialistPelin Batur, MD, walks us through the stages of menopause and what you may be able to expect during each one.

The menopause process is all about hormones. Your body begins to produce less of the hormone calledestrogen, which regulates your menstrual cycle, and your ovaries start running low on eggs. But it doesnt happen all at once.

Heres a quick overview of the three stages of menopause:

Dr. Batur explains each stage in greater detail, including the symptoms you might experience andhow to find relief.

You can think about perimenopause as the runway to the big event. It can start as early as a decade before menopause, though the average amount of time spent in perimenopause is four years.

During this time, your body is, little by little, winding down its naturalovulation process. The most common sign of perimenopause isirregular periods and menstrual cycles.

As your estrogen levels start to decrease, your periods and menstrual cycles may start getting a little wonky sometimes, closer together, sometimes skipping cycles, Dr. Batur explains. You may also have some of the typical menopausal symptoms.

Not everyone experiences noticeable symptoms during perimenopause, but they can include:

There are two stages to perimenopause early menopause transition and late menopause transition though theyre not always cut-and-dry and distinguishable from one another.

This first stage of perimenopause is the very beginning when your body is just starting to experience hormonal changes. During this time, your periods and menstrual cycles are still coming regularly, but you may notice other symptoms:

This is a natural phase of life, so if your symptoms are mild, you may be able to make do with lifestyle changes like getting more sleep and upping your cardio, Dr. Batur says. But if theyre really bothersome, speak to your healthcare provider, even if youre still having regular menstrual cycles.

The late menopause transition is when youre gettinga little closer to menopause. Youre more likely to start experiencing irregular periods and menstrual cycles.

During perimenopause, youre not ovulating as regularly, Dr. Batur says. You have up-and-down levels of estrogen, and you may not make progesterone as consistently, so you may skip a menstrual cycle and then have heavy bleeding during the next period because your uterine lining has thickened up from the impact of the estrogen.

Eventually, as you get closer and closer to menopause, you start skipping periods for months at a time, she continues.

If this happens, bring it up with your healthcare provider especially if youre in your early 40s or younger, which can be a sign ofpremature menopauseor a condition calledprimary ovarian insufficiency.

When youve gone a full 12 months without having your period, youve entered menopause (assuming you havent stopped bleeding because of another medical condition or a medication).

That typically happens around age 52, Dr. Batur shares, and then, you live the rest of your life in menopause, where youre no longer ovulating and you no longer have the ability to bear children.

Menopause symptoms typically last for seven to 10 years (though your timeframe may vary), and they can range from mild to severe. If youre in the latter camp, experiencing bothersome symptoms that you just cant shake, dont feel like you have to soldier on in silence.

Just saying, grin and bear it and eat healthier and lose some weight doesnt cut it for people who are really suffering during this time in their lives, Dr. Batur states. Your healthcare provider will also want to make sure that your symptoms arent related to other medical conditions.

Once you enter menopause, youre in menopause for the rest of your life; this is also called the postmenopause stage.

But now, youre at a higher risk for other health concerns. A decrease in estrogen is a risk factor in conditions like:

The older you get, the more tuned in your healthcare provider should be to menopauses impact on your health. But if theyre not bringing it up, you definitely should even if youre feeling fine, but especially if youre not.

The stages of menopause shouldnt make you feel miserable. If your symptoms are especially bothersome and having an impact on your quality of life, its time to ask for help.

Tell your Ob/Gyn or your primary care doctor, Hey listen, I think my hormones are going haywire, Dr. Batur advises. They can talk you through the options, which may include any of the following (or a combination of them):

Just remember: Theres no quick fix for the symptoms of menopause. If you raise concerns about themduring an annual visit, your healthcare provider may ask you to come back for another appointment so the two of you can go more in-depth about what youre experiencing and thats OK.

This is a very individual thing, and it can be very complicated, especially depending on your medical history, Dr. Batur says. Schedule another appointment, if you need it, and make sure your concerns are being addressed during dedicated time with your provider.

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What To Expect in Each Stage of Menopause - Health Essentials

The GenderGP Appraisal Pathway

This is a pathway designed by GenderGP which allows you to be in charge of your gender journey.

We believe that you are the expert in your gender, and by offering our expertise in healthcare, we can make sure that your medical transition is suited to what you need and is delivered in the safest way possible.

By going through this process you will be able to provide us with information about you and your health and your gender feelings. This will allow us to come to a joint agreement on the best treatment for you.

If you are in the UK or the EU, we can use our private prescription service. If you are outside the EU then we will carry out any necessary assessments and then issue you with a Treatment Summary for your provider so they can do blood testing and prescribe under our direct supervision.

We will make all the decisions on medication and blood results to keep you safe.

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How much does it cost to use our services - GenderGP

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SAN DIEGO, April 3, 2024 /PRNewswire/ -- Leading commercial organizations and patient advocacy groups in the field of cancer genetics today announced the founding of the Inter-Organization Cancer Genetics Clinical Evidence Coalition (INTERACT), a coalition whose mission is to increase evidence-based access to genetic testing for people with or at risk of hereditary cancers.

Founding laboratory members include organizer Ambry Genetics, a subsidiary of REALM IDx, Illumina, Myriad Genetics, and Quest Diagnostics. Volpara Health has also recently joined the coalition. Founding patient advocacy organization members include AliveAndKickn and FORCE. The coalition seeks to provide a collective voice in support of the progression of medical professional and industry guidelines for genetic testing for inherited mutations that increase cancer risk.

With growing insight into the role of genetic testing in cancer risk management and treatment, the population of individuals who benefit from knowing their genetic mutation status continues to increase. As leaders in the genetic testing and hereditary cancer field, the founding members believe it is their responsibility to help drive awareness and inform changes that will equalize access for those whose outcomes could benefit most from testing.

One of the primary objectives of INTERACT is to ensure policy and guidelines keep pace with the growing body of evidence surrounding inherited cancer risk.

Hereditary cancer genetic testing has been shown to improve outcomes by identifying those most at risk and informing management strategies. For instance, patients who test positive for a BRCA1 or BRCA2 mutation have up to 87% lifetime risk for breast cancer, and up to 40% lifetime risk for ovarian cancer.1,2 In addition, there are numerous other genes that increase risk for various forms of cancer. Armed with this information, patients and physicians can improve management through increased surveillance, chemoprevention, targeted therapeutics or risk-reducing surgical measures. As an example, studies have shown that prophylactic mastectomy in BRCA1/2 mutation carriers results in up to a 97% reduction in the risk for contralateral breast cancer, while salpingo-oophorectomy reduced ovarian cancer incidence by 69-100%.1,2

Despite the benefits of a patient and their provider knowing mutation status, disparities in access and uptake of cancer genetics services are well documented.3 INTERACT intends to improve access to genetic testing, with the goal of reaching vulnerable populations who may not currently be aware of their risk or their need for increased screening or other interventions.

"With Lynch syndrome, one of the most common hereditary cancer syndromes, patients have up to 80% lifetime risk for colorectal cancer4, but an estimated 95% of at-risk individuals have not been identified5," said Robin Dubin, Executive Director of AliveAndKickn. "To really improve survival rates with informed screening strategies, we need to help drive education and policies that support genetic testing for all those at risk."

Among the challenges to broadening access to genetic testing for hereditary cancer risk is a time lag in updating guidelines and medical policies after the publication of new medical literature. INTERACT will work to bring these differences to the attention of guideline committees and medical professional societies in an effort to bridge the gaps and reduce disparities in access to appropriate testing nationwide.

About INTERACT The mission of INTERACT is to bring together specialized genetic testing laboratories and patient advocacy groups to support the progression and evolution of medical policy and industry guidelines for cancer genetic testing. Our members are recognized institutions in the field of cancer genetics. Current commercial members include Ambry Genetics, a subsidiary of REALM IDx, Illumina, Myriad Genetics, Quest Diagnostics, and Volpara Health. Advocacy members include AliveAndKickn and FORCE: Facing Our Risk of Cancer Empowered. We seek to develop the evidence base and rationale to inform changes in cancer-related genetic testing policies to expand patient access to evidence-based testing.

For more information, visit: https://interactcoalition.org/

References:

Contact: [emailprotected]

SOURCE INTERACT Coalition

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INTERACT COALITION FORMED TO ADVANCE PATIENT ACCESS TO GENETIC TESTING FOR HEREDITARY ... - PR Newswire

Patients in England and Wales who have recently had an ischaemic stroke or transient ischaemic attack could be offered genetic testing to help inform their treatment, following backing from the National Institute for Health and Care Excellence (NICE).

The agency has launched a second consultation on recommendations that clinicians should offer CYP2C19 genotype testing when considering treatment with clopidogrel, an anti-platelet therapy currently recommended as a treatment option for patients at risk of a secondary stroke.

Approximately 35,850 people in England, Wales and Northern Ireland have a non-minor stroke every year.

An estimated 32% of people in the UK have at least one of the highlighted CYP2C19 gene variants, and evidence has suggested that those with these variants have an increased risk of another stroke when taking clopidogrel.

If the genotype test discovers that patients have one of the CYP2C19 gene variants, alternative stroke-prevention treatments would be offered.

Professor Jonathan Benger, chief medical officer at NICE, said: Recommending a genetic test that can offer personalised care to thousands of people who have a stroke each year will be a step forward in ensuring people receive the best possible treatment.

People who are currently taking clopidogrel will not receive retrospective testing and should continue with the treatment until they and their NHS clinician consider it appropriate to stop, NICE outlined.

It added that laboratory-based CYP2C19 genotype testing is its preferred option, followed by the Genedrive CYP2C19 ID Kit point-of-care test and, if neither of the first two options are available, the Genomadix Cube point-of-care test would be used.

The agencys committee has suggested that a phased rollout could be implemented when introducing laboratory-based testing, with testing set to initially be offered to people with a higher risk of stroke recurrence.

Juliet Bouverie, from the Stroke Association, said: Stroke devastates lives and leaves people with life-long disability.

We know that many stroke survivors spend the rest of their lives fearing another stroke, so its great to see that more people could be given appropriate help to significantly cut their risk of recurrent stroke.

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NICE backs post-stroke genetic testing to identify most suitable treatment options - PMLiVE

Sign up for CNNs Stress, But Less newsletter.Our six-part mindfulness guide will inform and inspire you to reduce stress while learning how to harness it.

There is no doubt that stress is a part of everyday life, but too much can have detrimental impacts on peoples physical and mental health.

I wanted to delve more into depth about the health impacts of stress during National Stress Awareness Month. What does stress do to the body? When does it become a problem, and what are some ways to cope with it? And what can people do with stressors such as a hard job or caregiving responsibilities that cant just go away?

To help us answer these questions, I had a conversation with CNN wellness expert Dr. Leana Wen. Wen is an emergency physician and adjunct associate professor at George Washington University. She previously served as Baltimores health commissioner.

CNN: What does stress do to a persons body?

Dr. Leana Wen: When people experience a perceived threat, a variety of hormones are released that make the heart beat faster and increase blood pressure and blood sugar. These hormones also divert energy away from other parts of the body, such as the immune system and digestive system. These are evolutionary adaptations that once helped people to respond to situations such as predators chasing after them. Such fight or flight responses are normal and may be helpful in modern-day life. For instance, they could help an athlete with a faster performance or a student with staying up to study for an exam.

The problem arises when the bodys stress response is continuous. A perpetual state of fight or flight could lead to many chronic problems. Individuals could experience anxiety and depression, and other mental health ailments. They could also have headaches, muscle tension, abdominal pain, sleep disturbances, decreased immunity to infections, and problems with memory and concentration. Chronic stress has also been linked to increased likelihoods of high blood pressure, diabetes, heart attack and stroke.

Story continues

CNN: Everyone experiences stress, so when does it become a problem?

Wen: Its natural for people to experience stress to discrete stressful events (those that have a clear onset such as the birth of a child, starting of a new job, a divorce or the death of a loved one) that happen in their lives. The problem is when stress becomes a chronic state of being.

Warning signs to look out for include signs or symptoms of mental health concerns or physical manifestations of stressfor instance, if someone starts having new heart palpitations, abdominal pain or headaches. In addition, some people may attempt to cope with stress by using alcohol or drugs. A change in substance use could be a red flag to look for underlying stressors.

People should also ask themselves if stress is negatively affecting their function at home, at work and with their friends. Someone who finds themselves unusually irritable and is lashing out at loved ones and colleagues may also be doing so because of excessive stress.

CNN: Why should we be aware of excessive stress and try to reduce it as a health priority?

Wen: We can think of stress as something in our lives that is modifiable, just like high blood pressure or high blood sugar. The stressor itself may not be able to be changed, just as we cannot change our genetic predisposition to hypertension or diabetes. However, our reaction to it is within our control. And its our reaction to the stressor that determines our health outcomes. If stress has detrimental effects on our health, just as high blood pressure and diabetes do, then we can and should look for ways to reduce these effects.

CNN: What are some ways we can cope with stress?

Wen: First, its important to clarify that there are good and bad ways to cope with stress. Some people may turn to these not-so-good ways because it may help them feel better in the short-term, but there are real risks. I mentioned drinking alcohol and using drugsobviously, these are not healthy coping strategies. Neither are binge-eating or smoking.

I think its really important to be self-aware. Be honest with yourself: When you have faced stressful situations in the past, have you turned to these unhealthy ways to cope? If so, be on the lookout and work to prevent these behaviors during stressful times.

Also, try to anticipate when there will be stressful situations. Is there a big deadline at work coming up? A family gathering that is likely to elicit negative emotions? A difficult conversation with a loved one? Knowing that a stressful event may occur can help you anticipate your reaction and plan accordingly.

I advise, too, that people make a list of stress relief techniques that have worked for them in the past. And try new techniques. Deep breathing exercises are something everyone can try and help both in the moment of the stressful encounter and after, for example, as is mindfulness meditation.

Im also a big fan of exercise. There is excellent scientific evidence that exercise is very effective at managing stress. Exercise reduces stress hormones and increases endorphins, which are feel-good neurotransmitters that can relax the body and improve mood.

CNN: What is your advice for people who have stressors in their livessuch as a hard job or caregiving responsibilitiesthat cant easily go away?

Wen: This is really hard, because of course it would be ideal to address the stressors themselves. But many people have stressful situations that they cant change.

It helps to be up front about that and acknowledge that changing the situation is not in your control. What is in your control, though, is your reaction to the situation.

Here is where self-awareness and self-care are so important. Learn to recognize when you are feeling especially stressed. Perhaps you feel tension in your neck and back muscles, or you have abdominal cramps or jitters. These are the times to practice deep breathing, meditation and other exercises that help you in the short-term.

For both short- and long-term benefit, its essential to make time for self-care. By that, I mean activities that you enjoy and that can take your mind off the stressful life situations. These could include taking a walk with a good friend, working in the garden, playing with your pets, reading a good book or otherwise participating in activities you enjoy. Think of the time you are putting aside for yourself as a kind of therapy; stress can make you unhealthy, so this is your way of giving yourself treatment to offset that stress.

Along those lines, knowing that stress is one factor that can impact your well-being, work to maximize the other aspects that contribute to overall health. Try to get adequate, restful sleep. Aim to eat healthy, whole foods and reduce your consumption of ultra-processed products. Make sure other chronic medical conditions, such as high blood pressure, are being treated. And do not wait to seek help from your mental health or primary care provider if the stress you are experiencing is leading to continuing mental health or physical distress.

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Genes affected by germline structural variation could conceivably influence cancer risk.

Researchers at Baylor College of Medicines Dan L Duncan Comprehensive Cancer Center and Human Genome Sequencing Center investigated the extent to which forms of genetic variation called germline or inherited structural variation (SV) influence gene expression in human cancers.

Structural variation is one type of genomic variation and can be beneficial, neutral or, if it affects functionally relevant regions of the genome, can seriously affect gene function and contribute to disease, including cancer, said corresponding author Dr. Chad Creighton, professor ofmedicineand co-director of cancer bioinformatics at theDan L Duncan Comprehensive Cancer Centerat Baylor.

Structural variations are larger differences in the genome that occur when a piece of DNA is duplicated, deleted, or switched around, which can impact genetic instructions encoded in DNA and affect the expression of nearby genes. Previous studies led by the researchers have shown that structural variations occurring in specific cell types, like breast cells, can strongly influence gene expression in ways that contribute to transforming a healthy breast cell into a cancer cell.

Its known that germline structural variation also can contribute to the molecular profile of cancers, Creighton said. Here we study the extent of its contribution. The study is published in Cell Reports Medicine.

The researchers worked with data developed by the Pan-Cancer Analysis of Whole Genomes consortium, which includes whole genome sequencing data from 2,658 cancers across 38 tumor types involving 20 major tissues of origin. The team integrated these data with RNA data to identify genes whose expression was associated with nearby germline structural variations.

We found most of the genes associated with germline structural variations would not necessarily have specific roles in cancer, but for some genes, the expression variation might be associated with other conditions, Creighton said.

At the same time, several genes affected by germline structural variation could conceivably contribute to cancer, for instance if these genes have an established cancer association or an association with patient survival.

This study shows that germline structural variation would represent a normal class of genetic variation passed down through generations and may play a significant role in cancer development. The researchers propose that the subset of genes with cancer-relevant associations arising in this study would represent strong candidates for further investigation on their value in genetic testing.

Fengju Chen, Yiqun Zhang and Fritz J. Sedlazeck also contributed to this work.

This study was supported by the National Institutes of Health grant P30CA125123.

By Ana Mara Rodrguez, Ph.D.

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Summary: Alzheimers disease, traditionally seen as a brain-centric condition, may have systemic origins and can be accelerated through bone marrow transplants from donors with familial Alzheimers to healthy mice.

A new study underscores the diseases potential transmission via cellular therapies and suggests screening donors for Alzheimers markers to prevent inadvertent disease transfer.

By demonstrating that amyloid proteins from peripheral sources can induce Alzheimers in the central nervous system, this research shifts the understanding of Alzheimers towards a more systemic perspective, highlighting the need for cautious screening in transplants and blood transfusions.

Key Facts:

Source: Cell Press

Familial Alzheimers disease can be transferred via bone marrow transplant, researchers show March 28 in the journalStem Cell Reports. When the team transplanted bone marrow stem cells from mice carrying a hereditary version of Alzheimers disease into normal lab mice, the recipients developed Alzheimers diseaseand at an accelerated rate.

The study highlights the role of amyloid that originates outside of the brain in the development of Alzheimers disease, which changes the paradigm of Alzheimers from being a disease that is exclusively produced in the brain to a more systemic disease.

Based on their findings, the researchers say that donors of blood, tissue, organ, and stem cells should be screened for Alzheimers disease to prevent its inadvertent transfer during blood product transfusions and cellular therapies.

This supports the idea that Alzheimers is a systemic disease where amyloids that are expressed outside of the brain contribute to central nervous system pathology, says senior author and immunologist Wilfred Jefferies, of the University of British Columbia.

As we continue to explore this mechanism, Alzheimers disease may be the tip of the iceberg and we need to have far better controls and screening of the donors used in blood, organ and tissue transplants as well as in the transfers of human derived stem cells or blood products.

To test whether a peripheral source of amyloid could contribute to the development of Alzheimers in the brain, the researchers transplanted bone marrow containing stem cells from mice carrying a familial version of the diseasea variant of the human amyloid precursor protein (APP) gene, which, when cleaved, misfolded and aggregated, forms the amyloid plaques that are a hallmark of Alzheimers disease.

They performed transplants into two different strains of recipient mice: APP-knockout mice that lacked an APP gene altogether, and mice that carried a normal APP gene.

In this model of heritable Alzheimers disease, mice usually begin developing plaques at 9 to 10 months of age, and behavioral signs of cognitive decline begin to appear at 11 to 12 months of age. Surprisingly, the transplant recipients began showing symptoms of cognitive decline much earlierat 6 months post-transplant for the APP-knockout mice and at 9 months for the normal mice.

The fact that we could see significant behavioral differences and cognitive decline in the APP-knockouts at 6 months was surprising but also intriguing because it just showed the appearance of the disease that was being accelerated after being transferred, says first author Chaahat Singh of the University of British Columbia.

In mice, signs of cognitive decline present as an absence of normal fear and a loss of short and long-term memory. Both groups of recipient mice also showed clear molecular and cellular hallmarks of Alzheimers disease, including leaky blood-brain barriers and buildup of amyloid in the brain.

Observing the transfer of disease in APP-knockout mice that lacked an APP gene altogether, the team concluded that the mutated gene in the donor cells can cause the disease and observing that recipient animals that carried a normal APP gene are susceptible to the disease suggests that the disease can be transferred to health individuals.

Because the transplanted stem cells were hematopoietic cells, meaning that they could develop into blood and immune cells but not neurons, the researchers demonstration of amyloid in the brains of APP knockout mice shows definitively that Alzheimers disease can result from amyloid that is produced outside of the central nervous system.

Finally the source of the disease in mice is a human APP gene demonstrating the mutated human gene can transfer the disease in a different species.

In future studies, the researchers plan to test whether transplanting tissues from normal mice to mice with familial Alzheimers could mitigate the disease and to test whether the disease is also transferable via other types of transplants or transfusions and to expand the investigation of the transfer of disease between species.

In this study, we examined bone marrow and stem cells transplantation. However, next it will be important to examine if inadvertent transmission of disease takes place during the application of other forms of cellular therapies, as well as to directly examine the transfer of disease from contaminated sources, independent from cellular mechanisms, says Jefferies.

Funding:

This research was supported by the Canadian Institutes of Health Research, the W. Garfield Weston Foundation/Weston Brain Institute, the Centre for Blood Research, the University of British Columbia, the Austrian Academy of Science, and the Sullivan Urology Foundation at Vancouver General Hospital.

Author: Kristopher Benke Source: Cell Reports Contact: Kristopher Benke Cell Reports Image: The image is credited to Neuroscience News

Original Research: The findings will appear in Stem Cell Reports

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Hereditary Alzheimer's Transmitted Via Bone Marrow Transplants - Neuroscience News

The Care Quality Commission (CQC) has registered Gender Plus Hormone Clinic to provide hormone treatments to 16 and 17-year-old children.

This paves the way for other private clinics to be registered, which would offer controversial medical treatments with lifelong consequences to vulnerable teenagers. The decision of the CQC to license a private clinic, creates a significant risk of a two tier approach, with less protection for those who seek help from the private sector. This further risks undermining the work of the Cass review for NHSE practice.

I want the court to set aside the registration by the CQC of Gender Plus Hormone Clinic to provide hormone treatment for teenagers. I also hope that this litigation will prevent the registration of other private clinics providing this controversial treatment. I want to ensure that those under 18 years old, do not suffer irreversible, lifelong harms both physical and psychological, from taking a controversial hormonal treatment which is not evidenced as safe or effective.

Why I am asking for this Judicial Review

I was in the NHS for nearly 40 years and I am now a psychotherapist in private practice. I have worked with people who present with issues around their gender identity for over 20 years. In my clinical experience of working with children and young people, I have not, to date, encountered a 16 to 17-year-old who I would have assessed to be sufficiently fully informed and psychologically ready to make such a life changing, potentially harmful decision. They are in the process of development from child to adult which involves significant mental and physical adjustments. Many of the young people with gender dysphoria/incongruence have no clear understanding of their underlying motivations to take cross, sex, hormones. However they are usually very aware of the discomfort they experience, and often hold a strong belief that the medication will help them feel better. They hope a change to their physical body will bring about a comfort in their mind. Some also receive strong messages from certain groups that medication is the answer to their difficulties which creates an urgent pressure on them and those around them for a solution. As a result, they are rarely able to give a full, in-depth psychological consideration to the implications and consequences of commencing a physical treatment, which is known to have serious, harmful side-effects, and, as yet has a very low level evidence base for it's efficacy and safety.

Under its current registration by the CQC, Gender Plus Hormone Clinic (GHPC) is not prevented from providing GnRH analogues (blockers) for the purpose of suspending puberty. There are some 16-year-olds who have not reached pubertal maturation. Further, the GPHC has said that it would prescribe puberty blockers alongside oestrogen therapy to achieve feminising effects. The NICE report (National Institute of Clinical Excellence) and the Cass review both state that this treatment model is not proven.

There is also considerable risk of complications due to this powerful medication. There are many known side-effects, including blood clots, gallstones, vaginal atrophy and male pattern baldness for females and potential loss of fertility, amongst many others.

The evidence base

The Cass review was commissioned by the NHS to provide a comprehensive review of the appropriate treatment for children and young people with gender dysphoria. The Cass Review sought advice from the National Institute for Health and Care Excellence (NICE) which conducted two separate evidence reviews.

Neither of them has found sufficient evidence to support the use of either puberty blockers or cross sex hormones as safe and effective.

In her interim report published in February 2022, Dr Cass has emphasised the gaps in the "evidence base regarding hormone treatment" (Para 1.41). Although some of her observations related specifically to puberty blockers, she also addressed cross-sex, hormones, and hormone treatment more generally. She said, among other things:

"The Review is not able to provide definitive advice on the use of puberty blockers and feminising/masculinising hormones at this stage, due to gaps in the evidence base; however, recommendations will be developed as our research programme progresses.

The lack of available high-level evidence was reflected in the recent NICE review into the use of puberty blockers and feminising/masculinising hormones commissioned by NHS England, with the evidence being too inconclusive to form the basis of a policy position(para 5.21)

At present we have the least information for the largest group of patients birth- registered females first presenting in early teens(para 5.11).

Your help:

I need your help to ensure that the registration of GPHC is cancelled and the other private clinics are unable to prescribe this controversial treatment to children under 18. We should not be careless or look away from the potential harms this medical treatment might cause to childrens previously healthy bodies.

Please support me with the legal fees required to mount a judicial review and challenge the CQC decision. I was the original claimant who started the Kiera Bell JR with Mrs A and our application on that occasion was successful in providing further scrutiny and attention in this area of paediatric healthcare. That judicial review potentially helped prevent irreversible harms to much younger children too as it led to a much wider scrutiny of the model of treatment in the GIDS.

I have assembled an expert legal team and will be lodging my claim with the High Court in the next few days. Please join me in seeking to protect vulnerable young people and share this crowdfunder link. I know these cases keep coming but we need to protect the next generation.

My X (twitter) handle is @sueevansprotect

Thank you very much.

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PROTECT TEENAGERS FROM HARMFUL AND IRREVERSIBLE MEDICAL TREATMENT - CrowdJustice

Abstract

Genome editing is the modification of genomic DNA at a specific target site in a wide variety of cell types and organisms, including insertion, deletion and replacement of DNA, resulting in inactivation of target genes, acquisition of novel genetic traits and correction of pathogenic gene mutations. Due to the advantages of simple design, low cost, high efficiency, good repeatability and short-cycle, CRISPR-Cas systems have become the most widely used genome editing technology in molecular biology laboratories all around the world. In this review, an overview of the CRISPR-Cas systems will be introduced, including the innovations, the applications in human disease research and gene therapy, as well as the challenges and opportunities that will be faced in the practical application of CRISPR-Cas systems.

Keywords: CRISPR, Cas9, Genome editing, Human disease models, Rabbit, Gene therapy, Off target effects

Genome editing is the modification of genomic DNA at a specific target site in a wide variety of cell types and organisms, including insertion, deletion and replacement of DNA, resulting in inactivation of target genes, acquisition of novel genetic traits and correction of pathogenic gene mutations [1], [2], [3]. In recent years, with the rapid development of life sciences, genome editing technology has become the most efficient method to study gene function, explore the pathogenesis of hereditary diseases, develop novel targets for gene therapy, breed crop varieties, and so on [4], [5], [6], [7].

At present, there are three mainstream genome editing tools in the world, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) nucleases systems [8], [9], [10]. Due to the advantages of simple design, low cost, high efficiency, good repeatability and short-cycle, CRISPR-Cas systems have become the most widely used genome editing technology in molecular biology laboratories all around the world [11], [12]. In this review, an overview of the CRISPR-Cas systems will be introduced, including the innovations and applications in human disease research and gene therapy, as well as the challenges and opportunities that will be faced in the practical application of CRISPR-Cas systems.

CRISPR-Cas is an adaptive immune system existing in most bacteria and archaea, preventing them from being infected by phages, viruses and other foreign genetic elements [13], [14]. It is composed of CRISPR repeat-spacer arrays, which can be further transcribed into CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), and a set of CRISPR-associated (cas) genes which encode Cas proteins with endonuclease activity [15]. When the prokaryotes are invaded by foreign genetic elements, the foreign DNA can be cut into short fragments by Cas proteins, then the DNA fragments will be integrated into the CRISPR array as new spacers [16]. Once the same invader invades again, crRNA will quickly recognize and pair with the foreign DNA, which guides Cas protein to cleave target sequences of foreign DNA, thereby protecting the host [16].

CRISPR-Cas systems can be classified into 2 classes (Class 1 and Class 2), 6 types (I to VI) and several subtypes, with multi-Cas protein effector complexes in Class 1 systems (Type I, III, and IV) and a single effector protein in Class 2 systems (Type II, V, and VI) [17], [18]. The classification, representative members, and typical characteristics of each CRISPR-Cas system are summarized in [10], [12], [15], [16], [17], [18].

Summary of CRISPR-Cas systems.

Type II CRISPR-Cas9 system derived from Streptococcus pyogenes (SpCas9) is one of the best characterized and most commonly used category in numerous CRISPR-Cas systems [18], [19]. The main components of CRISPR-Cas9 system are RNA-guided Cas9 endonuclease and a single-guide RNA (sgRNA) [20]. The Cas9 protein possesses two nuclease domains, named HNH and RuvC, and each cleaves one strand of the target double-stranded DNA [21]. A single-guide RNA (sgRNA) is a simplified combination of crRNA and tracrRNA [22]. The Cas9 nuclease and sgRNA form a Cas9 ribonucleoprotein (RNP), which can bind and cleave the specific DNA target [23]. Furthermore, a protospacer adjacent motif (PAM) sequence is required for Cas9 proteins binding to the target DNA [20].

During genome editing process, sgRNA recruits Cas9 endonuclease to a specific site in the genome to generate a double-stranded break (DSB), which can be repaired by two endogenous self-repair mechanisms, the error-prone non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway [24]. Under most conditions, NHEJ is more efficient than HDR, for it is active in about 90% of the cell cycle and not dependent on nearby homology donor [25]. NHEJ can introduce random insertions or deletions (indels) into the cleavage sites, leading to the generation of frameshift mutations or premature stop codons within the open reading frame (ORF) of the target genes, finally inactivating the target genes [26], [27]. Alternatively, HDR can introduce precise genomic modifications at the target site by using a homologous DNA repair template [28], [29] (). Furthermore, large fragment deletions and simultaneous knockout of multiple genes could be achieved by using multiple sgRNAs targeting one single gene or more [30], [31].

Mechanism of genome editing. Double-strand break (DSB) induced by nucleases can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. NHEJ can introduce random insertions or deletions (indels) of varying length at the site of the DSB. Alternatively, HDR can introduce precise genomic modifications at the target site by using a homologous DNA donor template.

CRISPR-Cas systems have become the most favorite genome editing tool in the molecular biology laboratory since they were confirmed to have genome editing capabilities in 2012 [23]. They have made numerous achievements in the field of correcting pathogenic mutations, searching for essential genes for cancer immunotherapy, and solving key problems in organ xenotransplantation [5], [32], [33]. Unfortunately, there are still some limitations which need to solve in CRISPR-Cas systems, such as potential off-target effects, limited genome-targeting scope restricted by PAM sequences, and low efficiency and specificity [34], [35]. Therefore, many research teams have been trying to improve this tool.

By introducing two point mutations, H840A and D10A, into HNH and RuvC nuclease domain, researchers have obtained a nuclease dead Cas9 (dCas9) [36]. The dCas9 lacks DNA cleavage activity, but DNA binding activity is not affected. Then, by fusing transcriptional activators or repressors to dCas9, the CRISPR-dCas9 system can be used to activate (CRISPRa) or inhibit (CRISPRi) transcription of target genes [37], [38]. Additionally, dCas9 can be fused to various effector domains, which enables sequence-specific recruitment of fluorescent proteins for genome imaging and epigenetic modifiers for epigenetic modification [39], [40]. Furthermore, this system is easy to operate and allows simultaneous manipulation of multiple genes within a cell [38].

In order to improve the efficiency of site-directed mutagenesis, base editing systems containing dCas9 coupled with cytosine deaminase (cytidine base editor, CBE) or adenosine deaminase (adenine base editor, ABE) have been developed [41], [42]. It can introduce CG to TA or AT to GC point mutations into the editing window of the sgRNA target sites without double-stranded DNA cleavage [41], [42]. Since base editing systems avoid the generation of random insertions or deletions to a great extent, the results of gene mutation are more predictive. However, owing to the restriction of base editing window, base editing systems are not suitable for any target sequence in the genome. Accordingly, C-rich sequences, for example, would produce a lot of off-target mutations [43]. Therefore, researchers have always been trying to develop and optimize novel base editing systems to overcome this drawback [44]. At present, base editing systems have been widely used in various cell lines, human embryos, bacteria, plants and animals for efficient site-directed mutagenesis, which may have broad application prospects in basic research, biotechnology and gene therapy [45], [46], [47]. In theory, 3956 gene variants existing in Clin var database could be repaired by base substitution of C-T or G-A [42], [48].

An NGG PAM at the 3 end of the target DNA site is essential for the recognization and cleavage of the target gene by Cas9 protein [20]. Besides classical NGG PAM sites, other PAM sites such as NGA and NAG also exist, but their efficiency of genome editing is not high [49]. However, such PAM sites only exist in about one-sixteenth of the human genome, thereby largely restricting the targetable genomic loci. For this purpose, several Cas9 variants have been developed to expand PAM compatibility.

In 2018, David Liu et al.[50] developed xCas9 by phage-assisted continuous evolution (PACE), which can recognize multiple PAMs (NG, GAA, GAT, etc.). In the latter half of the same year, Nishimasu et al. developed SpCas9-NG, which can recognize relaxed NG PAMs [51]. In 2020, Miller et al. developed three new SpCas9 variants recognizing non-G PAMs, such as NRRH, NRCH and NRTH PAMs [52]. Later in the same year, Walton et al. developed a SpCas9 variant named SpG, which is capable of targeting an expanded set of NGN PAMs [53]. Subsequently, they optimized the SpG system and developed a near-PAMless variant named SpRY, which is capable of editing nearly all PAMs (NRN and NYN PAMs) [53].

By using these Cas9 variants, researchers have repaired some previously inaccessible disease-relevant genetic variants [51], [52], [53]. However, there are still some drawbacks in these variants, such as low efficiency and cleavage activity [50], [51]. Therefore, they should be further improved by molecular engineering in order to expand the applications of SpCas9 in disease-relevant genome editing.

In addition to editing DNA, CRISPR-Cas systems can also edit RNA. Class 2 Type VI CRISPR-Cas13 systems contain a single RNA-guided Cas13 protein with ribonuclease activity, which can bind to target single-stranded RNA (ssRNA) and specifically cleave the target [54]. To date, four Cas13 proteins have been identified: Cas13a (also known as C2c2), Cas13b, Cas13c and Cas13d [55]. They have successfully been applied in RNA knockdown, transcript labeling, splicing regulation and virus detection [56], [57], [58]. Later, Feng Zhang et al. developed two RNA base edting systems (REPAIR system, enables A-to-I (G) replacement; RESCUE system, enables C-to-U replacement) by fusing catalytically inactivated Cas13 (dCas13) with the adenine/cytidine deaminase domain of ADAR2 (adenosine deaminase acting on RNA type 2) [59], [60].

Compared with DNA editing, RNA editing has the advantages of high efficiency and high specificity. Furthermore, it can make temporary, reversible genetic edits to the genome, avoiding the potential risks and ethical issues caused by permanent genome editing [61], [62]. At present, RNA editing has been widely used for pre-clinical studies of various diseases, which opens a new era for RNA level research, diagnosis and treatment.

Recently, Anzalone et al. developed a novel genome editing technology, named prime editing, which can mediate targeted insertions, deletions and all 12 types of base substitutions without double-strand breaks or donor DNA templates [63]. This system contains a catalytically impaired Cas9 fused to a reverse transcriptase and a prime editing guide RNA (pegRNA) with functions of specifying the target site and encoding the desired edit [63]. After Cas9 cleaves the target site, the reverse transcriptase uses pegRNA as a template for reverse transcription, and then, new genetic information can be written into the target site [63]. Prime editing can effectively improve the efficiency and accuracy of genome editing, and significantly expand the scope of genome editing in biological and therapeutic research. In theory, it is possible to correct up to 89% known disease-causing gene mutations [63]. Nevertheless, as a novel genome editing technique, more research is still needed to further understand and improve prime editing system.

So far, as a rapid and efficient genome editing tool, CRISPR-Cas systems have been extensively used in a variety of species, including bacteria, yeast, tobacco, Arabidopsis, sorghum, rice, Caenorhabditis elegans, Drosophila, zebrafish, Xenopus laevis, mouse, rat, rabbit, dog, sheep, pig and monkey [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], as well as various human cell lines, such as tumor cells, adult cells and stem cells [79], [80]. In medical field, the most important application of CRISPR-Cas systems is to establish genetically modified animal and cell models of many human diseases, including gene knockout models, exogenous gene knock-in models, and site directed mutagenesis models [80], [81].

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Establishing animal models of human diseases

Animal models are crucial tools for understanding gene function, exploring pathogenesis of human diseases and developing new drugs. However, traditional methods for generating animal models are complex, costly and time-consuming, which severely limit the application of animal models in basic medical research and preclinical studies [82]. Since the discovery of CRISPR-Cas systems, a series of genetically modified animal models have successfully been generated in a highly efficient manner [72], [73], [74], [75], [76], [77], [78].

Among numerous model animals, mice are widely used for scientific studies and recognized as the most important model animals in human disease research [83]. So far, researchers have successfully generated many genetically modified mouse models, such as cancer, cardiovascular disease, cardiomyopathy, Huntington's disease, albino, deafness, hemophilia B, obesity, urea cycle disorder and muscular dystrophy [84], [85], [86], [87], [88], [89], [90], [91], [92], [93]. Nevertheless, owing to the great species differences between humans and rodents, they cant provide effective assessment and long-term follow-up for research and treatment of human diseases [94]. Therefore, the application of larger model animals, such as rabbits, pigs and non-human primates, is becoming more and more widespread [74], [77], [78]. With the development of CRISPR-Cas systems, generating larger animal models for human diseases has become a reality, which greatly enriches the disease model resource bank.

Our research focuses on the generation of genetically modified rabbit models using CRISPR-Cas systems. Compared with mice, rabbits are closer to humans in physiology, anatomy and evolution [95]. In addition, rabbits have a short gestation period and less breeding cost. All these make them suitable for studies of the cardiovascular, pulmonary and metabolism diseases [95], [96]. Nowadays, we have generated a series of rabbit models for simulating human diseases, including congenital cataracts, duchenne muscular dystrophy (DMD), X-linked hypophosphatemia (XLH), etc (summarized in ) [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [107], [108], [109], [110], [111], [112], [113], [114]. Take the generation of PAX4 gene knockout rabbits as an example, the procedure we used to establish genetically modified rabbit models is summarized in and .

CRISPR-Cas system mediated rabbit models of human diseases.

Generation of PAX4 gene knockout (KO) rabbits using CRISPR-Cas9 system. (A) Schematic diagram of the sgRNA target sites located in the rabbit PAX4 locus. PAX4 exons are indicated by yellow rectangles; target sites of the two sgRNA sequences, sgRNA1 and sgRNA2, are highlighted in green; protospacer-adjacent motif (PAM) sequence is highlighted in red. Primers F and R are used for mutation detection in pups. (B) Microinjection and embryo transfer. First a mixture of Cas9 mRNA and sgRNA is microinjected into the cytoplasm of the zygote at the pronuclear stage. Then the injected embryos are transferred into the oviduct of recipient rabbits. After 30days gestation, PAX4 KO rabbits are born. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Summary of the PAX4 KO rabbits generated by CRISPR-Cas9 system.

In addition, the pig is an important model animal extensively used in biomedical research. Compared with mice, their body/organ size, lifespan, anatomy, physiology, metabolic profile and immune characteristics are more similar to those of humans, which makes the pig an ideal model for studying human cardiovascular diseases and xenotransplantation [115]. At present, several genetically modified pig models have been successfully generated, including neurodegenerative diseases, cardiovascular diseases, cancer, immunodeficiency and xenotransplantation model [116], [117], [118], [119], [120], [121], [122].

To date, non-human primates are recognized as the best human disease models. Their advantage is that their genome has 98% homology with the human genome; also, they are highly similar to humans in tissue structure, immunity, physiology and metabolism [123]. Whats more, they can be infected by human specific viruses, which makes them very important models in infectious disease research [124]. Nowadays, researchers have generated many genetically modified monkey models, such as cancer, muscular dystrophy, developmental retardation, adrenal hypoplasia congenita and Oct4-hrGFP knockin monkeys [125], [126], [127], [128], [129].

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Establishing cell models of human diseases

It was found that the efficiency of CRISPR-Cas mediated genome editing is higher in vitro than in vivo, thus the use of genetically modified cell models can greatly shorten the research time in medical research [130]. Until now, researchers have used CRISPR-Cas systems to perform genetic manipulations on various cell lines, such as tumor cells, adult cells and stem cells, in order to simulate a variety of human diseases [79], [80].

Fuchs et al. generated the RPS25-deficient Hela cell line by knocking out ribosomal protein eS25 (RPS25) gene using CRISPR-Cas9 system [131]. Drost et al. edited four common colorectal cancer-related genes (APC, P53, KRAS and SMAD4) in human intestinal stem cells (hISCs) by CRISPR-Cas9 technology [132]. The genetically modified hISCs with 4 gene mutations possessed the biological characteristics of intestinal tumors and could simulate the occurrence of human colorectal cancer [132]. Jiang et al. induced site-specific chromosome translocation in mouse embryonic stem cells by CRISPR-Cas9, in order to establish a cell and animal model for subsequent research on congenital genetic diseases, infertility, and cancer related to chromosomal translocation [133].

In addition, induced pluripotent stem cells (iPSCs) have shown great application prospect in disease model establishment, drug discovery and patient-specific cellular therapy development [134]. iPSCs have the ability of self-renewal and multiple differentiation potential, which are of great significance in disease model establishment and regenerative medicine research [135]. In recent years, by combining CRISPR-Cas systems with iPSC technology, researchers have generated numerous novel and reliable disease models with isogenic backgrounds and provided new solutions for cell replacement therapy and precise therapy in a variety of human diseases, including neurodegenerative diseases, acquired immunodeficiency syndrome (AIDS), -thalassemia, etc [134], [135], [136].

With the development of CRISPR-Cas systems and the discovery of novel Cas enzymes (Cas12, Cas13, etc.), CRISPR-based molecular diagnostic technology is rapidly developing and has been selected as one of the world's top ten science and technology advancements in 2018 [137].

Unlike Cas9, Cas13 enzymes possess a collateral cleavage activity, which can induce cleavage of nearby non-target RNAs after cleavage of target sequence [54]. Based on the collateral cleavage activity of Cas13, Feng Zhang et al.[138] developed a Cas13a-based in vitro nucleic acid detection platform, named SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing). It is composed of Cas13a, sgRNA targeting specific RNA sequences and fluorescent RNA reporters. After Cas13a protein recognizes and cleaves the target RNA, it will cut the report RNA and release the detectable fluorescence signal, so as to achieve the purpose of diagnosis [138]. Researchers have used this method to detect viruses, distinguish pathogenic bacteria, genotype human DNA and identify tumor DNA mutations [137], [138]. Later, Feng Zhang et al. improved SHERLOCK system and renamed it as SHERLOCKv2, which can detect four virus at the same time [139].

In addition to Cas13, Cas12 enzymes are also found to possess collateral cleavage activity [140]. Doudna et al.[141] developed a nucleic acid detection system based on Cas12a (also known as Cpf1), named DETECTR (DNA endonuclease-targeted CRISPR trans reporter). DETECTR has been used to detect cervical cancer associated HPV subtypes (HPV16 and HPV18) in either virus-infected human cell lines or clinical patient samples [141]. Furthermore, Doudna et al. are trying to use the newly discovered Cas14 and CasX proteins in molecular diagnosis, which may further enrich the relevant techniques of CRISPR-based molecular diagnosis [142], [143].

CRISPR-based molecular diagnostic technology has incomparable advantages over traditional molecular diagnostic methods, such as high sensitivity and single-base specificity, which is suitable for early screening of cancer, detection of cancer susceptibility genes and pathogenic genes [137], [144]. Meanwhile, CRISPR diagnostics is inexpensive, simple, fast, without special instrument, and is suitable for field quick detection and detection in less-developed areas [137], [144]. At present, many companies are trying to develop CRISPR diagnostic kits for family use, to detect HIV, rabies, Toxoplasma gondi, etc.

CRISPR-Cas9 system enables genome-wide high-throughput screening, making it a powerful tool for functional genomic screening [145]. The high efficiency of genome editing with CRISPR-Cas9 system makes it possible to edit multiple targets in parallel, thus a mixed cell population with gene mutation can be produced, and the relationship between genotypes and phenotypes could be confirmed by these mutant cells [146]. CRISPR-Cas9 library screening can be divided into two categories: positive selection and negative selection [147]. It has been utilized to identify genes associated with cancer cell survival, drug resistance and virus infection in various models [148], [149], [150]. Compared with RNAi-based screening, high-throughput CRISPR-Cas9 library screening has the advantages of higher transfection efficiency, minimal off-target effects and higher data reproducibility [151]. At present, scientists have constructed human and mouse genome-wide sgRNA libraries, and they have been increasingly improved according to different requirements [152], [153]. In the future, CRISPR-Cas9-based high-throughput screening technology will definitely get unprecedented development and application.

Gene therapy refers to the introduction of foreign genes into target cells to treat specific diseases caused by mutated or defective genes [154]. Target cells of gene therapy are mainly divided into two categories: somatic cells and germ line cells. However, since germ line gene therapy is complicated in technique as well as involves ethical and security issues, today gene therapy is limited to somatic cell gene therapy [155]. Traditional gene therapy is usually carried out by homologous recombination or lentiviral delivery. Nevertheless, the efficiency of homologous recombination is low, and lentiviral vectors are randomly inserted into the recipient genome, which may bring potential security risks to clinical applications [156]. Currently, with the rapid development of CRISPR-Cas systems, they have been widely applied in gene therapy for treating various of human diseases, monogenic diseases, infectious diseases, cancer, etc [155], [156], [157]. Furthermore, some CRISPR-mediated genome-editing therapies have already reached the stage of clinical testing. briefly summarizes the ongoing clinical trials of gene therapy using genome-editing technology, including ZFN, TALEN and CRISPR-Cas systems.

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Monogenic diseases

Monogenic diseases refer to the genetic diseases caused by mutations of a single allele or a pair of alleles on a pair of homologous chromosomes [158]. There are more than 6600 known monogenic diseases around the world, -thalassaemia, sickle cell disease (SCD), hemophilia B (HB), retinitis pigmentosa (RP), leber congenital amaurosis type 10 (LCA10), duchenne muscular dystrophy (DMD), hutchinson-gilford progeria syndrome (HGPS), hereditary tyrosinemia (HT), cystic fibrosis (CF), etc [159]. Most of the monogenic diseases are rare diseases lacking of effective treatment, which will greatly affect the life quality of patients. Nowadays, many animal models of monogenic diseases have been treated with CRISPR-mediated gene therapy. Furthermore, even some CRISPR clinical trials for monogenic diseases are going on [160].

Summary of clinical trials of gene therapy using genome-editing technology.

-Thalassaemia, a hereditary hemolytic anemia disease, is one of the most common and health-threatening monogenic diseases in the world. It is characterized by mutations in the -globin (HBB) gene, leading to severe anemia caused by decreased hemoglobin (Hb) level [161]. For the moment, the only way to cure -thalassemia is hematopoietic stem cell transplantation (HSCT). Yet, high cost of treatment and shortage of donors limit its clinical application [162]. Other therapy, for example, blood transfusion, can only sustain the life of patients but cant cure the disease [161]. To better treat -thalassemia, researchers have turned their attention to gene therapy. A major technical idea is to repair the defective -globin gene of iPSCs from patients with -thalassemia by CRISPR-Cas9 technology, then red blood cells can be produced normally and the disease could be cured [163], [164]. Besides, reactivating fetal hemoglobin (HbF) expression has also been proposed to be an effective method to treat -thalassemia through knockout of BCL11A gene, which suppresses the expression of fetal hemoglobin [165], [166].

Additionally, CRISPR-Cas systems have also been used for the treatment of other hematologic diseases, such as sickle cell disease (SCD) and hemophilia B (HB). SCD is a monogenic disease caused by a single-nucleotide mutation in human -globin gene, leading to a substitution of glutamic acid by valine and the production of an abnormal version of -globin, which is known as hemoglobin S (HbS) [167]. CRISPR-Cas9 system has been used to treat SCD by repairing the -globin gene mutation or reactivating HbF expression [168], [169]. HB is an X-linked hereditary bleeding disorder caused by deficiency of coagulation factor IX, and the most common treatment for hemophilia B is supplement blood coagulation factor [170], [171]. Huai et al. injected naked Cas9-sgRNA plasmid and donor DNA into the adult mice of F9 mutation HB mouse model for gene correction [172]. Meanwhile, Cas9/sgRNA were also microinjected into germline cells of this HB mouse model for gene correction. Both in vivo and ex vivo experiment were sufficient to remit the coagulation deficiency [172]. Guan et al. corrected the F9 Y371D mutation in HB mice using CRISPR-Cas9 mediated in situ genome editing, which greatly improved the hemostatic efficiency and increased the survival of HB mice [173].

Duchenne muscular dystrophy (DMD) is an X-chromosome recessive hereditary disease, with clinical manifestations of muscle weakness or muscle atrophy due to a progressive deterioration of skeletal muscle function [174]. It is usually caused by mutations in the DMD gene, a gene encoding dystrophin protein [174]. Deletions of one or more exons of the DMD gene will result in frameshift mutations or premature termination of translation, thereby normal dystrophin protein can not be synthesized [175]. Currently, there is no effective treatment for DMD. Conventional drug treatment can only control the disease to a certain extent, but can not cure it. It was found that a functional truncated dystrophin protein can be obtained by removing the mutated transcripts with CRISPR-Cas9 system [176], [177], [178]. In addition, base editing systems can also be applied in DMD treatment by repairing single base mutation or inducing exon skipping by introducing premature termination codons (PTCs) [179].

Retinitis pigmentosa (RP) is a group of hereditary retinal degenerative diseases characterized by progressive loss of photoreceptor cells and retinal pigment epithelium (RPE) function [180]. RP has obvious genetic heterogeneity, and the inheritance patterns include autosomal dominant, autosomal recessive, and X-linked recessive inheritance [180]. To date, there is still no cure for RP. In recent years, with the rapid development of gene editing technology, there has been some progress in the treatment of RP. Several gene mutations causing RP have been corrected by CRISPR-Cas9 in mouse models to prevent retinal degeneration and improve visual function, for example, RHO gene, PRPF31 gene and RP1 gene [181], [182].

Leber Congenital Amaurosis type 10 (LCA10) is an autosomal retinal dystrophy with severe vision loss at an early age. The most common gene mutation found in patients with LCA10 is IVS26 mutation in the CEP290 gene, which disrupts the coding sequence by generating an aberrant splice site [183]. Ruan et al. used CRISPR-Cas9 system to knock out the intronic region of the CEP290 gene and restored normal CEP290 expression [184]. In addition, subretinal injection of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing [185], [186].

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare lethal genetic disorder with the characteristic of accelerated aging [187]. A point mutation within exon 11 of lamin A gene activates a cryptic splice site, leading to the production of a truncated lamin A called progerin [188]. However, CRISPR-Cas based gene therapy has opened up a broad prospect in HGPS treatment. Administration of AAV-delivered CRISPR-Cas9 components into HGPS mice can reduce the expression of progerin, thereby improved the health condition and prolonged the lifespan of HGPS mice [189], [190]. In addition, Suzuki et al. repaired G609G mutation in a HGPS mouse model via single homology arm donor mediated intron-targeting gene integration (SATI), which ameliorated aging-associated phenotypes and extended the lifespan of HGPS mice [191].

CRISPR-Cas systems have also showed their advantages in gene therapy of hereditary tyrosinemia (HT) and cystic fibrosis (CF). HT is a disorder of tyrosine metabolism caused by deficiency of fuarylacetoacetate hydrolase (Fah) [192]. Yin et al. corrected a Fah mutation in a HT mouse model by injecting CRISPR-Cas9 components into the liver of the mice [193]. Then, the wild-type Fah protein in the liver cells began to express and the body weight loss phenotype was rescued [193]. CF, an autosomal recessive inherited disease with severe respiratory problems and infections, has a high mortality rate at an early age [194]. It is caused by mutations in the CFTR gene, which encodes an epithelial chloride anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR) [194]. Until now, genome editing strategies have been carried out in cell models to correct CFTR mutations. In cultured intestinal stem cells and induced pluripotent stem cells from cystic fbrosis patients, the CFTR homozygous 508 mutation has been corrected by CRISPR-Cas9 technology, leading to recovery of normal CFTR expression and function in differentiated mature airway epithelial cells and intestinal organoids [195], [196].

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Infectious diseases

In recent years, gene therapy has gradually been applied to the treatment of viral infectious diseases. Transforming host cells to avoid viral infection or preventing viral proliferation and transmission are two main strategies for gene therapy of viral infectious diseases [197].

Human immunodeficiency virus (HIV), a kind of retrovirus, mainly attacks the human immune system, especially the CD4 T lymphocytes. When human cells are invaded by HIV, the viral sequences can be integrated into the host genome, blocking cellular and humoral immunity while causing acquired immunodeficiency syndrome (AIDS) [198]. There is still no known cure for AIDS but it could be treated. Although antiretroviral therapy can inhibit HIV-1 replication, the viral sequences still exist in the host genome, and they could be reactivated at any time [199]. CRISPR-Cas9 system can target long terminal repeat (LTR) and destruct HIV-1 proviruses, thus it is possible to completely eliminate HIV-1 from genome of infected host cells [200], [201]. In addition, resistance to HIV-1 infection could be induced by knockout of the HIV co-receptor CCR5 gene in CD4 T cells [202], [203].

Cervical cancer is the second most common gynecologic malignant tumor. The incidence is increasing year by year and young people are especially prone to this disease. It was found that the occurrence of cervical cancer is closely related to HPV (human papillomavirus) infection [204]. HPV is a double-stranded cyclic DNA virus, E6 and E7 genes located in HPV16 early regions are carcinogenic genes [205]. Researchers designed sgRNAs targeting E6 and E7 genes to block the expression of E6 and E7 protein, subsequently the expression of p53 and pRb was restored to normal, finally increasing tumor cells apoptosis and suppressing subcutaneous tumor growth in in vivo experiments [206], [207], [208]. Moreover, HPV virus proliferation was blocked through cutting off E6/E7 genes, and the virus in the bodies could be eliminated [206], [207], [208].

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Cancer

Cancer is the second leading cause of death worldwide after cardiovascular diseases, and it is also a medical problem that needs to be solved urgently. A variety of genetic or epigenetic mutations have been accumulated in the cancer genome, which can activate proto-oncogenes, inactivate tumor suppressors and produce drug resistance [209], [210]. So far, CRISPR-Cas systems have been used to correct the oncogenic genome/epigenome mutations in tumor cells and animal models, resulting in inhibition of tumor cell growth and promotion of cell apoptosis, thereby inhibiting tumor growth [211], [212], [213].

In addition, immunotherapy is considered to be a major breakthrough in cancer treatment, especially chimeric antigen receptor-T (CAR-T) cell therapy, which has a significantly therapeutic effect on leukemia, lymphoma and certain types of solid tumors [214], [215], [216]. CAR-T cells are genetically manipulated, patient-specific T cells, which express receptors targeting antigens specially expressed on tumor cells, for example, CD19 CAR-T cells for B cell malignancies. Then these cells will be transfused back to patients to fight against cancer [217]. However, CAR-T cell therapy is complex, time-consuming and expensive, and it is greatly limited by the quality and quantity of autologous T cells. Therefore, researchers have used CRISPR-Cas9 system to develop universal CAR-T cells, such as simultaneously removing endogenous T cell receptor gene and HLA class I encoding gene on T cells of healthy donors and introducing CAR sequence [218], [219], [220]. Thereby, it could be used in multiple patients without causing graft versus host reaction (GVHR). In addition, CRISPR-Cas mediated genome editing has also been used to enhance the function of CAR-T cells by knocking out genes encoding signaling molecules or T cell inhibitory receptors, such as programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) [221], [222].

Though CRISPR-Cas mediated efficient genome editing technologies have been broadly applied in a variety of species and different types of cells, there are still some important issues needed to be addressed during the process of application, such as off-target effects, delivery methods, immunogenicity and potential risk of cancer.

It was found that designed sgRNAs will mismatch with non-target DNA sequences and introduce unexpected gene mutations, called off-target effects [223]. Off-target effects seriously restrict the widespread application of CRISPR-Cas mediated genome editing in gene therapy, for it might lead to genomic instability and increase the risk of certain diseases by introducing unwanted mutations at off-target sites [224]. At present, several strategies have been used to predict and detect off-target effects, online prediction software, whole genome sequencing (WGS), genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), discovery of in situ cas off-targets and verification by sequencing (DISCOVER-Seq), etc [225]. Furthermore, to minimize off-target effects, researchers have systematically studied the factors affecting off-target effects and developed a number of effective approaches.

(1)

Rational design and modification of sgRNAs

The specific binding of sgRNA with the target sequence is the key factor in CRISPR-Cas mediated genome editing. Rational design of highly specific sgRNAs might minimize off-target effects [224]. The length and GC content of sgRNAs, and mismatches between sgRNA and its off-target site will all affect the frequency of off-target effects [226]. In addition, on the basis of rational design of sgRNAs, the specificity of CRISPR-Cas systems can be further improved by modifying sgRNAs, such as engineered hairpin sgRNAs and chemical modifications of sgRNAs [227], [228].

(2)

Modification of Cas9 protein

As we know, the interaction between Cas9 and DNA affects the stability of DNA-Cas9/sgRNA complex as well as tolerance to mismatch [229]. Therefore, high-fidelity SpCas9 variants have been developed by introducing amino substitution(s) into Cas9 protein in order to destabilize the function structure of the CRISPR complex [230]. Researchers have developed several highly effective Cas9 mutants, high-fidelity Cas9 (SpCas9-HF1), enhanced specificity Cas9 (eSpCas9), hyper-accurate Cas9 (HypaCas9), etc [231], [232], [233]. All of them can significantly reduce off-target effects while retain robust target cleavage activity.

(3)

Adoption of double nicking strategy

Recently, a double-nicking strategy has been developed to minimize off-target effects, which employs two catalytic mutant Cas9-D10A nickases and a pair of sgRNAs to produce a cleavage on each strand of the target DNA, thus forming a functional double strand break [234]. Additionally, it was proven that the fusion protein generated by combining dCas9 with Fok nuclease can also reduce off-target effects [235]. Only when the two fusion protein monomers are close to each other to form dimers, can they perform the cleavage function [235]. This strategy could greatly reduce DNA cleavage at non-target sites.

(4)

Anti-CRISPRs

Off switches for CRISPR-Cas9 system was first discovered by Pawluk et al. in 2016. They identified three naturally existing protein families, named as anti-CRISPRs, which can specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis[236]. Later, Rauch et al. discovered four unique type IIA CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages, and two of them (AcrllA2 and AcrllA4) can block SpCas9 when assayed in Escherichia coli and human cells [237]. Recently, Doudna et al. discovered two broad-spectrum inhibitors of CRISPR-Cas9 system (AcrllC1 and AcrllC3) [238]. Therefore, in order to reduce off-target effects, the anti-CRISPRs could be used to prevent the continuous expression of Cas9 protein in cells to be edited.

(5)

Others

The concentration of Cas9/sgRNA can also affect the frequency of off-target mutations [239]. Thus, the optimal concentration of Cas9 and sgRNA needs to be determined by pre-experiment. Besides, the formulation of CRISPR-Cas9 can affect the frequency of off-target mutations as well. Cas9 nucleases can be delivered into target cells in 3 different forms: DNA expression plasmid, mRNA or recombination protein [240]. Currently, the use of Cas9/sgRNA ribonucleoprotein complexes (Cas9-RNPs), which are composed of purified Cas9 proteins in combination with sgRNA, is becoming more and more widespread. It was found that delivery as plasmid usually produces more off-targets than delivery as RNPs, since the CRISPR-Cas system is active for a shorter time without Cas9 transcription and translation stages [241], [242].

Nowadays, how to effectively deliver CRISPR-Cas components to specific cells, tissues and organs for precisely directed genome editing is still a major problem in gene therapy. Ideal delivery vectors should have the advantages of non-toxicity, well targeting property, high efficiency, low cost, and biodegradability [35], [156]. At present, three main delivery methods have been employed in delivering CRISPR-Cas components, including physical, viral and non-viral methods [243]. Physical methods are the simplest way to deliver CRISPR-Cas components, including electroporation, microinjection and mechanical cell deformation. They are simple and efficient, which can also improve the expression of genes, and being widely applied in in vitro experiments [243], [244]. In addition, viral vectors, such as adenovirus, adeno-associated virus (AAV) and lentivirus viral vectors, are being widely used for both in vitro/ex vivo and in vivo delivery due to their high delivery efficiency. They are commonly used for gene delivery in gene therapy, and some of them have been approved for clinical use [245], [246]. However, safety issue of viral vectors is still a major problem needed to be solved in pre-clinical trials. Therefore, researchers have turned their attention to non-viral vectors, for instance, liposomes, polymers and nanoparticles [247]. Based on the advantages of safety, availability and cost-effectiveness, they are becoming a hotspot for the delivery of CRISPR-Cas components [248].

Since all these delivery methods have both advantages and disadvantages, its necessary to design a complex of viral vectors and non-viral vectors, which combines the advantages of both vectors. Along with the deepening of research, various carriers could be modified by different methods to increase the delivery efficiency and reduce the toxicity [249]. In addition, more novel vectors, such as graphene and carbon nanomaterials (CNMs), could also be applied in the delivery of CRISPR-Cas components [250], [251].

Since the components of CRISPR-Cas systems are derived from bacteria, host immune response to Cas gene and Cas protein is regarded as one of the most important challenges in the clinical trials of CRISPR-Cas system [156], [252]. It was found that in vivo delivery of CRISPR-Cas components can elicit immune responses against the Cas protein [252], [253]. Furthermore, researchers also found that there were anti-Cas9 antibodies and anti-Cas9 T cells existing in healthy humans, suggesting the pre-existing of humoral and celluar immune responses to Cas9 protein in humans [254]. Therefore, how to detect and reduce the immunogenicity of Cas proteins is a major challenge will be faced in clinical application of CRISPR-Cas systems. Researchers are trying to handle this problem by modifying Cas9 protein or using Cas9 homologues [255].

Recently, two independent research groups found that CRISPR-Cas mediated double-stranded breaks (DSBs) can activate the p53 signaling pathway [256], [257]. This means that genetically edited cells are likely to become potential cancer initiating cells, and clinical treatment with CRISPR-Cas systems might inadvertently increase the risk of cancer [256], [257], [258]. Although there is still no direct evidence to confirm the relationship between CRISPR-Cas mediated genome editing and carcinogenesis, these studies once again give a warning on the application of CRISPR-Cas systems in gene therapy. It reminds us that there is still a long way to go before CRISPR-Cas systems could be successfully applied to humans.

CRISPR-Cas mediated genome editing has attracted much attention since its advent in 2012. In theory, each gene can be edited by CRISPR-Cas systems, even genes in human germ cells [259]. However, germline gene editing is forbidden in many countries including China, for it could have unintended consequences and bring ethical and safety concerns [260].

However, in March 2015, a Chinese scientist, Junjiu Huang, published a paper about gene editing in human tripronuclear zygotes in the journal Protein & Cell, which brings the ethical controversy of human embryo gene editing to a climax [261]. Since then, genome editing has been challenged by ethics and morality, and legal regulation of genome editing has triggered a heated discussion all around the world.

Then, on Nov. 28, 2018, the day before the opening of the second international human genome editing summit, Jiankui He, a Chinese scientist from the Southern University of Science and Technology, announced that a pair of gene-edited babies, named Lulu and Nana, were born healthy in China this month. They are the worlds first gene-edited babies, whose CCR5 gene has been modified, making them naturally resistant to HIV infection after birth [262]. The announcement has provoked shock, even outrage among scientists around the world, causing widespread controversy in the application of genome editing.

The society was shocked by this breaking news, for it involves genome editing in human embryos and propagating into future generations, triggering a chorus of criticism from the scientific community and bringing concerns about ethics and security in the use of genome editing. Therefore, scientists call on Chinese government to investigate the matter fully and establish strict regulations on human genome editing. Global supervisory system is also needed to ensure genome editing of human embryos moving ahead safely and ethically [263].

Since CRISPR-Cas mediated genome editing technologies have provided an accessible and adaptable means to alter, regulate, and visualize genomes, they are thought to be a major milestone for molecular biology in the 21st century. So far, CRISPR-Cas systems have been broadly applied in gene function analysis, human gene therapy, targeted drug development, animal model construction and livestock breeding, which fully prove their great potential for further development. However, there are still some limitations to overcome in the practical applications of CRISPR-Cas systems, and great efforts still need to be made to evaluate their long-term safety and effectiveness.

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