CRISPR Gene Editing Has Been Used to Cure Mice of Sickle Cell Disease


IN BRIEF
  • The results are promising and could lead to a treatment for this disease that afflicts about 100,000 Americans.
  • While human tests are still a ways off, CRISPR could one day be used to effectively treat a number of ailments, from high cholesterol to HIV.

CORRECTING A MUTATION

Gene editing shows promise as a new treatment for sickle cell disease, according to a study published in the online journal Science Translational Medicine.

Experts from the University of California, Berkeley, UCSF Benioff Children’s Hospital Oakland Research Institute (CHORI), and the University of Utah School of Medicine have found success in correcting the blood cell mutation in tests of the blood of both mice and human sickle cell patients using CRISPR-Cas9, a genome “scissor” that can cut out and edit a DNA sequence.

After CRISPR was used to correct the mutated hematopoietic stem cells — precursor cells that mature into the hook-shaped hemoglobin characteristic of sickle cell disease, the corrected blood stem cells produced healthy hemoglobin. Following reintroduction into the mice, the genetically engineered stem cells remained in circulation for at least four months — a significant indication that any potential therapy would be lasting.

 The tests of blood from afflicted humans showed that the proportion of corrected stem cells was high enough to produce substantial benefit for the patients, so the researchers are hoping to one day be able to reinfuse the human patients with the edited strain of cells as it could alleviate symptoms of sickle cell disease, including anemia and pain caused by vessel blockages.

While the results are promising and could lead to a treatment for this disease that afflicts about 100,000 Americans, the researchers emphasize that future testing on mice and safety analyses would need to be conducted before human trials could begin.

A CRISPR’D FUTURE

“Sickle cell disease is just one of many blood disorders caused by a single mutation in the genome,” said Jacob Corn, senior author on the study. “It’s very possible that other researchers and clinicians could use this type of gene editing to explore ways to cure a large number of diseases.”

Indeed, developments in gene-editing  technology within such fields as medicine, agriculture, and biology are taking us further into what some are calling “the age of CRISPR.”

It’s already being researched as a treatment for many other ailments and disorders— β-thalassemia, severe combined immunodeficiency (SCID)Wiskott-Aldrich syndrome, even HIV — so this one type of technology could end up being something of a panacea for what ails us, as long as what ails us is genetic.

CRISPR gene editing tech brings countless opportunities and challenges.


Dr. Thomas Doetschman, Ph.D., examines the embryonic cells used to study and implant mutated and disease genes; if the mutated gene successfully imbeds itself into a sperm or egg cell, the resulting rat that is born will be studied to research the effects of that same disease genes in humans. CRISPR CAS9 is technology that allows the splicing of genes to both remove and replace particular DNA strands. CRISPR can affect either just the patient or his descendants as well, depending on the technique used.

A new genome editing technology known as CRISPR has the potential to revolutionize the way scientists study diseases and genetics.

“I think it’s a really useful tool for science, in fact it’s sort of revolutionizing the speed at which we can accomplish certain things in the laboratory and it has tremendous potential for therapeutic applications,” said Kimberly McDermott, a research associate professor of medicine and an associate professor of cellular and molecular medicine, cancer biology and genetics.

Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR-Cas9, is based off a bacterial immune system, said Thomas Doetschman, professor of cancer biology, genetics and cellular and molecular medicine.

When bacteria become infected by a virus, they take pieces of the virus’s DNA and incorporate it into their own genome. This allows the bacteria to recognize and attack the virus if it ever appears again. This system allows them to destroy the virus, but it also allows them to destroy DNA, Doetschman said.

In developing CRISPR, scientists took a hint from the bacteria.

“What it [CRISPR] actually does is causes a mutation at that site, in the DNA, and then repairs it,” Doetschman said. “And you can repair it in different ways, such that you can actually modify the sequence of the DNA.”

This has enormous implications for the study of genetics and combating human diseases. And while it may sound exciting, human gene editing isn’t all fun and games.

There are two ways the CRISPR technology can be used in humans, Doetschman said. The first way is to alter somatic cells, which don’t get passed down to the next generation. This would only affect the patient who is receiving the treatment. The second way, known as the germline, can have serious long-lasting effects. Altering genes in the germline can produce permanent changes in the patient that will then be passed on to their children.

“There’s two completely different ways of doing this, and the real concern, the big concern, is that it be used by some unscrupulous people to try to change the germline of people, so that you can create progeny that will all have this kind of modification,” Doetschman said.

CRISPR isn’t just for humans; it can be used to edit plant cells as well.

“It could alter genes in a plant so that the plant either becomes resistant to or susceptible to agents that might otherwise kill the plant,” Doetschman said. This could mean disease-resistant plants or increased nutritional content.

One of CRISPR’s greatest contributions is in the realm of research, specifically for understanding normal development and disease processes, McDermott said.

For example, in the future scientists may be able to grow human organs from the patient’s own cells, using CRISPR.

Recent studies on mice and rats have introduced the possibility of using a model organism, such as a pig, to grow human organs, McDermott said.

Another exciting possibility available through CRISPR involves induced pluripotent stem cells, Doetschman said. This process essentially works as a time machine for your cells.

Doetschman describes it as the ability to put your own cells, such as skin cells, in culture and de-differentiate those cells back down to the pluripotent “master key” stem cell, using CRISPR. Once your adult cells are transformed into stem cells, you can make the genetic modifications you’d like, such as correcting a mutation, and then re-differentiate the cells back into the cell type of the tissue you want to correct.

These cells could potentially be engrafted  back into the patient’s disease tissue, Doetschman said.

Dr. Thomas Doetschman, Ph.D., describes a few of the many functions performed in the workspace pictured, which can effectively seal itself to create a sterile and airtight environment in which researchers can operate. CRISPR technology may redefine the future of genetics.

When it comes to working with human therapeutics, safety and regulations are extremely important, McDermott said.

As scientists, their primary concern is to minimize and prevent harm in every way possible. One of these regulations is a patent that was recently issued to the MIT and Harvard-affiliated Broad Institute, one of the centers responsible for creating CRISPR technology.

Despite heavy public controversy surrounding the patent, Doetschman said the patent is a good thing, because it will allow scientists to ensure that CRISPR research is carried out in a safe way, especially in regards to human use.

“I think from a scientist’s perspective, the thing that we’re really focusing on is trying to listen to our colleagues but also the public in general about what are the fears of this technology,” McDermott said. “Of course when you start to edit genes and mutate genes there’s a lot of concerns about what might happen.”

As for the future of human genetics research, both Doetschman and McDermott remain optimistic. CRISPR improves both the efficiency and the accuracy of genome research.

McDermott said while scientists may have had the ability to make mutations in cells in the past, the results were usually inefficient and could produce off-target effects.

CRISPR might not be the cure to every disease, but it is the key to unlock many avenues of research, Doetschman said.

“In terms of the research end of science and medical research, it’s expanding tremendously the scientist’s ability to ask questions about genetic disease,” Doetschman said.

HIV Can Mutate to Evade Attempts To Modify It With CRISPR Gene Editing


IN BRIEF

HIV can defeat efforts to cripple it with CRISPR gene editing technology, researchers say. And the very act of editing – involving snipping at the virus’s genome – may introduce mutations that help it resist attack.

There’s a negative side to the news that a second team of scientists successfully modified human embryos, making them HIV-resistant. The team was able to modify some but not all embryos in the study. And it seems that HIV can actually defeat our efforts to cripple it with CRISPR gene-editing technology.

Ultimately, it seems that CRISPR itself may introduce mutations that help HIV resist attack; however, the team behind the work thinks that these problems may be surmountable.

RESISTING ATTACK

When HIV infects a T cell, its genome is inserted into the cell’s DNA, and it hijacks its DNA-replicating machinery to churn out more copies of the virus. But a T cell equipped with a DNA-shearing enzyme called Cas9 can find, cut, and cripple the invader’s genome.

That method did seem to work, at least for a short period of time, when a team led by virologist Chen Liang, at McGill University in Montreal, Canada, infected T cells that had been given the tools to stop HIV. But two weeks later, Liang’s group saw that the T cells were pumping out copies of virus particles that had escaped the CRISPR attack.

Liang’s team believes that the mutations occurred when Cas9 cut the viral DNA. When DNA is cut, its host cell tries to repair the break; in doing so, it sometimes introduces or deletes DNA letters. These ‘indels’ usually inactivate the gene that was cut — which is how CRISPR works. But sometimes this doesn’t happen.

Occasionally, Liang thinks, some of the indels made by the T cell’s machinery leave the genome of the invading HIV able to replicate and infect other cells. And worse, the change in sequence means the virus can’t be recognized and targeted by T cells with the same machinery. This means it becomes resistant to any future attack.

ALTERING HUMAN GENES

Liang thinks that the challenges seen in this study can be overcome. He suggests inactivating several essential HIV genes at once, or using CRISPR in combination with HIV-attacking drugs. Gene editing therapies that make T cells resistant to HIV invasion (by altering human, not viral, genes) would also be harder for the virus to combat.

A clinical trial is under way to test this approach using another gene-editing tool, zinc-finger nucleases.

CRISPR Gene Editing Has Repaired a Blood-Borne Disease


IN BRIEF
  • Researchers from Stanford have used the CRISPR-Cas9 gene editing technique to fix the gene defects that cause sickle cell disease.
  • Approximately 70,000 – 100,000 individuals in the United States have sickle cell disease and 3 million have sickle cell trait.

GENE THERAPY

Gene editing remains a widely controversial topic due to the large potential for both benefit and “accidents.” Despite this, scientists are still hard at work developing gene edits that can solve a wide variety of diseases.

A new study may have found the key to solving a painful, and potentially fatal, genetic defect. Researchers from Stanford used the CRISPR-Cas9 gene editing technique to fix the gene defects that cause sickle cell disease.

Sickle cell disease is actually a group of related diseases that cause the formation of hemoglobin S, or sickle-shaped hemoglobin. That shape results in red blood cells becoming tangled in each other. Enough tangling, and blood vessels could be blocked, causing pain and even death.

To correct the issue, one has to repair the genes that code for abnormal hemoglobin. Using CRISPR, the researchers were able to do this. They took hematopoietic stem cells from patients that have the disease and edited them to repair their genome.

After, the repaired stem cells are concentrated and injected to healthy mice. Once there, the stem cells find their way into bone marrow and start producing more of their healthy variants. Sixteen weeks after injection, the researchers found healthy red blood cells thriving in the bone marrow.

ONE OF MANY

This is just one of the increasing number of diseases that have met their match thanks to CRISPR. The gene editing technique is composed of an enzyme that can splice DNA sequences and guide RNA that take them to the specific sequences that need splicing.

“There’s already a lot of active research going on using the CRISPR technology to fix diseases like Duchenne muscular dystrophy or cystic fibrosis or Huntington’s disease,” says Jennifer Doudna, a pioneer of the technology, to CNBC.

But even if ways to cure using CRISPR are abound, researchers are treading lightly on implementation, since editing genes is risky and can have disastrous consequences. Ultimately, we will need to have a serious debate on how to go about the changing the very blueprint of our bodies.

First results of CRISPR gene editing of normal embryos released


Artwork of human embryo with four cells
Disease-causing mutations could be removed in early embryos

A team in China has corrected genetic mutations in at least some of the cells in three normal human embryos using the CRISPR genome editing technique. The latest study is the first to describe the results of using CRISPR in viable human embryos, New Scientist can reveal.

While this study – which attempted to repair the DNA of six embryos in total – was very small, the results suggest CRISPR works much better in normal embryos than it did in previous tests on abnormal embryos that could not develop into children.

“It is encouraging,” says Robin Lovell-Badge of the Francis Crick Institute in London, who has contributed to several major reports on human genome editing. The numbers are far too low to make strong conclusions though, he cautions.

 The CRISPR gene editing technique is a very efficient way of disabling genes, by introducing small mutations that disrupt the code of a DNA sequence. CRISPR can also be used to repair genes, but this is much more difficult.

Until now, results have only been published from experiments in which the CRISPR technique was used in abnormal embryos, made when two sperm fertilise the same egg. The idea behind this work was that it was more ethical to test the technique on embryos that could never fully develop.

In the first attempt to fix genes in human embryos, fewer than 1 in 10 cells were successfully repaired – an efficiency rate that is too low to make the method practical. A second study published in 2016 also had a low rate of efficiency. However, because these embryos were very genetically abnormal, these experiments may not have given an accurate indication of how well the technique would work in healthier embryos.

The Chinese team behind the latest study, at the Third Affiliated Hospital of Guangzhou Medical University, first carried out experiments with abnormal embryos, and found the repair rate was very low. But they had more success when they tried to repair mutations in normal embryos derived from immature eggs donated by people undergoing IVF.

Genetic disease

Immature eggs like these are usually discarded by IVF clinics, as the success rate is much lower than with mature eggs. However, children have been born from such immature eggs.

Jianqiao Liu and his team matured donated immature eggs, and fertilised each by injecting sperm from one of two men with a hereditary disease. They then injected the CRISPR machinery into these single-cell embryos before they started dividing.

The first sperm donor had a mutation called G1376T in the gene for the G6PD enzyme. This is a common cause of favism in China, a disorder in which eating certain foods such as fava beans can trigger the destruction of red blood cells.

In two of the resulting embryos, the G1376T mutation was corrected. But in one of the embryos, not all the cells were corrected. CRISPR turned off the G6PD gene in some of its cells rather than fixing it – making it what is known as a “mosaic”.

The second sperm donor had a mutation called beta41-42, which is one of the causes of the blood disease beta-thalassemia. Four of the resulting embryos carried the mutation. In one, CRISPR induced another mutation rather than fixing the beta41-42. In another, the mutation was successfully repaired in only some of the cells, creating another mosaic embryo.  It did not work at all in the other two embryos.

In total, the mutation in one embryo was corrected in every cell, and two were corrected in some of the cells.

While firm conclusions cannot be drawn based on just six embryos, these results are encouraging as they suggest CRISPR gene repair is more efficient in normal cells. “It does look more promising than previous papers,” says Fredrik Lanner of the Karolinska Institute in Sweden, whose team has begun using CRISPR to disable genes in human embryos to study embryonic development.

Preventing mosaics

Several other groups have begun editing the genomes of normal human embryos or plan to start soon. There are rumours that another three or four studies on the use of CRISPR in human embryos have been completed but not yet published. It isn’t clear why this is the case, but the controversy surrounding the area may have made both researchers and journals wary.

The results so far, however, show the technology is far from the point where it could be safely used for editing embryos.

To make it safer to use gene editing to prevent children inheriting disease-causing mutations, researchers will need to find a way to prevent mosaicism. Edited embryos would always be tested before being implanted in a woman, but if they are mosaics such tests cannot guarantee the resulting child will be disease-free.

“This would need to be solved before the methods could be used clinically to correct a disease,” says Lovell-Badge. Progress is already being made: at least two teams have already found ways of reducing the risk of mosaicism in animals.

Mosaicism could also be avoided by editing the genomes of sperm and eggs prior to IVF, rather than embryos. This is expected to become possible in people in the next few years.

There are also a few diseases where mosaicism might not matter, Lovell-Badge points out, such as metabolic liver diseases where only 20 per cent function is enough to keep people healthy.

However, a major report on gene editing by the US National Academy of Sciences recently concluded that trials of germline gene editing should be allowed only if they meet a number of criteria – the first being “the absence of reasonable alternatives”.

Yet almost all inherited diseases can already be prevented by existing forms of screening, such as testing IVF embryos and implanting only disease-free ones. There are only a small number of cases where this method – called preimplantation genetic diagnosis – will not work because none of a couple’s embryos will be disease-free.

CRISPR Gene Editing Used to Treat Lung Cancer Treatment for First Time Ever


Researchers in China have made the headlines this month as they become the first ever to make changes to a human’s genetic code. To do this, they simply injected the patient with gene-edited cells using the CRISPR technique. This is the first time ever that CRISPR has been used in this way, and as controversial as it may be, it’s still a proud moment for China.

The patient received the treatment as part of a trial to try and treat lung cancer. The ten patients taking part in the trial have all been diagnosed with metastatic non-small-cell lung cancer and have not responded to any standard treatment. They all also have a life expectancy of fewer than six months.

CRISPR is a great technique for gene editing that allows scientists to hone in on a single gene and repair it, amend it, or remove it. It’s something can be used in various areas ranging from agriculture to medicine, and this is just the beginning as far as China is concerned. The reason it hasn’t been used on any humans until now is purely down to ethics. But, Lu You and the team gained ethical approval to go ahead from West China Hospital’s review board.

China will continue to lead pave the way as far as CRISPR work is concerned, but the U.S. will soon be catching up as the first U.S. CRISPR trial is looking to be launched by the end of the year.

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