Lakshmi Supriya is a freelance science writer based in Bengaluru.
Snip, snip. The defective genes in your body are cut and replaced with a normal gene, and just like that, genetic diseases can be treated. Now, the new technology of gene editing that has taken therapeutics by storm, called CRISPR, has moved another step forward. Researchers have found a new, more efficiency, way of delivering the different components needed for the editing.
The origin of CRISPR dates back to the early 1990s, when Francisco Mojica discovered that DNA fragments of some microbes called archaea had several copies of a roughly palindromic sequences of bases – the building blocks of DNA – that were different from the sequences that are generally found in archaea. Called clustered regularly interspaced palindromic repeats (CRISPR), he found such sequences in several species of bacteria over the next decade.
More research by several groups suggested that these sequences helped the bacteria fight against attacks by viruses or other foreign bodies by incorporating parts of the viral DNA into the CRISPR sequence. These foreign DNA are called spacers and they act as a memory bank, allowing the bacteria to recognise the virus as an invader when it next attacks. Another key component of the bacteria’s defence system, the Cas9 protein, is responsible for chopping up the DNA of the invader, and is guided to the right position on the DNA by a piece of RNA.
Inspired by this natural defence mechanism in bacteria, CRISPR was modified to serve as a tool to edit genome sequences. By changing the sequence of the guiding RNA, the Cas9 protein could be directed to any desired location on the DNA. Once the particular genome sequence has been cut, either the body’s natural DNA repair mechanisms will fix it by simply gluing back the ends – or a specific sequence can be supplied to fill in the gap. A simple cut and paste that has now edited the DNA.
Although the technology is the simplest yet for gene editing, delivering all the components – Cas9, the guide RNA and the repair DNA – together is a challenge.
Cas9 is a set of proteins produced by bacteria, and delivering them has been a challenge. They cannot be simply isolated and injected into the system. Typically, a particular type of virus – known as the adeno-associated virus – has been used as a delivery vehicle for this protein. The virus was chosen because most humans are immune to it, and modified such that it can produce the protein.
However, there are several disadvantages of using a virus. One of the biggest problems is the chance of editing the genome in the wrong place because the virus does not stop producing the protein once the correction has been done at the desired location. The greater the amount of protein in the system, the greater the chance is that it will cut DNA, even if it is not at the right location. In addition, viruses are small, so a large number is required to produce sufficient editing. This issue becomes worse for human therapies because numerous copies of the virus will be required – often beyond clinically acceptable levels.
In a recent study, researchers found a way around this problem. Instead of using a virus, they used nanoparticles to deliver all the components simultaneously. “This study makes a major advance by demonstrating the delivery of all three [components], with promising results in human cells and in mice,” says Chase Beisel, a professor at North Carolina State University, who works on CRISPR systems and was not involved in the study.
In a method dubbed CRISPR-Gold, all three components can be attached around a gold nanoparticle and enclosed using a polymer, keeping it all together in one package. This package can be delivered into a variety of cells. Once it reaches a cell, the polymer breaks apart to release all the components.
“Gold is particularly easy to work with because of its facile reactivity with thiols,” says Niren Murthy, a professor of bioengineering at the University of California, Berkeley, and one of the authors of the study. Taking advantage of this fact, Murthy and colleagues designed DNA with thiol groups, which are molecules of sulphur and hydrogen. To this, they attached the repair DNA and the Cas9 proteins along with the guide RNA, ensuring that the nanoparticle now has all the components required for gene editing in a single package. Different types of cells easily take up gold nanoparticles, allowing easy delivery of the gene-editing agents.
“CRISPR-Gold and, more broadly, CRISPR-nanoparticles, open a new way for safer, accurately controlled delivery of gene-editing tools,” says Irina Conboy, a professor of bioengineering at the University of California, Berkeley, and one of the authors of the study, in a statement.
Using this system, researchers showed that they could edit genes in human embryonic stem cells and mouse muscle cells in the lab. In addition, they were able to remove a genetic mutation that causes a disease called Duchenne muscular dystrophy. This disease is a perfect candidate for the CRISPR technology because it is caused by a mutation in one specific gene, which prevents the body from producing dystrophin, a protein required for muscular development. It occurs in children and causes extreme muscular weakness. Starting with muscle loss in the legs, it progresses up the body. Within a few years, afflicted children cannot walk or stand, and may also suffer from intellectual disabilities. There is no known cure for the disease.
Injecting CRISPR-Gold into the muscles of mice afflicted with this disease significantly increased their muscle function almost twofold. Analysis showed that the mutated gene had been removed and that very low levels of untargeted DNA had been removed, about 0.005-0.2% for five different genomic DNA sequences tested.
However, the fraction of mutant DNA that was repaired is still very low, only about 0.8%. “While this frequency may be acceptable for diseases that can be reversed by editing a fraction of the cells, other diseases will require much more efficient means of editing,” says Beisel. Murthy speculates that about a 5% efficiency of repair is needed for therapeutic benefits. “We anticipate that we can achieve this by doing multiple injections,” he says.
Another concern is if the addition of a new system into the body would cause any adverse immune reactions. Testing the mice two weeks after injection showed that there had been no adverse immune reaction in them.
Although the system has effectively shown that non-viral delivery of the gene-editing components is possible, Murthy says they are working on improving it. One limitation of the method today is that it requires direct intramuscular injection to the diseased muscles. CRISPR-Gold can potentially be used for treating genetic diseases where tissue can be accessed with a direct injection. “However, developing formulations that can be injected intravenously would increase the types of diseases that could be treated with CRISPR-Gold,” he says.
Murthy also thinks that the size of the gold nanoparticles – about 100-200 nanometers wide – is too big for editing muscle after intravenous delivery. To bring the technology to market, Murthy and colleagues have formed a company called GenEdit and are now focusing on improving the rate of gene repair for muscular dystrophy.
However, Beisel imagines that the technology need not be limited to treating genetic diseases in humans. They could be used in gene therapy in other animals and plants, such as making disease-resistant plants or fatter animals for meat consumption. “Time will tell whether these nanoparticles would be effective for other cells, such as animals, plants, fungi or bacteria.”