Fungi Join the Fight Against Malaria

SLAP. You’re sitting outside at a summer bonfire and you catch a mosquito feasting on your leg. Groaning, you realize this probably means a couple days of itching and a swollen welt. Usually in North Carolina, you’re going to be just fine and should only seek medical attention if you experience complications. However, for many, particularly in Sub-Saharan Africa, a mosquito bite can mean something particularly dangerous: the possibility of infection with malaria.

Cells infected with Plasmodium falciparum possess characteristic purple dots, often connected in a “headphones” shape. Source: Steven Glenn, Laboratory and Consultation Division, USCDCP (PIXIO)

Malaria is a parasitic infection caused by Plasmodium falciparum, a single-celled organism that lives in the salivary gland of Aedes aegypti mosquitoes and is considered the deadliest human parasite. The World Health Organization reported in 2018 that progress in reducing cases of malaria globally has stalled, with nearly 435,000 people, primarily Sub-Saharan African children, dying from the disease per year. Much of this lack of progress can be attributed to mosquitoes developing a tolerance for commonly used chemical insecticides. Recent work from scientists at the University of Maryland in collaboration with researchers in Burkina Faso has hacked biology to create a new tool to combat the problem of malaria-carrying mosquitoes. The tool? Genetically modified fungus that wipes out mosquitoes with a deadly dose of spider toxin.

To develop their tool, the researchers used a type of fungus that already occurs in malaria-affected areas and targets mosquitoes: Metarhizium pingshaense. This species of fungus is entomopathogenic, which means that the fungus is a parasite to insect hosts and kills or disables them. In the wild, this fungus can kill mosquitoes, albeit quite slowly. It takes over a week in the lab for Metarhizium to kill a mosquito, by which time the mosquito could spread malaria and reproduce. To speed up the process, the scientists used genetic engineering to give Metarhizium a deadly tool: a gene from the Australian Blue Mountains Funnel-web spider that creates a potent toxin. In the wild, the spider’s fangs deliver a neurotoxin that disrupts the way neurons send signals in the brain. This causes symptoms including dangerously elevated heart rate and blood pressure and difficulty breathing. If bitten by a funnel web spider, death can occur in humans rapidly. However, to ensure the toxin only affects mosquitos, the scientists disabled the toxin from being made by the fungus until the right moment — when the fungus reaches the bloodstream of the mosquito.

The Australian Funnel Web spider is considered to be one of the deadliest spiders on Earth. Source: Brenda Clarke (flickr)
A cockroach infected with a species of Metarhizium. Source: Chengshu Wang and Yuxian Xia, PLoS Genetics Jan. 2011 (Wikimedia Creative Commons)

The fungus was tested in a unique environment in Burkina Faso: the MosquitoSphere, an enclosure where genetically modified organisms are permitted to be tested.

To conduct the experiment, two populations of 1500 mosquitoes were released in the MosquitoSphere. To expose the insects to the fungus, the investigators mixed the Metarhizium spores with sesame oil and spread the mixture onto black cotton sheets. When mosquitoes landed on the sheets, they were exposed to the toxic fungal spores. The mosquito population exposed to the unmodified Metarhizium had no problem reproducing before the fungus proved fatal, with their population totaling nearly 2500 after 45 days. In contrast, the population of mosquitoes exposed to the genetically modified Metarhizium after 45 days shrunk to a mere 13 individuals. A population of 13 mosquitoes indicated that the mosquitoes were dying more quickly than they could breed, therefore producing an unsustainable, or collapsed population. The striking results of the experiments in the MosquitoSphere encouraged the investigators to conclude that the genetically modified Metarhizium can function as a potent mosquito-killing bio-pesticide and cause an interbreeding colony of mosquitoes to collapse in a little over a month.

Despite success in the MosquitoSphere, the fungus still needs to undergo more testing to be considered safe for humans and the environment. If it is approved, a formulation of the fungal spores could be used to protect dwellings and mosquito netting from malaria-carrying mosquitoes. This study is a promising sign for the use of genetically modified bio-pesticides to wipe out disease-causing insects, which could prove invaluable for malaria prevention. 

Peer editor: Elise Hickman

Are some of us “immune” to genome editing?

https://www.flickr.com/photos/nihgov/27669625144

Some of us may have characteristics we would like to change. We may want to be taller, skinnier, or smarter. While many traits can be changed during our lifetime, others simply cannot. There are certain ‘fates’ we cannot avoid, such as inherited diseases. For example, if you inherit two copies of a gene called CFTR that has mutations, you are likely to develop Cystic Fibrosis. Although your life choices can help accommodate the disease, there is currently no cure for Cystic Fibrosis and many other genetic disorders. However, with the development of genome editing, the potential to alter the entirety of our genetic information, there is hope in the ability to one day eradicate genetic diseases once and for all.

There are currently several genome editing methods available, but the most popular method is the CRISPR-Cas9 system, which was recently and famously used to edit the germline of babies born in China. Editing the germline means that the changes made to the genome will be inherited throughout following generations. Although this practice is still officially banned around the world, CRISPR-Cas9 was used to change two twins’ genomes. This tool allows us to edit parts of the genome by either removing, adding, or changing a given segment of DNA sequence. It is the go-to editing technique used by scientists because it can generate changes quickly, efficiently, and accurately  at a relatively low cost. There are two major components of this system – Cas9 and a guide RNA (gRNA). Cas9 is an enzyme that acts as ‘molecular scissors’ and cuts both strands of DNA at a specific location, which allows the removal, addition, or change of DNA sequence. The sequence where Cas9 cuts is determined by the gRNA, which, as its name implies, is an RNA molecule that ‘guides’ Cas9 to a specific place in the genome through complementary base pairing.

The CRISPR-Cas9 system was originally discovered in bacteria as a defense mechanism against viral infection. The most commonly used versions of Cas9 for genome editing come from two bacterial species – Staphylococcus aureus and Streptococcus pyogenes. Both species of bacteria frequently live on the body as harmless colonizers, but in some instances, they can cause disease. If you have been infected with either S. aureus or S. pyogenes, you likely developed adaptive immunity to the bacterium in order to protect yourself against future infections. A recent study from Stanford University published in the journal Nature Medicine examined this phenomenon, asking whether healthy humans possessed adaptive immunity against Cas9 from S. aureus and S. pyogenes.

First off, you may be wondering why adaptive immunity to Cas9 would matter at all. When we are exposed to ‘invaders,’ such as bacteria, our body develops adaptive immunity in the form of antibodies and T cells that target specific components (also called antigens) of the invader. For example, if you’ve eaten raw eggs (in cookie dough, let’s say), you may have suffered from an upset stomach caused by infection with Salmonella. If you are then exposed to Salmonella again in your lifetime, both antibodies and T cells that developed in response to the first infection should act quickly to suppress and clear the infection. Similarly, if you were previously infected with S. aureus, your body may negatively respond to receiving modified human cells producing Cas9 originating from S. aureus for the purpose of genome editing but the antibodies and T cells generated during the initial infection may quickly clear these cells. Our own body may think it is protecting us, although in reality it may be thwarting our efforts to make a change in our genome that is intended to have a positive effect. Even worse, this unintentional stimulation of our immune system could lead to an overwhelming response that can lead to organ failure and potentially death. Unfortunately, there is a documented case of a patient death due to an immune response to gene therapy using viruses. It may therefore be necessary to screen each patient before the start of therapy to determine whether they possess immune components that would make this form of therapy inefficient or even deadly.

The Stanford study mentioned above examined this phenomenon by testing for the presence of both antibodies and T cells against Cas9 from S. aureus and S. pyogenes in healthy donors. The authors found that ~75% of donors produced antibodies against Cas9 from S. aureus and ~60% of donors produced antibodies against Cas9 from S. pyogenes. These results come from analyzing blood from more than 125 adult donors. The scientists also used several approaches to examine T cell responses. In this case, 78% and 67% of donors were positive for antigen-specific T cells again Cas9 from S. aureus or S. pyogenes, respectively.

In summary, the authors of this study provide evidence for existing adaptive immunity to Cas9 from S. aureus and S. pyogenes in a large number of healthy adults. Two additional studies examined adaptive immunity against Cas9 with varying results. However, all three studies suggest that pre-existing immunity to Cas9 needs to be carefully considered and addressed before the CRISPR-Cas9 system moves toward clinical trials as a therapy for genetic diseases. In other words, scientists need to address whether the presence of antibodies and/or T cells against Cas9 would lead to destruction of cells carrying Cas9 for the purposes of genome editing. Other potential solutions include using Cas9 derived from bacterial species that do not cause human infections and thus should not trigger an adaptive immune response, or suppressing the immune system while administering CRISPR-Cas9.

CRISPR-Cas9 genome editing has the potential to transform our lives in many ways. While this technology certainly holds promise for the treatment of genetic disease, the Stanford study highlights the need for more research before we can conclude that CRISPR-Cas9 is effective and safe. Optimization of this system may improve its performance in clinical trials and lead to future approval for the treatment of human disease.

Peer edited by Keean Braceros and Isabel Newsome

Your Guide to the CRISPR Babies

Imagine a future in which we can edit genes like a sentence in Microsoft Word. We could highlight, delete, and correct a section of a gene known to cause disease, virtually eliminating the devastating genetic illnesses that cost the world billions of dollars and countless hours of heartache every year. This is a future envisioned by many scientists working on the CRISPR-Cas9 system of gene editing. These researchers have used the technology to cure everything from liver disease to cataracts in mice. Ethical concerns have limited these experiments to model organisms and until recently, on-demand gene editing in humans seemed like more of a science fiction fantasy than a potential reality. All of that appears to have changed, however, with a Chinese scientist named He Jiankui claiming to have successfully used CRISPR to delete an HIV-related gene in two human babies late last year. This claim has stirred up quite a bit of controversy, to put it lightly, in the scientific community, but why is it such a big deal? Let’s take a closer look at the experiment and the hopes and concerns it brings to light.

A CRISPR introduction

CRISPR, short for CRISPR-Cas9, stands for “clusters of regularly interspaced palindromic repeats.” The major player in the CRISPR system is Cas9, which is a protein that acts like molecular scissors to chop up DNA. This system first evolved as a bacterial defense mechanism that targeted Cas9 to common sequences found in viruses, allowing the bacteria to chew up viral DNA and prevent infection. However, scientists then discovered that Cas9 could be targeted to just about any sequence of DNA they wanted. The secret lies in how Cas9 targets specific DNA sequences: the protein works together with a piece of RNA called guide RNA that matches up to the target DNA sequence. Modify this RNA and you can cause Cas9 to cut up whatever DNA you choose. Thanks to DNA repair, scientists can even provide a template sequence, say to correct a bad version of the gene, that can be incorporated into the DNA cut by Cas9 to fix a genetic mutation. These manipulations can be done in cells in a dish or even by injecting the CRISPR components into animals.

An overview of using CRISPR to delete a segment of DNA. Made using biorender.io.

The first CRISPR humans?

Until last November, using CRISPR to induce inheritable changes in live organisms was restricted to animals like fruit flies and mice. However, on November 26, 2018, the Associated Press reported that Chinese scientist He Jiankui claimed to have successfully used CRISPR to delete a gene associated with HIV infection in two human embryos. The edited embryos were created using in vitro fertilization (IVF), checked for successful editing after CRISPR treatment, and implanted back into the women who donated them. Allegedly, the edited embryos resulted in the birth of healthy twin girls. The gene Jiankui claims to have deleted encodes the protein CCR5, a human cell surface receptor that the HIV virus requires to infect immune cells. Essentially, deletion of CCR5 results in no HIV infection. This manipulation, Jiankui says, could protect the children from acquiring HIV and has broad implications for the management of HIV from a public health perspective. From a basic science perspective, the birth of children from CRISPR-edited embryos is an incredible achievement. However, as with almost every method of genome editing, the ethical controversy of Jiankui’s work has become just as important as the scientific implications.

Ethical and scientific concerns

One of the largest outstanding questions surrounding the alleged CRISPR babies is the fact that they are just that: alleged. Jiankui has not published the results of his manipulations in a peer-reviewed scientific journal and the identities of the children’s parents have been kept private to protect them from media scrutiny and potential backlash. However, let’s say that Jiankui actually did successfully delete the CCR5 gene in embryos that went on to become healthy human babies. What’s the problem with that? First, although some people have naturally occurring mutations that inactivate CCR5, the changes that Jiankui made in the embryos do not appear to mimic those natural mutations, leading to concerns about potential unintended consequences. Additionally, a well-known issue with CRISPR is its potential for off-target effects, or making changes in genes other than the targeted one. Finally, even if CCR5 was correctly inactivated with no off-target effects, the naturally occurring inactivating CCR5 mutations have been shown to have negative health effects, such as increasing the risk for infection and complications of West Nile virus. The scientific consensus is that using CRISPR to make such drastic, inheritable changes is unsafe simply because we don’t know enough about it yet. Ethically, a large concern with gene editing in humans is the old slippery slope argument: if we can delete disease-causing genes, what’s to stop people from editing embryos to select for traits like eye color or intelligence? Along the same lines, proponents of CRISPR gene editing in humans speak of “curing” conditions with a probable genetic basis like autism. However, advocacy organizations like Autism Speaks say this is a fundamental misunderstanding of this complex condition and that many people with autism view it as an inextricable part of themselves, not a disease to be cured. For these reasons and more, the larger scientific community has condemned Jiankui’s alleged experiments as risky and unethical. More extensive experiments in model organisms and strict ethical guidelines are needed before scientists can even think about bringing CRISPR into the mainstream.  Although this technology seems like it could be a magic bullet for genetic editing, it’s clear that the way forward is uncertain and each new advance creates more questions than it does answers. For now, at least, a world of CRISPR on-demand is still a distant future.

Peer edited by Rachel Battaglia and Breanna Turman.

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Putting the Brakes on CRISPR

CRISPR-Cas9, more commonly referred to as CRISPR, has been one of the hottest terms in science over the last few years. For goodness sake, Jennifer Lopez is the executive producer of the prospective NBC bio-terror drama CRISPR, which is centered around the gene-editing technique. Starting in 2012, CRISPR began its rise to becoming the newest and most promising gene-editing tool. It has since transformed the way many labs conduct research and has turned into a multi-billion dollar industry. Even though CRISPR has become more mainstream with seemingly limitless applications, recent research has shown we must move forward cautiously and be patient as this technology matures. 

 

CRISPR Therapeutics currently is the largest biotechnology company that specializes in gene editing via CRISPR. After initially opening at $15 per share on October 19, 2016, CRISPR Therapeutics climbed to $74 per share and had a market cap of $3.47 billion on May 30, 2018. The company’s surge was in response to their announcement in April 2018 that they were moving forward with an Investigational New Drug (IND) application with their partner Vertex Pharmaceuticals Inc. The two companies also had plans to start a Phase 1/2 clinical trial to treat adults with sickle cell disease using CRISPR gene-editing technology in both the United States and Europe. On May 30, 2018, CRISPR Therapeutics announced that the FDA put a clinical hold on its IND application pending the resolution of certain questions by the FDA. The announcement offers no insight into the questions or concerns of the FDA, but this news was enough to spook investors. CRISPR Therapeutics’ stock fell 20.2% in June and 19.1% in July. Currently, it is trading about $48 per share and its market cap is valued at $2.295 billion.

Only part of the CRISPR Therapeutics decline can be attributed to the FDA’s announcement. Three separate studies published this summer negatively influenced investors outlook on CRISPR Therapeutics, as well as the two other major biotechs specializing in CRISPR, Editas Medicine and Intellia Therapeutics. The first study, which was published June 11 in Nature Medicine, highlights how double stranded breaks in DNA caused by Cas9, the molecular scissors of the CRISPR-Cas9 system, are toxic to human pluripotent stem cells (hPSCs). hPSCs, cell lines similar to an early embryo that are capable of differentiating into other cell types, depend on the tumor suppressor protein p53 for its toxic response to prevent growth of aberrant cells. Given that p53 mutations are prevalent in hPSCS, there is also a concern that hPSCs engineered using CRISPR-Cas9 could cause cancer. A second study, published in the same issue of Nature Medicine on June 11, used human retinal pigment epithelial cells and reached a conclusion similar to the previous study, CRISPR-Cas9 induces a p53-dependent DNA damage response. In addition, this group also found CRISPR-Cas9 causes cell cycle arrest. Both studies clearly indicate that it is crucial to monitor p53 function when developing cell-based therapies using CRISPR-Cas9.

A third study, published July 16 in Nature Biotechnology, further casts a cloud of uncertainty over CRISPR. This study revealed that CRISPR-Cas9 causes “significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitor and a human differentiated cell line.” Even though these three articles were all released in the last two months and were the primary reason investors in CRISPR-based companies have been more reluctant to invest, two other studies also show negative effects of CRISPR. One shows an adaptive immune response to Cas9 and the other shows major genomic rearrangements from in a mouse model following the use of CRISPR-Cas9 also show negative effects of CRISPR.

Even though it has been a disappointing summer for companies specializing in CRISPR, CRISPR Therapeutics, Editas Medicine, and Intellia Therapeutics remain adamant that the future looks bright. For many people, these studies might be more a speed bump rather than a road block.

Peer edited by Justine Grabiec.

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