Tag Archives: muscular dystrophy

Lifesaving moths and nanomagnets

Rice University bioengineers use a magnetic field to activate nanoparticle-attached baculoviruses in a tissue. The viruses, which normally infect alfalfa looper moths, are modified to deliver gene-editing DNA code only to cells that are targeted with magnetic field-induced local transduction. Courtesy of the Laboratory of Biomolecular Engineering and Nanomedicine

Kudos to whomever put that diagram together! That’s a lot of well conveyed information.

Now for the details about how this technology might save lives. From a November 13, 2018 news item on Nanowerk,

A new technology that relies on a moth-infecting virus and nanomagnets could be used to edit defective genes that give rise to diseases like sickle cell, muscular dystrophy and cystic fibrosis.

Rice University bioengineer Gang Bao has combined magnetic nanoparticles with a viral container drawn from a particular species of moth to deliver CRISPR/Cas9 payloads that modify genes in a specific tissue or organ with spatial control.

A November 12, 2018 Rice University news release (also on EurekAlert published on November 13, 2018), which originated the news item, provides detail,

Because magnetic fields are simple to manipulate and, unlike light, pass easily through tissue, Bao and his colleagues want to use them to control the expression of viral payloads in target tissues by activating the virus that is otherwise inactivated in blood.

The research appears in Nature Biomedical Engineering. In nature, CRISPR/Cas9 bolsters microbes’ immune systems by recording the DNA of invaders. That gives microbes the ability to recognize and attack returning invaders, but scientists have been racing to adapt CRISPR/Cas9 to repair mutations that cause genetic diseases and to manipulate DNA in laboratory experiments.

CRISPR/Cas9 has the potential to halt hereditary disease – if scientists can get the genome-editing machinery to the right cells inside the body. But roadblocks remain, especially in delivering the gene-editing payloads with high efficiency.

Bao said it will be necessary to edit cells in the body to treat many diseases. “But efficiently delivering genome-editing machinery into target tissue in the body with spatial control remains a major challenge,” Bao said. “Even if you inject the viral vector locally, it can leak to other tissues and organs, and that could be dangerous.”

The delivery vehicle developed by Bao’s group is based on a virus that infects Autographa californica, aka the alfalfa looper, a moth native to North America. The cylindrical baculovirus vector (BV), the payload-carrying part of the virus, is considered large at up to 60 nanometers in diameter and 200-300 nanometers in length. That’s big enough to transport more than 38,000 base pairs of DNA, which is enough to supply multiple gene-editing units to a target cell, Bao said.

He said the inspiration to combine BV and magnetic nanoparticles came from discussions with Rice postdoctoral researcher and co-lead author Haibao Zhu, who learned about the virus during a postdoctoral stint in Singapore but knew nothing about magnetic nanoparticles until he joined the Bao lab. The Rice team had previous experience using iron oxide nanoparticles and an applied magnetic field to open blood vessel walls just enough to let large-molecule drugs pass through.

“We really didn’t know if this would work for gene editing or not, but we thought, ‘worth a shot,'” Bao said.

The researchers use the magnetic nanoparticles to activate BV and deliver gene-editing payloads only where they’re needed. To do this, they take advantage of an immune-system protein called C3 that normally inactivates baculoviruses.

“If we combine BV with magnetic nanoparticles, we can overcome this deactivation by applying the magnetic field,” Bao said. “The beauty is that when we deliver it, gene editing occurs only at the tissue, or the part of the tissue, where we apply the magnetic field.”

Application of the magnetic field allows BV transduction, the payload-delivery process that introduces gene-editing cargo into the target cell. The payload is also DNA, which encodes both a reporter gene and the CRISPR/Cas9 system.

In tests, the BV was loaded with green fluorescent proteins or firefly luciferase. Cells with the protein glowed brightly under a microscope, and experiments showed the magnets were highly effective at targeted delivery of BV cargoes in both cell cultures and lab animals.

Bao noted his and other labs are working on the delivery of CRISPR/Cas9 with adeno-associated viruses (AAV), but he said BV’s capacity for therapeutic cargo is roughly eight times larger. “However, it is necessary to make BV transduction into target cells more efficient,” he said.

Here’s a link to and a citation for the paper,

Spatial control of in vivo CRISPR–Cas9 genome editing via nanomagnets by Haibao Zhu, Linlin Zhang, Sheng Tong, Ciaran M. Lee, Harshavardhan Deshmukh, & Gang Bao. Nature Biomedical Engineering (2018) DOI: https://doi.org/10.1038/s41551-018-0318-7 Published: 12 November 2018

This paper is behind a paywall.

Minimalist DNA nanodevices perform bio-analytical chemistry inside live cells

A comparison of minimalist versus baroque architecture is one of the more startling elements in this March 24, 2016 news item on Nanowerk about a scientist working with DNA (deoxyribonucleic acid) nanodevices,

Some biochemistry laboratories fashion proteins into complex shapes, constructing the DNA nanotechnological equivalent of Baroque or Rococo architecture. Yamuna Krishnan, however, prefers structurally minimalist devices.

“Our lab’s philosophy is one of minimalist design,” said Krishnan, a professor of chemistry at the University of Chicago. “It borders on brutalist. Functional with zero bells and whistles. There are several labs that design DNA into wonderful shapes, but inside a living system, you need as little DNA as possible to get the job done.”

That job is to act as drug-delivery capsules or as biomedical diagnostic tools.

A March 24, 2016 University of Chicago news release by Steve Koppes, which originated the news item, provides some background information before launching into the latest news,

In 2011, Krishnan and her group, then at the National Centre for Biological Sciences in Bangalore, India, became the first to demonstrate the functioning of a DNA nanomachine inside a living organism. This nanomachine, called I-switch, measured subcellular pH with a high degree of accuracy. Since 2011, Krishnan and her team have developed a palette of pH sensors, each keyed to the pH of the target organelle.

Last summer, the team reported another achievement: the development of a DNA nanosensor that can measure the physiological concentration of chloride with a high degree of accuracy.

“Yamuna Krishnan is one of the leading practitioners of biologically oriented DNA nanotechnology,” said Nadrian Seeman, the father of the field and the Margaret and Herman Sokol Professor of Chemistry at New York University. “These types of intracellular sensors are unique to my knowledge, and represent a major advance for the field of DNA nanotechnology.”

Chloride sensor

Chloride is the single most abundant, soluble, negatively charged molecule in the body. And yet until the Krishnan group introduced its chloride sensor—called Clensor—there was no effective and practical way to measure intracellular stores of chloride.

“What is especially interesting about this sensor is that it is completely pH independent,” Seeman said, a significant departure from Krishnan’s previous scheme. “She spent a number of years developing pH sensors that work intra-cellularly and provide a fluorescent signal as a consequence of a shift in pH.”

The ability to record chloride concentrations is important for many reasons. Chloride plays an important role in neurobiology, for example. But calcium and sodium—both positively charged ions—tend to grab most of the neurobiological glory because of their role in neuron excitation.

“But if you want your neuron to fire again, you have to bring it back to its normal state. You have to stop it firing,” Krishnan said. This is called “neuronal inhibition,” which chloride does.

“It’s important in order to reset your neuron for a second round of firing, otherwise we would all be able to use our brains only once,” she said.

Under normal circumstances, the transport of chloride ions helps the body produce thin, freely flowing mucus. But a genetic defect results in a life-threatening disease: cystic fibrosis. Clensor’s capacity to measure and visualize protein activity of molecules like the one related to cystic fibrosis transmembrane could lead to high-throughput assays to screen for chemicals that would restore normal functioning of the chloride channel.

Nine diseases

“One could use this to look at chloride ion channel activity in a variety of diseases,” Krishnan said. “Humans have nine chloride ion channels, and the mutation of each of these channels results in nine different diseases.” Among them are osteopetrosis, deafness, muscular dystrophy and Best’s macular dystrophy.

The pH-sensing capabilities of the I-switch, meanwhile, are important because cells contain multiple organelles that maintain specific values of acidity. Cells need these different microenvironments to carry out specialized chemical reactions.

“Each subcellular organelle has a specific resting value of acidity, and that acidity is crucial to its function,” Krishnan said. “When the pH is not the value that it’s meant to be, it results in a range of different diseases.”

There are 70 rare diseases called lysosomal storage disorders, which are progressive and often fatal. Each one—including Batten disease, Niemann-Pick disease, Pompe disease and Tay-Sachs disease—represents a different way a lysosome can go bad. She likened a defective lysosome to a garbage bin that never gets emptied.

“The lysosome is basically responsible for chewing up all the garbage and making sure it’s either reused or got rid of. It’s the most acidic organelle in the cell.” And that acidity is crucial for the degradation process.

Although there are 70 lysosomal storage diseases, small molecule drugs are available for only a few of them. These existing treatments—enzyme-replacement therapies—are expensive and are only palliative treatments. One goal of Krishnan’s group is to demonstrate the utility of their pH sensors to discover new biological insights into these diseases. Developing small molecule drugs—which are structurally simpler and easier to manufacture than traditional biological drugs—could help significantly.

“If we can do this for one or two lysosomal diseases, there’ll be hope for the other 68,” Krishnan said.

Here are links to and citations for the 2015 and 2011 papers,

A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells by Sonali Saha, Ved Prakash, Saheli Halder, Kasturi Chakraborty, & Yamuna Krishnan. Nature Nanotechnology 10, 645–651 (2015)  doi:10.1038/nnano.2015.130 Published online 22 June 2015

An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism by Sunaina Surana, Jaffar M. Bhat, Sandhya P. Koushika, & Yamuna Krishnan. Nature Communications 2, Article number: 340  doi:10.1038/ncomms1340 Published 07 June 2011

The 2015 paper is behind a paywall but the 2011 paper is open access.