Tag Archives: Sabrina Richards

Hit and run gene therapy?

The approach looks promising but there’s a still long way to go before this ‘simpler, gentler’ approach to gene therapy will make its way into any treatments. From an August 30, 2017 news item on Nanowerk,

A new biomedical tool using nanoparticles that deliver transient gene changes to targeted cells could make therapies for a variety of diseases — including cancer, diabetes and HIV — faster and cheaper to develop, and more customizable.

The tool, developed by researchers at Fred Hutchinson Cancer Research Center and tested in preclinical models, is described in a paper published August 30 [2017] in Nature Communications.

This animation demonstrates the approach,

Biodegradable nanoparticles (orange) carry short-lived gene therapy to specific cells (light teal). Animation by Kimberly Carney / Fred Hutch News Service

An August 30, 2017 Fred Hutchinson Cancer Research Center (Fred Hutch) news release (from news release received via email; also on EurekAlert) by Sabrina Richards, which originated the news item, elucidates further (Note: Some links and notes have been removed),

“Our goal is to streamline the manufacture of cell-based therapies,” said lead author DR. MATTHIAS STEPHAN [6], a faculty member in the Fred Hutch Clinical Research Division and an expert in developing biomaterials. “In this study, we created a product where you just add it to cultured cells and that’s it — no additional manufacturing steps.”

Stephan and his colleagues developed a nanoparticle delivery system to extend the therapeutic potential of messenger RNA, which delivers molecular instructions from DNA to cells in the body, directing them to make proteins to prevent or fight disease.

The researchers’ approach was designed to zero in on specific cell types — T cells of the immune system and blood stem cells — and deliver mRNA directly to the cells, triggering short-term gene expression. It’s called “hit-and-run” genetic programming because the transient effect of mRNA does not change the DNA, but it is enough to make a permanent impact on the cells’ therapeutic potential.

Stephan and colleagues used three examples in the Nature Communications paper to demonstrate their technology:

* Nanoparticles carried a gene-editing tool to T cells of the immune system that snipped out their natural T-cell receptors, and then was paired with genes encoding a “chimeric antigen receptor” or CAR, a synthetic molecule designed to attack cancer.
* Targeted to blood stem cells, nanoparticles were equipped with mRNA that enabled the stem cells to multiply and replace blood cancer cells with healthy cells when used in bone marrow transplants.
* Nanoparticles targeted to CAR-T cells and containing foxo1 mRNA, which signals the anti-cancer T cells to develop into a type of “memory” cell that is more aggressive and destroys tumor cells more effectively and maintains anti-tumor activity longer.

Other attempts to engineer mRNA into disease-fighting cells have been tricky. The large messenger molecule degrades quickly before it can have an effect, and the body’s immune system recognizes it as foreign — not coming from DNA in the nucleus of the cell — and destroys it.

Stephan and his Fred Hutch collaborators devised a workaround to those hurdles.

“We developed a nanocarrier that binds and condenses synthetic mRNA and protects it from degradation,” Stephan said. The researchers surrounded the nanoparticle with a negatively charged envelope with a targeting ligand attached to the surface so that the particle selectively homes in and binds to a particular cell type.

The cells swallow up the tiny carrier, which can be loaded with different types of manmade mRNA. “If you know from the scientific literature that a signaling pathway works in synergy, you could co-deliver mRNA in a single nanoparticle,” Stephan said. “Every cell that takes up the nanoparticle can express both.”

The approach involves mixing the freeze-dried nanoparticles with water and a sample of cells. Within four hours, cells start showing signs that the editing has taken effect. Boosters can be given if needed. Made from a dissolving biomaterial, the nanoparticles are removed from the body like other cell waste.

“Just add water to our freeze-dried product,” Stephan said. Since it’s built on existing technologies and doesn’t require knowledge of nanotechnology, he intends for it to be an off-the-shelf way for cell-therapy engineers to develop new approaches to treating a variety of diseases.

The approach could replace labor-intensive electroporation, a multistep cell-manufacturing technique that requires specialized equipment and clean rooms. All the handling ends up destroying many of the cells, which limits the amount that can be used in treatments for patients.

Gentler to cells, the nanoparticle system developed by the Fred Hutch team showed that up to 60 times more cells survive the process compared with electroporation. This is a critical feature for ensuring enough cells are viable when transferred to patients.

“You can imagine taking the nanoparticles, injecting them into a patient and then you don’t have to culture cells at all anymore,” he said.

Stephan has tested the technology is cultured cells in the lab, and it’s not yet available as a treatment. Stephan is looking for commercial partners to move the technology toward additional applications and into clinical trials where it could be developed into a therapy.

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

Hit-and-run programming of therapeutic cytoreagents using mRNA nanocarriers by H. F. Moffett, M. E. Coon, S. Radtke, S. B. Stephan, L. McKnight, A. Lambert, B. L. Stoddard, H. P. Kiem, & M. T. Stephan. Nature Communications 8, Article number: 389 (2017) doi:10.1038/s41467-017-00505-8 Published online: 30 August 2017

This paper is open access.

Nanodiamonds as imaging devices

Two different teams have recently published studies in Science magazine (Feb. 1, 2013 issue) about their work with nanodiamonds, flaws, and imaging in what seems to be a case of synchronicity as there are no obvious connections between the teams.

Sabrina Richards writes in her Jan. 31, 2013 article for The Scientist about the possibility of taking snapshots of molecules at some time in the future (Note: Links have been removed),

A miniscule diamond flaw—just two atoms different—could someday enable researchers to image single molecules without resorting to time-consuming and technically exacting X-ray crystallography. The new approach, published today (January 31 [sic]) in Science, relies on a single electron to detect perturbation in molecular magnetic fields, which can provide clues about the structures of proteins and other molecules.

The work was inspired by magnetic resonance imaging (MRI), which uses electromagnetic coils to detect the magnetic fields emitted by hydrogen atom protons.  But traditional MRI requires many trillions of protons to get a clear image—of a brain, for example—preventing scientists from visualizing anything much smaller than millimeters-wide structures. To detect just a few protons, such as those of a single molecule, scientists would need an atomic-scale sensor.

To construct such a sensor, physicists Daniel Rugar at IBM Research and David Awschalom at the University of California, Santa Barbara, turned to diamonds. A perfect diamond, made entirely of carbon atoms covalently bonded to each other, has no free electrons and therefore no magnetic properties, explained Hammel. But a special kind of defect, known as a nitrogen-vacancy (NV) center, confers unique magnetic properties.

Jyllian Kemsley’s Jan. 31, 2013 article for C&EN (Chemical and Engineering News) discusses the work from both teams and describes the technique they used,

To downscale NMR [aka MRI], both groups used a detector made of diamond with a site defect called a single nitrogen-vacancy (NV) center, in which a nitrogen atom and a lattice hole replace two adjacent carbon atoms. Prior work had determined that NV centers are sensitive to the internal magnetic fields of the diamond. The new research demonstrates that the fluorescence of such centers can be used to detect magnetic fields emanating from just outside the diamond. Both groups were able to use NV centers to detect nuclear polarization of hydrogens in poly(methyl methacrylate) with a sample volume lower limit of about (5 nm)3. Further development is necessary to extract structural information.

Still, nothing much has happened with this technique as Richards notes in her article,

So far, the study is “just a proof of principle,” noted Awschalom. The researchers haven’t actually imaged any molecules yet, but simply detected their presence. Still, Awschalom said, “we’ve shown it’s not a completely ridiculous idea to detect external nuclear magnetic fields with one electron.” …

Here’s a citation and a link to the article,

Nanoscale Nuclear Magnetic Resonance with a Nitrogen-Vacancy Spin Sensor by H. J. Mamin, M. Kim, M. H. Sherwood, C. T. Rettner, K. Ohno, D. D. Awschalom, D. Rugar. Science 1 February 2013: Vol. 339 no. 6119 pp. 557-560 DOI: 10.1126/science.1231540

The other research is described in a Feb. 14, 2013 news item on Azonano,

Magnetic resonance imaging (MRI) reveals details of living tissues, diseased organs and tumors inside the body without x-rays or surgery. What if the same technology could peer down to the level of atoms? Doctors could make visual diagnoses of a person’s molecules – examining damage on a strand of DNA, watching molecules misfold, or identifying a cancer cell by the proteins on its surface.

It is remarkably  similar work as Kemsley notes not helped by the fact that the one line description for both articles in Science magazine’s Table of Contents is identical.  (One line description: The optical response of the spin of a near-surface atomic defect in diamond can be used to sense proton magnetic fields.) The City College of New York City Feb. 13, 2013 news release, which originated the Azonano news item about the other team, offers more details,

 … Dr. Carlos Meriles, associate professor of physics at The City College of New York, and an international team of researchers at the University of Stuttgart and elsewhere have opened the door for nanoscale MRI. They used tiny defects in diamonds to sense the magnetic resonance of molecules. They reported their results in the February 1 [2013] issue of Science.

“It is bringing MRI to a level comparable to an atomic force microscope,” said Professor Meriles, referring to the device that traces the contours of atoms or tugs on a molecule to measure its strength. A nanoscale MRI could display how a molecule moves without touching it.

“Standard MRI typically gets to a resolution of 100 microns,” about the width of a human hair, said Professor Meriles. “With extraordinary effort,” he said, “it can get down to about 10 microns” – the width of a couple of blood cells. Nanoscale MRI would have a resolution 1,000 to 10,000 times better.

To try to pick up magnetic resonance on such a small scale, the team took advantage of the spin of protons in an atom, a property usually used to investigate quantum computing. In particular, they used minute imperfections in diamonds.

Diamonds are crystals made up almost entirely of carbon atoms. When a nitrogen atom lodges next to a spot where a carbon atom is missing, however, it creates a defect known as a nitrogen-vacancy (NV) center.

“These imperfections turn out to have a spin – like a little compass – and have some remarkable properties,” noted Professor Meriles. In the last few years, researchers realized that these NV centers could serve as very sensitive sensors. They can pick up the magnetic resonance of nearby atoms in a cell, for example. But unlike the atoms in a cell, the NVs shine when a light is directed at them, signaling what their spin is. If you illuminate it with green light it flashes red back.

“It is a form of what is called optically detected magnetic resonance,” he said. Like a hiker flashing Morse code on a hillside, the sensor “sends back flashes to say it is alive and well.”

“The NV can also be thought of as an atomic magnet. You can manipulate the spin of that atomic magnet just like you do with MRI by applying a radio frequency or radio pulses,” Professor Meriles explained. The NV responds. Shine a green light at it when the spin is pointing up and it will respond with brighter red light. A down spin gives a dimmer red light.

In the lab, graduate student Tobias Staudacher — the first author in this work — used NVs that had been created just below the diamond’s surface by bombarding it with nitrogen atoms. The team detected magnetic resonance within a film of organic material applied to the surface, just as one might examine a thin film of cells or tissue.

“Ultimately,” said Professor Meriles, “One will use a nitrogen-vacancy mounted on the tip of an atomic force microscope – or an array of NVs distributed on the diamond surface – to allow a scanning view of a cell, for example, to probe nuclear spins with a resolution down to a nanometer or perhaps better.”

Here’s a citation and a link to this team’s study,

Nuclear Magnetic Resonance Spectroscopy on a (5-Nanometer)3 Sample Volume by T. Staudacher, F. Shi, S. Pezzagna, J. Meijer, J. Du, C. A. Meriles, F. Reinhard1, J. Wrachtrup. Science 1 February 2013: Vol. 339 no. 6119 pp. 561-563 DOI: 10.1126/science.1231675

Both articles are behind paywalls.