Tag Archives: gene-editing platform

Nanoparticle-based delivery platform for CRISPR-Cas9 (gene-editing technology)

A February 18, 2018 King Abdullah University of Science and Technology (KAUST; Saudi Arabia) news release (also on EurekAlert but published on Feb. 20, 2018) describes a new technology for delivering CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 into cells,

A new delivery system for introducing gene-editing technology into cells could help safely and efficiently correct disease-causing mutations in patients.

The system, developed by KAUST scientists, is the first to use sponge-like ensembles of metal ions and organic molecules to coat the molecular components of the precision DNA-editing technology known as CRISPR/Cas9, allowing efficient release of the genome-editing machinery inside the cell.

“This method presents an easy and economically feasible route to improve on the delivery problems that accompany RNA-based therapeutic approaches,” says Niveen Khashab, the associate professor of chemical sciences at KAUST who led the study. “This may permit such formulations to be eventually used for treating genetic diseases effectively in the future.”

CRISPR/Cas9 has a double delivery problem: For the gene-editing technology to work like a molecular Swiss Army knife, both a large protein (the Cas9 cutting enzyme) and a highly charged RNA component (the guide RNA used for DNA targeting) must each get from the outside of the cell into the cytoplasm and finally into the nucleus, all without getting trapped in the tiny intracellular bubbles that are known as endosomes.

To solve this problem, Khashab and her lab turned to a nano-sized type of porous material known as a zeolitic imidazolate framework, which forms a cage-like structure into which other molecules can be placed. The researchers encapsulated the Cas9 protein and guide RNA in this material and then introduced the resulting nanoparticles into hamster cells.

The encapsulated CRISPR-Cas9 constructs were not toxic to the cells. And because particles in the coating material become positively charged when absorbed into endosomes, they caused these membrane-bound bubbles to burst, freeing the CRISPR-Cas9 machinery to travel to the nucleus, home to the cell’s genome. There the gene-editing technology could get to work.

Using a guide RNA designed to target a gene that caused the cells to glow green under fluorescent light, Khashab and her team showed that they could reduce the expression of this gene by 37 percent over four days with their technology. “These cage-like structures are biocompatible and can be triggered on demand, making them smart options to overcome delivery problems of genetic materials and proteins,” says the study’s first author Shahad Alsaiari, a Ph.D. student in Khashab’s lab.

The researchers’ plan to test their system in human cells and in mice, and eventually, they hope, in clinical trials.

The zeolitic imidazolate framework forms a cage-like scaffold over the CRISPR/Cas9 machinery.. Reprinted (adapted) with permission from Alsaiari, S.K., Patil, S., Alyami, M., Alamoudi, K.O., Aleisa, F.A., Merzaban, J., Li M. & Khashab, N.M. Endosomal escape and delivery of CRISPR/Cas9 genome editing machinery enabled by nanoscale zeolitic imidazolate framework. Journal of the American Chemical Society 140, 143–146 (2018). © 2018 American Chemical Society; KAUST Xavier Pita and Heno Huang ][downloaded from https://discovery.kaust.edu.sa/en/article/475/a%250adelivery-platform-for-gene-editing-technology]

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

Endosomal Escape and Delivery of CRISPR/Cas9 Genome Editing Machinery Enabled by Nanoscale Zeolitic Imidazolate Framework by Shahad K. Alsaiari, Sachin Patil, Mram Alyami, Kholod O. Alamoudi, Fajr A. Aleisa, Jasmeen S. Merzaban, Mo Li, and Niveen M. Khashab. J. Am. Chem. Soc., 2018, 140 (1), pp 143–146 DOI: 10.1021/jacs.7b11754 Publication Date (Web): December 22, 2017

Copyright © 2017 American Chemical Society

This paper is behind a paywall.

Is there a risk of resistance to nanosilver?

Anyone who’s noticed how popular silver has become as an antibacterial, antifungal, or antiviral agent may have wondered if resistance might occur as its use becomes more common. I have two bits on the topic, one from Australia and the other from Canada.

Australia

Researchers in Australia don’t have a definitive statement on the issue but are suggesting more caution (from a March 31, 2017 news item on Nanowerk),

Researchers at the University of Technology Sydney [UTS] warn that the broad-spectrum antimicrobial effectiveness of silver is being put at risk by the widespread and inappropriate expansion of nanosilver use in medical and consumer goods.

As well as their use in medical items such as wound dressings and catheters, silver nanoparticles are becoming ubiquitous in everyday items, including toothbrushes and toothpaste, baby bottles and teats, bedding, clothing and household appliances, because of their antibacterial potency and the incorrect assumption that ordinary items should be kept “clean” of microbes.

Nanobiologist Dr Cindy Gunawan, from the ithree institute at UTS and lead researcher on the investigation, said alarm bells should be ringing at the commercialisation of nanosilver use because of a “real threat” that resistance to nanosilver will develop and spread through microorganisms in the human body and the environment.

A March 31 (?), 2017 University of Technology Sydney press release by Fiona McGill, which originated the news item, expands on the theme,

Dr Gunawan and ithree institute director Professor Liz Harry, in collaboration with researchers at UNSW [University of New South Wales] and abroad, investigated more than 140 commercially available medical devices, including wound dressings and tracheal and urinary catheters, and dietary supplements, which are promoted as immunity boosters and consumed by throat or nasal spray.

Their perspective article in the journal ACS Nano concluded that the use of nanosilver in these items could lead to prolonged exposure to bioactive silver in the human body. Such exposure creates the conditions for microbial resistance to develop.

E. coli bacteria. Photo: Flickr/NIAID

 

The use of silver as an antimicrobial agent dates back centuries. Its ability to destroy pathogens while seemingly having low toxicity on human cells has seen it widely employed, in treating burns or purifying water, for example. More recently, ultra-small (less than 10,000th of a millimetre) silver nanoparticles have been engineered for antimicrobial purposes.  Their commercial appeal lies in superior potency at lower concentrations than “bulk” silver.

“Nanosilver is a proven antimicrobial agent whose reliability is being jeopardised by the commercialisation of people’s fear of bacteria,” Dr Gunawan said.

“Our use of it needs to be far more judicious, in the same way we need to approach antibiotic usage. Nanosilver is a useful tool but we need to be careful, use it wisely and only when the benefit outweighs the risk.

“People need to be made aware of just how widely it is used, but more importantly they need to be made aware that the presence of nanosilver has been shown to cause antimicrobial resistance.”

What is also needed, Dr Gunawan said, is a targeted surveillance strategy to monitor for any occurrence of resistance.

Professor Harry said the findings were a significant contribution to addressing the global antimicrobial resistance crisis.

“This research emphasises the threat posed to our health and that of the environment by the inappropriate use of nanosilver as an antibacterial, particularly in ordinary household and consumer items,” she said.

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

Widespread and Indiscriminate Nanosilver Use: Genuine Potential for Microbial Resistance by Cindy Gunawan, Christopher P. Marquis, Rose Amal, Georgios A. Sotiriou, Scott A. Rice⊥, and Elizabeth J. Harry. ACS Nano, Article ASAP DOI: 10.1021/acsnano.7b01166 Publication Date (Web): March 24, 2017

Copyright © 2017 American Chemical Society

This paper is behind a paywall.

Meanwhile, researchers at the University Calgary (Alberta, Canada) may have discovered what could cause resistance to silver.

Canada

This April 25, 2017 news release on EurekAlert is from the Experimental Biology Annual Meeting 2017,

Silver and other metals have been used to fight infections since ancient times. Today, researchers are using sophisticated techniques such as the gene-editing platform Crispr-Cas9 to take a closer look at precisely how silver poisons pathogenic microbes–and when it fails. The work is yielding new insights on how to create effective antimicrobials and avoid the pitfalls of antimicrobial resistance.

Joe Lemire, a postdoctoral fellow at the University of Calgary, will present his work in this area at the American Society for Biochemistry and Molecular Biology annual meeting during the Experimental Biology 2017 meeting, to be held April 22-26 in Chicago.

“Our overarching goal is to deliver the relevant scientific evidence that would aid policymakers in developing guidelines for when and how silver could be used in the clinic to combat and control infectious pathogens,” said Lemire. “With our enhanced mechanistic understanding of silver toxicity, we also aim to develop novel silver-based antimicrobial therapies, and potentially rejuvenate other antibiotic therapies that bacteria have come to resist, via silver-based co-treatment strategies.”

Lemire and his colleagues are using Crispr-Cas9 genome editing to screen for and delete genes that allow certain bacterial species to resist silver’s antimicrobial properties. [emphasis mine] Although previous methods allowed researchers to identify genes that confer antibiotic resistance or tolerance, Crispr-Cas9 is the first technology to allow researchers to cleanly delete these genes from the genome without leaving behind any biochemical markers or “scars.”

The team has discovered many biological pathways involved in silver toxicity and some surprising ways that bacteria avoid succumbing to silver poisoning, Lemire said. While silver is used to control bacteria in many clinical settings and has been incorporated into hundreds of commercial products, gaining a more complete understanding of silver’s antimicrobial properties is necessary if we are to make the most of this ancient remedy for years to come.

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Joe Lemire will present this research at 12-2:30 p.m. Tuesday, April 25, [2017] in Hall F, McCormick Place Convention Center (poster B379 939.2) (abstract). Contact the media team for more information or to obtain a free press pass to attend the meeting.

About Experimental Biology 2017

Experimental Biology is an annual meeting comprised of more than 14,000 scientists and exhibitors from six host societies and multiple guest societies. With a mission to share the newest scientific concepts and research findings shaping clinical advances, the meeting offers an unparalleled opportunity for exchange among scientists from across the U.S. and the world who represent dozens of scientific areas, from laboratory to translational to clinical research. http://www.experimentalbiology.org #expbio

About the American Society for Biochemistry and Molecular Biology (ASBMB)

ASBMB is a nonprofit scientific and educational organization with more than 12,000 members worldwide. Founded in 1906 to advance the science of biochemistry and molecular biology, the society publishes three peer-reviewed journals, advocates for funding of basic research and education, supports science education at all levels, and promotes the diversity of individuals entering the scientific workforce. http://www.asbmb.org

Lemire’s co-authors for the work presented at the 2017 annual meeting are: Kate Chatfield-Reed (The University of Calgary), Lindsay Kalan (Perelman School of Medicine), Natalie Gugala (The University of Calgary), Connor Westersund (The University of Calgary), Henrik Almblad (The University of Calgary), Gordon Chua (The University of Calgary), Raymond Turner (The University of Calgary).

For anyone who wants to pursue this research a little further, the most recent paper I can find is this one from 2015,

Silver oxynitrate: An Unexplored Silver Compound with Antimicrobial and Antibiofilm Activity by Joe A. Lemire, Lindsay Kalan, Alexandru Bradu, and Raymond J. Turner. Antimicrobial Agents and Chemotherapy 05177-14, doi: 10.1128/AAC.05177-14 Accepted manuscript posted online 27 April 2015

This paper appears to be open access.