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Lipid Nanoparticles Enable In Vivo Delivery of Genome-Editing Proteins


NEW YORK (GenomeWeb) – Scientists from Tufts University and Harvard University have demonstrated delivery of pre-complexed CRISPR/Cas9-guide RNA ribonuceloproteins using lipid nanoparticle technology. They also used the low-toxicity technology to deliver Cre recombinase to mouse brain in vivo, suggesting that it could be used to deliver CRISPR/Cas9 for in vivo therapeutic applications.

Led by first author Ming Wang and co-senior author Qiaobing Xu of Tufts and co-senior author David Liu of Harvard, the researchers successfully delivered two genome-editing proteins into mammalian cells using synthesized, self-assembling, bioreducible, lipid-like nanoparticles.

Using these lipid nanoparticles to deliver a ribonucleoprotein (RNP) consisting of a Cas9 protein complexed with a guide RNA (gRNA), they were able to achieve genome-editing efficiencies comparable with commercially available lipid-based transfection systems, in particular Thermo Fisher Scientific's Lipofectamine 2000 (LPF2K). Three of the combinatorially synthesized lipid nanoparticles delivered Cas9-gRNA RNPs to reach editing efficiencies approaching 70 percent of cells or more, while transfection with LPF2K led to editing efficiencies of approximately 75 percent.

The scientists published their results last week in the Proceedings of the National Academy of Sciences.

As CRISPR/Cas9 systems become more specific, with fewer off-target effects, and are shown to be able to erase harmful genetic mutations in human diseases, delivery into the cell is drawing more attention as a looming hurdle. Simply put, it's not easy to get CRISPR/Cas9 into human tissues with low risk and high efficiency. As firms like Intellia Therapeutics develop CRISPR-based therapies, they're already thinking about ex vivo- and in vivo-based therapies as separate problems.

Delivery hurdles can be further parsed into what to deliver to the cell (i.e. mRNA, plasmid, RNP) and how to deliver it (i.e. viral vectors, electroporation, lipid-based transfection).

"People have investigated using messenger RNA and viruses to deliver [CRISPR/Cas9]," Xu told GenomeWeb. "But we thought the protein is the most straightforward method. Protein delivery is safer than viral delivery vectors; if you use a virus, then the viral vectors can get an immune response and the host can generate neutralizing antibodies."

Xu added that the virus can also potentially introduce unwanted mutations, although CRISPR/Cas9 can also introduce off-target edits on its own.

For Xu, all these advantages open the door for the technology to deliver CRISPR/Cas9 for in vivo therapeutic applications. His lab has been working on protein delivery using the lipid nanoparticles since before 2012 and has published studies demonstrating delivery of cytotoxic proteins and cellular factors for cancer therapy and functional enzymes for enzyme replacement therapy.

That work caught the eye of some of Liu's students, Xu said, and the two began collaborating on delivering genome-editing proteins.

The lipid nanoparticles and genome-editing proteins self-assemble through electrostatic interactions. "If you throw those things together in an Eppendorf tube, they will assemble," Xu said.

The package — lipid nanoparticles on the outside, proteins on the inside — can then simply be pipetted into a dish of cells and the contents will find their way inside. "The nanoparticles adhere on the [cell] surface and the cell will eat it through the endocytosis pathway," Xu said. "Because it's a positively charged lipid, the lipid can escape from the endosome and release the cargo in the cytosome."

Lipid-based delivery of the CRISPR/Cas9 system has been around since it was first used for genome editing. In a 2013 methods paper in Nature Protocols, Fei Ann Ran, a postdoc in Feng Zhang's lab at the Massachusetts Institute of Technology, included Lipofectamine in her transfection protocols, although it was to deliver CRISPR plasmids and mRNAs, rather than the Cas9 RNP.

In addition, scientists from Thermo Fisher Scientific last year published a study in the Journal of Biotechnology detailing the use of several reagents in the Lipofectamine portfolio to deliver Cas9 mRNA and RNPs.

The Tufts and Harvard team first showed Xu's lipid nanoparticle technology could deliver a genome-editing protein by transfecting HeLa-DsRed cells. After establishing that it could successfully deliver Cre recombinase, they packaged Cas9 RNPs targeting the EGFP reporter gene in GFP-expressing HEK cells to test genome-editing efficiency.

The scientists synthesized 12 lipids in a combinatorial manner to find those that would best transfect the cells. "Our combinatorial synthesis allows facile generation of lipids with chemically diverse head groups, enabling study of the structure-activity relationship of the head groups," they wrote in the study. Of the 12 different lipid-packaging materials, five led to genome-editing efficiencies of 50 percent or more and three approached efficiencies of 70 percent or more. They found that Cas9 RNP delivered by LPF2K achieved efficiency of greater than 70 percent.

But, Xu said that beating Lipofectamine was not their goal. "[It] has toxic side effects and is not good for in vivo delivery," he said.

As part of the PNAS study, the researchers demonstrated in vivo delivery of Cre recombinase, but not CRISPR/Cas9, by directly injecting mice brains. The injected brains showed a strong reporter signal, which was "confined to the injection site with minimal diffusion," the authors wrote.

Xu added that his lab's lipid nanoparticles have low immunogenicity and toxicity and the injected animals appeared to tolerate the injection.

For Xu, it's an important step forward. He said his lab will now turn to maximum tolerance and pharmacokinetics and pharmacodynamics studies to better assess the toxicity in animals. He suggested that the combinatorial synthesis model described in the paper could be important because it provides multiple options. Some might yield higher efficiency; some lower toxicity. Some might offer the golden combination of both.

"You have more choices," he said.