Structures are alternatives to viral vectors for gene therapy using the CRISPR-Cas9 system

Original source: Physics World

Researchers have developed a new non-viral nanocapsule to deliver a gene-editing payload into biological cells. The capsule, which is made of a biodegradable polymer, is a version of the CRISPR-Cas9 with guide RNA. The structure could help overcome some of the problems associated with viral vector delivery of gene editing tools.

CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats) genome editing could potentially be used to treat many genetic diseases, including those currently without a cure. Most delivery technologies for CRISPR require viral vectors, however.

Although viral vectors are very efficient (viruses have, after all, billions of years of experience in invading cells), they can cause undesirable immune responses in the body. They also need to be altered to carry gene editing-machinery, rather than their own viral genes, into cells to alter their DNA (to correct a problem in the genetic code, for example, that causes a disease). This process, which needs to be adapted to each type of new cell target, can be time-consuming and complex.

In recent years, researchers have begun developing non-viral vectors, which are typically easier and cheaper to produce and scale up. Many of these are based on cationic liposomal components or polymers and can successfully encapsulate CRISPR-Cas9. They are beset with problems though, including the fact that they are relatively large (more than 100 nm across), can only accommodate a low gene-editing payload, are unstable and, most importantly, highly cytotoxic.

The Cas9/sgRNA ribonucleoprotein nanocapsule

A team led by Shaoqin (Sarah) Gong of the University of Wisconsin-Madison in the US has now developed the Cas9/sgRNA ribonucleoprotein (RNP) nanocapsule (NC) to address these challenges. The RNP NCs are very small (around 25 nm in size) and are very stable in the extracellular space, including the bloodstream, thanks to their covalent nature. They also have high RNP loading content, good biocompatibility and high editing efficiency.

“Unlike other previous RNP delivery nanosystems that typically contain multiple copies of the RNP, the RNP nanocapsule we report on normally contains just one RNP per nanoparticle,” explains Gong. “What is more, we can conveniently modify the surfaces of the RNP NCs with various targeted ligands, such as peptides, so that they can be used to target different organs/cells and treat different types of diseases.”

The RNP NCs are also relatively straightforward to make and they can be lyophilized (freeze-dried), which makes it easier to purify, store, transport and dose them, she adds. And last but not least, the Cas9 protein and sgRNA are present in a 1:1 molar ratio. The RNP only survives for a short time within the target cell, thus producing less off-target effects.

“This is important since editing the wrong tissue in the body after injecting gene therapies is of grave concern,” says team member Krishanu Saha, who co-chairs a steering committee for a consortium on gene editing in the US. “If reproductive organs are inadvertently edited, the patient would pass on the gene edits to their children and every subsequent generation.”

The researchers made their nanocapsules by enriching monomers with different charges and functionalities around the Cas9 RNP complex. They then polymerized the structure to form the nanocapsule. “As mentioned, this polymer coating is stable in the bloodstream/extracellular space, but it falls apart inside cells so that the RNP can edit the cell genome,” explains Gong.

Gene editing experiments

She and her colleagues tested out their delivery capsules in gene editing experiments on murine retinal pigment epithelium (RPE) tissue and skeletal muscle. “We locally injected our nanocapsules into subretinal spaces or skeletal muscles. We found that the capsules efficiently delivered their gene-editing machinery and modified the appropriate target genes in the tissue in question. Furthermore, by functionalizing the surface of the nanocapsules, we were able to modulate the extent and efficiency of the gene-editing process.”

Xiaoyuan (Shawn) Chen, senior investigator at the National Institute of Biomedical Imaging and Bioengineering (NIBIB) at the US National Institutes of Health (NIH), who was not involved in this work, says that the new technique is a “cool” way of using relatively small sized nanocapsules for high efficiency loading. “The crosslinking the researchers employed makes the particles stable during the delivery phase but readily releases the payload inside the cytosol thanks to cleavage of the linkers by a molecule called glutathione. The imidazole groups present also allow efficient endosomal escape through a proton sponge effect.

“Although the current study has only attempted local delivery for RPE cells and skeletal muscle cells, the same principle may be used to deliver RNP targeting to other organs.”

Broadening the applications of CRISPR-Cas9 gene-editing technology

The Wisconsin-Madison team believes that its work will facilitate the development of safe and efficient delivery nanosystems for the CRISPR-Cas9 genome editing tools, for both in vitro and in vivo applications. “In particular, it will broaden the applications of this gene-editing technology and so help us better understand and treat various genetic diseases,” Gong tells Physics World.

“We now plan to apply this technique to deliver various CRISPR genome editing machineries to treat brain and eye diseases and are currently working with several clinical collaborators to this end.”

Full details of the current study are reported in Nature Nanotechnology 10.1038/s41565-019-0539-2. The researchers have also filed a patent on the nanoparticles they have made.