Darkness, as in “gloom of night,” is one of those things that shouldn’t stay couriers from the “swift completion of their appointed rounds.” But what if the couriers are viral vectors or nanoparticles that deliver agents for CRISPR genome editing? Maybe these delivery vehicles would work better in the light, if only they were given the chance. Then, deliveries in the dark, now the rule, would become the exception.
To create CRISPR delivery vehicles that respond to light, scientists from the University of New South Wales (UNSW) Sydney decided to modify liposomes—bubble-like vesicles—so that their membranes would contain verteporfin, a molecule best known as a photosensitizing drug. When exposed to laser light, verteporfin generates reactive oxygen species that impair membranes. This action, which has been useful in treating certain eye diseases, now has another purpose: Helping liposomal CRISPR delivery vehicles release their contents when and where required.
The UNSW Sydney scientists reported their findings in a paper (“Spatial and Temporal Control of CRISPR-Cas9-Mediated Gene Editing Delivered via a Light-Triggered Liposome System”) that was recently published in the journal ACS Applied Materials and Interfaces. This paper detailed results from experiments with cell lines and animal models.
“We demonstrated its efficient protein release by respectively assessing the targeted knockout of the eGFP gene in human HEK293/GFP cells and the TNFAIP3 gene in TNFα-induced HEK293 cells,” the article’s authors wrote. “We further validated our results at a single-cell resolution using an in vivo eGFP reporter system in zebrafish (77% knockout).”
CRISPR gene therapy agents are typically delivered by viral vectors. However, viral vectors are less than ideal delivery vehicles because they can be toxic and provoke adverse immune responses.
For a more benign alternative, the UNSW Sydney scientists suggest their newly developed own light-triggered liposomes. That is, besides providing targeted delivery, the light-triggered liposomes are much safer than viruses.
“Liposomes are already well established as an extremely effective drug delivery system,” said Wei Deng, PhD, a senior research fellow at UNSW Sydney and the senior author of the current study. “These ‘bubbles’ are relatively simple to prepare. Then they can be filled with appropriate medication and injected into the body.”
Deng added that liposomes are the most common and well-established drug delivery vehicles. “The traditional delivery vehicle of CRISPR is based on viruses,” she added, “but they create their own problems because it is difficult to predict the reaction of patients to the viruses.”
“Unlike the traditional liposome-based delivery systems, our liposomes can be ‘turned on’ under light illumination,” Deng explained. “When light is shone onto the liposomes, they can be disrupted at once, immediately releasing the entire payload.”
Deng’s team demonstrated in both cell lines and animal models that when the liposomes are triggered by an LED light, they eject the CRISPR contents, which then go to work looking for genes of interest. Deng says the light can activate the liposomes up to a centimeter below the surface of the skin.
But what if the problem area is a deep-seated tumor? Deng said that future studies will use X-rays to achieve the same effect. “We fully expect that we will be able to carry out X-ray triggering of CRISPR delivery in deep tissue at depths greater than one centimeter,” she declared. “Our past research has already indicated that liposomes can be triggered by X-rays.
“CRISPR technology has created a very promising tool for developing targeted gene therapies and cell-based therapies. Its outcomes would largely increase with the desired delivery system, and in this context, our findings may provide such a system.”
Looking ahead, Deng said that she and her colleagues are interested in carrying out research that shows X-rays can be used to deliver CRISPR gene-splicing molecules for deep cancer treatment.
“To advance studies that use X-ray light, we need to find a proper animal model, that is, one that might help us translate this technology for something like breast cancer treatment. So, we’re looking for collaborators who have this sort of expertise.”