Scientists develop new drug delivery system for treating genetic diseases

A team of researchers led by Harvard and Broad Institute scientists has developed a new drug delivery system using engineered DNA-free virus-like particles (eVLPs) to package and deliver therapeutic levels of gene-editing proteins to animal models of disease. The team utilized eVLPs to edit a gene in mice that is associated with high cholesterol levels and to partially restore vision in mice with a point mutation that causes genetic blindness.

Because eVLPs enable safer in vivo delivery of gene-editing agents than some methods used in the clinic, with comparable or higher efficiencies, this new platform is a promising technology for the delivery of therapeutic macromolecules in living animals with minimized risk of off-target editing or DNA integration.

In the paper, published in Cell, the researchers detail how they developed virus-like particles to deliver base editors, proteins that make programmable single-letter changes in DNA, and CRISPR-Cas9 nuclease, a protein that cuts DNA at targeted sites in the genome. The authors identified factors that influence virus-like particle delivery efficiency and demonstrated that engineering virus-like particles can overcome multiple structural limits to their potency. The safe and efficient delivery of gene-editing agents to cells in living humans and animals is a major challenge. The team's eVLPs are the first virus-like particles to deliver therapeutic levels of base editors to a variety of cell types in adult animals.

The delivery of therapeutic macromolecules into mammalian cells in animals and eventually in patients is one of the most important challenges in life sciences. There is often a very steep drop-off between in vitro and in vivo delivery, so we made the decision early on that our new delivery technology would need to show good efficacy in animal models."

David Liu, paper's senior author, Thomas Dudley Cabot Professor of the Natural Sciences, and core faculty member of the Broad Institute

This work was led by members of Liu's lab, including postdoctoral fellow Samagya Banskota, and graduate student Aditya Raguram, in collaboration with research teams led by Krzysztof Palczewski at the University of California, Irvine, and Kiran Musunuru at the Perelman School of Medicine at the University of Pennsylvania.

This new delivery system finds a novel use for virus-like particles and builds on the success of base editors, which the Liu Lab developed in 2016 to rewrite individual DNA bases such as the mutations that cause thousands of genetic diseases.

Virus-like particles, assemblies of viral proteins that can infect cells but lack viral genetic material, have long been studied as drug delivery vehicles. Because they are able to carry molecular cargo and lack viral genetic material, they are able to exploit the efficiency and tissue targeting advantages of viral delivery without the drawbacks of using actual viruses, which can insert their genetic material into the cell's genome and potentially cause cancer. However, existing VLP delivery strategies have had limited therapeutic efficacy in vivo.

To successfully deploy VLPs, the team identified delivery limitations and systematically engineered the components of VLPs to overcome cargo packaging, release, and localization bottlenecks. In doing so, they developed fourth-generation eVLPs that packaged 16 times more cargo proteins than previous designs and enabled an eight- to 26-fold increase in editing efficiency in cells and animals.

The team tested their optimized eVLP system to deliver base editors to the liver in mice, where they efficiently edited a gene that can lower "bad" cholesterol levels. A single injection of eVLPs resulted in an average of 63 percent editing of the target gene and a 78 percent drop in its protein levels, which substantially reduce the risk of coronary heart disease.

"The cholesterol target is particularly interesting because it is not only relevant to patients with a rare genetic disease," Raguram said. "We are hopeful this is one example of genome editing being able to benefit a large population because cholesterol levels impact the health of billions of people."

The researchers also used a single eVLP injection to correct disease-causing point mutation in mice with a genetic retinal disorder. They corrected the point mutation efficiently with less-off target editing than other base editing delivery techniques, resulting in the partial restoration of vision.

The team also injected eVLPs directly into the brain in mice and observed around 50 percent editing efficiency in cells exposed to the eVLPs.

Going forward, Banskota is optimistic that eVLPs will be utilized by scientists quite easily because of the system's relative simplicity and its versatility.

"Because our system is relatively simple and easily engineered, it allows other scientists to adopt and build upon this technology quickly," Banskota said. "Beyond carrying gene-editors, eVLPs have the ability to transport other macromolecules with lots of therapeutic potential."