The CRISPR-Cas9 gene editing system has been hailed as a promising solution to a range of genetic diseases, but the technology has proven difficult to deliver into cells. One strategy is to open the cell membrane using an electric shock, but that can accidentally kill the cell. Another is to use viruses as couriers. Problem is, viruses can cause off-target side effects.
Scientists at Fred Hutchinson Cancer Research Center are proposing an alternative delivery vehicle: gold. They developed gold nanoparticles that can be packed with all the CRISPR components necessary to make clean gene edits. When they tested the gold nanoparticles in lab models of inherited blood disorders and HIV, between 10% and 20% of the targeted cells were successfully edited, with no toxic side effects. They reported their findings in the journal Nature Materials.
“We engineered the gold nanoparticles to quickly cross the cell membrane, dodge cell organelles that seek to destroy them and go right to the cell nucleus to edit genes,” said Reza Shahbazi, Ph.D., a Fred Hutch postdoctoral researcher, in a statement.
Gold nanoparticles have several attributes Shahbazi and colleagues found appealing, including microscopic spheres on their surface that adhere easily to different types of molecules. They started with laboratory-grade liquid gold, which they mixed into a substance that prompts gold ions to form particles. They selected particles that were 19 nanometers wide—large enough to hold the CRISPR components but small enough to easily pass through cell walls.
The team then used the gold nanoparticles to deliver CRISPR to blood stem cells. In one experiment, they sought to disrupt the gene CCR5 to make cells resistant to HIV. In the second, they created a gene mutation that can protect against blood disorders, including sickle cell disease.
Each gold nanoparticle contained four CRISPR components, including the enzyme needed to make the DNA cuts. But instead of using the most popular enzyme, Cas9, the Fred Hutch researchers chose Cas12a, which they believed would lead to more efficient edits. Plus, Cas12a only needs one molecular guide, while Cas9 requires two.
Other research groups are examining the potential of gold as an alternative delivery vehicle for CRISPR. In fact, last December, University of California, Berkeley spinoff GenEdit raised $8.5 million in seed funding to develop its gold-based CRISPR technology for treating Duchenne muscular dystrophy. The Berkeley team showed they could correct the mutated dystrophin gene in mouse models by injecting their system, dubbed CRISPR-Gold, into muscles.
The Fred Hutch researchers believe they are the first to target blood stem cells with gold nanoparticles carrying CRISPR components. They observed that the cells swallowed the nanoparticles within six hours and began the gene-editing process within 48 hours. In mice, gene editing peaked eight weeks after injection, and the edited cells were still in circulation 22 weeks after the treatment, they reported.
What’s more, the scientists found edited cells in the animals’ spleens and thymus, as well as in their bone marrow—a sign that dividing blood cells could deliver the treatment throughout the body without the need for a second treatment, they believe.
The Fred Hutch team is now working on boosting the effectiveness of the gold-based CRISPR delivery system so that 50% or more of the targeted cells are edited, according to the statement. The researchers are also looking for a commercial partner to bring the technology into people, with a goal of starting clinical trials in the next few years.