Encapsulated gene therapy
Nanocapsules will deliver CRISPR-Cas9 system for glioblastoma therapy to the brain
Yulia Panchenko, PCR.news
Most modern systems for delivering CRISPR-Cas9 to the brain use viral vectors that have disadvantages such as low capacity, immunogenicity and the danger of inducing mutations. The main problem of delivery systems to the brain is overcoming the blood—brain barrier. An international team of scientists has obtained nanocapsules for noninvasive delivery of the therapeutic Cas9/hydRNA complex. The authors have shown the effectiveness of a new system for the treatment of glioblastoma in mice.
The nanocapsule consists of a thin polymer shell with ligands that bind LRP-1 receptors contained in large quantities on endothelial cells of the blood-brain barrier and on glioblastoma. These nanocapsules are formed by in situ polymerization. Their average hydrodynamic diameter is 31 nm, each capsule contains one Cas9/hydRNA complex. The polymer shell contains disulfide bonds, which are destroyed inside the cell in the presence of a large amount of glutathione, which releases the load. The capture of nanocapsules by the cell and the release of the Cas9/hydRNA complex were demonstrated on the cell line of glioblastoma U87MG. It also showed the ability to edit the luciferase gene.
The PLK1 gene, which plays an important role in mitosis and is expressed in large quantities in many tumors, was chosen as the editing target. Inhibition of PLK1 suppresses the proliferation of glioblastoma cells and leads to apoptosis. The authors packed hydRNA targeting PLK1 into nanocapsules and added it to U87MG cells. The editing efficiency was 36.6%, while the decrease in protein expression reached 53%. After 72 hours, apoptosis was started in 37.4% of the cells.
Researchers have demonstrated in vitro models that nanocapsules overcome the blood-brain barrier well and can penetrate deep into glioblastoma. After that, we moved on to tests on BALB/c mice. The study of pharmacokinetics showed that nanocapsules injected intravenously can exist in the blood for a long time, while the free Cas9/hydRNA complex is eliminated quickly.
With intravenous administration, nanocapsules overcome the blood-brain barrier (probably by transcytosis) and penetrate into the tumor located in the mouse brain. The authors also demonstrated the knockout of the luciferase gene in vivo. Similar results were obtained in immunocompetent C57BL/6 mice.
With the introduction of a therapeutic complex with a hydRNA targeting PLK1, tumor growth in the brain of mice slowed down. At the same time, the weight did not significantly decrease, unlike controls in which weight loss correlated with rapid tumor growth. After therapy, the average survival was 68 days versus 22-24 days in controls. The authors confirmed that the slowing of tumor growth was due to a decrease in PLK1 expression. The efficiency of gene editing was 33.8%. Good results have also been shown in mice with xenografts obtained from patients.
In order to check the security of the system, the researchers identified sites where inappropriate editing could potentially occur. The frequency of mutations in these sites was below 0.5% in both tumor and normal tissues, including kidney and liver tissues. Thus, the safety of a new CRISPR-Cas9 delivery method for glioblastoma therapy in mice has been demonstrated.
Article by Zou et al. Blood-brain barrier–penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy is published in the journal Science Advances.
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