Osteosarcoma-specific delivery of CRISPR/Cas9 by aptamer-functionalized lipopolymer for therapeutic genome editing of VEGFA in a patient-derived xenograft model

Osteosarcoma (OS) is a highly aggressive pediatric cancer, characterized by frequent lung metastasis and pathologic bone destruction. Vascular endothelial growth factor A (VEGFA), highly expressed in OS, not only contributes to angiogenesis within tumor microenvironment via paracrine stimulation of vascular endothelial cells, but also acts as an autocrine survival factor for tumor cells, thus making it a promising therapeutic target for OS. A monoclonal anti-VEGFA antibody is now in phase II trial for treating OS patients. However, anti-VEGFA antibody could only deactivate extracellular VEGFA, but have little effect on intracellular VEGFA expression and self-growth stimulation in OS cells. Theoretically, inhibiting the VEGFA gene at genome DNA level in OS cells could efficiently decrease VEGFA production. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 nuclease (CRISPR/Cas9) is a raising genome editing technology and is appropriate for VEGFA gene disruption in OS cells. However, a major bottleneck to achieve its therapeutic potential is the lack of in vivo targeted delivery systems.

Aptamers are single-stranded DNA (ssDNA) or RNA oligonucleotides. They could specifically recognize target cells and have been widely used for in vivo targeted delivery of therapeutics. Recently, our group has reported an OS aptamer LC09, which exhibits high specificity to both mouse and human OS cells. We constructed a CRISPR/Cas9 plasmid targeting mouse VEGFA gene, and encapsulated the CRISPR/Cas9 plasmid into PEG-PEI-Cholesterol (PPC) lipopolymer, which is a non-virus plasmid carrier under clinical trial with proved safety. The surface of the PPC particles was finally modified with the aptamer LC09 for OS cell recognition. The in vitro data demonstrated that LC09 modification facilitated the uptake and the VEGFA gene silencing efficiency of CRISPR/Cas9 in mouse OS cells, resulting in subsequently reduced VEGFA expression, cell migration and invasion, and triggering apoptosis of mouse OS cells. In a syngeneic orthotopic OS mouse model, LC09 facilitated selective distribution of CRISPR/Cas9 in both orthotopic OS tumor and lung metastasis, leading to effective VEGFA genome editing in tumor, decreased VEGFA expression and secretion, inhibited orthotopic OS malignancy and lung metastasis, as well as reduced angiogenesis and bone lesion with no detectable toxicity.

To further demonstrate the translational potential of this aptamer-based CRISPR/Cas9 in vivo delivery system, we examined the uptake of LC09 in human primary OS cells collected from OS patients after surgery in comparison with human primary osteoblasts. LC09 remained its high specificity and uptake in human primary OS cells. We then postulate that in an orthotopic patient-derived xenograft (PDX) mouse model of osteosarcoma, LC09 could facilitate the selective accumulation of a human VEGFA gene-targeting CRISPR/Cas9 (hCRISPR/Cas9) in OS tissues, leading to subsequent disruption of VEGFA in OS cells, and inhibition of tumor growth and metastasis. The following aims will be achieved to prove our hypothesis:
(1) To construct and optimize a CRISPR/Cas9 system targeting human VEGFA gene (hCRISPR/Cas9);
(2) To examine the LC09-mediated changes in the distribution of hCRISPR/Cas9 in OS and non- tumor tissue in an orthotopic PDX mouse model;
(3) To detect the LC09-mediated changes in VEGFA editing of hCRISPR/Cas9 in an orthotopic PDX mouse model;
(4) To evaluate the LC09-mediated changes in anti-tumor and anti-metastasis activity of hCRISPR/Cas9 in an orthotopic PDX mouse model;
(5) To assess anti-angiogenesis, anti-bone disruption activity and safety of LC09-PPC- hCRISPR/Cas9 in an orthotopic PDX mouse model.

The proposed work would reveal the translational value of this novel aptamer-functionalized osteosarcoma-targeting delivery system of CRISPR/Cas9 to achieve genome editing for silencing tumor-promoting genes.