Osteosarcoma (OS) is a highly aggressive pediatric cancer, characterized by frequent lung metastasis and pathologic bone destruction. Vascular endothelial growth factor A (VEGFA) that is highly expressed in OS contributes to angiogenesis within the tumour microenvironment. However, it also acts as an autocrine survival factor for tumour cell themselves, making it a promising therapeutic target for OS.
Clustered regulated interspaced short palindromic repeat (CRISPR) – associated Cas9 nuclease is a versatile genome editing technology and holds tremendous promise for cancer treatment because of its high specificity and efficiency towards the treatment.
However, a major bottleneck to achieve the therapeutic potential of the CRISPR/Cas9 is the lack of in vivo tumour-targeted delivery systems. Aptamers selected by cell – based SELEX could specifically recognize target cells and have been widely used for in vivo targeted delivery of therapeutics.
Taking advantage of highly specific aptamers, Liang et al. demonstrated a novel CRISPR/ Cas9 based genome editing approach for the treatment of OS. The authors used OS cell-specific aptamer (LC09) and developed a LC09 – functionalized PEG-PEI-Cholesterol (PPC) lipopolymer encapsulating CRISPR/Cas9 plasmids encoding VEGFA gRNA and Cas9 (LC09-PPC-CRISPR/Cas9). Results demonstrated LC09 aptamer achieved:
- Highest binding ability and specificity to target OS cells as compared to negative control cells (Figure 1)
- Tumour-specific delivery of CRISPR/Cas9 in both orthotopic OS and lung metastasis
- Facilitated VEGFA genome editing in tumour
- Decreased VEGFA expression and secretion
- Inhibited orthotopic OS malignancy and lung metastasis
- Reduced angiogenesis and bone lesion
- No detectable toxicity in a syngeneic orthotopic OS mouse model
Figure 1. Binding ability of enriched ssDNA LC09 aptamer to target OS cells K7M2 and negative cells (normal hepatocytes PBMCs) as determined by flow cytometry.
In vitro treatment and characterization of OS cells with different CRISPR/Cas9 formulations was performed and analysed using various microscopy methods. Briefly mouse OS cells (K7M2) were cultured in medium with various CRISPR/Cas9 formulations followed by incubation with endothelial cells (Figure 2a). LC09 mediated CRISPR/Cas9 formulation dramatically inhibited viability, migration and invasion of K7M2 cells (Figure 2c, top row). Moreover, aptamer mediated conditioned medium (LC09-PPC-CRISPR/Cas9) showed weaker proliferation, migration, invasion and tube formation of endothelial cells in OS cells (Figure 2c, bottom 3 rows).
Figure 2. Schematic diagram showing experimental design (a). Proliferation of endothelial cells post incubation with various CRISPR/Cas9 formulations (b and c).
In vivo angiogenesis by Matrigel plug assay, immunohistochemistry and bone destruction by microCT in OS tissues showed mice treated with aptamer-mediated formulation (LC09-PPC-CRISPR/Cas9) exhibited:
- Less neovascularization in subcutaneous OS region (Figure 3a);
- Decreased expression of endothelial cell activation markers (CD34 and vWF) (3b) and
- Relieved bone destruction in OS tissues (3c)
Figure 3. In vivo anti-angiogenesis and anti-bone destruction of different CRISPR/Cas9 formulations in OS tissues.
In summary, the OS cell specific aptamer LC09 facilitated selective distribution of CRISPR/Cas9 in both orthotopic OS tissue and metastatic OS tissue of lung lesion enabling promising in vivo tumour targeted delivery system for therapeutic CRISPR/Cas9. Beyond that, aptamers possess excellent properties such as:
- Small size
- High binding affinity
- Greater specificity
- Good biocompatibility
- High stability and low immunogenicity
Aptamers therefore contribute to wide applications in the biomedical field including cancer therapy. If you want to explore how aptamers can be used for your CRISPR research, discovery and development projects then please contact Aptamer group using the form below.
Liang C, Li F, Wang L, et al. Tumor cell-targeted delivery of CRISPR/Cas9 by aptamer-functionalized lipopolymer for therapeutic genome editing of VEGFA in osteosarcoma. Biomaterials. 2017;147:68–85. doi:10.1016/j.biomaterials.2017.09.015