CRISPR/Cas9 therapeutics have been in vitro techniques historically. This review shows the promise of potential in vivo aptamer based delivery of CRISPR/Cas9 therapeutics.
Precise editing of DNA sequences in the human genome can correct mutations or introduce novel genetic functionality, thus providing promising solutions to various diseases. Amongst the various diverse gene editing technology to correct a gene, clustered regulated interspaced short palindromic repeat – associated Cas9 nuclease (CRISPR/Cas9) has demonstrated to be a versatile and rapid technology. It is powered by ‘guide RNA’ (gRNA) that directs the Cas9 protein to the desired DNA locus for DNA cleavage (Figure 1). The cell’s repair mechanism can then be co-opted to make edits to the genome. The CRISPR/Cas9 has shown to hold promise for cancer treatment because of its high specificity and efficiency towards the treatment.
Figure 1. Schematic representation of CRISPR/Cas9 plasmids for gRNA delivery and gene editing (Adapted from https://www.bio-connect.nl/crispr-cas9-knockout-plasmids/cnt/page/4136)
For transient transfections, each of gRNA and Cas9 can be delivered as plasmid DNA or RNA via non-viral vectors such as lipids, polymers, antibodies and hybrid materials. However, these non-viral vectors still suffer from their relatively low delivery efficiency, thereby limiting the therapeutic potential of CRISPR/Cas9 plasmids especially in vivo.
Aptamers have been shown as a promising tool in the development of targeted delivery systems, because of their unique binding properties that prove them advantageous over other technologies including:
- Fast and easy tissue penetration because of the small molecular weight and size.
- Improved binding affinity and stability as they are chemically modifiable.
- Efficient targeted delivery systems allowing development of multifunctional molecules.
- Less likely to instigate an immune reaction in vivo as they are immunologically inert.
- More consistent end product because of negligible batch-to-batch variation with a robust oligonucleotide synthesis process.
These advantages make aptamers more desirable for developing efficient delivery systems for carrying CRISPR/Cas9 plasmids to the target cells or tissues both in vitro and in vivo. Considering these properties of aptamers, several research groups have developed aptamers that can aid in delivering the CRISPR/Cas9 plasmid to the target cells and tissues. We review two of such promising findings here that can be effective in the future cancer treatment.
Aptamers enhance targeted delivery of CRISPR/Cas9 plasmid in tumour cells in vitro
Focal adhesion kinase (FAK) has shown to be expressed in the vast majority of cancer types. Down regulating the FAK protein can result in cell apoptosis preventing cancer invasion and metastasis of tumour cells.
In this view, Liu et al., 2019 developed a multiple functionalized targeting delivery system (DS-ANalg) that could efficiently deliver Cas9/sgRNA plasmids to the targeted tumour cell nuclei. DS-ANalg was constructed using gene editing technology that involved Cas9/sgRNA and an aptamer AS1411 with high specificity for nucleolin in the tumour cell membrane and nuclei.
The Cas9/sgRNA plasmids were compacted by protamine in the presence of calcium ions to form nanosized cores, which was further decorated by peptide and AS1411 aptamer (Figure 2). The overall structure enhanced the plasmid loading capability and achieved effective genome editing in targeted tumour cells.
Figure 2. Schematic representation of peptide and aptamer functionalized gene delivery system (DS ANalg) delivering genome editing plasmid to the nucleus of cancer cells.
DS-ANalg showed higher uptake in cancerous HeLa cells compared to other control delivery systems because of the high specificity of the aptamer AS1411 to its target nucleolin (Figure 3). The DS-ANalg could specifically deliver the Cas9/sgRNA plasmid to the nuclei of the tumorous cells for knocking out the protein tyrosine kinase 2 (PTK2) gene to downregulate focal adhesion kinase (FAK). FAK knockout resulted in negative regulation on the PI3K/AKT signaling pathway, cell apoptosis and various proteins that play important roles in tumour progression. This delivery system provided a promising approach and effective strategy for tumour targeted genome editing with reduced side effects on normal cells in cancer gene therapy.
Figure 3. Left image: Cellular uptake of various delivery systems loaded with plasmid FAK showed higher uptake of DS-ANalg in target Hela cells as compared to other control delivery systems assessed by confocal microscopy and flow cytometry. Top right image: FAK knockout in target Hela cells was most effective with DS-ANalg delivery system demonstrating highest gene editing efficiency. Bottom right image: The cell migration and invasion assay on FAK knockout Hela cells showed reduced migration and 90% supressed invasion as assessed by wound healing and transwell assay, respectively.
Aptamers enhance targeted delivery of CRISPR/Cas9 plasmid in tumour tissues in vivo
Osteosarcoma (OS) is a highly aggressive paediatric 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. Inhibition of VEGFA can therefore supress OS growth, metastasis and angiogenesis.
Taking advantage of highly specific aptamers, Liang et al. 2017 demonstrated a novel CRISPR/ Cas9 based genome editing approach for the treatment of OS. The authors used OS cell-specific aptamer (LC09) and constructed CRISPR/Cas9 plasmids encoding VEGFA gRNA and Cas9. They encapsulated the CRISPR/Cas9 plasmids into a non-virus plasmid, in which the LC09 aptamer was conjugated to PEG-PEI-Cholesterol (PPC) lipopolymer. The complex delivery system was called LC09-PPC-CRISPR/Cas9.
The aptamer LC09 helped in achieving efficient delivery of CRISPR/Cas9, leading to effective VEGFA genome editing in the tumour. 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 4a)
- Decreased expression of endothelial cell activation markers (CD34 and vWF) (4b)
- Relieved bone destruction in OS tissues (4c)
Figure 4. In vivo anti-angiogenesis and anti-bone destruction of different CRISPR/Cas9 formulations in OS tissues. Aptamer-mediated formulation (LC09-PPC-CRISPR/Cas9) exhibited promising results in OS therapy as compared to their controls.
In summary, the OS cell specific aptamer LC09 facilitated inhibition of orthotopic OS malignancy and lung metastasis, reduced angiogenesis and bone lesion with no detectable toxicity in the syngeneic orthotopic OS mouse model. The study demonstrated aptamer mediated approach for CRISPR/Cas9 genome editing could be a promising treatment for in vivo therapeutics.
Aptamers as effective gene delivery agents
Overall, integrating the internalisation properties and an endowed therapeutic potential, several aptamers can be used in new combination strategies for the development of such multifunctional molecules. These molecules, with the aid of aptamers, can enhance the effectiveness of the treatment with improved selectivity and modulating the gene response in cells and tissues.
Aptamer Group Ltd (AGL) offers the advantage of designing aptamers that are optimized for the conditions you want to use them in. This way they are engineered to bind to their target with high specificity and affinity. AGL has dedicated researchers to identify aptamers against novel and significant targets and continuously aims to conduct further research in prevention, diagnosis and treatment of cancer and other diseases. If you want to explore how aptamers can be used for your CRISPR research, discovery and development projects then please contact us 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
Liu BY, He XY, Xu C, Ren XH, Zhuo RX, Cheng SX. Peptide and Aptamer Decorated Delivery System for Targeting Delivery of Cas9/sgRNA Plasmid To Mediate Antitumor Genome Editing. ACS Appl Mater Interfaces. 2019;11(27):23870-23879. doi:10.1021/acsami.9b05772