Periodic Reporting for period 1 - CodeSphere (CodeSphere - Discovering Therapeutic Nanoparticles by Molecular Encoding)
Okres sprawozdawczy: 2017-09-01 do 2019-02-28
The project has focused on tagging nanoparticles for drug delivery. Such nanoparticles can be follow by tagging using DNA barcodes. Experimental work on the CodeSphere project has focused on the delivery of messenger RNA (mRNA) cargo to human cell lines. This was assessed via flow cytometry, using a commercial transfection reagent and various homemade lipid nanoparticle (LNP) formulations. Initial attempts to modify antibodies for use in decorating the NP surface were performed, to assess any potential antibody-mediated effect in the delivery to cell lines.
Work on the CodeSphere project was focused on exploring chemistries for antibody modification and attachment to NPs. Alongside this; various types of NPs were explored for the delivery of mRNA to cell lines. Including lipid, polymeric, peptide and carbohydrate NPs. Experimental work was pursued on the lipid and polymeric NPs as they showed most promise in the literature and from our preliminary results. Once conditions for transfection were established in human cell lines, lipid and polymeric NP-mediated transfection of mRNA in human whole PBMCs was assessed. There is great interest in developing NP therapies which specifically target T-cells in whole blood, and so, this became a primary focus. This involved an initial optimization of the transfection protocol, due to differences between cell lines and primary cells, in terms of sensitivity to cytotoxic lipid/polymer compounds. As our preliminary results indicate, delivery of mRNA to T-cells in a heterogeneous blood sample, with minimal cytotoxicity, is possible. Delivery to different immune cell subsets in human whole PBMCs is now being evaluated. Efforts were then re-focused on developing NPs which promote delivery to T-cells in a cell-specific manner. This is a major focus in current NP-based therapies and has yet to be achieved, with difficulties highlighted in the literature and in failed clinical trials. Some novel covalent/non-covalent techniques are currently being explored for antibody modification and attachment.
Also, additional effort has been applied to make proof of concept for the DNA barcoding strategy for tagging LNPs. This would allow the simultaneous assessment of formulation parameters for effective delivery of cargo to T-cells. Our data suggest that DNA oligonucleotides can be co-encapsulated, along with mRNA, and delivered to cell lines and primary cells in culture. Isolation of transfected cells with FACS and subsequent PCR/qPCR analysis indicates that the barcodes can also be recovered after co-encapsulation and delivery. A small proof of concept library is under development, whereby different mRNA-LNP complexes will be tagged with a unique barcode, allowing a basic screening of formulation parameters for delivery to T-cells in whole PBMCs.
Work on the CodeSphere project was focused on exploring chemistries for antibody modification and attachment to NPs. Alongside this; various types of NPs were explored for the delivery of mRNA to cell lines. Including lipid, polymeric, peptide and carbohydrate NPs. Experimental work was pursued on the lipid and polymeric NPs as they showed most promise in the literature and from our preliminary results. Once conditions for transfection were established in human cell lines, lipid and polymeric NP-mediated transfection of mRNA in human whole PBMCs was assessed. There is great interest in developing NP therapies which specifically target T-cells in whole blood, and so, this became a primary focus. This involved an initial optimization of the transfection protocol, due to differences between cell lines and primary cells, in terms of sensitivity to cytotoxic lipid/polymer compounds. As our preliminary results indicate, delivery of mRNA to T-cells in a heterogeneous blood sample, with minimal cytotoxicity, is possible. Delivery to different immune cell subsets in human whole PBMCs is now being evaluated. Efforts were then re-focused on developing NPs which promote delivery to T-cells in a cell-specific manner. This is a major focus in current NP-based therapies and has yet to be achieved, with difficulties highlighted in the literature and in failed clinical trials. Some novel covalent/non-covalent techniques are currently being explored for antibody modification and attachment.
Also, additional effort has been applied to make proof of concept for the DNA barcoding strategy for tagging LNPs. This would allow the simultaneous assessment of formulation parameters for effective delivery of cargo to T-cells. Our data suggest that DNA oligonucleotides can be co-encapsulated, along with mRNA, and delivered to cell lines and primary cells in culture. Isolation of transfected cells with FACS and subsequent PCR/qPCR analysis indicates that the barcodes can also be recovered after co-encapsulation and delivery. A small proof of concept library is under development, whereby different mRNA-LNP complexes will be tagged with a unique barcode, allowing a basic screening of formulation parameters for delivery to T-cells in whole PBMCs.