ANALYSIS OF MATERIALS FOR TISSUE DELIVERY
Described herein are compositions and methods for identifying materials suitable for delivery of an agent to a target tissue. These compositions and methods may simultaneously screen a library of materials for the ability to deliver an agent to a target. The compositions and methods may also be used to confirm that the agent is delivered in a manner sufficient for function of the agent.
1 . A method of characterizing delivery vehicles for delivery of an agent, comprising:
(a) formulating multiple lipid nanoparticle (LNP) delivery vehicles having different chemical compositions, wherein each different LNP delivery vehicle comprises:
(i) a biologically active molecule that generates a detectable signal when delivered by the LNP delivery vehicle to the cytoplasm of cells of at least two species of non-human mammals; and
(ii) a chemical composition identifier that identifies the chemical compositions of each of the LNP delivery vehicles;
(b) administering multiple LNP delivery vehicles to multiple tissues of at least one of the species of non-human mammal;
(c) sorting cells from the multiple tissues of the non-human mammal that generate the detectable signal from cells that do not generate the detectable signal, wherein the cells that generate the detectable signal are also sorted based on the presence or absence of a cell surface protein that is indicative of tissue type or cell type; and
(d) identifying the chemical composition identifier in the sorted cells that generate the detectable signal to determine the chemical composition of the LNP delivery vehicle in the sorted cells to correlate the chemical composition of the LNP delivery vehicles with the tissue or cell type containing the LNP delivery vehicles.
2 . The method of claim 1 , wherein the LNP delivery vehicles further comprise the agent to be delivered.
3 . The method of claim 1 or 2 , wherein the two species of non-human mammals are selected from mouse, rat and non-human primate.
4 . The method of any one of claims 1 - 3 , wherein the agent is a nucleic acid agent.
5 . The method of claim 4 , wherein the nucleic acid agent comprises RNA, DNA, or a combination of RNA and DNA.
6 . The method of any one of claims 1 - 5 , wherein the detectable signal is indicative of down regulation of a gene typically expressed in the cells.
7 . The method of claim 6 , wherein the down regulation results in reduced expression of one or more of: beta-2-microglobulin, CD47, CD81, AP2S1, LGALS9, ITGB1, ITGA5, CD45, TIE2, MGAT4B, MGAT2, VAMP3, and GPAA1.
8 . The method of claim 6 , wherein the biologically active molecule that generates a detectable signal is siRNA, an antisense oligonucleotide or a DNA transgene.
9 . The method of claim 8 , wherein the DNA transgene expresses a shRNA.
10 . The method of any one of claims 1 - 5 , wherein the detectable signal is indicative of up regulation of a gene typically expressed in the cells.
11 . The method of claim 9 , wherein the biologically active molecule that generates a detectable signal is mRNA or a DNA transgene.
12 . The method of claim 11 , wherein the mRNA is a modified mRNA that enhances generation of the detectable signal and/or decreases immunogenicity as compared to unmodified mRNA.
13 . The method of claim 12 , wherein the modified mRNA comprises one or more of a 5′-endcap, a 5′-untranslated region (UTR), a 3′-UTR, 3′-polyadenylation, codon optimization and base modifications.
14 . The method of any one of claims 1 - 13 , wherein the chemical composition identifier is a nucleic acid barcode.
15 . The method of claim 14 , further comprising sequencing the nucleic acid barcodes to identify the chemical compositions of the LNP delivery vehicles.
16 . The method of any one of claims 1 - 15 , wherein the non-human mammal to which the multiple LNP delivery vehicles are administered is a non-human primate.
17 . The method of any one of claims 1 - 15 , wherein the biologically active molecule that generates a detectable signal comprises a DNA transgene.
18 . The method of claim 17 , wherein the biologically active molecule that generates a detectable signal further comprises at least one selected from a cell-specific promoter, a small RNA promoter, a 3′-UTR, 3′-polyadenylation, a small RNA polymerase terminal, and the chemical composition identifier.
19 . The method of claim 18 , wherein the chemical composition identifier comprises a nucleic acid barcode and, optionally, a barcode tag.
20 . The method of claim 17 , wherein the biologically active molecule that generates a detectable signal is represented by at least one selected from:
5′-Promoter-Transgene-3′UTR & PolyA-Small RNA pol term-Tag for BC-Barcode-Small RNA Promoter-3′;
5′-Small RNA poly term-Tag for BC-Barcode-Small RNA Promoter-Promoter-Transgene-3′UTR & PolyA-3′;
5′-Small RNA Promoter-Barcode-Tag for BC-Small RNA pol term-Promoter-Transgene-3′UTR & PolyA-3′;
5′-Barcode-Promoter-Transgene-3′UTR & PolyA-3′; and
5′-Promoter-Transgene-3′UTR & PolyA-3′+Barcode.