- Document Number:
20240150751
- Appl. No:
18/279555
- Application Filed:
April 22, 2022
- نبذة مختصرة :
Provided herein are compositions and methods of use thereof for screening a plurality of uniquely identifiable therapeutic moieties by identifying one or more reporters indicative of a cell state. The methods include the administration of a library of expression cassettes, comprising a plurality of nucleic acid sequences, each encoding a different therapeutic moiety operably linked to a therapeutic moiety barcode; a plurality of nucleic acid sequences encoding one or more reporters that collectively, when expressed in a cell or an isolated nucleus, are indicative of a cell state or a likelihood of a cell state of the cell; and one or more sequences encoding a non-coding nuclear retention RNA motif.
- Claim:
1. A method for identifying a candidate therapeutic moiety comprising: (a) administering to an animal or an organoid a library of expression cassettes comprising: a plurality of nucleic acid sequences, each encoding a different therapeutic moiety operably linked to a therapeutic moiety barcode; a plurality of nucleic acid sequences encoding one or more reporters that collectively, when expressed in a cell or an isolated nucleus, are indicative of a cell state or a likelihood of a cell state of the cell; and one or more sequences encoding a non-coding nuclear retention RNA motif; and (b) identifying a candidate therapeutic moiety that results in a change in a cell state or a likelihood of a cell state of a cell of the animal or the organoid, thereby identifying a candidate therapeutic moiety.
- Claim:
2. (canceled)
- Claim:
3. The method of claim 1, further comprising isolating the nucleus of one or more cells comprising said one or more reporters.
- Claim:
4-5. (canceled)
- Claim:
6. The method of claim 1, further comprising enriching or sorting a population of nuclei of cells having the change in the cell state or the likelihood of the cell state, wherein enriching or sorting comprises enriching or sorting the population of cells or nuclei based on a level of the one or more reporters and wherein enriching or sorting comprises performing FACS, an affinity purification method, flow cytometry, or microfluidic sorting.
- Claim:
7. (canceled)
- Claim:
8. The method of claim 1, wherein identifying comprises identifying the candidate therapeutic moiety based on a presence of the therapeutic moiety barcode in the cell or nucleus and wherein identifying comprises performing single cell analysis, single nucleus analysis, RNA sequencing, single cell RNA sequencing, single nucleus RNA sequencing, droplet-based single cell RNA sequencing, droplet-based single nucleus RNA sequencing, bulk analysis, sequencing a population of nuclei, or sequencing a population of cells to determine an amount of the candidate therapeutic moiety present in the population of cells.
- Claim:
9. The method of claim 1, wherein identifying comprises a single cell or single nucleus RNA sequencing and/or (ii) droplet-based single cell or droplet-based single nucleus RNA sequencing.
- Claim:
10. (canceled)
- Claim:
11. The method of claim 1, wherein said one or more sequences encoding a non-coding nuclear retention RNA motif comprise a sequence encoding a 1ncRNA or a fragment thereof.
- Claim:
12. The method of claim 1, wherein said one or more sequences encoding a non-coding nuclear retention RNA motif comprise a sequence encoding a BMP2-OP1-responsive gene (BORG) or a fragment thereof.
- Claim:
13. The method of claim 1, wherein said one or more sequences encoding a non-coding nuclear retention RNA motif comprise one or more copies of a pentamer motif comprising SEQ ID NO:1.
- Claim:
14-15. (canceled)
- Claim:
16. The method of claim 1, wherein said one or more sequences encoding a non-coding nuclear retention RNA motif comprise one or more copies of a nucleic acid sequence comprising SEQ ID NO:2, 3, 4, 5, 6 or 7.
- Claim:
17-18. (canceled)
- Claim:
19. The method of claim 1, wherein said one or more sequences encoding a non-coding nuclear retention RNA motif comprise a JPX, PVT1, or NR2F1-AS1 sequence or a fragment thereof.
- Claim:
20. The method of claim 1, wherein said one or more sequences encoding a non-coding nuclear retention RNA motif comprise one or more nucleic acid sequences comprising SEQ ID NO:7.
- Claim:
21-26. (canceled)
- Claim:
27. The method of claim 1, wherein said one or more sequences encoding a non-coding nuclear retention RNA motif comprise a SIRLOIN sequence comprising a nucleic acid sequence comprising SEQ ID NO:9 or a fragment thereof.
- Claim:
28. The method of claim 1, wherein said one or more sequences encoding a non-coding nuclear retention RNA motif comprise one or more copies of a nucleic acid sequence comprising SEQ ID NO:8.
- Claim:
29. The method of claim 1, wherein said one or more sequences encoding a non-coding nuclear retention RNA motif comprise a sequence that binds an amino acid sequence comprising SEQ ID NO:10.
- Claim:
30. The method of claim 1, wherein said one or more sequences encoding a non-coding nuclear retention RNA motif comprise a sequence that encodes an enChr, wherein the enChr comprises one or more copies of a U1 snRNP recognition motif, and wherein the U1 snRNP recognition motif comprises one or more copies of a nucleic acid sequence comprising SEQ ID NO:11, 12, 13, or any combination thereof.
- Claim:
31-32. (canceled)
- Claim:
33. The method of claim 1, wherein said one or more sequences encoding a non-coding nuclear retention RNA motif comprise a sequence that encodes a U1 snRNP recognition motif, wherein the U1 snRNP recognition motif comprises one or more copies of a nucleic acid sequence comprising SEQ ID NO:11, 12, 13, or any combination thereof.
- Claim:
34. (canceled)
- Claim:
35. The method of claim 1, wherein each nucleic acid sequences, encoding a therapeutic moiety barcode is operably linked to nucleic acid sequence encoding a Polymerase III promoter.
- Claim:
36. The method of claim 35, wherein a capture sequence is further operably linked to the therapeutic moiety barcode under the control of the Polymerase III promoter, wherein the capture sequence has a sequence comprising any one of SEQ ID NOs:14-17.
- Claim:
37. (canceled)
- Claim:
38. The method of claim 35, wherein one or more molecular enrichment sequences are operably linked to the therapeutic moiety barcode under the control of the Polymerase III promoter, wherein the one or more molecular enrichment sequences have a sequence comprising any one of SEQ ID NOs:18-97.
- Claim:
39. (canceled)
- Claim:
40. The method of claim 35, wherein a unique genome identification (UGI) sequence is operably linked to the therapeutic moiety barcode region under the control of the Polymerase III promoter, wherein the UGI has a sequence comprising SEQ ID NO:98.
- Claim:
41. (canceled)
- Claim:
42. The method of claim 1, wherein the non-coding nuclear retention RNA motif is operably linked to one or more of said therapeutic moiety barcodes.
- Current International Class:
12; 12; 01
- الرقم المعرف:
edspap.20240150751
Processing Request
No Comments.