Mutant subgenomic promoter library and uses thereof

Patent metadata

Field	Value
Publication number	US-12098366-B2
Application number	US-202016784962-A
Country	US
Kind code	B2
Filing date	Feb 7, 2020
Priority date	Apr 9, 2019
Publication date	Sep 24, 2024
Grant date	Sep 24, 2024

Abstract

Disclosed herein is a subgenomic promoter library derived from alphavirus. Also provided herein are methods of using the subgenomic promoters to produce antibodies and other molecules.

First claim

What is claimed is:

1. An engineered subgenomic promoter library comprising a plurality of promoters, wherein each promoter comprises a nucleic acid sequence at least 70% identical to nucleic acid sequences of SEQ ID NOs: 1, 3-13, 15-22, 24-40, 43, 44, 46-50, 52-56, and 58-74.

2. The engineered subgenomic promoter library of claim 1 , wherein the promoter is a subgenomic promoter derived from an alphavirus.

3. The engineered subgenomic promoter library of claim 2 , wherein the alphavirus is Venezuela Equine Encephalitis virus, Semliki Forest virus, or Sindbis virus.

4. The engineered subgenomic promoter library of claim 1 , wherein each of the subgenomic promoters further comprises restriction endonuclease sites at the 5′ and 3′ ends.

5. The engineered subgenomic promoter library of claim 1 , wherein the library comprises engineered subgenomic promoters having differential activities.

6. An engineered nucleic acid comprising:
(i) a promoter, wherein the promoter comprises a nucleic acid sequence at least 70% identical to nucleic acid sequences of SEQ ID NOs: 1, 3-13, 15-22, 24-40, 43, 44, 46-50, 52-56, and 58-74, wherein the promoter is less than 200 nucleotides long; and
(ii) a transgene operably linked to the promoter of (i).

7. The engineered nucleic acid of claim 6 , wherein the transgene encodes for a therapeutic molecule or a detectable molecule.

8. The engineered nucleic acid of claim 7 , wherein the transgene encodes for a heavy chain or a light chain of an antibody.

9. An expression cassette comprising one or more engineered nucleic acid of claim 6 .

10. The expression cassette of claim 9 , wherein the expression cassette is an antibody expression cassette.

11. An antibody expression cassette, comprising
a first engineered nucleic acid comprising a first engineered subgenomic promoter, wherein the promoter comprises a nucleic acid sequence at least 70% identical to nucleic acid sequences of SEQ ID NOs: 1, 3-13, 15-22, 24-40, 43, 44, 46-50, 52-56, and 58-74 wherein the promoter is less than 200 nucleotides long operably linked to a first transgene, wherein the first transgene encodes a heavy chain of an antibody; and
a second engineered nucleic acid comprising a second engineered subgenomic promoter, wherein the promoter comprises a nucleic acid sequence at least 70% identical to nucleic acid sequences of SEQ ID NOs: 1, 3-13, 15-22, 24-40, 43, 44, 46-50, 52-56, and 58-74 wherein the promoter is less than 200 nucleotides long operably linked to a second transgene, wherein the second transgene encodes a light chain of an antibody.

12. A vector comprising one or more engineered nucleic acid of claim 6 .

13. The vector of claim 12 , wherein the vector is a plasmid, RNA replicon, linear double stranded DNA, viral vector, liposome, or nanoparticle.

14. The vector of claim 13 , wherein the one or more engineered nucleic acid is located at the subgenomic region of the RNA replicon.

15. A cell comprising the engineered nucleic acid of claim 6 .

16. The antibody expression cassette of claim 11 , wherein the first engineered subgenomic promoter and the second engineered subgenomic promoter have differential activities.

17. A vector comprising the antibody expression cassette of claim 11 .

18. The vector of claim 17 , wherein the vector is a plasmid, RNA replicon, linear double stranded DNA, viral vector, liposome, or nanoparticle.

19. A cell comprising the antibody expression cassette of claim 11 .

20. The engineered subgenomic promoter library of claim 1 , wherein the library comprises subgenomic promoter sequences set forth in SEQ ID NOs: 1, 3-13, 15-22, 24-40, 43, 44, 46-50, 52-56, and 58-74.

Assignees

• Massachusetts Inst Technology

Inventors

• Weiss Ron
• Huh Jin

Classifications

• C12N15/86

  Viral vectors · CPC title

• C12N15/1072

  Differential gene expression library synthesis, e.g. subtracted libraries, differential screening · CPC title

• C07K2317/76

  Antagonist effect on antigen, e.g. neutralization or inhibition of binding · CPC title

• C07K2317/21

  from primates, e.g. man · CPC title

• C07K16/2818

  against CD28 or CD152 · CPC title