IP Library Granted Patent US 12,421,626
Granted Patent B2
US 12,421,626 · App. 17/281,540 · Granted Sep 23, 2025

Cell-stored barcoded deep mutational scanning libraries and uses of the same

Inventors: Jesse Bloom (Seattle, WA); Adam S. Dingens (Seattle, WA); Katharine H. D. Crawford (Seattle, WA); Caelan Radford (Seattle, WA)
Assignees: Fred Hutchinson Cancer Center; University of Washington
C40B40/08
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Quick Facts
Patent No.
US 12,421,626
App. No.
17/281,540
Granted
Sep 23, 2025
Kind
B2
Abstract

Cell-stored barcoded viral protein deep mutational scanning libraries are described. The libraries can be used to map resistance mutations to therapeutic treatments. The libraries can be used to predict viruses that become resistant to therapeutic compounds and/or may more easily evolve to infect new species. The libraries can also be used to more safely study dangerous viruses that normally require high safety biocontainment facilities. The libraries include features that allow efficient collection and assessment of informative data, obviating many bottlenecks of previous approaches.

Claims (20)

1. A cell-stored barcoded deep mutational scanning library of variants of a viral protein comprising: storage cells, wherein at least 90% of the storage cells comprise a non-self-inactivating viral vector comprising a single homozygous barcoded variant nucleotide sequence encoding a viral protein variant from a set of homozygous barcoded variant nucleotide sequences in the library integrated into the storage cell's genome, wherein the set of homozygous barcoded variant nucleotide sequences collectively encode viral protein variants comprising at least 15 amino acid substitutions at at least 95% of amino acid positions of the viral protein.

2. The library of claim 1 , wherein the viral protein variants comprise viral entry protein variants.

3. The library of claim 1 , wherein the viral protein variants comprise viral gag pol variants.

4. The library of claim 1 , wherein the viral protein variants comprise viral Tat variants.

5. The library of claim 1 , wherein the viral protein variants comprise viral Rev variants.

6. The library of claim 1 , wherein the viral vector comprises a retroviral vector or a lentiviral vector.

7. The library of claim 1 , wherein the viral vector comprises a functional U3.

8. The library of claim 1 , wherein the viral vector comprises a gene encoding a reporter or selectable marker.

9. The library of claim 8 , wherein the gene encoding the reporter or selectable marker is linked to each barcoded variant sequence by a linker.

10. The library of claim 9 , wherein the linker is selected from Thosea asigna virus 2A, porcine teschovirus-1 P2A, equine rhinitis A virus E2A, and foot-and-mouth disease virus F2A.

11. The library of claim 1 , wherein the viral vector comprises a first promoter to express the reporter or selectable marker and a second promoter to express the viral variant protein.

12. The library of claim 1 , wherein each barcoded variant sequence comprises a barcode located after the stop codon of the variant sequence.

13. The library of claim 1 , wherein the storage cells are derived from 293T, HEK293T/17, HEK293F, HEK293S, HEK293SGH, EK293FTM, HEK293SGGD, GP2-293, HeLa, HeLa S3, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, COS-7, A549, MDCK, HepG2, C2C12, THP-1, HUDEP-2, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, BS-C-1, monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts, 10.1 mouse fibroblasts, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO—IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr-/-, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, Hepa1c1c7, HL-60, HMEC, HT-29, JY, K562, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT2, RenCa, RIN-5F, RMA/RMAS, Saos-2, Sf-9, SkBr3, T2, T-47D, T84, THP1, U373, U87, U937, VCaP, Vero, WM39, WT-49, X63, YAC-1, and YAR cells.

14. The library of claim 2 , wherein the storage cells further comprise plasmids comprising sequences encoding viral Gag Pol, Tat, and Rev proteins.

15. The library of claim 2 , wherein the storage cells further comprise a plasmid comprising a sequence encoding a functional unrelated viral entry protein.

16. The library of claim 3 , wherein the storage cells further comprise plasmids comprising sequences encoding Tat, Rev, and an entry protein.

17. The library of claim 4 , wherein the storage cells further comprise plasmids comprising sequences encoding an entry protein, Gag Pol, and Rev.

18. The library of claim 5 , wherein the storage cells further comprise plasmids comprising sequences encoding an entry protein, Gag Pol, and Tat.

19. The library of claim 1 , wherein the viral protein is derived from a virus selected from Chikungunya, Ebola, Hendra, hepatitis B, hepatitis C, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), influenza, Lassa, measles, Middle East respiratory syndrome coronavirus (MERS-COV), Nipah, Rabies, respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus (SARS-COV).

20. The library of claim 2 , wherein the viral entry protein variants are variants of a viral entry protein selected from influenza hemagglutinin (HA), HIV envelope (Env), Chikungunya E1 Env, Chikungunya E2 Env, Ebola glycoprotein (EBOV GP), Hendra F glycoprotein, Hendra G glycoprotein, hepatitis B large (L), hepatitis B middle (M), hepatitis B small(S), hepatitis C glycoprotein E1, hepatitis C glycoprotein E2, Lassa virus envelope glycoprotein (LASV GP), measles hemagglutinin glycoprotein (H), measles fusion glycoprotein F0 (F), MERS-COV Spike(S), Nipah fusion glycoprotein F0 (F), Nipah glycoprotein G, Rabies virus glycoprotein G (RABV G), RSV fusion glycoprotein F0 (F), RSV glycoprotein G, and SARS-COV Spike(S).

Assignments (4)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 12, 2025
From: DUSENBURY, KATHARINE; RADFORD, CAELAN
To: UNIVERSITY OF WASHINGTON
Reel/Frame 071396/0865 →
CONFIRMATORY LICENSE Recorded Nov 22, 2023
From: FRED HUTCHINSON CANCER RESEARCH CENTER
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 065666/0487 →
MERGER AND CHANGE OF NAME Recorded Jun 23, 2022
From: FRED HUTCHINSON CANCER RESEARCH CENTER; SEATTLE CANCER CARE ALLIANCE
To: FRED HUTCHINSON CANCER CENTER
Reel/Frame 060438/0369 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 10, 2021
From: BLOOM, JESSE; DINGENS, ADAM S.
To: FRED HUTCHINSON CANCER RESEARCH CENTER
Reel/Frame 057131/0837 →
Continuity (2)
Provisional Application 62692398 · Jun 29, 2018
Related Publication 20210363661A1 · Nov 25, 2021
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