# US 11,821,111 B2 · Barcoded influenza viruses and deep mutational scanning libraries including the same

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- Number: US 11,821,111 B2
- Document ID: US11821111B2
- Status: Granted
- Kind: B2
- Application number: 17/097,853
- Filed: 2020-11-13
- Published: 2021-05-20
- Granted: 2023-11-21
- Earliest claimed: 2019-11-15
- Assignees: Fred Hutchinson Cancer Center
- Applicants: Fred Hutchinson Cancer Center; University of Washington
- Inventors: Jesse Bloom; Allison Greaney; Andrea Loes; Adam S. Dingens
- Web page: https://patentgrep.com/patent/US11821111B2

## CPC

- C40B 50/06: Biochemical methods, e.g. using enzymes or whole viable microorganisms
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12N 2760/16021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2760/16121: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2760/16122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2760/16221: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2760/16222: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Methods to create barcoded influenza viruses without disrupting the function of the viral proteins and the proper packaging of the viral genome segments are described. The barcoded influenza viruses can be used within deep mutational scanning libraries to map influenza resistance mutations to therapeutic treatments. The libraries can also be used to predict influenza strains that may become resistant to therapeutic treatments and/or more easily evolve to infect new species. The libraries include features that allow efficient collection and assessment of informative data, obviating bottlenecks of previous approaches.

## Claims

### Claim 1

A method for barcoding an influenza virus genome segment that comprises:
a 3′ viral RNA genome packaging signal;
an open reading frame; and
a 5′ viral RNA genome packaging signal, comprising:
inserting a nucleic acid comprising a barcode and a copy of the 5′ viral RNA genome packaging signal between the end of the open reading frame and the non-coding portion of the 5′ viral RNA genome packaging signal,
wherein the method has minimal to no effects on viral fitness,
wherein the barcode comprises 5-100 nucleotides in length, and
wherein the open reading frame encodes hemagglutinin (HA), neuraminidase (NA), M1 matrix protein (M1), M2 ion channel protein (M2), nuclear protein (NP), nonstructural protein 1 (NS1), nonstructural protein 1 (NS2), or a subunit of an RNA-dependent RNA polymerase complex selected from PB1, PB2, and PA.

### Claim 2 (depends on 1)

The method of claim 1, further comprising inserting a nucleic acid comprising a copy of the 3′ viral RNA genome packaging signal between the non-coding portion of the 3′ viral RNA genome packaging signal and the beginning of the open reading frame.

### Claim 3 (depends on 1)

The method of claim 1, wherein the copy of the 5′ viral RNA genome packaging signal lacks a start codon.

### Claim 4 (depends on 1)

The method of claim 1, wherein the copy of the 5′ viral RNA genome packaging signal has at least 95% sequence identity with the 5′ viral RNA genome packaging signal.

### Claim 5 (depends on 2)

The method of claim 2, wherein the copy of the 3′ viral RNA genome packaging signal lacks a start codon.

### Claim 6 (depends on 2)

The method of claim 2, wherein the copy of the 3′ viral RNA genome packaging signal has at least 95% sequence identity with the 3′ viral RNA genome packaging signal.

### Claim 7 (depends on 1)

The method of claim 1, wherein the barcode is 18 nucleotides in length.