# US 12,173,305 B2 · cis and trans requirements for terminal resolution of human bocavirus 1

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- Number: US 12,173,305 B2
- Document ID: US12173305B2
- Status: Granted
- Kind: B2
- Application number: 16/304,064
- Filed: 2017-05-26
- Published: 2019-07-04
- Granted: 2024-12-24
- Earliest claimed: 2016-05-26
- Assignees: University of Iowa Research Foundation; University of Kansas
- Applicants: University of Iowa Research Foundation; University of Kansas
- Inventors: John F. Engelhardt; Ziying Yan; Jianming Qiu
- Web page: https://patentgrep.com/patent/US12173305B2

## CPC

- C12N 15/86: Viral vectors
- 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 2750/14321: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2750/14322: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2750/14343: viral genome or elements thereof as genetic vector
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A recombinant mutant BoV genome is provided, as well as methods of using the vector, e.g., to prepare helper-free virus.

## Claims

### Claim 1

A method of preparing helper-virus-free recombinant human bocavirus 1 (rhBoV1), comprising:
a) providing a mutant human bocavirus (BoV1) genome comprising
(i) a left-end hairpin (LEH), a 3′ untranslated region (UTR) and a truncated right-end hairpin (REH), wherein the truncated REH consists of the contiguous nucleotide sequence of 5357 to 5402 of SEQ ID NO: 1,
(ii) one or more mutations in each one of the human bocavirus NS2 protein, NS3 protein, NS4 protein, VP1 protein, VP2 protein, VP3 protein, NS1 protein and NP1 protein, such that human NS2, NS3, NS4, VP1, VP2, VP3, NS1 and NP1 proteins are not expressed from the genome when the genome is introduced to cells, wherein the one or more mutations include a deletion of one or more nucleotides, an insertion of one or more nucleotides, or a substitution of one or more nucleotides;
b) providing one or more vectors encoding at least human bocavirus NP1, NS1, VP1, VP2 and VP3, which vectors do not include one or more of LEH, a 3′ UTR and a REH;
c) introducing the mutant human bocavirus genome and the one or more vectors to HEK293 cells and culturing said HEK293 cells; and
d) collecting helper-free virus from the HEK293 cells of c).

### Claim 2 (depends on 1)

The method of claim 1, wherein the mutant genome of a) comprises an expression cassette encoding a heterologous gene product or a heterologous nucleic acid sequence for homologous recombination with selected sequences in a mammalian genome.

### Claim 3 (depends on 2)

The method of claim 2, wherein the heterologous gene product is a viral, bacterial, tumor, parasite, or fungal antigen.

### Claim 4 (depends on 2)

The method of claim 2, wherein the heterologous nucleic acid sequence is less than about 6 kb or wherein the heterologous nucleic acid sequence comprises CFTR sequences.

### Claim 5 (depends on 2)

The method of claim 2, wherein the heterologous gene product is a therapeutic gene product.

### Claim 6 (depends on 2)

The method of claim 2, wherein the heterologous gene product is cystic fibrosis transmembrane conductance regulator, β-globin, γ-globin, tyrosine hydoxylase, glucocerebrosidase, aryl sulfatase A, factor VIII, dystrophin, alpha 1-antitrypsin, surfactant protein SP-D, SP-A or SP-C, erythropoietin, HBOV protein, influenza virus protein, RSV protein, a neutralizing antibody or an antigen binding fragment thereof, SARS virus protein, DNA endonuclease, zinc finger nuclease, a fusion protein including a DNA binding domain linked to a nuclease, or a cytokine.

### Claim 7 (depends on 6)

The method of claim 6, wherein the cytokine is selected from the group consisting of IFN-alpha, IFN-gamma, INF, IL-1, IL-17, and IL-6.

### Claim 8

A method of preparing helper-virus-free recombinant human bocavirus 1 (rhBoV1), comprising:
a) providing a HEK293 cell that expresses human bocavirus VP1, VP2 and VP3 and bocavirus NP1 and NS1, but does not express one or more of human bocavirus NS2, NS3 or NS4;
b) providing a mutant human BoV1genome comprising
(i) a left-end hairpin (LEH), a 3′ untranslated region (UTR) and a truncated right-end hairpin (REH), wherein the truncated REH consists of the contiguous nucleotide sequence of 5357 to 5402 of SEQ ID NO: 1,
(ii) one or more mutations in each one of the human bocavirus NS2 protein, NS3 protein, NS4 protein, VP1 protein, VP2 protein, VP3 protein, NS1 protein and NP1 protein, such that human NS2, NS3, NS4, VP1, VP2, VP3, NS1 and NP1 proteins are not expressed from the genome when the genome is introduced to cells, wherein the one or more mutations include a deletion of one or more nucleotides, an insertion of one or more nucleotides, or a substitution of one or more nucleotides;
c) introducing the mutant BoV1 genome into the HEK293 cell of step (a) and culturing the HEK293 cell so as to express the mutant human BoV1 genome in the HEK293 cell; and
d) collecting helper-free virus from the culture of c).

### Claim 9 (depends on 8)

The method of claim 8, wherein the mutant genome of b) comprises an expression cassette encoding a heterologous gene product or a heterologous nucleic acid sequence for homologous recombination with selected sequences in a genome of a mammal.

### Claim 10 (depends on 9)

The method of claim 9, wherein the heterologous gene product is a viral, bacterial, tumor, parasite, or fungal antigen.

### Claim 11 (depends on 9)

The method of claim 9, wherein the heterologous nucleic acid sequence is less than about 6 kb or wherein the heterologous nucleic acid sequence comprises CFTR sequences.

### Claim 12 (depends on 9)

The method of claim 9, wherein the heterologous gene product is a therapeutic gene product.

### Claim 13 (depends on 9)

The method of claim 9, wherein the heterologous gene product is cystic fibrosis transmembrane conductance regulator, β-globin, γ-globin, tyrosine hydoxylase, glucocerebrosidase, aryl sulfatase A, factor VIII, dystrophin, alpha 1-antitrypsin, surfactant protein SP-D, SP-A or SP-C, erythropoietin, HBOV protein, influenza virus protein, RSV protein, a neutralizing antibody or an antigen binding fragment thereof, SARS virus protein, DNA endonuclease, zinc finger nuclease, a fusion protein including a DNA binding domain linked to a nuclease, or a cytokine.

### Claim 14 (depends on 13)

The method of claim 13, wherein the cytokine is selected from the group consisting of IFN-alpha, IFN-gamma, INF, IL-1, IL-17, and IL-6.

### Claim 15

A method of preparing helper-virus-free recombinant human bocavirus 1 (rhBoV1), comprising:
a) providing a mutant human BoV1 genome comprising
(i) a left-end hairpin (LEH), a heterologous nucleic acid sequence for homologous recombination with selected sequences in a genome of a mammal, a 3′ untranslated region (UTR) and a truncated right-end hairpin (REH), wherein the truncated REH consists of the contiguous nucleotide sequence of 5357 to 5402 of SEQ ID NO: 1,
(ii) one or more mutations in each one of the human bocavirus NS2 protein, NS3 protein, NS4 protein, VP1 protein, VP2 protein, VP3 protein, NS1 protein and NP1 protein, such that human NS2, NS3, NS4, VP1, VP2, VP3, NS1 and NP1 proteins are not expressed from the genome when the genome is introduced to cells, wherein the one or more mutations include a deletion of one or more nucleotides, an insertion of one or more nucleotides, or a substitution of one or more nucleotides;
b) providing one or more vectors encoding at least human bocavirus NP1, NS1, VP1, VP2 and VP3, which vectors do not include one or more of LEH, a 3′ UTR and a REH;
c) introducing the mutant human BoV1 genome and the one or more vectors to HEK293 cells and culturing said HEK293 cells; and
d) collecting helper-free virus from the cells of c).

### Claim 16

A method of preparing helper-virus-free recombinant human bocavirus 1 (rhBoV1), comprising:
a) providing a HEK293 cell that expresses human bocavirus VP1, VP2 and VP3 and bocavirus NP1 and NS1, but does not express one or more of human bocavirus NS2, NS3 or NS4;
b) providing a mutant human BoV1 genome comprising
(i) a left-end hairpin (LEH), a heterologous nucleic acid sequence for homologous recombination with selected sequences in a genome of a mammal, a 3′ untranslated region (UTR) and a truncated right-end hairpin (REH), wherein the truncated REH consists of the contiguous nucleotide sequence of 5357 to 5402 of SEQ ID NO: 1,
(ii) one or more mutations in each one of the human bocavirus NS2 protein, NS3 protein, NS4 protein, VP1 protein, VP2 protein, VP3 protein, NS1 protein and NP1 protein, such that human NS2, NS3, NS4, VP1, VP2, VP3, NS1 and NP1 proteins are not expressed from the genome when the genome is introduced to cells, wherein the one or more mutations include a deletion of one or more nucleotides, an insertion of one or more nucleotides, or a substitution of one or more nucleotides;
c) introducing the mutant BoV1 genome into the HEK293 cell of step (a) and culturing the HEK293 cell so as to express the mutant human BoV1 genome in the host HEK293 cell; and
d) collecting helper free virus from the culture of c).