cis and trans requirements for terminal resolution of human bocavirus 1

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).

2. 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.

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

4. 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.

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

6. 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.

7. 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.

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).

9. 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.

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

11. 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.

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

13. 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.

14. 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.

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).

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).