Chimeric bocavirus capsid proteins

Abstract

The present invention provides chimeric bocavirus capsid proteins comprising amino acid sequences from AAV and bocavirus, nucleic acids encoding said proteins, plasmids, host cells and compositions as well as vectors comprising said capsids; and their use in gene therapy.

Description

[0001] CHIMERIC BOCAVIRUS CAPSID PROTEINS

[0002] FIELD OF THE INVENTION

[0003] The present invention provides chimeric bocavirus capsid proteins comprising amino acid sequences from AAV and bocavirus, nucleic acids encoding said proteins, plasmids, host cells and compositions as well as vectors comprising said capsids; and their use in gene therapy.

[0004] Background of the invention

[0005] Adeno-associated viruses (AAV) belong to the genus dependoparvovirus of the subfamily Parvovirinae, family Parvoviridae and are composed of a single-stranded DNA genome that is encapsidated in a 25 nm non-enveloped capsid. Gene therapy vectors based on wild-type AAV have proven safe and efficacious in numerous clinical trials. For the engineering of recombinant AAV (rAAV) gene therapy vectors with enhanced organ or cell specificity and / or transduction efficiency, a wide variety of techniques have been developed to modify and improve the properties of AAV capsids, ranging from ancestral reconstruction and peptide display to directed molecular evolution (Becker et al. Pathogens 2022). Among the last, a very powerful and versatile approach is DNA shuffling, where chimeric AAV capsid sequences consisting of amino acid sequences from different AAV serotypes are created via cap gene fragmentation and reassembly based on partial homologies (Grimm et al. J Virol 2008).

[0006] More recently, another genus of the Parvovirinae subfamily, the bocaparvoviruses (bocaviruses) has been identified in humans. The 5,543 nucleotide genome of human bocavirus 1 (HBoVl) encodes three open reading frames that enable the expression of non- structural proteins (NS1-NS4 and NP1), structural proteins (VP1, VP2 and VP3) and a small non-coding RNA. The structural proteins are encoded by one mRNA and are produced by differential splicing and translation from three different start codons. The full length VP1 has 671 amino acids with a N-terminal VP1 unique domain (VPlu, 90 amino acids). The VP3 protein, which consists of the C-terminal portion of VP1, has 542 amino acids. The VP2 protein has 581 amino acids and consists of all of VP3 and a VP1 / VP2 shared domain of 39 amino acids. The larger genome size of bocavirus compared to AAV means that bocaviruses have a larger packaging capacity. Due to their larger packaging capacity compared to AAV, bocaviruses are of great interest as a delivery vector for gene therapy. Bocaviruses are especially attractive for gene therapy of as of yet untreatable genetic diseases caused by non- or only partially functional genes having sizes larger than 4.5 kilobases. Three further serotypes of human bocavirus (HBoV2, HBoV3, HBoV4) and one gorilla bocavirus (GBoVl) have been described.

[0007] Bocaviruses have also been studied for their suitability as hybrid parvoviral gene therapy vectors, harboring cross-genera combinations of recombinant AAV2 genomes with capsid shells from different bocavirus serotypes. For example, Yan et al. (Molecular Therapy 2013 (21) 2:2181-2194) created a cross-genera pseudotype virus containing a 5.5 kilobases recombinant AAV2 genome packaged within a wildtype HBoVl capsid.

[0008] A broader use of bocavirus vectors is however prevented by limited tissue targeting options and, in particular, their low transduction efficiency compared to AAV vectors.

[0009] Thus, there is a need for bocavirus-based vectors that demonstrate improved transduction efficiency and tissue targeting, while retaining the advantageous features of bocaviruses such as their increased packaging capacity compared to AAV.

[0010] Objectives and Summary of the Invention

[0011] To improve transduction efficiency of bocavirus vectors, a series of rationally designed chimeric capsid proteins composed of amino acid sequences from both bocavirus and AAV viral proteins were designed. However, given the low sequence identity between the AAV and bocavirus genera (VP1 capsids <40% identity based on amino acid sequence alignment) it was not expected that their mixing would result in viable capsids at all. The inventors have surprisingly found that by swapping amino acid sequence of bocavirus with the corresponding sequence of AAV, chimeric bocavirus capsid proteins with improved properties can be generated. In particular, the inventors have surprisingly found that by swapping an amino acid domain of bocavirus VP1 with the corresponding sequence of AAV VP1 resulted in a chimeric capsid protein that retained the transduction properties of AAV while also displaying the increased packaging capacity of bocavirus. It was hitherto unknown if AAV and a bocavirus sequence, in particular an AAV and bocavirus VP1 sequences could be swapped and / or combined. It was not known that an inter-species chimeric capsid protein would be functional, especially considering the low amino acid sequence percentage identity between AAV VP1 and bocavirus VP1 of only about 33-34% (for example, AAV2 VP1 and GBoVl share an amino acid sequence percentage identity of 33,76% when using processing-in-memory alignment and 29.9% when using Smith-Waterman alignment online tool by EMBL's European Bioinformatics Institute at www.ebi.ac.uk).

[0012] Thus, in a first aspect, the invention provides a chimeric bocavirus capsid protein comprising a bocavirus amino acid sequence, in particular a bocavirus Viral Protein (VP) amino acid sequence, and an adeno-associated virus (AAV) amino acid sequence, in particular an AAV VP amino acid sequence.

[0013] In one embodiment, the chimeric bocavirus capsid protein of the invention improves the transduction efficiency of a recombinant bocavirus or bocavirus-like particle compared to a wild-type bocavirus capsid protein.

[0014] Chimeric Bocavirus Capsid Proteins

[0015] The inventors have performed a series of sequence swaps between bocavirus and AAV VP1. The resulting chimeric capsid proteins are schematically depicted in Figures 2, 6 and 9. The inventors have replaced portions of bocavirus VP1 with portions of AAV VP1. Hence, in one embodiment, the chimeric bocavirus capsid protein is VP1.

[0016] In one embodiment, the VP1 protein comprises, in order from N-terminus to C-terminus, an N-terminal VPlu domain, a VP1 / VP2 domain, and a VP3 domain. The VP3 domain may be further divided into an unstructured VP3 domain, which is adjacent to the VP1 / VP2 domain, and a structured VP3 domain, which includes the C-terminus.

[0017] In one embodiment, the AAV VP amino acid sequence comprises at least 20 consecutive amino acids of an AAV VP1. In another embodiment, the AAV VP amino acid sequence comprises at least 30 consecutive amino acids, at least 40 consecutive amino acids, at least 50 consecutive amino acids, at least 60 consecutive amino acids, at least 70 consecutive amino acids, at least 80 consecutive amino acids, at least 90 consecutive amino acids, at least 100 consecutive amino acids, at least 110 consecutive amino acids, or at least 120 consecutive amino acids of an AAV VP1.

[0018] In another embodiment, the AAV VP amino acid sequence comprises at least 150 consecutive amino acids, at least 200 consecutive amino acids, at least 250 consecutive amino acids, at least 300 consecutive amino acids, at least 350 consecutive amino acids, at least 400 consecutive amino acids, at least 450 consecutive amino acids, or at least 500 consecutive amino acids of an AAV VP1.

[0019] In one embodiment, the AAV VP amino acid sequence comprises two fragments of at least 20 consecutive amino acids of an AAV VP1 each. In another embodiment, the AAV VP amino acid sequence comprises two fragments of at least 30 consecutive amino acids, at least 40 consecutive amino acids, at least 50 consecutive amino acids, at least 60 consecutive amino acids, at least 70 consecutive amino acids, at least 80 consecutive amino acids, at least 90 consecutive amino acids, at least 100 consecutive amino acids, at least 110 consecutive amino acids, or at least 120 consecutive amino acids of an AAV VP1 each.

[0020] In one embodiment, the AAV VP amino acid sequence comprises at least 20 consecutive amino acids of an AAV VPlu domain. In another embodiment, the AAV VP amino acid sequence comprises at least 30 consecutive amino acids, at least 40 consecutive amino acids, at least 50 consecutive amino acids, at least 60 consecutive amino acids, at least 70 consecutive amino acids, at least 80 consecutive amino acids, at least 90 consecutive amino acids, at least 100 consecutive amino acids, at least 110 consecutive amino acids, or at least 120 consecutive amino acids of an AAV VPlu domain. In a series of experiments, increasing portions of the N-terminus of bocavirus VP1 were replaced with increasing portions of the N-terminus of AAV VP1. The N-terminus of VP1 comprises fragments of the VPlu domain or the complete VPlu domain.

[0021] Hence, in one embodiment, the AAV VP amino acid sequence comprises the N terminus of the AAV VPlu domain.

[0022] The inventors were able to show that a virus with a chimeric capsid protein comprising an AAV VPlu domain exhibits improved transduction efficiency compared to a wild-type bocavirus or a bocavirus without an AAV VPlu domain. Hence, in one embodiment, the AAV VP amino acid sequence comprises a complete AAV VPlu domain.

[0023] The inventors also investigated whether a virus carrying a chimeric capsid protein comprising less than a complete AAV VPlu domain retains improved transduction efficiency compared to a wild-type bocavirus or a bocavirus without any amino acid sequence from an AAV VPlu domain. Hence, in one embodiment, the AAV VP amino acid sequence of the chimeric bocavirus capsid protein comprises an amino acid sequence from an AAV VP1 that includes the N-terminus of VPlu.

[0024] In another embodiment, the AAV VP amino acid sequence comprises at least the first 20 amino acids from the N-terminus of an AAV VPlu domain. In another embodiment, the AAV VP amino acid sequence comprises at least the first 30 amino acids, at least the first 40 amino acids, at least the first 50 amino acids, at least the first 60 amino acids, at least the first 70 amino acids, at least the first 80 amino acids, at least the first 90 amino acids, at least the first 100 amino acids, at least the first 110 amino acids, or at least the first 120 amino acids of an AAV VPlu domain.

[0025] Hence, in one embodiment, the AAV VP amino acid sequence of the chimeric bocavirus capsid protein comprises the first 43 amino acids of the N terminus of the AAV VPlu domain. In another embodiment, the AAV VP amino acid sequence of the chimeric bocavirus capsid protein comprises at least the first 43 amino acids of the N terminus of the AAV VPlu domain. Since bocavirus and AAV VP proteins are not exactly the same size, the swapped sequences may differ in size. For example, in one embodiment, a 90 amino acid bocavirus VPlu domain may be replaced by a 138 amino acid AAV VPlu domain. In another embodiment, the first 10 amino acids of a bocavirus VPlu domain may be replaced by the first 43 amino acids of an AAV VPlu domain. Exemplarily, such a chimeric bocavirus capsid protein is set forth in SEQ ID NO: 73 (Fig. 9 construct NGH A2.2).

[0026] The swapped VP amino acid sequence of the chimeric bocavirus capsid protein may also comprise other domains of VP1, or fragments of other domains. The chimeric bocavirus capsid protein further comprises a VP amino acid sequence from a VP1 / 2 domain of a Parvoviridae. The VP1 / 2 domain may be an AAV VP1 / 2 domain or a bocavirus VP1 / 2 domain.

[0027] Hence, in one embodiment, the AAV VP amino acid sequence of the chimeric bocavirus capsid protein comprises the VPlu and VP1 / 2 amino acid sequence of an AAV VP.

[0028] Thus, in one embodiment, the AAV VP amino acid sequence comprises at least the first 130 amino acids, at least the first 140 amino acids, at least the first 150 amino acids, at least the first 160 amino acids, at least the first 170 amino acids, at least the first 180 amino acids, at least the first 190 amino acids, or at least the first 200 amino acids of an AAV VP1. In one particular embodiment, the AAV VP amino acid sequence comprises the first 201 amino acids of an AAV VP1, corresponding to the VPlu and VP1 / 2 domains.

[0029] In one embodiment, the bocavirus VP amino acid sequence comprises at least 20 consecutive amino acids of a bocavirus VP1. In another embodiment, the bocavirus VP amino acid sequence comprises at least 30 consecutive amino acids, at least 40 consecutive amino acids, at least 50 consecutive amino acids, at least 60 consecutive amino acids, at least 70 consecutive amino acids, at least 80 consecutive amino acids, at least 90 consecutive amino acids, at least 100 consecutive amino acids, at least 110 consecutive amino acids, or at least 120 consecutive amino acids of a bocavirus VP1. In another embodiment, the bocavirus VP amino acid sequence comprises at least 150 consecutive amino acids, at least 200 consecutive amino acids, at least 250 consecutive amino acids, at least 300 consecutive amino acids, at least 350 consecutive amino acids, at least 400 consecutive amino acids, at least 450 consecutive amino acids, or at least 500 consecutive amino acids of a bocavirus VP1.

[0030] In one embodiment, the bocavirus VP amino acid sequence comprises two fragments of at least 20 consecutive amino acids of a bocavirus VP1 each. In another embodiment, the bocavirus VP amino acid sequence comprises two fragments of at least 30 consecutive amino acids, at least 40 consecutive amino acids, at least 50 consecutive amino acids, at least 60 consecutive amino acids, at least 70 consecutive amino acids, at least 80 consecutive amino acids, at least 90 consecutive amino acids, at least 100 consecutive amino acids, at least 110 consecutive amino acids, or at least 120 consecutive amino acids of a bocavirus VP1 each.

[0031] In another aspect of the invention, the chimeric bocavirus capsid protein comprises an amino acid sequence of an AAV VP1 as described above, and a bocavirus VP amino acid sequence, wherein the bocavirus VP amino acid sequence comprises an amino acid sequence from a bocavirus VP3 domain.

[0032] In one embodiment, the invention provides a chimeric bocavirus capsid protein, consisting of a bocavirus VP amino acid sequence, and an AAV VP amino acid sequence.

[0033] In a particular embodiment, the chimeric bocavirus capsid protein consists of any one of the amino acid sequences of an AAV VP1 as described above, and a bocavirus VP amino acid sequence from a bocavirus VP3 domain.

[0034] Therefore, in one embodiment, the chimeric bocavirus capsid protein comprises the VPlu domain of AAV VP1 and the VP3 domain of bocavirus. Exemplarily, such a chimeric bocavirus capsid protein is set forth in SEQ ID NO: 3. In one particular embodiment, the chimeric bocavirus capsid protein consists of the VPlu domain of AAV VP1 and the VP1 / 2 domain and VP3 domain of bocavirus. In another embodiment, the chimeric bocavirus capsid protein comprises the VPlu domain and VP1 / 2 domain of AAV VP1 and the VP3 domain of bocavirus. Exemplarily, such a chimeric bocavirus capsid protein is set forth in SEQ ID NO: 6. In another embodiment, the chimeric bocavirus capsid protein consists of the VPlu domain and VP1 / 2 domain of AAV VP1 and the VP3 domain of bocavirus.

[0035] The VP3 domain of bocavirus can be separated into an unstructured region and a structured region. The unstructured region consists of the first 32 amino acids of bocavirus VP3. Hence, the structured region of VP3 is an N-terminally truncated VP3. In one embodiment, the bocavirus VP amino acid sequence is an N-terminally truncated VP3. In another embodiment the bocavirus VP amino acid sequence is an N-terminally truncated VP3 that is truncated by up to 32 amino acids.

[0036] In one embodiment, the truncated (unstructured) region of VP3 is replaced by the unstructured region of an AAV VP3. In a particular embodiment, the first 32 amino acids of bocavirus VP3 are replaced by the first 14 amino acids of AAV VP3.

[0037] Hence, in one embodiment, the chimeric bocavirus capsid protein comprises the VPlu domain, VP1 / 2 domain and the unstructured region of the VP3 domain of AAV VP1. Thus, in one embodiment, the chimeric bocavirus capsid protein comprises up to 216 amino acids from AAV VP1. In a particular embodiment, the chimeric bocavirus capsid protein comprises 216 amino acids from AAV VP1.

[0038] Therefore, in one embodiment, the chimeric bocavirus capsid protein comprises the VPlu domain, VP1 / 2 domain and the unstructured region of the VP3 domain of AAV VP1 and the N-terminally truncated VP3 domain of bocavirus that is truncated by 32 amino acids. Exemplarily, such a chimeric bocavirus capsid protein is set forth in SEQ ID NO: 9. In another embodiment, the chimeric bocavirus capsid protein consists of the VPlu domain, VP1 / 2 domain and the unstructured region of the VP3 domain of AAV VP1 and the N-terminally truncated VP3 domain of bocavirus that is truncated by 32 amino acids. In one embodiment, the chimeric bocavirus capsid protein comprises between 20 and 216 consecutive amino acids from AAV VP1. In another embodiment, the chimeric bocavirus capsid protein comprises between 30 and 216 consecutive amino acids from AAV VP1, between 40 and 216 consecutive amino acids from AAV VP1, between 50 and 216 consecutive amino acids from AAV VP1, between 60 and 216 consecutive amino acids from AAV VP1, between 70 and 216 consecutive amino acids from AAV VP1, between 80 and 216 consecutive amino acids from AAV VP1, between 90 and 216 consecutive amino acids from AAV VP1, between 100 and 216 consecutive amino acids from AAV VP1, between 110 and 216 consecutive amino acids from AAV VP1, between 120 and 216 consecutive amino acids from AAV VP1, between 130 and 216 consecutive amino acids from AAV VP1, between 140 and 216 consecutive amino acids from AAV VP1, between 150 and 216 consecutive amino acids from AAV VP1, between 160 and 216 consecutive amino acids from AAV VP1, between 170 and 216 consecutive amino acids from AAV VP1, between 180 and 216 consecutive amino acids from AAV VP1, between 190 and 216 consecutive amino acids from AAV VP1, between 200 and 216 consecutive amino acids from AAV VP1, or between 210 and 216 consecutive amino acids from AAV VP1.

[0039] In one embodiment, the chimeric bocavirus capsid protein comprises between 20 and 216 consecutive amino acids from the N-terminus of AAV VP1. In another embodiment, the chimeric bocavirus capsid protein comprises between 30 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 40 and 216 consecutive amino acids from the N- terminus of AAV VP1, between 50 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 60 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 70 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 80 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 90 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 100 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 110 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 120 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 130 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 140 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 150 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 160 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 170 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 180 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 190 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 200 and 216 consecutive amino acids from the N-terminus of AAV VP1, or between 210 and 216 consecutive amino acids from the N-terminus of AAV VP1.

[0040] In one embodiment, the chimeric bocavirus capsid protein comprises between 20 and 216 amino acids from AAV VP1 and the remaining amino acid sequence of the chimeric bocavirus capsid protein is from bocavirus. In another embodiment, the chimeric bocavirus capsid protein comprises between 30 and 216 amino acids from AAV VP1, between 40 and 216 amino acids from AAV VP1, between 50 and 216 amino acids from AAV VP1, between 60 and 216 amino acids from AAV VP1, between 70 and 216 amino acids from AAV VP1, between 80 and 216 amino acids from AAV VP1, between 90 and 216 amino acids from AAV VP1, between 100 and 216 amino acids from AAV VP1, between 110 and 216 amino acids from AAV VP1, between 120 and 216 amino acids from AAV VP1, between 130 and 216 amino acids from AAV VP1, between 140 and 216 amino acids from AAV VP1, between 150 and 216 amino acids from AAV VP1, between 160 and 216 amino acids from AAV VP1, between 170 and 216 amino acids from AAV VP1, between 180 and 216 amino acids from AAV VP1, between 190 and 216 amino acids from AAV VP1, between 200 and 216 amino acids from AAV VP1, or between 210 and 216 amino acids from AAV VP1 and the remaining amino acid sequence of the chimeric bocavirus capsid protein is from bocavirus VP protein.

[0041] In one embodiment, the chimeric bocavirus capsid protein comprises between 20 and 216 consecutive amino acids from the N-terminus of AAV VP1 and the remaining amino acid sequence of the chimeric bocavirus capsid protein is from bocavirus. In another embodiment, the chimeric bocavirus capsid protein comprises between 30 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 40 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 50 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 60 and 216 consecutive amino acids from the N- terminus of AAV VP1, between 70 and 216 consecutive amino acids from the N-terminus of

[0042] AAV VP1, between 80 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 90 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 100 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 110 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 120 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 130 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 140 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 150 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 160 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 170 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 180 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 190 and 216 consecutive amino acids from the N-terminus of AAV VP1, between 200 and 216 consecutive amino acids from the N-terminus of AAV VP1, or between 210 and 216 consecutive amino acids from the N-terminus of AAV VP1 and the remaining amino acid sequence of the chimeric bocavirus capsid protein is from bocavirus VP protein.

[0043] Further Chimeric Bocavirus Capsid Proteins

[0044] The AAV VP amino acids sequence may be from any AAV. In one embodiment, the AAV VP amino acid sequence is from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13. The AAV VP amino acid sequence may be from a wild-type, recombinant, engineered or synthetic AAV.

[0045] In one embodiment, the AAV VP amino acid sequence is at least 70% identical to the amino acid sequence of AAV2. In another embodiment, the AAV VP amino acid sequence is at least 70% identical to the amino acid sequence of AAV8. In a preferred embodiment, the AAV VP amino acid sequence is from AAV2 or AAV8. In a particularly preferred embodiment, the AAV VP amino acid sequence is from AAV2.

[0046] In one embodiment, the bocavirus VP amino acid sequence is from a mammalian bocavirus. In a preferred embodiment, the bocavirus VP amino acid sequence is from a primate bocavirus. In a more preferred embodiment, bocavirus VP amino acid sequence is from a human bocavirus or a gorilla bocavirus.

[0047] In particularly preferred embodiment, the bocavirus VP amino acid sequence from a primate bocavirus is selected from the group consisting of HBoVl, HBoV2, HBoV3, HBoV4 and GBoVl. In another embodiment, the bocavirus VP amino acid sequence is from GBoVl.

[0048] Therefore, in one embodiment, the chimeric bocavirus capsid protein comprises the VPlu domain of AAV2 VP1 and the VP3 domain of bocavirus GBoVl. This chimeric bocavirus capsid protein is set forth in SEQ ID NO: 3. In one particular embodiment, the chimeric bocavirus capsid protein consists of the VPlu domain of AAV2 VP1 and the VP1 / 2 domain and VP3 domain of bocavirus GBoVl.

[0049] In another embodiment, the chimeric bocavirus capsid protein comprises the VPlu domain of AAV2 VP1 and the VP3 domain of bocavirus HBoVl. This chimeric bocavirus capsid protein is set forth in SEQ ID NO: 63. In one particular embodiment, the chimeric bocavirus capsid protein consists of the VPlu domain of AAV2 VP1 and the VP1 / 2 domain and VP3 domain of bocavirus HBoVl.

[0050] In another embodiment, the chimeric bocavirus capsid protein comprises the VPlu domain and VP1 / 2 domain of AAV2 VP1 and the VP3 domain of bocavirus GBoVl. This chimeric bocavirus capsid protein is set forth in SEQ ID NO: 6. In a particular embodiment, the chimeric bocavirus capsid protein consists of the VPlu domain and VP1 / 2 domain of AAV2 VP1 and the VP3 domain of bocavirus GBoVl.

[0051] In another embodiment, the chimeric bocavirus capsid protein comprises the VPlu domain and VP1 / 2 domain of AAV2 VP1 and the unstructured region of the VP3 domain of AAV2 VP3 and the N-terminally truncated VP3 domain of bocavirus GBoVl that is truncated by 32 amino acids. This chimeric bocavirus capsid protein is set forth in SEQ ID NO: 9. In a particular embodiment, the chimeric bocavirus capsid protein consists of the VPlu domain, VP1 / 2 domain of AAV VP1 and the unstructured region of the VP3 domain of AAV2 VP3 and the VP3 domain of bocavirus GBoVl that is N-terminally truncated by 32 amino acids. In a second aspect, the invention provides a recombinant bocavirus comprising any one of the chimeric bocavirus capsid proteins described above. Furthermore, the invention provides a bocavirus-like particle comprising the chimeric bocavirus capsid protein of the invention.

[0052] Hence, in one aspect, the invention provides a recombinant bocavirus or bocavirus-like particle having a capsid comprising the chimeric bocavirus capsid protein of the invention.

[0053] In one embodiment, the recombinant bocavirus or bocavirus-like particle further comprises a transgene packaged within the capsid. The transgene may be a virus genome or a recombinant virus genome carrying a therapeutic gene.

[0054] In one embodiment, the recombinant bocavirus or bocavirus-like particle comprises the genome of a heterologous virus packaged within the capsid. In a preferred embodiment, the genome is from a virus of the family Parvoviridae. In a particularly preferred embodiment, the genome is from a dependoparvovirus. In one embodiment, the genome is from an AAV virus. The genome may be a recombinant or a wild-type genome .

[0055] In one embodiment, the genome comprises at least one ITR from a virus of the family Parvoviridae. Preferably, the genome comprises at least one ITR from a virus of the subfamily Parvovirinae, more preferably from a virus of the group consisting of AAV or bocavirus. In another embodiment, the genome comprises at least one synthetic ITR.

[0056] In one embodiment, the genome comprises at least one ITR from bocavirus.

[0057] In one embodiment, the genome comprises two ITRs flanking a transgene.

[0058] In one embodiment, the genome comprises ITRs from an AAV virus. In one embodiment, the genome comprises ITRs from an AAV virus and no additional AAV genes. In another embodiment, the genome comprises ITRs from an AAV virus and a heterologous transgene. In a particular embodiment, the genome comprises ITRs are from AAV2.

[0059] Packaged within the recombinant bocavirus or bocavirus-like particle comprising the chimeric bocavirus capsid protein of the invention may be a cargo that is not a transgene. Nucleic acids, plasmids and host cells

[0060] In another aspect, the invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric bocavirus capsid protein of the invention.

[0061] In another aspect, the invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the recombinant bocavirus or bocavirus-like particle of the invention.

[0062] Particularly, the invention provides isolated nucleic acids comprising a nucleic acid sequence as set forth in the SEQ ID NOs provided in the column entitled “Full length chimeric nucleotide sequence” of Tables 3 and 5.

[0063] Furthermore, the invention also provides a plasmid comprising any one of the isolated nucleic acid molecules of the invention.

[0064] The invention also provides a host cell comprising a chimeric bocavirus capsid protein of the invention. Furthermore, another aspect of the invention is a host cell comprising a recombinant bocavirus or bocavirus-like particle of the invention. A further aspect of the invention is a host cell comprising the any one of the isolated nucleic acids of the invention. And yet another aspect of the invention is a host cell comprising a plasmid of the invention.

[0065] In one embodiment, the host cell is a mammalian cell. In another embodiment, the host cell is a non-human mammalian cell.

[0066] Compositions and medical uses

[0067] In another aspect, the invention provides a composition comprising the chimeric bocavirus capsid protein of the invention and optionally one or more excipients. In another aspect, the invention provides a composition comprising the recombinant bocavirus or bocavirus-like particle of the invention, the isolated nucleic acid of the invention, the plasmid of the invention or the host cell of the invention and optionally one or more excipients. In another aspect, the invention relates to the chimeric bocavirus capsid protein of the invention for use as a medicament.

[0068] In another aspect, the invention provides the recombinant bocavirus or bocavirus-like particle of the invention, the isolated nucleic acid of the invention, the plasmid of the invention, the host cell of the invention or the composition of the invention for use as a medicament.

[0069] In another aspect, the invention provides the recombinant bocavirus or bocavirus-like particle of the invention, the isolated nucleic acid of the invention, the plasmid of the invention, the host cell of the invention or the composition of the invention for use as a medicament.

[0070] The chimeric bocavirus capsid protein of the invention is especially suitable for gene therapy, or treatment of a genetic disease. Because the chimeric bocavirus capsid protein has an improved packaging capacity in comparison to conventionally gene therapy vectors, such as AAV virus vectors, the chimeric bocavirus capsid protein of the invention enables gene therapy for larger genes than the ones that could be packaged in AAV virus vectors, which have a packaging capacity of about 4.7 to 4.9 kilobases.

[0071] Hence, in one aspect, the invention relates to the chimeric bocavirus capsid protein of the invention, the recombinant bocavirus or bocavirus-like particle of the invention, the isolated nucleic acid of the invention, the plasmid of the invention, the host cell of the invention, or the composition of the invention for use in the treatment of a genetic disease. In one embodiment, the genetic disease is caused by a defective gene.

[0072] In one further aspect, the invention relates to the chimeric bocavirus capsid protein of the invention, the recombinant bocavirus or bocavirus-like particle of the invention, the isolated nucleic acid of the invention, the plasmid of the invention, the host cell of the invention, or the composition of the invention for use in gene therapy. In one embodiment, the gene therapy is administered to a patient with a defective gene. The gene therapy replaces the defective gene with a healthy, i.e. wildtype variant of the gene.

[0073] In another embodiment, the gene therapy is administered to a patient with a gene that is aberrantly expressed. The aberrant expression may be reduced expression or increased expression.

[0074] Thus, in one embodiment, the invention relates to the chimeric bocavirus capsid protein of the invention, the recombinant bocavirus or bocavirus-like particle of the invention, the isolated nucleic acid of the invention, the plasmid of the invention, the host cell of the invention, or the composition of the invention for use in gene therapy, wherein the gene therapy comprises treatment with a gene of interest with a length of at least 1 kilobases, at least 1,5 kilobases, at least 2 kilobases, at least 2,5 kilobases, at least 3 kilobases, at least

[0075] 3.5 kilobases, at least 4 kilobases, at least 4,5 kilobases, at least 5 kilobases, or at least 5.5 kilobases.

[0076] In one embodiment, the defective gene has a length of about 1 to about 5.5 kilobases. In another embodiment, the defective gene has a length of about 1 to about 5.5 kilobases, about 1,5 to about 5.5 kilobases, about 2 to about 5.5 kilobases, about 2,5 to about 5.5 kilobases, about 3 to about 5.5 kilobases, about 3,5 to about 5.5 kilobases, about 4 to about

[0077] 5.5 kilobases, about 4,5 to about 5.5 kilobases, about 5 to about 5.5 kilobases. In a preferred embodiment, the defective gene has a length of about 4 to about 5.5 kilobases.

[0078] In one embodiment, the defective gene has a length of at least 1 kilobases, at least 1,5 kilobases, at least 2 kilobases, at least 2,5 kilobases, at least 3 kilobases, at least 3,5 kilobases, at least 4 kilobases, at least 4,5 kilobases, at least 5 kilobases, or at least 5.5 kilobases.

[0079] In another embodiment, the defective gene has a length of more than 4.8 kilobases. In another embodiment, the defective gene has a length of more than 4.5 kilobases. In one embodiment, the disease-causing gene has a length of about 1 to about 5.5 kilobases. In another embodiment, the disease-causing gene has a length of about 1 to about 5.5 kilobases, about 1,5 to about 5.5 kilobases, about 2 to about 5.5 kilobases, about

[0080] 2.5 to about 5.5 kilobases, about 3 to about 5.5 kilobases, about 3,5 to about 5.5 kilobases, about 4 to about 5.5 kilobases, about 4,5 to about 5.5 kilobases, about 5 to about 5.5 kilobases. In a preferred embodiment, the disease-causing gene has a length of about 4 to about 5.5 kilobases.

[0081] In one embodiment, the disease-causing gene has a length of at least 1 kilobases, at least

[0082] 1.5 kilobases, at least 2 kilobases, at least 2,5 kilobases, at least 3 kilobases, at least 3,5 kilobases, at least 4 kilobases, at least 4,5 kilobases, at least 5 kilobases, or at least 5.5 kilobases.

[0083] In another embodiment, the disease-causing gene has a length of more than 4.8 kilobases. In another embodiment, the disease-causing gene has a length of more than 4.5 kilobases.

[0084] In one embodiment, gene therapy comprises delivery of a transgene with a total length of about 1 to about 5.5 kilobases, preferably with a length of about 4 to about 5.5 kilobases to a cell. The transgene comprises the gene of interest and optionally one or more further components. The gene of interest is a functional variant, i.e. not disease-associated, version of a defective gene or disease-causing gene. For example, the gene of interest may be a wild-type gene.

[0085] The transgene may comprise one or more components selected from the group consisting of genes of interest, regulatory sequences, signalling sequences, selection markers, ITRs and / or viral genes.

[0086] In another embodiment, the gene therapy comprises delivery of a transgene with a length of at least 4.8 kilobases.

[0087] In one embodiment, the total size of the transgene is less than 6 kilobases. In one embodiment, the transgene comprises a healthy gene of interest (GOI) and a regulatory element. The regulatory element may be a promoter and / or enhancer. Hence, in one embodiment, the gene of interest has a maximal length of about 5.8 kilobases while the regulatory element has a length of at least 0.3 kilobases.

[0088] In another embodiment, the transgene comprises more than one gene and a regulatory element. In one embodiment, the transgene comprises two genes and at least one regulatory element with a total combined length of 6 kilobases.

[0089] In one embodiment, the defective gene has a mutation selected from the group consisting of insertion, deletion, insertion-deletion, missense mutation, truncation mutation, stop mutation, nonsense mutation, frameshift mutation, repeat expansion. In another embodiment, the defective gene exhibits reduced or modified expression.

[0090] In another embodiment, the invention relates to the use of a chimeric bocavirus capsid protein of the invention, the recombinant bocavirus or bocavirus-like particle of the invention, the isolated nucleic acid of the invention, the plasmid of the invention, the host cell of the invention or the composition of the invention in the manufacture of a medicament.

[0091] In another embodiment, the invention relates to the use of a chimeric bocavirus capsid protein of the invention, the recombinant bocavirus or bocavirus-like particle of the invention, the isolated nucleic acid of the invention, the plasmid of the invention, the host cell of the invention or the composition of the invention in the manufacture of a medicament for the treatment of a disease.

[0092] In another embodiment, the invention relates to a method of treating a disease comprising administering the recombinant bocavirus or bocavirus-like particle of the invention.

[0093] In one embodiment, the disease is a genetic disease.

[0094] Figure Legends

[0095] Figure 1: Schematic representation of 2-component bocavirus structural protein expression cassettes, showing transcription start sites, ORF and translated protein domains of Viral Protein 1 (VP1), VP2 and VP3 of gorilla Bocavirus. hEPO: human erythropoietin, bGH poly(A): bovine growth hormone polyadenylation, TSS: transcription start site. The T7 promoter, DNA sequence that serves as a recognition site for the T7 RNA polymerase enzyme. CMV (Cytomegalovirus) promoter initiates gene expression, M130L point mutation to abolish expression of VP3 from the VP1 / VP2 plasmid (see Examples).

[0096] Figure 2: Schematic representation of VP protein domains from AAV2, GBoVl, and chimeras NGB (next generation bocavirus) A2.1-2.3 and NGB Bl.1-1.3. VPlu: unique Viral Protein 1 domain, VP1 / 2: shared domain of Viral Protein 1 and Viral Protein 2, VP3: Viral Protein 3. Numbers indicate amino acid residues added (plus) or removed (minus).

[0097] Figure 3: Downstream yields for vectors carrying chimeras NGB A2.1-2.3 and NGB Bl.1-1.3 as determined by ITR qPCR in purified vector genomes (VG) per liter culture.

[0098] Figure 4: Silver-staining of iodixanol-gradient purified viruses made with chimeric capsid proteins. (1) NGB A2.1, (2) NGB A2.2, (3) NGB Bl.l, (4) NGB Bl.2, (5) NGB Bl.3, (6) NGB A2.3, (7) NGA Bl.l, (8) NGA B1.2, (9) NGA B1.3, (10) NGA B1.4, (11) GBoVl Control, (12) AAV2. All other constructs were produced using a sc (self-complementary) payload.

[0099] Figure 5: Transduction profiles in Huh7 cells of the chimeras NGB A2.1-2.3 and NGB Bl.l- 1.3 and the GBoVl control, all carrying a GFP reporter, or untransduced Huh7 cells (negative control). A: Fluorescent microscopy images showing GFP signal produced by Huh7 cells transduced with chimeras NGB A2.1-2.3 and NGB Bl.1-1.3 or GBoVl control. Scalebar 700pm.

[0100] B: Quantification of transduction as determined by GFP expression compared to untransduced cells.

[0101] Figure 6 Schematic representation of VP1 protein domains from AAV2, GBoVl, and chimeras NGA (next generation AAV) Bl.1-1.4.

[0102] Figure 7: Downstream yields for vectors carrying chimeras NGA Bl.1-1.4 as determined by ITR qPCR in purified vector genomes (VG) per liter culture.

[0103] Figure 8: Expression of chimeras NGA Bl.1-1.4, AAV2, or a GBoVl control, all carrying a GFP reporter in Huh7 cells or untransduced Huh7 cells (negative control). A: Fluorescent microscopy images showing GFP signal produced by Huh7 cells transduced with chimeras NGA Bl.1-1.4. Scalebar 700 pm. B: Quantification of transduction as determined by GFP expression compared to untransduced cells.

[0104] Figure 9: Schematic representation of VP1 protein domains from AAV2, GBoVl, AAV8, HBoVl, and chimeras NGH A2.1 (next generation human bocavirus), NGB A8.1, NGB A2.4, NGA Bl.5, NGH (next generation human bocavirus) A2.2, and NGB A8.2.

[0105] Figure 10: Silver-staining of iodixanol-gradient purified viruses with carrying chimeric capsid proteins (1,2) NGH A2.1, (3,4) NGB A8.1, (5,6) NGB A2.4, (7,8) NGH A2.2, (9,10) NGA B1.5, (11,12) NGB A8.2, (13,14) NGB A2.1.

[0106] Figure 11: Upstream and downstream titers for vectors carrying chimeras NGH-A2.1, NGB A8.1, NGB-A2.4, NGA-B1.5, NGH-A2.2, NGB-A8.2, NGB A2.1 (control) as determined by ITR qPCR in purified vector genomes (VG) per liter culture.

[0107] Figure 12: Expression of chimeras (NGH-A2.1, NGB A8.1, NGB-A2.4, NGA-B1.5, NGH-A2.2, NGB-A8.2, NGB A2.1), all carrying a GFP reporter in Huh7 cells. A: Fluorescent microscopy images showing GFP signal produced by Huh7 cells transduced with chimeras (NGH-A2.1, NGB A8.1, NGB-A2.4, NGA-B1.5, NGH-A2.2, NGB-A8.2, NGB A2.1). B: Quantification of transduction as determined by GFP expression compared to untransduced cells. Statistics were performed using Kruskal-Wallis test with Dunn's multiple comparisons test, p-value * <0.05, ** <0.01, *** <0.001, **** <0.0001.

[0108] Figure 13: The NGB-A2.1 peptide insertion constructs were transfected into HEK293T cells for virus production, followed by iodixanol purification and PGR quantification. Upstream and downstream titers of NGB-A2.1 peptide insertion constructs are shown. VR: variable region, A2: peptide GNYSRGVDA (SEQ ID NO: 93), HA: YPYDVPDYA (SEQ ID NO: 95), vg / ml: virus genomes per ml.

[0109] Figure 14 NGB-A2.1 peptide insertion constructs transduced into Huh7 cells. NGB-A2.1 capsids display peptide insertion at various variable regions (VR1, VR3.2, VR3.3, VR4, VR5) and carry a genetic GFP reporter payload. Huh7 cells were transduced with NGB A2.1 peptide insertion variants at an MOI (multiplicity of infection) of 2 x 105in the presence of IpM Doxorubicin. Transduction efficiency is determined by the percentage of GFP expressing Huh7 cells (Y axis). VR: variable region, A2: peptide GNYSRGVDA (SEQ ID NO: 93), HA: YPYDVPDYA (SEQ ID NO: 95).

[0110] DETAILED DESCRIPTION OF THE INVENTION

[0111] Definitions

[0112] Before the invention is described in detail with respect to some of its preferred embodiments, the following general definitions are provided.

[0113] The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

[0114] The present invention will be described with respect to particular embodiments and with reference to certain figures but the invention is not limited thereto but only by the claims.

[0115] Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

[0116] Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated. The terms “about” or “approximately” in the context of the present invention denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ± 10%, and preferably of ± 5%. Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used.

[0117] As used herein, the term „Parvoviridae" refers to a family of viruses. Parvoviridae are small, icosahedral, non-enveloped viruses of 18-26 nm in diameter that contain a single molecule of linear, negative- or positive-sense single-standed DNA (ssDNA). Parvoviridae are among the smallest, simplest eukaryotic viruses and fall into two groups: defective viruses that are dependent on a helper virus for replication; and autonomous, replication-competent viruses. The family Parvoviridae comprises two subfamilies, the Parvovirinae, which infect vertebrates and the densovirinae, which infect invertebrates. Currently, eight genera are associated with the subfamily Parvovirinae, which are the genera amdoparvovirus, aveparvovirus, bocaparvovirus (bocavirus), copiparvovirus, dependoparvovirus (e.g. adeno-associate virus), erythroparvovirus (e.g. B19 virus), protoparvovirus (e.g. canine parvovirus, feline parvovirus), and tetraparvovirus. The term “parvovirus” used herein exclusively refers to the genus protoparvovirus and does not refer to the subfamily Parvovirinae.

[0118] The term “adeno-associated virus” or "AAV" as used herein refers to a virus of the family Parvoviridae, subfamily Parvovirinae, genus dependoparvovirus. “AAV” may be used to refer to the naturally occurring wild-type virus itself or derivatives thereof. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant, synthetic or engineered variants, except where required otherwise. Furthermore, "AAV" refers to both the genetic components of the virus, e.g., the genome (positive or negative) and RNA transcripts thereof (either sense or antisense), proteins encoded by the genome (including structural and nonstructural proteins), and viral particles.

[0119] Adeno-associated viruses (AAV) are non-pathogenic, helper-dependent members of the Parvoviridae family. One of the identifying characteristics of this group of viruses is the encapsidation of a single-stranded DNA (ssDNA) genome that can be either the sense or antisense strand. In the case of AAV, the separate plus or minus polarity strands are packaged with equal frequency, and either is infectious. The small (about 4.8 kilobases) ssDNA genome consists of two open reading frames, Rep and Cap, flanked by two 145 base ITRs (inverted terminal repeats). Rep and Cap are translated to produce multiple distinct proteins (e.g., Rep78, Rep68, Rep52, and Rep40, required for the AAV life cycle; and VP1, VP2, and VP3, the capsid proteins). When constructing a nucleic acid to be delivered using AAV, the exogenous nucleic acid (e.g., a transgene) is placed between the two ITRs, and Rep and Cap typically are supplied in trans. Being helper dependent, the adeno-associated viruses generally require a helper virus for a productive infection.

[0120] The term “adeno-associated virus” or "AAV" as used herein refers to a virus of the family Parvoviridae, subfamily Parvovirinae, genus dependoparvovirus. “AAV” may be used to refer to the naturally occurring wild-type virus itself or derivatives thereof. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant, synthetic or engineered variants. Furthermore, "AAV" refers to both the genetic components of the virus, e.g., the genome (positive or negative) and RNA transcripts thereof (either sense or antisense), proteins encoded by the genome (including structural and nonstructural proteins), and viral particles. The term "adeno-associated virus" (AAV), includes but is not limited to, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including serotypes 3 A and 3B), AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9, AAV serotype 10, AAV serotype 11, AAV serotype 12, AAV serotype 13, AAVrh8, AAVrhIO, AAVrh.74, snake AAV, caprine AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, those AAV serotypes and clades disclosed by Gao et al. (J. Virol. 78:6381 (2004)) and Moris et al. (Virol. 33:375 (2004)), and any other AAV now known or later discovered.

[0121] “Recombinant AAV” or “rAAV” refers to a virus that is devoid of the rep gene and is used as vector for in vitro or in vivo gene delivery. rAAVs typically comprise an encapsidated genome which carries a therapeutic gene expression cassette in place of the genes necessary for virus production. The AAV genome is flanked by two ITRs at the ends that serve as the viral origins of replication and the packaging signal. In a rAAV vector, the only sequences of viral origin are the ITRs, which are needed to guide genome replication and packaging during vector production. The ITR-flanked rAAV genome can be cloned into plasmids and manipulated using standard molecular cloning techniques. As used herein, the term “bocavirus” refers to a virus of the family Parvoviridae, subfamily Parvovirinae, genus bocaparvovirus and may be used to refer to the naturally occurring wildtype virus or non-naturally occuring derivatives thereof. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant, synthetic or engineered variants, except where indicated otherwise. Furthermore, "bocavirus" refers to both the genetic components of the virus, e.g., the genome (positive or negative) and RNA transcripts thereof (either sense or antisense), proteins encoded by the genome (including structural and nonstructural proteins), and viral particles.

[0122] As used herein, the term "serotype" refers to a virus, such as AAV or bocavirus, which is identified by and distinguished from other viruses of the same genus based on capsid protein reactivity with defined antisera. For example, serotype AAV2 is used to refer to an AAV which contains capsid proteins encoded from the cap gene of AAV 2 and a genome containing 5' and 3' ITR sequences from the same AAV2 serotype.

[0123] There are four known serotypes of human bocavirus (H BoV), HBoVl, HBoV2, HBoV3, and HBoV4. However, included in bocavirus are serotypes derived from other non-human primates and other mammals such as swine bocavirus or gorilla bocavirus (GBoV).

[0124] As used herein, the term “recombinant bocavirus” refers to a non-naturally occurring bocavirus. This includes for example a bocavirus comprising capsid proteins from bocavirus and a heterologous ssDNA genome. The term may also refer to a non-naturally occurring bocavirus wherein parts of the capsid protein amino acid sequence has been altered or replaced by capsid protein amino acid sequence from a different Parvoviridae. The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. For example, a recombinant bocavirus may comprise a capsid shell containing VP proteins of bocavirus origin and a genome comprising ITRs from AAV and a therapeutic transgene. The term “bocavirus-like particle” refers to a virus-like particle composed of capsid proteins derived from bocavirus. Virus-like particles are synthesized by expression of capsid proteins which self-assemble into a virus-like structure, i.e.a capsid shell, but do not contain any viral genetic material. They are not pathogenic or infective, but can mimic the epitopes of the natural virus. Bocavirus-like particles may comprise wild-type or non-naturally occurring, recombinant capsid proteins. This may include amino acid sequences from bocavirus VP1, VP2 or VP3, or full length VP1, VP2 or VP3 proteins. They may also include amino acid sequences from VP1, VP2 or VP3 proteins of other Parvoviridae, such as AAV. Bocavirus-like particles can carry “cargo” or “payload”. A cargo may be a nucleic acid encoding a polypeptide or an RNA agent, such as one or more of dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, IncRNA, piRNA, and snoRNA. Bocavirus-like particles can also be used as a nanocarrier, to display antigenic epitopes or deliver small molecules.

[0125] As used herein, the term “capsid protein” refers to a protein product of an open reading frame (ORF) encoded by the ssDNA genome of Parvoviridae, often termed “cap” ORF. The cap ORF encodes three capsid proteins called “viral proteins” (VP) Viral Protein 1 (VP1), VP2 and VP3, that are produced via differential splicing of the mRNA and use of alternate translational start codons. All VPs share a common C-terminal VP3 amino acid sequence, which is also called the VP3 common region or VP3 domain. The N-terminal region of VP2 which does not overlap with VP3 is called the VP1 / VP2 common region or VP1 / VP2 domain. The N-terminal region of VP1 which does not overlap with VP2 or VP3 is called VP1 unique (VPlu) region or VPlu domain. The full length VP1 has 671 amino acids with a N-terminal VP1 unique domain (VPlu, 90 amino acids). The VP3 protein, which consists of the C-terminal portion of VP1, has 542 amino acids. The VP2 protein has 581 amino acids and consists of all of VP3 and a VP1 / VP2 shared domain of 39 amino acids.

[0126] The term “domain” is used to describe a protein fragment or region that is a defined part of a protein. A domain may be structurally or functionally defined. Herein, the term “domain” may also refer to the amino acid sequence portion of a protein that is exchanged or swapped between two different viruses. For example, as used herein, the term “domain” refers to the VP3 common amino acid sequence (VP3 domain), the VP1 / VP2 common amino acid sequence (VP1 / VP2 domain) and the VPlu unique amino acid sequence (VPlu domain), respectively.

[0127] The term “truncated” or “truncated protein” as used herein refers to an amino acid sequence or a nucleic acid sequence encoding an amino acid sequence that comprises less than a full- length protein or protein domain.

[0128] As used herein, the term “fusion protein” refers to a continuously translated chain of amino acids that is the product of two or more distinct nucleic acid elements.

[0129] "Packaging" as used herein refers to a series of subcellular events that results in the assembly and encapsidation of a cargo, e.g. a viral genome or recombinant viral genome or transgene. Thus, when a suitable polynucleotide is introduced into a packaging cell line under appropriate conditions, it can be assembled into a viral particle. The encapsidating protein shell is referred to as capsid. The capsid or shell has an inner and outer surface. The outer surface of the capsid is the part of the shell that is in contact with the environment.

[0130] As used herein, the term “amino acid sequence” refers to a continuous chain of consecutive amino acids. As used herein, an amino acid sequence from a protein may comprise a complete open reading frame, a single domain, or a fragment of a domain, or may comprise consecutive amino acids from two or more domains. For example, a bocavirus VP amino acid sequence may comprise a continuous chain of consecutive amino acids from the VP3 domain, but may comprise less than to complete VP3 domain. A bocavirus VP amino acid sequence may also for example comprise a continuous chain of consecutive amino acids spanning the VP1 / VP2 and VP3 domains.

[0131] As used herein, the term “chimeric” when used to describe a protein, for example a capsid protein, refers to a protein that is encoded by nucleic acid sequences that originally coded for two or more separate proteins. Hence, the protein may be a fusion protein composed of amino acid sequences from two or more proteins that do not naturally occur fused together. In some cases, the amino acid sequences of the two or more source proteins are fused together; in other cases, an amino acid sequence of one protein replaces a homologous amino acid sequence in another protein; combinations of these two options are also envisioned. Particularly, as used herein, “chimeric capsid protein” refers to a capsid protein that comprises amino acid sequences of two or more different species of virus from the subfamily Parvovirinae. Replacing a homologous sequence of a protein from one species with that of another species is herein also referred to as “swapping”. Thus, a VPlu domain of bocavirus VP1 protein may be swapped for a VPlu domain from an AAV virus VP1 protein, resulting in a chimeric VP1 protein with both bocavirus and AAV amino acid sequences.

[0132] The term "chimeric virus" or "chimeric viral particle" does not refer to a chimeric capsid protein as described herein, but is commonly used to refer to a viral particle composed of at least one capsid protein containing an encapsidated polynucleotide (e.g. a viral genome) which is from a different virus. The process of producing viruses by combining the genome of one virus with the envelope or capsid of another virus of a different serotype or species is also referred to as “pseudotyping”. A chimeric virus comprising a bocavirus (BoV) capsid protein and a recombinant heterologous parvovirus genome, such as a recombinant AAV (rAAV) genome as disclosed in WO2014168953A1 does not refer to a chimeric capsid protein as described herein. However, the application also encompasses chimeric virus comprising a bocavirus (BoV) capsid protein and a recombinant heterologous parvovirus genome, such as a recombinant AAV (rAAV) genome which comprise a chimeric capsid protein as described herein.

[0133] As used herein, “tropism” refers to the specificity of a capsid protein present in a virus or virus-like particle for infecting a particular type of cell or tissue. The tropism of a capsid for a particular type of cell or tissue may be determined by measuring the ability of a virus or viruslike particle comprising the capsid protein to infect or to transduce a particular type of cell or tissue, using standard assays that are well-known in the art.

[0134] The term “viral vector” or “vector”, used interchangeably herein, refers to a virus that has been engineered to deliver genetic material to cells, in a process called "transduction" or "transducing”. Transduction is a process for the introduction of an exogenous polynucleotide, e.g., a transgene in a viral vector, into a host cell leading to expression of the polynucleotide, e.g., the transgene, in the cell. Altered expression or persistence of a polynucleotide introduced via the virus can be determined by methods well known to the art including, but not limited to, protein expression, e.g., by ELISA, flow cytometry and Western blot, measurement of and DNA and RNA production by hybridization assays, e.g., Northern blots, Southern blots and gel shift mobility assays. Other methods used for the introduction of the exogenous polynucleotide include well-known techniques such as transfection, lipofection, viral infection, transformation, and electroporation, as well as non-viral gene delivery techniques. The introduced polynucleotide may be stably or transiently maintained in the host cell. A viral vector may also be referred to as “virion” or “viral particle”.

[0135] "Gene expression" or "expression" refers to the process of gene transcription, translation, and post-translational modification.

[0136] A "helper virus" for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpes viruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used.

[0137] Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. An "infectious" virus or viral particle is one that comprises a polynucleotide component, which it is capable of delivering into a cell for which the viral species is trophic. The term does not necessarily imply any replication capacity of the virus.

[0138] The term "polynucleotide" or “nucleic acid sequence” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A nucleic acid sequence is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the nucleic acid sequence is RNA). Thus, the term polynucleotide sequence or nucleic acid sequence is the alphabetical representation of a polynucleotide molecule. A polynucleotide may comprise modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term nucleic acid sequence, as used herein, refers interchangeably to double- and single-stranded polynucleotide molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a nucleic acid sequence encompasses both the doublestranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0139] As used herein, “isolated” refers to a nucleic acid molecule or a nucleic acid sequence that has been substantially separated, produced apart from, or purified away from other biological components in the cell or tissue of an organism in which the component occurs, such as other cells, chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins. Isolated proteins or nucleic acids, or cells containing such, in some examples are at least 50% pure, such as at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% pure.

[0140] "Host cells," "cell lines," "cell cultures," "packaging cell line" and other such terms denote higher eukaryotic cells, e.g., mammalian cells, such human cells, useful in the present invention. These cells can be used as recipients for recombinant vectors, viruses or other transfer polynucleotides, and include the progeny of the original cell that was transduced. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or in genomic complement) to the original parent cell.

[0141] A "plasmid" or "expression vector" as used herein is an expression construct used for cloning and gene expression, comprising a region which encodes a polypeptide or RNA of interest. A "plasmid" is a type of vector, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. In the present specification, “plasmid”, "plasmid vector" and "expression vector" can be used interchangeably as the plasmid is the most commonly used form of vector. The term "expression vector" means a vector capable of directing expression of a particular nucleotide sequence in an appropriate host cell. An expression vector comprises a regulatory nucleic acid element operably linked to a nucleic acid of interest, which is optionally operably linked to a termination signal and / or other regulatory element. The combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an "expression cassette," a large number of which are known and available in the art or can be readily constructed from components that are available in the art.

[0142] A “viral vector” or “vector” as described above, is a different type of vector that is used for gene delivery and gene expression, but as opposed to a plasmid, it means a transgene that is packaged within a viral capsid and enters the cell by transduction or infection.

[0143] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation with a labeling component. The term “amino acid sequence” as used herein likewise refers to a continuous string of amino acids. This term can be used interchangeably with the terms “polypeptide” or “protein”. The term “amino acid sequence” may refer to a full-length protein, to a domain of a protein, to a functional region of a protein, or to a fragment or portion of a protein, wherein the fragment may be of any length.

[0144] A “fragment” or “portion” of an amino acid sequence can be understood to mean an amino acid sequence of reduced length relative to a reference amino acid sequence and comprising, consisting essentially of, or consisting of an amino acid sequence of contiguous amino acids identical or almost identical to the reference amino acid sequence. Such an amino acid fragment or portion according to the disclosure can be, where appropriate, included in a larger amino acid sequence of which it is a constituent. In one embodiment, the amino acid sequence fragment may be reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more amino acids in reference to the full-length amino acid sequence. In one embodiment, the amino acid sequence fragment may be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the reference amino acid sequence. A “fragment” or “portion” of a nucleotide sequence can be understood to mean a nucleic acid sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, or consisting of a nucleic acid or nucleotide sequence of contiguous nucleotides identical or almost identical to the reference nucleic acid sequence. In one embodiment, the nucleic acid fragment may be reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more nucleotides in reference to the full-length nucleic acid sequence. Such a nucleic acid sequence fragment or portion according to the disclosure can be, where appropriate, included in a larger polynucleotide of which it is a constituent. In an aspect, a fragment or portion of a nucleotide sequence or nucleic acid sequence can comprise the sequence encoding an exon having one or more mutations. In one embodiment, the nucleic acid fragment may be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the reference nucleic acid sequence.

[0145] “Sequence identity” or “percentage identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they are optimally aligned. For example, sequence similarity or identity can be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences can have “substantial sequence identity” if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, preferably 90%, 95%, 98%, 99% or more. Such sequences are also referred to as “variants” herein, e.g., other variants of a missing, deficient, and / or mutant protein or enzyme. I

[0146] Alternatively, the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80-85%, preferably 85-90%, more preferably 90-95%, and most preferably 98-100% sequence identity to the reference sequence over a defined length of the molecules, as determined using the methods above. As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.

[0147] A “recombinant” polynucleotide or amino acid sequence as used interchangeably herein can refer to a nucleotide or an amino acid sequence that is the product of various combinations of cloning, restriction and / or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide or amino acid sequence found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide.

[0148] Different nucleic acids or proteins having homology can be referred to as “homologues” The term homologue includes homologous sequences from the same and other species and orthologous sequences from the same and other species. “Homology” refers to the level of similarity between two or more nucleic acid and / or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids or proteins.

[0149] "Heterologous" means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared. For example, a polynucleotide or nucleic acid sequence introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide or nucleic acid sequence (and, when expressed, can encode a heterologous polypeptide or amino acid sequence).

[0150] The term “composition” as used herein refers to a mixture comprising a therapeutically effective amount of the agent according to the present invention, i.e. a bocavirus or bocavirus-like particle comprising the chimeric bocavirus capsid protein of the invention, and one or more excipients. The term “excipient” as used herein may also be referred to as “pharmaceutically acceptable carrier”, or “pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,”, or “pharmaceutically acceptable vehicle,” used interchangeably herein, refer to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type.

[0151] A pharmaceutically acceptable carrier is essentially non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier will not inhibit otherwise adversely affect the function of the agent according to the present invention. Suitable carriers include, but are not limited to water, dextrose, glycerol, saline, ethanol, and any combination thereof. The carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants, which enhance the effectiveness of the formulation.

[0152] As used herein, the term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.).

[0153] The words “treat” or “treating” or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In an aspect, the terms cover any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease. For example, in an aspect, treating a disease or disorder can reduce the severity of an established a disease or disorder in a subject by 1 %-100% as compared to a control. In an aspect, treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a disease or disorder (such as a genetic disease or disorder). For example, treating a disease or disorder can reduce one or more symptoms of a disease or disorder in a subject by 1 %-l 00% as compared to a control. In an aspect, treating can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms of an established a disease or disorder. It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of a disease or disorder. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of a disease or disorder.

[0154] An "individual" or "subject" treated in accordance with this invention refers to vertebrates, particularly members of a mammalian species, and includes but is not limited to domestic animals, sports animals, and primates, including humans. In one embodiment, the subject treated in accordance with this invention is a mammal. In one embodiment, the subject treated in accordance with this invention is a human. In another embodiment, the subject treated in accordance with this invention is a non-human mammal.

[0155] “Wild-type”, “control” or “reference” gene expression, mRNA or protein levels are determined by a control sample, cell or organisms, or by averaging the expression levels from multiple control samples, cells or organisms. In the context of the present invention, the term “wild-type” or "control" refers to a cell or organism that is healthy or a sample from a subject that is healthy or to a cell or organism with a specific disease that is different from the disease to be treated.

[0156] The term “genetic disease” or “genetic disorder” as used herein refers to a disease caused by one or more mutations in a single gene (monogenic) or in multiple genes (polygenic). The genetic disease may be autosomal dominant, autosomal recessive, X-linked dominant, X- linked recessive, Y-linked or mitochondrial. The term “gene therapy” as used herein refers to the alteration of endogenous gene expression by introduction of a therapeutic agent. Most commonly, gene therapy involves the introduction of foreign, i.e. heterologous, DNA or RNA into a cell or organism. This results in an increase or decrease of gene expression or in the replacement of a defective gene. The introduction of a heterologous nucleic acid can be achieved with a vector, such as a bocavirus vector comprising the chimeric bocavirus capsid protein of the invention.

[0157] The term “defective gene” as used herein refers to an alteration in the genetic code that leads to a defective gene product, such as a truncated or missense protein, or an altered expression level of a gene product. The alteration subsequently leads to a phenotypic symptom or increases the likelihood of a phenotypic symptom which can result in disease. Most commonly, the defect is caused by a genetic mutation. Known genetic mutations include, for example, insertion, deletion, insertion-deletion, miss-sense mutation, truncation mutation, stop mutation, nonsense mutation, frameshift mutation, and repeat expansion. The mutation may also occur in a non-coding region, for example a regulatory region.

[0158] As used herein, “Peptide display” refers to the expression of short peptides (also called insertion peptides or targeting peptides) into the capsid protein of a virus, to be displayed on the outside of the virus. Short peptides refers to peptides with a length of up to 20 amino acids.

[0159] The term “insertion site” as used herein describes a location in the nucleic acid sequence of a protein in which a genetic alteration, e.g. an exogenous nucleic acid sequence, can be inserted. In the context of this specification, the inserted nucleic acid sequence encodes a peptide of at least 4 amino acids. For example, insertion site 332 / 333 of VR III of VP3 means that within the coding DNA sequence of VP3, the location of the insertion site is between amino acids 332 and amino acid 333 of VR III.

[0160] The term “variable region” or “VR” as used herein refers to a specific region on the AAV or bocavirus capsid protein that is not conserved between serotypes or between virus species and shows a high degree of variability in its amino acid sequence and / or structure. These regions are structurally distinct and are located on the capsid surface. VRs are commonly identified by Roman numerals, but Arabic numerals are used e.g. in Figure legends herein for convenience.

[0161] Chimeric Capsid Proteins

[0162] The capsids of AAV, bocavirus and other Parvoviridae are formed by a mixture of the three overlapping gene products VP1, VP2 and VP3 that are encoded by the ca open reading frame. All VPs share a common C-terminal VP3 sequence. The capsid proteins form an icosahedral structure. The outside layer of the capsid, also called shell, is formed by VP3 protein. The N- terminal domains specific to VP1 and VP2 proteins point towards the center of the icosahedron. The capsid composition ratio of Parvoviridae is estimated to be 1:1:10 of VP1:VP2:VP3, as determined by mass spectrometry studies.

[0163] The chimeric bocavirus capsid protein of the invention comprises an amino acid sequence from a bocavirus VP and an amino acid sequence from an AAV VP. The amino acid sequence from a bocavirus VP and an amino acid sequence from an AAV VP form a fusion protein. This chimeric fusion protein comprises all sequences necessary to form a bocavirus capsid. Hence, in one embodiment, the chimeric bocavirus capsid protein of the invention can assemble into a capsid structure.

[0164] Thus the term “viral protein”, abbreviated “VP”, comprises VP1, VP2 and VP3.

[0165] In one embodiment, the chimeric bocavirus capsid protein of the invention comprises a bocavirus VP1 sequences in which amino acid residues are replaced by the corresponding amino acid sequence from AAV VP1.

[0166] In one embodiment, at least 20 amino acid residues of bocavirus VP1 are replaced by the corresponding amino acid residues of AAV VP1. In another embodiment, at least 40 amino acid residues of bocavirus VP1 are replaced by the corresponding amino acid residues of AAV VP1.

[0167] The corresponding amino acid sequences of bocavirus VP1 and AAV VP1 may not be the same length. In a preferred embodiment, the chimeric bocavirus capsid protein comprises a VP3 amino acid sequence from a bocavirus VP and a VPlu amino acid sequence from an AAV VP. Hence, in one embodiment, the chimeric bocavirus capsid protein is a fusion protein comprising a VP3 amino acid sequence from a bocavirus VP and a VPlu amino acid sequence from an AAV VP.

[0168] The VPlu amino acid sequence is N-terminal from the VP3 amino acid sequence within the VP1 protein, in a continuous polypeptide chain. Between the VPlu and the VP3 amino acid sequences, there may a VP1 / VP2 amino acids sequence from either AAV or bocavirus. In a preferred embodiment, the chimeric bocavirus capsid protein comprises a VP3 amino acid sequence from a bocavirus VP, a VP1 / VP2 amino acid sequence from a bocavirus VP, and a VPlu amino acid sequence from an AAV VP. Hence, in one embodiment, the chimeric bocavirus capsid protein is a fusion protein comprising a VP3 amino acid sequence from a bocavirus VP, a VP1 / VP2 amino acid sequence from a bocavirus VP, and a VPlu amino acid sequence from an AAV VP.

[0169] The VPlu domain of AAV2 is defined as the first 138 amino acids of VP1 counted from the N- terminus of AAV2 VP1 and is set forth in SEQ ID NO: 1. The VPlu domain of AAV8 is defined as the first 137 amino acids of VP1 counted from the N-terminus of AAV8 VP1 and is set forth in SEQ ID NO: 64. The VP3 domain of GBoVl is defined as the C-terminal 542 amino acids of GBoVl VP1 and is set forth in SEQ ID NO: 5 .The VP3 plus VP1 / VP2 domain of GBoVl is defined as the C-terminal 581 amino acids of GBoVl VP1 and is set forth in SEQ ID NO: 2.

[0170] In a preferred embodiment, the chimeric bocavirus capsid protein is a fusion protein comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 3. In another embodiment, the chimeric bocavirus capsid protein is a fusion protein comprising an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 3.

[0171] AAV VP Amino Acid Sequences Adeno-associated viruses of any serotype are suitable to prepare the chimeric bocavirus capsid protein of the invention, since the various serotypes are functionally and structurally related, even at the genetic level (see, e.g., Blacklow, 1988; and Rose, 1974). All AAV serotypes exhibit similar replication properties mediated by homologous rep genes; and all generally bear three related capsid proteins such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive crosshybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to ITRs. The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. Among the various AAV serotypes, AAV2 is most commonly employed.

[0172] In one embodiment, the AAV VP amino acid sequence comprises an AAV VPlu amino acid sequence. In one embodiment, the AAV VP amino acid sequence consists of an AAV VPlu amino acid sequence.

[0173] In one embodiment, the AAV VP amino acid sequence comprises an AAV VPlu and a AAV VP1 / 2 amino acid sequence. In one embodiment, the AAV VP amino acid sequence consists of an AAV VPlu and an AAV VP1 / 2 amino acid sequence.

[0174] In one embodiment, the AAV VP amino acid sequence is from an AAV selected from the group consisting of wildtype, recombinant, synthetic and engineered variant of AAV.

[0175] In one embodiment, the AAV VP amino acid sequence is from an hybrid AAV capsid, optionally selected from the group consisting of mosaic, chimeric and combinatorial variant of AAV. Mosaic variants have multiple subunits of various serotypes in their capsid. Chimeric variants are produced by capsid protein domain swapping and / or DNA shuffling. Combinatorial variants have mixed genomes and are produced by DNA shuffling and error- prone PCR methods.

[0176] In one embodiment, the AAV VP amino acid sequence is from a non-naturally occurring AAV. In one embodiment, the AAV VP amino acid sequence is from a wildtype variant of AAV selected from the group consisting of primate AAV, snake AAV, caprine AAV, avian AAV, bovine AAV, canine AAV, equine AAV, bovine AAV, goat AAV, and shrimp AAV.

[0177] In one embodiment, the AAV VP amino acid sequence is from a recombinant variant of AAV that is based on an AAV selected from the group consisting of primate AAV, snake AAV, caprine AAV, avian AAV, bovine AAV, canine AAV, equine AAV, bovine AAV, goat AAV, and shrimp AAV.

[0178] In one embodiment, the AAV VP amino acid sequence is from an AAV selected from the group consisting of primate AAV, snake AAV, caprine AAV, avian AAV, bovine AAV, canine AAV, equine AAV, bovine AAV, goat AAV, shrimp AAV, and synthetic AAV.

[0179] In one embodiment, the AAV VP amino acid sequence is from a wildtype AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13.

[0180] In one embodiment, the AAV VP amino acid sequence is from a recombinant AAV based on an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13.

[0181] In one embodiment, the AAV VP amino acid sequence is from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrhIO, AAVrh.74. In another embodiment, the AAV VP amino acid sequence is from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13. In another embodiment, the AAV VP amino acid sequence is from an AAV selected from AAV2 or AAV8, preferably AAV2.

[0182] In one embodiment, the AAV VP amino acid sequence comprises an AAV VPlu amino acid sequence and is from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13. In another embodiment, the AAV VP amino acid sequence comprises an AAV VPlu amino acid sequence and is from an AAV selected from AAV2 or AAV8, preferably AAV2. In one embodiment, the AAV VP amino acid sequence is AAV2 VPlu. In another embodiment, the AAV VP amino acid sequence is AAV8 VPlu. In another embodiment, the AAV VP amino acid sequence is AAV2 or AAV8 VPlu.

[0183] In one embodiment, the AAV VP amino acid sequence comprises the first 43 amino acid sequences of an AAV VPlu amino acid sequence and is from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13. In another embodiment, the AAV VP amino acid sequence comprises the first 43 amino acid sequences of an AAV VPlu amino acid sequence and is from an AAV selected from AAV2 or AAV8, preferably AAV2. In one embodiment, the AAV VP amino acid sequence is the first 43 amino acid sequences of AAV2 VPlu. In another embodiment, the AAV VP amino acid sequence is the first 43 amino acid sequences of AAV8 VPlu. In another embodiment, the AAV VP amino acid sequence is the first 43 amino acid sequences of AAV2 or the first 43 amino acid sequences of AAV8 VPlu.

[0184] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence of AAV2. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence of AAV2.

[0185] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence of AAV8. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence of AAV8.

[0186] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1.

[0187] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 31. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 31.

[0188] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 4. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0189] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 34. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 34.

[0190] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 7. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7.

[0191] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 37. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 37.

[0192] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 64. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 64.

[0193] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 79. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 79.

[0194] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 61. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 61.

[0195] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 76. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 76.

[0196] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 67. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 67.

[0197] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 82. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 82.

[0198] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 74. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 74.

[0199] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 89. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 89.

[0200] In one embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, and SEQ ID NO: 74. Preferably, the AAV VP amino acid sequence comprises an amino acid sequence at least 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ I D NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, and SEQ ID NO: 74. More preferably, the AAV VP amino acid sequence comprises an amino acid sequence at least 70% identical to an amino acid sequence set forth in SEQ ID NO: 67 or SEQ ID NO: 74. In another embodiment, the AAV VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, and SEQ ID NO: 74. Preferably, the AAV VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, and SEQ I D NO: 74. More preferably, the AAV VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to an amino acid sequence set forth in SEQ ID NO: 67 or SEQ ID NO: 74.

[0201] Bocavirus VP Amino Acid Sequences

[0202] In one embodiment, the bocavirus VP amino acid sequence comprises a bocavirus VP3 amino acid sequence. In one embodiment, the bocavirus VP amino acid sequence consists of a bocavirus VP3 amino acid sequence.

[0203] In one embodiment, the bocavirus VP amino acid sequence comprises a truncated bocavirus VP3 amino acid sequence, such as an N-terminally truncated VP3 amino acid sequence. In one embodiment, the N-terminally truncated bocavirus VP3 amino acid sequence may be truncated by between 1 and 32 amino acid sequences, for example by between 2 and 32 amino acid sequences, between 3 and 32 amino acid sequences, between 4 and 32 amino acid sequences, between 5 and 32 amino acid sequences, between 6 and 32 amino acid sequences, between 7 and 32 amino acid sequences, between 8 and 32 amino acid sequences, between 9 and 32 amino acid sequences, between 10 and 32 amino acid sequences, between 11 and 32 amino acid sequences, between 12 and 32 amino acid sequences, between 13 and 32 amino acid sequences, between 14 and 32 amino acid sequences, between 15 and 32 amino acid sequences, between 16 and 32 amino acid sequences, between 17 and 32 amino acid sequences, between 18 and 32 amino acid sequences, between 19 and 32 amino acid sequences, between 20 and 32 amino acid sequences, between 21 and 32 amino acid sequences, between 22 and 32 amino acid sequences, between 23 and 32 amino acid sequences, between 24 and 32 amino acid sequences, between 25 and 32 amino acid sequences, between 26 and 32 amino acid sequences, between 27 and 32 amino acid sequences, between 28 and 32 amino acid sequences, between 29 and 32 amino acid sequences, between 30 and 32 amino acid sequences, or between 31 and 32 amino acid sequences. Preferably, the N-terminally truncated bocavirus VP3 amino acid sequence may be truncated by 32 amino acid sequences.

[0204] In one embodiment, the bocavirus VP amino acid sequence comprises a bocavirus VP3 and a bocavirus VP1 / 2 amino acid sequence. In one embodiment, the bocavirus VP amino acid sequence consists of a bocavirus VP3 and a bocavirus VP1 / 2 amino acid sequence.

[0205] In one embodiment, the bocavirus VP amino acid sequence is from a mammalian bocavirus. In a preferred embodiment, the bocavirus VP amino acid sequence is from a primate bocavirus. In one embodiment, the primate bocavirus is selected from the group consisting of HBoVl, HBoV2, HBoV3, HBoV4 and GBoVl. In one embodiment, the bocavirus VP amino acid sequence is from HBoVl. In one embodiment, the bocavirus VP amino acid sequence is from HBoV2. In one embodiment, the bocavirus VP amino acid sequence is from HBoV3. In one embodiment, the bocavirus VP amino acid sequence is from HBoV4. In one embodiment, the bocavirus VP amino acid sequence is from GBoVl. Preferably, the bocavirus VP amino acid sequence is from GBoVl.

[0206] Hence, in a preferred embodiment, the bocavirus VP amino acid sequence comprises a bocavirus VP3 amino acid sequence, wherein the bocavirus is selected from the group consisting of HBoVl, HBoV2, HBoV3, HBoV4 and GBoVl. In an even more preferred embodiment, the bocavirus VP amino acid sequence comprises the bocavirus VP3 amino acid sequence from GBoVl. In another embodiment, the bocavirus VP amino acid sequence consists of the bocavirus VP3 amino acid sequence from GBoVl.

[0207] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0208] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 32. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 32.

[0209] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 5. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 5.

[0210] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 35. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 35.

[0211] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 8. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8.

[0212] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 38. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 38.

[0213] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 62. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 62.

[0214] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 77. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 77.

[0215] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 65. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 65.

[0216] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 80. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 80.

[0217] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 69. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 69.

[0218] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 84. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 84.

[0219] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 91. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 91.

[0220] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 92. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 92.

[0221] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 72. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 72.

[0222] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 87. In another embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 87.

[0223] In one embodiment, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 91, and SEQ ID NO: 72. Preferably, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 91 and SEQ ID NO: 72. More preferably, the bocavirus VP amino acid sequence comprises an amino acid sequence at least 70% identical to an amino acid sequence set forth in SEQ ID NO: 91 or SEQ ID NO: 72.

[0224] In another embodiment, the bocavirus VP amino acid sequence consists of an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 91 and SEQ ID NO: 72. Preferably, the bocavirus VP amino acid sequence consists of an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 91 and SEQ ID NO: 72. More preferably, the bocavirus VP amino acid sequence consists of an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to an amino acid sequence set forth in SEQ ID NO: 91 or SEQ ID NO: 72.

[0225] Chimeric Capsid VP Amino Acid Sequences In one aspect, the invention relates to a chimeric bocavirus capsid protein comprising a bocavirus Viral Protein (VP) amino acid sequence, and an adeno-associated virus (AAV) VP amino acid sequence.

[0226] The chimeric bocavirus capsid protein of the invention is capable of forming a capsid. The capsid comprises an outer shell that has an outer surface. In one embodiment, the outer surface of the capsid that is produced from the chimeric bocavirus capsid protein consists of amino acid sequences from bocavirus. The outer surface of the capsid is made up of VP3 protein. Hence, in one embodiment, the structured VP3 domain of the chimeric bocavirus capsid protein is from bocavirus.

[0227] In one embodiment, the chimeric bocavirus capsid protein comprises a bocavirus VP3 amino acid sequence, and an AAV VPlu amino acid sequence. Preferably, in one embodiment, the chimeric bocavirus capsid protein comprises a bocavirus VP3 amino acid sequence, and the first 43 amino acid sequences of an AAV VPlu amino acid sequence.

[0228] In one embodiment, the chimeric bocavirus capsid protein comprises an HBoVl VP3 amino acid sequence, and an AAV2 or AAV8 VPlu amino acid sequence. Preferably, in one embodiment, the chimeric bocavirus capsid protein comprises an HBoVl VP3 amino acid sequence, and the first 43 amino acid sequences of an AAV2 or AAV8 VPlu amino acid sequence.

[0229] In one embodiment, the chimeric bocavirus capsid protein comprises an HBoV2 VP3 amino acid sequence, and an AAV2 or AAV8 VPlu amino acid sequence. Preferably, in one embodiment, the chimeric bocavirus capsid protein comprises an HBoV2 VP3 amino acid sequence, and the first 43 amino acid sequences of an AAV2 or AAV8 VPlu amino acid sequence.

[0230] In one embodiment, the chimeric bocavirus capsid protein comprises an HBoV3 VP3 amino acid sequence, and an AAV2 or AAV8 VPlu amino acid sequence. Preferably, in one embodiment, the chimeric bocavirus capsid protein comprises an HBoV3 VP3 amino acid sequence, and the first 43 amino acid sequences of an AAV2 or AAV8 VPlu amino acid sequence.

[0231] In one embodiment, the chimeric bocavirus capsid protein comprises an HBoV4 VP3 amino acid sequence, and an AAV2 or AAV8 VPlu amino acid sequence. Preferably, in one embodiment, the chimeric bocavirus capsid protein comprises an HBoV4 VP3 amino acid sequence, and the first 43 amino acid sequences of an AAV2 or AAV8 VPlu amino acid sequence.

[0232] In one embodiment, the chimeric bocavirus capsid protein comprises a GBoVl VP3 amino acid sequence, and an AAV2 or AAV8 VPlu amino acid sequence. Preferably, in one embodiment, the chimeric bocavirus capsid protein comprises a GBoVl VP3 amino acid sequence, and the first 43 amino acid sequences of an AAV2 or AAV8 VPlu amino acid sequence.

[0233] In one embodiment, the chimeric bocavirus capsid protein comprises an N-terminally truncated bocavirus VP3 amino acid sequence, and an AAV2 VPlu amino acid sequence. Preferably, in one embodiment, the chimeric bocavirus capsid protein comprises an N- terminally truncated bocavirus VP3 amino acid sequence, and the first 43 amino acid sequences of an AAV2 VPlu amino acid sequence.

[0234] In one embodiment, the chimeric bocavirus capsid protein comprises an N-terminally truncated bocavirus VP3 amino acid sequence, and an AAV8 VPlu amino acid sequence. Preferably, in one embodiment, the chimeric bocavirus capsid protein comprises an N- terminally truncated bocavirus VP3 amino acid sequence, and the first 43 amino acid sequences of an AAV8 VPlu amino acid sequence.

[0235] In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 3. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 33. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 33.

[0236] In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 6. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 6.

[0237] In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 36. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 36.

[0238] In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 9. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 9.

[0239] In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 39. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 39. In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 63. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 63.

[0240] In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 78. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 78.

[0241] In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 66. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 66.

[0242] In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 81. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 81.

[0243] In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 68. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 68. In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 83. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 83.

[0244] In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 73. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 73.

[0245] In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 88. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 88.

[0246] In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 75. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 75.

[0247] In one embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 90. In another embodiment, the chimeric bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 90. The chimeric bocavirus capsid protein of the invention improves the transduction efficiency of a recombinant bocavirus or bocavirus-like particle compared to a wild type bocavirus capsid protein. In one embodiment, the chimeric bocavirus capsid protein of the invention improves the transduction efficiency of a recombinant bocavirus or bocavirus-like particle by at least 5% compared to a wild type bocavirus capsid protein. In another embodiment, the chimeric bocavirus capsid protein of the invention improves the transduction efficiency of a recombinant bocavirus or bocavirus-like particle by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to a wild type bocavirus capsid protein. In a preferred embodiment, the chimeric bocavirus capsid protein of the invention improves the transduction efficiency of a recombinant bocavirus or bocavirus-like particle by at least 50% compared to a wild type bocavirus capsid protein. In another embodiment, the chimeric bocavirus capsid protein of the invention improves the transduction efficiency of a recombinant bocavirus or bocavirus-like particle by more than 95% compared to a wild type bocavirus capsid protein.

[0248] The improved transduction efficiency can be determined by standard methods. For example, the level of transgene expression in the transduced cells can serve as a measure of transduction efficiency. The level of transgene expression may be determined at the protein or mRNA level. For example, if the transgene is a fluorescent protein, the transgene expression level may be determined by counting cells positive for the fluorescent protein using fluorescent microscopy. Alternatively, the protein expression levels can be determined by Western Blot. Another method to determine transduction efficiency is by using an antibody or probe specific for bocavirus VP3 protein and quantifying the signal.

[0249] In one embodiment, the chimeric bocavirus capsid protein of the invention improves the transduction efficiency of a recombinant bocavirus or bocavirus-like particle by at least 50%, wherein the transduction efficiency is determined by transgene expression levels. In one embodiment, the transgene in a fluorescent protein. In another embodiment, the chimeric bocavirus capsid protein of the invention improves the transduction efficiency of a recombinant bocavirus or bocavirus-like particle by at least 50%, wherein the transduction efficiency is determined using an anti-bocavirus VP3 antibody and quantifying the positive signal.

[0250] Nucleic acids, plasmids and host cells

[0251] The invention also provides a plasmid comprising any one of the isolated nucleic acid molecules of the invention.

[0252] The nucleic acid molecule of the invention can be packaged into one or more vectors, e.g., plasmids or viral vectors. In some embodiments, the plasmids or viral vectors are delivered to the tissue of interest by, e.g., intramuscular injection, intravenous administration, transdermal administration, intranasal administration, oral administration, or mucosal administration. Such delivery may be either via a single dose or multiple doses. One skilled in the art understands that the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choices, the target cells, organisms, tissues, the general conditions of the subject to be treated, the degrees of transformation / modification sought, the administration routes, the administration modes, the types of transformation / modification sought, etc.

[0253] Delivery of virus or virus-like particle comprising the chimeric bocavirus capsid protein of the invention, e.g. the bocavirus or bocavirus-like particle of the invention may mean the introduction of an expression vector, i.e. a plasmid, that enables production of said virus or virus-like particle.

[0254] The invention also provides a host cell comprising a chimeric bocavirus capsid protein of the invention. Furthermore, another aspect of the invention is a host cell comprising a recombinant bocavirus or bocavirus-like particle of the invention. A further aspect of the invention is a host cell comprising the any one of the isolated nucleic acids of the invention. And yet another aspect of the invention is a host cell comprising a plasmid of the invention. In one embodiment, the host cell is a mammalian cell. In another embodiment, the host cell is a non-human mammalian cell.

[0255] Compositions and medical uses

[0256] A virus or virus-like particle comprising the chimeric bocavirus capsid protein of the invention, e.g. the bocavirus or bocavirus-like particle of the invention, can be used for administration to an individual for purposes of gene therapy or vaccination. Suitable diseases for therapy include but are not limited to those induced by viral, bacterial, or parasitic infections, various malignancies and hyperproliferative conditions, autoimmune conditions, and congenital deficiencies.

[0257] Gene therapy can be conducted to enhance the level of expression of a particular protein either within or secreted by the cell. A virus or virus-like particle comprising the chimeric bocavirus capsid protein of the invention, e.g. the bocavirus or bocavirus-like particle of the invention may be used to genetically alter cells either for gene marking, replacement of a missing or defective gene, or insertion of a therapeutic gene.

[0258] Alternatively, a polynucleotide may be provided to the cell by a virus or virus-like particle comprising the chimeric bocavirus capsid protein of the invention, e.g. the bocavirus or bocavirus-like particle of the invention that decreases the level of expression. This may be used for the suppression of an undesirable phenotype, such as the product of a gene amplified or overexpressed during the course of a malignancy, or a gene introduced or overexpressed during the course of a microbial infection. Expression levels may be decreased by supplying a therapeutic or prophylactic polynucleotide comprising a sequence capable, for example, of forming a stable hybrid with either the target gene or RNA transcript (antisense therapy), capable of acting as a ribozyme to cleave the relevant mRNA or capable of acting as a decoy for a product of the target gene.

[0259] Vaccination can be conducted to protect cells from infection by infectious pathogens. As the traditional vaccine methods, a virus or virus-like particle comprising the chimeric bocavirus capsid protein of the invention, e.g. the bocavirus or bocavirus-like particle of the invention of this invention may be used to deliver transgenes encoding viral, bacterial, tumor or fungal antigen and their subsequent expression in host cells. The antigens, which expose to the immune system to evoke an immune response, can be in the form of virus-like particle vaccines or subunit vaccines of virus-coding proteins. Alternatively, as the method of passive immunization, a virus or virus-like particle comprising the chimeric bocavirus capsid protein of the invention, e.g. the bocavirus or bocavirus-like particle of the invention of this invention might be used to deliver genes encoding neutralizing antibodies and their subsequent expression in host non-hematopoietic tissues. The vaccine-like protection against pathogen infection can be conducted through direct provision of neutralizing antibody from vector- mediated transgene expression, bypassing the reliance on the natural immune system for mounting desired humoral immune responses.

[0260] The introduction of a virus or virus-like particle comprising the chimeric bocavirus capsid protein of the invention, e.g. the bocavirus or bocavirus-like particle of the invention may involve use of any number of delivery techniques (both surgical and non-surgical) which are available and well known in the art. Such delivery techniques, for example, include vascular catheterization, cannulization, injection, inhalation, endotracheal, subcutaneous, inunction, topical, oral, percutaneous, intra-arterial, intravenous, and / or intraperitoneal administrations.

[0261] In particular, for delivery of a virus or virus-like particle comprising the chimeric bocavirus capsid protein of the invention, e.g. the bocavirus or bocavirus-like particle of the invention to a tissue, any physical or biological method that will introduce a virus or virus-like particle comprising the chimeric bocavirus capsid protein of the invention, e.g. the bocavirus or bocavirus-like particle of the invention to a host cell or organism can be employed.

[0262] The chimeric bocavirus capsid protein or the bocavirus or bocavirus-like particle of the invention can be administered systemically, regionally, or locally, or by any route, e.g., by injection, infusion, orally (e.g., ingestion or inhalation), or topically (e.g., transdermally). Such delivery and administration include intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous, intracavity, intracranial, transdermal (topical), parenteral, e.g., transmucosal or rectal. Exemplary routes of administration and delivery include intravenous (i.v.) , intraperitoneal (i.p.), intraarterial, intramuscular, parenteral, subcutaneous, intrapleural, topical, dermal, intradermal, transdermal, parenteral (e.g., transmucosal), intracranial, intraspinal, buccal (digestive organ), mucosal, respiratory, intranasal, intubation, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity, iontophoresis, intraocular, ocular, i ntragl a nd u I a r, intraorgan, and intralymphatic.

[0263] For purposes of intramuscular injection, solutions in an adjuvant such as sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose. A dispersion of viral particles can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.

[0264] Compositions of this invention may be used in vivo az well as ex vivo, in vivo gene therapy comprises administering the vectors, e.g. the virus or virus-like particle comprising the chimeric bocavirus capsid protein of the invention, e.g. the bocavirus or bocavirus-like particle of the invention directly to a subject. Pharmaceutical compositions can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to use. For administration into the respiratory tract, one mode of administration is by aerosol, using a composition that provides either a solid or liquid aerosol when used with an appropriate aerosolubilizer device. Another mode of administration into the respiratory tract is using a flexible fiberoptic bronchoscope to instill the vectors.

[0265] Typically, the viral vectors are in a pharmaceutically suitable pyrogen-free buffer such as Ringer's balanced salt solution (pH 7.4) . Although not required, pharmaceutical compositions may optionally be supplied in unit dosage form suitable for administration of a precise amount. The decision of whether to use in vivo or ex vivo therapy, and the selection of a particular composition, dose, and route of administration will depend on a number of different factors, including but not limited to features of the condition and the subject being treated. The assessment of such features and the design of an appropriate therapeutic or prophylactic regimen is ultimately the responsibility of the prescribing physician.

[0266] The outer shell of the chimeric bocavirus capsid protein comprises the structured region of bocavirus VP3. Therefore, any method used to detect bocavirus VP3 can also be used to detect the chimeric bocavirus capsid protein of the invention or a bocavirus or bocavirus-like particle of the invention. Such detection methods are known in the art. The outer shell, i.e. the VP3 protein, also determines the reactivity and tropism of the virus or virus-like particle. For these reasons, a virus or virus-like particle with a bocavirus VP3 domain is classified as a bocavirus.

[0267] In some embodiments of the invention, a sample is tested for the chimeric bocavirus capsid protein by determining whether the chimeric bocavirus capsid protein polypeptide or a nucleic acid encoding the polypeptide is present in the sample. Various methods are available to the skilled person for testing the sample, for example testing for hybridisation of a nucleic acid that encodes the chimeric bocavirus capsid protein to a specific primer or probe, or testing for binding of the chimeric bocavirus capsid protein to a specific binding member. Detection of the presence of the chimeric bocavirus capsid protein of the invention or the nucleic acid of the invention in the sample indicates that the sample is positive for the chimeric bocavirus capsid protein.

[0268] For example, the sample may be tested by being contacted with a specific binding member such as an antibody under appropriate conditions for specific binding. The binding member may optionally be labelled with a detectable label. Examples of suitable labels are described elsewhere herein. For example, the label may be a fluorescent label. Antibodies can be labelled with e.g. coloured latex, colloidal gold or colloidal selenium for detection by eye, or with an enzyme producing a detectable, e.g. coloured, product when a substrate is added. Binding may then be determined, e.g. using a reporter system. Where a panel of antibodies is used, different reporting labels may be employed for each antibody so that binding of each can be determined. Testing for binding of chimeric bocavirus capsid protein polypeptide to a specific binding member may employ e.g. immunofluorescence (IF), immunochromatography, or an enzyme immunoassay (EIA, ELISA).

[0269] The cargo packaged within the chimeric bocavirus capsid protein may be a transgene. In one embodiment, the transgene is the genome of a heterologous virus of the subfamiliy Parvovirinae. In one embodiment, the transgene is an AAV genome. In one embodiment, the transgene is not a bocavirus genome.

[0270] In another embodiment, the transgene is the wild-type or corrected version of a gene that is defective in the subject to the treated. In this case, a recombinant AVV genome (rAAV) is produced that comprises the transgene. In one embodiment, the transgene is a polynucleotide encoding a therapeutically relevant protein.

[0271] The transgene is integrated by recombinant techniques into or in place of the AAV genomic coding region (i.e., in place of the AAV rep and cap genes), but is generally flanked on either side by AAV inverted terminal repeat (ITR) regions. This means that an ITR appears both upstream and downstream from the coding sequence, either in direct juxtaposition, e.g., (although not necessarily) without any intervening sequence of AAV origin in order to reduce the likelihood of recombination that might regenerate a replication-competent AAV genome. However, a single ITR may be sufficient to carry out the functions normally associated with configurations comprising two ITRs and vector constructs with only one ITR can thus be employed in conjunction with packaging and production methods.

[0272] In some embodiments, the present invention provides kits having a packaging material and one or more components therein. Kits typically include a label or package insert including a description of the components or instructions for use of the components therein in vitro, in vivo, or ex vivo. A kit may contain a series of such components, e.g., a chimeric bocavirus capsid protein of the invention, a bocavirus or bocavirus-like particle of the invention, and optionally one or more additional active agents, e.g., another compound, agent, drug, or composition. Further Embodiments

[0273] The invention is also described by the following items:

[0274] 1. A chimeric bocavirus capsid protein, comprising a bocavirus Viral Protein (VP) amino acid sequence, and an adeno-associated virus (AAV) VP amino acid sequence.

[0275] 2. The chimeric bocavirus capsid protein of item 1, wherein the chimeric bocavirus capsid protein consists of a bocavirus VP amino acid sequence and an AAV VP amino acid sequence.

[0276] 3. The chimeric bocavirus capsid protein of any one of items 1 or 2, wherein an amino acid sequence of bocavirus VP is replaced by the corresponding amino acid sequence of AAV VP.

[0277] 4. The chimeric bocavirus capsid protein of any one of items 1 to 3, wherein an amino acid sequence of at least 20 amino acids of bocavirus VP is replaced by the corresponding amino acid sequence of AAV VP, preferably an amino acid sequence of at least 40 amino acids.

[0278] 5. The chimeric bocavirus capsid protein of any one of items 1 to 4, wherein the chimeric bocavirus capsid protein is VP1.

[0279] 6. The chimeric bocavirus capsid protein of any one of the previous items, wherein the AAV VP amino acid sequence comprises at least 20 amino acids of an AAV VPlu domain.

[0280] 7. The chimeric bocavirus capsid protein of any one of the previous items, wherein the AAV VP amino acid sequence comprises the N terminus of the AAV VPlu domain, preferably wherein the AAV VP amino acid sequence comprises at least the first 43 amino acids of the N terminus of the AAV VPlu domain. The chimeric bocavirus capsid protein of any one of the previous items, wherein the bocavirus VP amino acid sequence comprises an amino acid sequence from a bocavirus VP3 domain. The chimeric bocavirus capsid protein of any one of the previous items, wherein the amino acid sequence from a bocavirus VP3 domain is a full-length bocavirus VP3 domain or an N-terminal truncated bocavirus VP3 domain. The chimeric bocavirus capsid protein of any one of the previous items, wherein the chimeric bocavirus capsid protein further comprises an VP amino acid sequence from a VP1 / 2 domain of a Parvoviridae, preferably wherein the VP1 / 2 domain is a AAV VP1 / 2 domain or a bocavirus VP1 / 2 domain. The chimeric bocavirus capsid protein of any one of the previous items, wherein the AAV VP amino acid sequence is from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13. The chimeric bocavirus capsid protein of any one of the previous items, wherein the AAV VP amino acid sequence is from AAV2 or AAV8, preferably AAV2. The chimeric bocavirus capsid protein of any one of the previous items, wherein the bocavirus VP amino acid sequence is from a primate bocavirus. The chimeric bocavirus capsid protein of item 13, wherein the primate bocavirus is a human bocavirus or a gorilla bocavirus. 15. The chimeric bocavirus capsid protein of item 13, wherein the mammalian bocavirus is selected from the group consisting of HBoVl, HBoV2, HBoV3, HBoV4 and GBoVl, preferably, wherein the mammalian bocavirus is GBoVl.

[0281] 16. The chimeric bocavirus capsid protein of any one of the previous items, wherein the AAV VP amino acid sequence comprises a sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 67.

[0282] 17. The chimeric bocavirus capsid protein of any one of the previous items, wherein the amino acid sequence from a bocavirus VP3 domain comprises a sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 8.

[0283] 18. The chimeric bocavirus capsid protein of any one of the previous items, wherein the chimeric bocavirus capsid protein can assemble into a viral capsid structure.

[0284] 19. A recombinant bocavirus or bocavirus-like particle having a capsid comprising the chimeric bocavirus capsid protein of any one of the previous items.

[0285] 20. The recombinant bocavirus or bocavirus-like particle of item 19, further comprising a transgene packaged within the capsid.

[0286] 21. The recombinant bocavirus or bocavirus-like particle of any one of items 19 or 20, comprising the genome of a heterologous virus packaged within the capsid, preferably the genome of a virus of the genus Parvoviridae, more preferably the genome of an AAV virus, wherein the genome is a recombinant or a wild-type genome.

[0287] 22. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric bocavirus capsid protein as defined in any one of items 1 to 19. 23. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the recombinant bocavirus or bocavirus-like particle as defined in any one of items 20 or 21.

[0288] 24. A plasmid comprising the isolated nucleic acid molecule of any one of items 22 or 23.

[0289] 25. An isolated host cell comprising the chimeric bocavirus capsid protein of any one of items 1 to 18, the recombinant bocavirus or bocavirus-like particle of any one of items 19 to 21, the isolated nucleic acid of item 22 or 23, or the plasmid of item 24.

[0290] 26. The isolated host cell according to item 25, wherein the host cell is a mammalian cell, preferably a human cell.

[0291] 27. A composition comprising the chimeric bocavirus capsid protein of any one of items 1 to 18, the recombinant bocavirus or bocavirus-like particle of item 19 to 21, the isolated nucleic acid of items 22 or 23, the plasmid of item 24, or the host cell of items 25 or 26, and optionally one or more excipients.

[0292] 28. The chimeric bocavirus capsid protein of any one of items 1 to 18, the recombinant bocavirus or bocavirus-like particle of item 19 to 21, the isolated nucleic acid of items 22 or 23, the plasmid of item 24, the host cell of items 25 or 26, or the composition of item 27 for use as a medicament.

[0293] 29. The chimeric bocavirus capsid protein of any one of items 1 to 18, the recombinant bocavirus or bocavirus-like particle of item 19 to 21, the isolated nucleic acid of items 22 or 23, the plasmid of item 24, the host cell of items 25 or 26, or the composition of item 27 for use in the treatment of a genetic disease.

[0294] 30. The chimeric bocavirus capsid protein of any one of items 1 to 18, the recombinant bocavirus or bocavirus-like particle of item 19 to 21, the isolated nucleic acid of items 22 or 23, the plasmid of item 24, the host cell of items 25 or 26, or the composition of item 27 for use in gene therapy in a patient with a defective gene. The chimeric bocavirus capsid protein, the recombinant bocavirus or bocavirus-like particle, the isolated nucleic acid, the plasmid, the host cell, or the composition for use of item 30, wherein the defective gene has a mutation selected from the group consisting of insertion, deletion, insertion-deletion, miss-sense mutation, truncation mutation, stop mutation, nonsense mutation, frameshift mutation, repeat expansion. The chimeric bocavirus capsid protein, the recombinant bocavirus or bocavirus-like particle, the isolated nucleic acid, the plasmid, the host cell, or the composition for use of items 30 or 31, wherein the defective gene has a length of about 1 to about 5.5 kilobases, preferably a length of about 4 to about 5.5 kilobases. The chimeric bocavirus capsid protein, the recombinant bocavirus or bocavirus-like particle, the isolated nucleic acid, the plasmid, the host cell, or the composition for use of items 30 to 32, wherein the defective gene has a length of more than 4.8 kilobases. The chimeric bocavirus capsid protein of any one of items 1 to 18, wherein the AAV VP amino acid sequence comprises a sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 74. The chimeric bocavirus capsid protein of any one of items 1 to 18, wherein the amino acid sequence from a bocavirus VP3 domain comprises a sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 62. A chimeric bocavirus capsid protein comprising a sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 68. A chimeric bocavirus capsid protein comprising a sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 73. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 61. 39. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 64.

[0295] 40. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 67.

[0296] 41. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 74.

[0297] 42. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 60% identical to the amino acid sequence set forth in SEQ ID NO: 61.

[0298] 43. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 60% identical to the amino acid sequence set forth in SEQ ID NO: 64.

[0299] 44. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 60% identical to the amino acid sequence set forth in SEQ ID NO: 67.

[0300] 45. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 60% identical to the amino acid sequence set forth in SEQ ID NO: 74.

[0301] 46. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 61.

[0302] 47. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 64.

[0303] 48. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 67.

[0304] 49. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 74.

[0305] 50. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 61.

[0306] 51. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 64.

[0307] 52. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 67.

[0308] 53. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 74. 54. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 61.

[0309] 55. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 64.

[0310] 56. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 67.

[0311] 57. The chimeric bocavirus capsid protein of item 1 comprising a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 74.

[0312] 58. The chimeric bocavirus capsid protein of any one of the previous items, wherein the AAV VP amino acid sequence comprises a sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 67.

[0313] 59. The chimeric bocavirus capsid protein of any one of the previous items, wherein the amino acid sequence from a bocavirus VP3 domain comprises a sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 8.

[0314] 60. The chimeric bocavirus capsid protein of any one of items 1 to 18, the recombinant bocavirus or bocavirus-like particle of item 19 to 21, the isolated nucleic acid of items 22 or 23, the plasmid of item 24, the host cell of items 25 or 26, or the composition of item 27 for use in a method of diagnosis.

[0315] 61. A kit comprising a nucleic acid encoding the chimeric bocavirus capsid protein according to any one of items 1 to 59 and a nucleic acid encoding a transgene.

[0316] 62. The kit according to item 61, wherein the nucleic acid encoding a transgene has a size of at least 4.5 kilobases, preferably at least 4.8 kilobases.

[0317] Examples

[0318] Example 1: Construction of chimeric plasmids

[0319] Chimeric capsid protein amino acid sequence design: VP1, VP2, and VP3 nucleotide sequences for AAV2 (NC_001401), AAV8 (NC_006261.1), GBoVl (HM145750.1) and HBoVl (JQ923422) were retrieved from Genbank, and assembled according to the design of chimeric constructs. The generated protein sequences were back-translated to DNA sequences using codon optimization for human codon usage including sequence modification for ablation of restriction sites used for cloning and removal of cryptic splice and cloning sites. Finally, these reading frames were provided as split expression systems with ORFs for VP1 / VP2 (VP1A3) and VP2 / VP3 separated (see Figure 1). The construct GBoV-VPl carries a M130L mutation of the VP3 start codon to prevent translation of VP3 in this construct.

[0320] Respective vectors were generated by using an non-structural protein 1 (NPl)-independent strategy for capsid expression (Yan et al. Human Gene Therapy. 2019) comprising the following 5 plasmid system for chimeric capsid protein expression and packaging of AAV- genome payloads: Adenohelper, AAV2Rep, VP1 A3, VP2 / VP3, self-complimentary CMV-GFP.

[0321] The expression system was adapted from Yan et al., 2019, splitting the capsid viral protein reading frames for VP1 and VP2 / VP3. Using a codon-adaptive index, AAV2 / GBoVl VP1, VP2, and VP3 reading frames were synthesized to closely match Homo sapiens codonusage. Enhancements were made using the CMV promoter / enhancer and a 5’ human erythropoietin (EPO) intron and bovine growth hormone polyadenylation (bGH polyA) signal, similar to Yan et al., 2019. Vectors were combined with either a pVAX or pTwist system. 3D cell culture runs in Biostat bioreactors evaluated the GBoV 5-plasmid system's mediumscale performance.

[0322] Vector purification was performed by iodixanol density gradient centrifugation using methods established for AAV purification (Strobel et al. Human Gene Therapy Methods. 2015).

[0323] Quality Control of vectors comprised qPCR titer and silver staining SDS-PAGE for VP1 / VP2 / VP3 to determine expression levels and ratios of the proteins (see Figures 4 and 10).

[0324] Hepatocyte derived carcinoma cell line Huh-7 were cultivated in DMEM, 2mM L-Glutamine, high glucose (Thermo Fisher, 41965-047) with 10% Fetal bovine serum, heat inactivated (Gibco, 10101-145) (hereafter “culture medium”) at constant 37° C in humidified 5% CO2 atmosphere. Cells were routinely passaged at 90% confluence by trypsinization using Trypsin-EDTA (0.05%) (Thermo Fisher, 25300-054).

[0325] 5xl04cells were seeded in CELLSTAR®-96-Well-Plates with micro-clear bottom (Greiner, M0562-32EA) 24 hours prior to transduction in culture medium and incubated at 37° C with 5% CO2. On the day of the transduction the culture medium was replaced with 50pL of fresh culture medium per well. Infection mixes were prepared to MOIs ranging from IxlO4to 2xl05based on the formula vector prep [iiL] =cell number*M01*repUcates * 1.2 and filled with vector genome s / iL growth medium to 150^1, * replicates * 1.2 - vector prep [p.L]. Following this, 150pL of infection mix were added to each well. Finally, 50pL of 5pM Doxorubicin in culture medium were added to give a final Doxorubicin concentration of IpM. The cells were then returned to the incubator and maintained at 37° C with 5% CO2. 24 hours post transduction, the cells were washed with lx PBS (Thermo Fisher, 14190-094), after which 150pL fresh growth medium was added per well. GFP expression was assessed with a Celigo Imaging Cytometer (Nexcelom) and a Discover Echo Revolution Automated Fluorescence Microscope (Bico) every 24 hours post-transduction, while cells were kept in lx PBS. At the final timepoint (72h-144h post transduction), flow cytometric acquisition was carried out on a CytoFLEX SRT Benchtop Cell Sorter (Beckman Coulter) or a BD FACSCanto™ II (BD Biosciences).

[0326] Virus copy number (VCN) and mRNA levels were determined by qPCR for selected candidates (data not shown).

[0327] Example 2: Design of Chimeric Capsid Proteins with AAV2-GBoVl Swapped Sequences

[0328] In these constructs, a part of the GBoVl VP1 sequence was replaced by the respective AAV2 sequence, while the remaining sequence of GBoVl VP1 remained intact.

[0329] In constructs NGB (next generation bocavirus) A2.1-3, increasing portions of the N-terminal GBoVl VP1 sequence were replaced by N-terminal AAV2 VP1 sequence, resulting in chimeric capsids (see Figure 2).

[0330] • NGB A2.1: Exchange of VPlu • NGB A2.2: Exchange of VPlu plus VP1 / 2

[0331] • NGB A2.3: Exchange of VPlu plus VP1 / 2 plus the unstructured region of VP3 (replacement of N-terminal 32 amino acids of GBoVl VP3 by the respective 14 amino acids of AAV2) Constructs NGB Bl.1-3 all maintain the N-terminal sequence of VPlu from GBoVl, but various portions of the VP1 / 2 region are swapped for the respective sequence from AAV2. This could potentially allow for a better externalization of VPlu (see Figure 2).

[0332] • NGB Bl.1: Exchange of VP1 / 2

[0333] • NGB Bl.2: Exchange of VPl / 2u but without the N-terminal 17 amino acids of GBoVl and replacement by the likewise shortened AAV2 VP1 / 2 domain

[0334] • NGB Bl.3: Exchange of VPl / 2u but without the N-terminal 17 amino acids of GBoVl (as in construct NGB Bl.l) plus the unstructured region of VP3 (replacement of N-terminal 32 amino acids of GBoVl VP3 by the respective 14 amino acids of AAV2). Figure 2 shows a schematic representation of constructs NGB A2.1-A2.3 and B1.1-B1.3.

[0335] Table 1 shows the respective SEQ ID NOs of amino acid sequences used, Table 2 shows the respective SEQ ID NOs of nucleotide sequences encoding the amino acids.

[0336] Table 1: Amino Acid sequences used in chimera construction for chimeras NGB (next generation bocavirus) A2.1-A2.3, NGB B1.1-B1.3 and NGA (next generation AAV) B1.1-B1.4

[0337] Table 2: Nucleotide sequences used in chimera construction for chimeras NGB A2.1-A2.3,

[0338] NGB B1.1-B1.3 and NGA B1.1-B1.4

[0339] Example 3: Recombinant Vector Formation with AAV2- GBoVl Chimeric Capsids

[0340] All six chimeric constructs NGB A2.1- NGB A2.3 and NGB Bl.l- NGB Bl.3 were proficient in recombinant vector formation. Compared to a GBoVl control vector, purified vector yields from the downstream process were in a similar range for constructs NGB-A2.1, NGB-A2.2, NGB-A2.3, NBG Bl.2 and lower for constructs NGB-B1.1 and NGB-B1.3 (Figure 3).

[0341] Further quality control of vector material by silver staining of iodixanol gradient purified viruses with chimeric capsid proteins showed that all variants gave rise to comparable products (see Figure 4).

[0342] Example 4: In Vitro Transduction of Virus Encapsidated with AAV2- GBoVl Chimeric Capsid

[0343] Vectors were generated as described above and Huh7 cells were transduced in vitro. GBoVl was used as a control virus.

[0344] As shown in Figure 5, the VPlu domain of AAV2 can indeed increase the transduction efficiency of a virus with at least the VP3 sequence from GBoVl. The highest efficiency was seen for chimera NGB A2.1, which maintained GBoVl’s VP1 / 2 and VP3 sequence.

[0345] The additional presence of the VP1 / 2 region of AAV2 (chimera NGB A2.2) or even the unstructured region of VP3 of AAV2 (chimera NGB A2.3) only showed a moderate activity increase compared to the control virus with GBoVl capsid. This was probably caused by a structural incompatibility to efficiently externalize VPlu. Keeping GBoVl’s VPlu domain and only exchanging the VP1 / 2 domain, irrespective of N- terminal or C-terminal extensions, (chimeras NGB Bl.l- NGB Bl.3) failed to improve transduction properties compared to a virus with a GBoVl capsid (see Figure 5). This result suggests that AAV VPlu is likely crucial for improving transduction.

[0346] Example 5: Design of Chimeric Capsid Proteins with GBoVl-AAV2 Swapped Sequences

[0347] In these constructs, a part of the AAV2 VP1 sequence was replaced by the respective GBoVl sequence, while the remaining sequence of AAV2 VP1 remained intact (see Figure 6).

[0348] Using the same rational as in Example 1 above, the effect of replacing the N-terminal domains of AAV2 with those of GBoVl was tested.

[0349] In constructs NGA Bl.l - Bl.4, increasing portions of the N-terminal AAV2 capsid are replaced by that of GBoVl, resulting in chimeric capsids.

[0350] • Construct NGA Bl.l: Exchange of VPlu

[0351] • Construct NGA Bl.2: Exchange of VPlu plus the N-terminal 17 amino acids of VP1 / 2.

[0352] • Construct NGA Bl.3: Exchange of VPlu plus the N-terminal 37 amino acids of VP1 / 2 (predicted NLS from GBoVl).

[0353] • Constructs NGA Bl.4: Exchange of VPlu plus VP1 / 2.

[0354] AAV assembly protein 2 (AAP2) gene in reading frame 2 of AAV is maintained in NGA Bl.2 and Bl.3, but not in Bl.4. AAP2 reading frame starts at nucleotide position 383.

[0355] Table 1 shows the respective SEQ ID NOs of amino acid sequences used, Table 2 shows the shows the respective SEQ ID NOs of nucleotide sequences encoding the amino acids.

[0356] Example 6: Recombinant Vector Formation with Chimeric Capsids

[0357] All four chimeric constructs NGA Bl.l- NGA Bl.4 were proficient in recombinant vector formation. Compared to a virus with GBoVl wild-type capsid, purified vector yields from the downstream process a similar range for constructs NGA Bl.1-1.4 (Figure 7). Further QC of vector material showed that all variants gave rise to comparable products (data not shown).

[0358] Example 7: In Vitro Transduction of Virus Encapsidated with GBoVl-AAV2 Chimeric Capsid

[0359] In vitro biopotency studies using Huh-7 cells revealed that the VPlu domain of GBoVl retains all functions required for transduction in a heterologous, trans-genera (i.e. AAV) context, see NGA Bl.l, Figure 8. However, compared to wild-type AAV2, the transduction of viruses with chimeric capsids NGA Bl.l and NGA Bl.2 was reduced (see Figure 8).

[0360] The additional presence of increasing portions of GBoVl’s VP1 / 2 domain (and concomitantly decreasing portions of AAV2’s VP1 / 2 domain) resulted in a further gradual activity decline of the vectors (constructs NGA Bl.2-1.4). While construct NGA Bl.2 harboring 17 amino acids of GBoV’s VP1 / 2 showed almost the same activity as construct NGA Bl.l, the swapping of 37 amino acids of VP1 / 2 in construct NGA Bl.3 already led to a substantial activity loss; a complete exchange of the VP1 / 2 domain as in construct NGA Bl.4 led to an inactive vector.

[0361] In contrast to constructs NGA Bl.1-1.3, very low yields in vector particle formation were noted for construct NGA Bl.4. This was most likely due to the absence of AAV assembly protein 2 (AAP2). This protein is encoded in an alternative reading frame within the VP1 / 2 - VP3 region of AAV2 and critically required for AAV2 capsid assembly (Sonntag et al. 2010 PNAS; Grosse et al. 2017 J Virol). For construct NGA Bl.3, lack of transduction was probably caused by a structural incompatibility to correctly assemble into viral particles and / or to externalize VPlu.

[0362] Example 8: Design of Chimeric Capsid Proteins Comprising Sequences from AAV8 or AAV2 and GBoVl or HBoVl

[0363] The inventors have shown that VPlu of AAV2 boosts transduction of viruses comprising chimeric capsids with sequences from AAV2 and GBoVl. To put this discovery on a broader basis further constructs were designed that produced chimeras with different AAV serotypes and different bocaviruses.

[0364] NGH A2.1: Resembles NGB A2.1 but using sequences from HBoVl instead of GBoVl. Hence, in this construct the VP1 domain of HBoVl is replaced by AAV2 VPlu, while the rest of the VP1 sequence is from HBoVl.

[0365] NGB A8.1: Resembles NGB A2.1 but using sequences from AAV8 instead of AAV2. Hence, in this construct the VP1 domain of GBoVl is replaced by AAV8 VPlu, while the rest of the VP1 sequence is from GBoVl.

[0366] The main difference between VPlu of GBoVl and AAV2 is AAV2’s N-terminal extension of 43 amino acids, a feature that is conserved between AAV family members. This domain of AAV8 seems to be sufficient to boost avian AAV (Molecular Therapy Vol 30 Issue 4 Supplement pl-592 Abstract 436). According to Popa-Wagner et al. 2012 J Virol 86(17), the presence of 3 N-terminal PDZ domains in AAV2 (contained within the 43 aa) is important for the nuclear import of AAV2.

[0367] NGB A2.4: Resembles NGB A2.1 but using only the N-terminal 43 amino acids of AAV2 VPlu and the remaining sequence from GBoVl. The GBoVl sequence starts from amino acid 10, which represents the start of a region that is homologous between AAV2 and GBoVl (phospholipase 2 (PLA2) domain).

[0368] NGA Bl.5: Replacement of only the homologous region of VPlu (amino acids 44-138) by that of GBoVl (amino acids 10-90), the remaining sequences are from AAV2.

[0369] NGH A2.2: N-terminal 43 amino acids of AAV2 VPlu fused to HBoVl.

[0370] NGB A8.2: N-terminal 43 amino acids of AAV8 VPlu fused to GBoVl.

[0371] A schematic representation of these constructs is depicted in Figure 9. Table 3 shows the respective SEQ ID NOs of amino acid sequences used, Table 4 shows the respective SEQ ID NOs of nucleotide sequences encoding the amino acids. Table 3: Amino acid (AA) sequences used in chimera construction for chimeras NGB A3.1-

[0372] A3.6

[0373] Table 4: Nucleic acid (NA) sequences used in chimera construction for chimeras NGB A3.1- A3.6

[0374] Example 9: Recombinant Vector Formation with further AAV2- GBoVl Chimeric Capsids and Capsids comprising AAV8 and / or HBoVl domains

[0375] Quality control of vector material by silver staining of iodixanol gradient purified viruses with chimeric capsid proteins showed that all variants gave rise to comparable products (see Figure 10).

[0376] Example 10: In Vitro Transduction of Virus Encapsidated with AAV2- GBoVl Chimeric Capsids and Capsids comprising AAV8 and / or HBoVl domains

[0377] The chimeric constructs NGH-A2.1, NGB-A8.1, NGB-A2.4, NGA-B1.5, NGH-A2.2 and NGB- A8.2 were proficient in recombinant vector formation. Compared to NGB-A2.1 capsid which served as control, purified vector yields from the downstream process a similar range these constructs (Figure 11).

[0378] Next, expression of of chimeras NGH-A2.1, NGB A8.1, NGB-A2.4, NGA-B1.5, NGH-A2.2, NGB-A8.2, NGB A2.1 all carrying a GFP reporter was tested in Huh7 cells as described above. As shown in Figure 12, the chimeras were able to transduce Huh7 cells.

[0379] Example 11: Insertion of short peptides into chimera NGB-A2.1

[0380] To investigate the suitability of the chimeras described herein for peptide display, short peptides were inserted into chimera NGB-A2.1 at different positions. The insertion sites used are located in variable regions (VR) 1, VR 3, VR 4 and VR 5. Table 5 column 1 shows the variable region in which an insertion site is located. The insertion site is defined by the two adjacent amino acids, i.e. the peptide is inserted between the two listed amino acids. The amino acids numbering is shown relative to the starting point of counting amino acids. For example, since VP1, VP2 and VP3 are encoded in the same transcript, and the amino acid of VP1 also includes VP3, numbering of VP1 can be used to define an insertion site in VP3. Column 2 shows the position of the insertion site in NGB A2.1 according to the numbering of VP1 (SEQ ID NO: 33). Alternatively, the insertion site can be counted according to the numbering of VP3, with the start codon of VP3 counted as the first amino acid. Column 3 shows the position in NGB A2.1 VP3 when counting from the start of VP3 (SEQ ID NO: 5).

[0381] Since all insertion sites are located in VP3, and chimera NGB-A2.1 carries VP3 from GBoVl, the insertion sites can also be found in GBoVl. Column 4 shows the position of the insertion site in GBoVl VP1 (SEQ ID NO: 117) and column 5 shows the position of the insertion site in GBoVl VP3 counted from the start of VP3 (SEQ ID NO: 5).

[0382] Table 5: Insertion sites used for peptide display

[0383] The 9mer peptides used as insertion peptides in this proof-of-principle study are “A2”: GNYSRGVDA (SEQ ID NO: 93, encoded by a nucleic acid according to SEQ ID NO: 94) derived from Borner et al. 2020 (Mol Ther. 2020 Apr 8;28(4) : 1016 -1032) and “HA”: YPYDVPDYA (SEQ ID NO: 95 encoded by a nucleic acid according to SEQ ID NO: 96), a human influenza hemagglutinin derived tag. Table 6 shows the SEQ ID NOs of the constructs.

[0384] Table 6: SEQ ID NOs of peptide insertion constructs. AA: amino acid sequences, NA: nucleic acid sequence, A2: GNYSRGVDA peptide, HA: YPYDVPDYA peptide.

[0385] Transduction of the constructs was performed as described above for example 4. Figure 13 shows the upstream and downstream titers of the constructs. Figure 14 shows the transduction efficiency of the constructs, which was determined by calculating the % of GFP cells after FACS sorting.

[0386] Conclusions

[0387] The inventors were able to show that chimeric capsid proteins comprising amino acid sequences from AAV and bocavirus can form functional capsid shells when transduced into cells. Recombinant bocavirus comprising a chimeric bocavirus capsid protein with an N-terminal amino acid sequence from AAV that includes VPlu and a bocavirus VP3 resulted in improved transduction efficiency compared to recombinant bocavirus comprising wild-type bocavirus capsid proteins (Constructs NGB A2.1-A2.3). In contrast, a recombinant AAV comprising a bocavirus VPlu and an AAV VP3 exhibited a decrease in transduction efficiency compared to a virus comprising wild-type AAV capsid proteins (Constructs NGA B1.1-B1.4). Including the VP1 / VP2 domain of AAV in these constructs improved their transduction efficiency (Constructs NGA Bl.l, Bl.2).

[0388] Recombinant bocavirus comprising a chimeric bocavirus capsid protein comprising an AAV VP1 / VP2 domain or a fragment thereof and a bocavirus VPlu and VP3 or structured domain of VP3 were able to transduce cells with a similar efficiency to wild-type bocavirus capsid protein (Constructs NGB B1.1-B1.3).

[0389] The inventors were able to demonstrate that replacing amino acid sequences of a bocavirus VP1 with the corresponding amino acid sequences of a AAV VP1 results in a chimeric bocavirus capsid protein that can form a functional capsid shell and the resulting virus can efficiently transduce mammalian cells.

[0390] Furthermore, the inventors showed that the AAV VPlu domain is able to confer improved transduction efficiency to a bocavirus VP1 capsid protein compared to a wild-type bocavirus capsid protein.

[0391] Replacing the VPlu domain of a bocavirus VP1 with a VPlu domain from AAV resulted in a chimeric bocavirus capsid protein that showed improved transduction efficiency compared to a wild-type bocavirus virus. At the same time, retaining the bocavirus VP3 domain ensured that the packaging capacity of the virus remains unchanged.

[0392] In order to create a chimeric bocavirus capsid protein with a minimal AAV VP fragment, the inventors also produced constructs that only included the first 43 amino acids of the AAV VP1 protein, while the rest of the VP1 sequence is from bocavirus. These constructs were able to efficiently transduce Huh7 cells.

[0393] The chimeric bocavirus capsid proteins are also suitable for peptide display.

[0394] Thus, the inventors were able to produce a virus with improved transduction efficiency compared to wild-type bocavirus with a transgene packaging capacity of up to 5.5 kilobases. This allows for the first time a gene therapeutical approach additionally for genes up to 5.5 kb and thus, to effectively treat diseases of hitherto unmet medical need.

Claims

Claims1. A chimeric bocavirus capsid protein, comprising a bocavirus Viral Protein (VP) amino acid sequence and an adeno-associated virus (AAV) VP amino acid sequence.

2. The chimeric bocavirus capsid protein of any one of the previous claims, wherein the AAV VP amino acid sequence comprises the at least 20 amino acids of an AAV VPlu domain.

3. The chimeric bocavirus capsid protein of any one of the previous claims, wherein the bocavirus VP amino acid sequence comprises an amino acid sequence from a bocavirus VP3 domain.

4. The chimeric bocavirus capsid protein of any one of the previous claims, wherein the chimeric bocavirus capsid protein further comprises an VP amino acid sequence from a VP1 / 2 domain of a Parvoviridae, preferably wherein the VP1 / 2 domain is a AAV VP1 / 2 domain or a bocavirus VP1 / 2 domain.

5. The chimeric bocavirus capsid protein of any one of the previous claims, wherein the AAV VP amino acid sequence is from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, preferably wherein the AAV VP amino acid sequence is from AAV2 or AAV8.

6. The chimeric bocavirus capsid protein of any one of the previous claims, wherein the bocavirus VP amino acid sequence is from a primate bocavirus, preferably wherein the primate bocavirus is a human bocavirus or a gorilla bocavirus.

7. The chimeric bocavirus capsid protein of claim 6, wherein the mammalian bocavirus is selected from the group consisting of H BoVl, HBoV2, H BoV3, HBoV4 and GBoVl, preferably, wherein the mammalian bocavirus is GBoVl.

8. The chimeric bocavirus capsid protein of any one of the previous claims, wherein the AAV VP amino acid sequence comprises an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 67.

9. The chimeric bocavirus capsid protein of any one of the previous claims, wherein the amino acid sequence from a bocavirus VP3 domain comprises an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ I D NO: 8.

10. The chimeric bocavirus capsid protein of any one of the previous claims, wherein the amino acid sequence of the chimeric bocavirus capsid protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO:3.

11. A recombinant bocavirus or bocavirus-like particle having a capsid comprising the chimeric bocavirus capsid protein of any one of the previous claims, optionally further comprising a transgene packaged within the capsid.

12. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric bocavirus capsid protein as defined in any one of claims 1 to 10.

13. A plasmid comprising the isolated nucleic acid molecule of claim 12.

14. A host cell comprising the chimeric bocavirus capsid protein of any one of claims 1 to 10, the recombinant bocavirus or bocavirus-like particle of claim 11, the isolated nucleic acid of claim 12, or the plasmid of claim 13.

15. A composition comprising the chimeric bocavirus capsid protein of any one of claims 1 to 10, the recombinant bocavirus or bocavirus-like particle of claim 11, the isolated nucleic acid of claim 12, the plasmid of claim 13, or the host cell of claim 14, and optionally one or more excipients.

16. The chimeric bocavirus capsid protein of any one of claims 1 to 10, the recombinant bocavirus or bocavirus-like particle of claim 11, the isolated nucleic acid of claim 12, theplasmid of claim 13, or the host cell of claim 14, or the composition of claim 15 for use as a medicament.

17. The chimeric bocavirus capsid protein of any one of claims 1 to 10, the recombinant bocavirus or bocavirus-like particle of claim 11, the isolated nucleic acid of claim 12, the plasmid of claim 13, or the host cell of claim 14, or the composition of claim 15, for use in gene therapy.

18. The chimeric bocavirus capsid protein, the recombinant bocavirus or bocavirus-like particle, the isolated nucleic acid, the plasmid, the host cell, or the composition for use of claim 17, wherein the gene therapy comprises delivery of a transgene with a length of about 1 to about 5.5 kilobases, preferably a length of about 4 to about 5.5 kilobases, more preferably a length of at least 4.8 kilobases.

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