Novel bocavirus capsid peptide insertion sites

Abstract

The present invention provides recombinant bocavirus capsid proteins comprising peptide inserts, viral vectors comprising said capsid proteins, nucleic acids encoding said capsid proteins and viral vectors, plasmids, host cells and compositions as well as vectors comprising said capsid proteins; and their use as a medicament and in gene therapy.

Description

[0001] Novel Bocavirus Capsid Peptide Insertion Sites

[0002] FIELD OF THE INVENTION

[0003] The present invention provides recombinant bocavirus capsid proteins comprising peptide inserts, viral vectors comprising said capsid proteins, nucleic acids encoding said capsid proteins and viral vectors, plasmids, host cells and compositions as well as vectors comprising said capsid proteins; and their use as a medicament and in gene therapy.

[0004] Background of the invention

[0005] The use of Parvoviridae in gene therapy vector engineering is highly appealing due to their safety, efficiency, and specificity, with adeno-associated virus (AAV) being a prime example. More recently, another genus of the Parvoviridae family, the bocaparvoviruses (bocaviruses) has been identified in humans. Human bocavirus 1 (HBoVl) is an increasingly popular gene therapy vector candidate due to its ability to transduce polarized human airway epithelia (pHAEs) when cross-packaged with AAV2 genomes.

[0006] 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 larger genome size of bocavirus compared to AAV means that bocaviruses have a larger packaging capacity. Due to this larger packaging capacity compared to AAV, bocaviruses are of great interest as a delivery vector for gene therapy.

[0007] Bocaviruses are especially attractive for gene therapy 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. The structure of HBoVl, 3 and 4 capsids has been described using cryo-electron microscopy (Mietzsch et al. 2017, Journal of Virology 91(11)). 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.

[0008] Thus, there is a need for bocavirus-based gene therapy viral 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.

[0009] Objectives and Summary of the Invention

[0010] The present invention solves the above-mentioned problem by providing recombinant bocavirus capsid proteins and bocavirus vectors with improved transduction efficiency and cell specificity. In particular, the inventors identified insertion sites that are suitable to display short peptides, such as targeting peptides, on the surface of bocaviruses. Since peptide display has not previously been employed in bocaviruses, the inventors systematically investigated the abilities of the gorilla bocavirus capsid to tolerate peptides inserted on its surface.

[0011] The exploration of the potential of bocavirus capsids for peptide display is expected to accelerate their in vivo screening and utilization as unique gene therapy vectors.

[0012] The inventors were able to successfully introduce peptides into the bocavirus capsid without disrupting assembly of the virus or viral particle. The inventors surprisingly found that the most promising insertion sites in bocavirus were different from the preferred insertion sites known for the closely related AAV.

[0013] Hence, in one aspect, the invention is directed to a recombinant bocavirus capsid protein, comprising at least one heterologous peptide insert of at least 4 amino acids in length within viral protein 3 (VP3).

[0014] Variable regions are short stretches of amino acids in the capsid proteins of Parvoviridae that are structurally different between viruses and virus serotypes. These regions are thought to contribute to transduction efficiency and tropism of the virus (Govindasamy et al. 2006, J Virol, 80(23):11556-70). In one embodiment, the peptide insert is inserted at an insertion site located within a variable region (VR) of VP3. In one particular embodiment, the insertion site is located within a VR selected from the group consisting of VR I, VR III, VR IV, VR V, VR VIII and VR IX of VP3.

[0015] GBoVl variants that display model peptides on the surface using different insertion sites have been generated and subjected to manufacturability tests. Viable variants were tested for in vitro transduction efficacy resulting in a feasibility ranking of the insertion sites. Reporter-encoding vectors were produced and their performance in two cell types, Huh7, and primary human liver cells, was tested. Eight preferred insertion sites were selected based on these results.

[0016] In one embodiment, the insertion site is located immediately following an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 selected from the group consisting of amino acid S209 of VR I, amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, amino acid G408 of VR IV, amino acid T461 of VR V, amino acid T521 of VR VIII, and amino acid T636 in VR IX.

[0017] The amino acid sequence of gorilla bocavirus 1 VP1 (SEQ ID NO: 1) may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 22. In a preferred embodiment, the insertion site is located immediately following an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 selected from the group consisting of amino acid D332 of VR III; amino acid G333 of VR III; or amino acid T334 of VR III.

[0018] Importantly, the recombinant capsid needs to be capable of forming a functional capsid, i.e. encapsidation of the virus needs to be unperturbed by the peptide insert. Therefore, the insertion sites are selected such that they did not impede functionality of the virus or viral particle. Hence, in one embodiment, the recombinant bocavirus capsid protein of the invention is capable of forming a functional capsid.

[0019] Bocavirus have been isolated from a range of mammals including primates, bats, cattle and dogs. Hence, in one embodiment, the bocavirus is a mammalian bocavirus. In a preferred embodiment, the recombinant bocavirus capsid protein is from a primate bocavirus, more preferably from human or gorilla bocavirus.

[0020] The peptide insert typically consists of a peptide and flanking amino acids. Flanking amino acids maintain flexibility and promote efficient display of the peptide epitope on the surface of the capsid.

[0021] In one embodiment, the peptide has a length of at least 4 amino acids. The peptide insert comprises contiguous amino acids. Hence, in a preferred embodiment, the peptide insert comprises at least 4 contiguous amino acids.

[0022] In one embodiment, the peptide insert may be from a heterologous protein that is not a bocavirus protein.

[0023] Peptide insertions into bocavirus capsid proteins have not been described previously. Therefore, exemplary model peptides that have been used in AAV were chosen as a proof or principle to validate the insertion site. These peptides have been described for example by Borner et al. (Borner et al. 2019, Molecular Therapy 28(4):1016-1032). In one embodiment, the peptide comprises an RGD (Arg-Gly-Asp) motif. In another embodiment, the peptide comprises a NXXRXXX motif.

[0024] Hence, in one particular embodiment, the peptide is selected from the group consisting of XXXRGDXXX and NXXRXXX.

[0025] Furthermore, in one embodiment, the peptide insert may be flanked at each end by a linker comprising at least one amino acid. In one embodiment, the linker has a length of between one amino acid and four amino acids. The linker may be different length at each end of the peptide (i.e. N-terminus and C-terminus), or it may be the same length.

[0026] In another aspect, the invention also relates to a recombinant bocavirus or bocavirus-like particle comprising the recombinant bocavirus capsid protein of the invention.

[0027] Upon formation of the recombinant bocavirus or bocavirus-like particle, the recombinant bocavirus capsid protein forms part of the capsid of the virus. The peptide insert is located within VP3, which forms the outer shell of the capsid. Importantly, after encapsidation, the peptide insert is arranged such that it points away from the capsid to the outside, i.e. does not reach into the lumen. Hence, in one embodiment, the peptide insert is surface exposed within the capsid.

[0028] The recombinant bocavirus may further comprise a gene expression cassette comprising a gene of interest, i.e. a transgene. Alternatively, packaged within the recombinant bocavirus or bocavirus-like particle comprising the recombinant bocavirus capsid protein of the invention may be a cargo that is not a transgene.

[0029] In one embodiment, the bocavirus or bocavirus-like particle is a mammalian bocavirus or bocavirus-like particle, preferably a primate bocavirus or bocavirus-like particle.

[0030] In another aspect, the invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the recombinant bocavirus capsid protein of the invention. 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.

[0031] Furthermore, the invention also provides a plasmid vector comprising any one of the isolated nucleic acid molecules of the invention.

[0032] The invention also provides a host cell comprising a recombinant 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 vector of the invention.

[0033] In one embodiment, the host cell is a mammalian cell. In another embodiment, the host cell is a non-human mammalian cell. In one embodiment, the host cell is an isolated host cell.

[0034] In another aspect, the invention provides a composition comprising the recombinant bocavirus capsid protein of the invention and optionally one or more excipients.

[0035] 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 vector of the invention or the host cell of the invention and optionally one or more excipients.

[0036] In another aspect, the invention relates to the recombinant bocavirus capsid protein of the invention for use as a medicament.

[0037] 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. In one embodiment, the invention relates to the use of the recombinant bocavirus capsid protein of the invention for the manufacture of a medicament.

[0038] In one embodiment, the invention relates to the use of 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 the manufacture of a medicament.

[0039] The recombinant bocavirus capsid protein of the invention and the recombinant bocavirus or bocavirus-like particle of the invention are especially suitable for gene therapy, or treatment of a genetic disease. Gene therapy for larger genes than the ones that can be packaged in AAV virus vectors, which have a packaging capacity of less than 4.8 kilobases, is feasible, because bocavirus has an improved packaging capacity of at least 5.5 kilobases in comparison to conventionally gene therapy vectors, such as AAV virus vectors,

[0040] Hence, in another aspect, the invention provides the recombinant 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 for use in gene therapy.

[0041] 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.

[0042] 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.

[0043] In another embodiment, the gene therapy comprises delivery of a transgene with a length of at least 4.5 kilobases.

[0044] In another embodiment, the gene therapy comprises delivery of a transgene with a length of at least 4.8 kilobases. Figure Legends

[0045] Figure 1: Schematic representation of 2-component bocavirus structural protein expression cassettes designed to express GBoVl Viral Protein 1 (VP1) and VP2 (pVAX EVT001 expression plasmid) and GBoVl VP2 and VP3 (pTwist EVT002 expression plasmid), showing transcription start sites, ORF (grey), and translated protein domains (black) of VP1, VP2, and VP3 of GBoVl. hEPO: human erythropoietin, bGH polyA: bovine growth hormone polyadenylation, TSS: transcription start site, CMVe: Cytomegalovirus enhancer, CMVp: Cytomegalovirus promoter; T7: Bacteriophage T7. M130L: point mutation in the VP3 start codon. Expression cassettes are shown schematically and are not drawn to scale.

[0046] Figure 2: Cloning strategy for inserting peptides into GBoVl in six variable regions (VRs). Primers for each VR were designed using NEBuilder®. The Master Template was synthesized as a linearized DNA fragment carrying the peptide insert. Fragment and linearized plasmid with matching overlaps were produced by PGR amplification and ligated to form the final plasmid based on the matching overhangs.

[0047] Figure 3: Schematic overview of the final recombinant bocavirus capsid proteins of GBoVl that have been cloned and manufactured, with peptide insertions. The schematic representations of GBoVl with llmer peptide insertions are presented on the left side, while the GBoVl capsid protein variants with 9mer peptide insertions are shown on the right side.

[0048] Figure 4: A: Schematic overview of the proviral plasmids used in this study. Shown are five plasmids needed for bocavirus vector production, VP1(VP2): VP1 / VP2 expression plasmid, VP2+VP3: VP2 / VP3 expression plasmid, Ad5H: Adenohelper Plasmid, Rep2: AAV2 replication plasmid, AAV-GFP Proviral: self-complimentary CMV-GFP (sc-GFP) plasmid, HEK293 / NRC: National Research Council of Canada. B: GBoVl peptide insertion plasmids were transfected into HEK293T cell for virus production, followed by iodixanol purification, and 5pl per lane loaded on an SDS-PAGE. Silver-staining of iodixanol-gradient purified GBoVl peptide insertions using two different protocols: Protocol 1 (Cell media + Cell pellet) and Protocol 2 (Cell pellet) (see Examples), d3 : day 3, d5: day 5. From left to right lane 2 to 26.

[0049] Figure 5: Upstream and downstream yields of GBoVl peptide insertion variants. A: Upstream titers presented in vector genomes per ml, GBoVl 9mer (white bars) and llmer (black bars) peptide insert capsid protein variants compared to wild-type AAV6 (line) and GBoVl (dashed line). B: Downstream titers presented in total vector genomes, GBoVl 9mer (white bars) and llmer (black bars) peptide insert capsid protein variants compared to wildtype AAV6 (line) and GBoVl (dashed line).

[0050] Figure 6: In vitro transduction of Huh7 cells with peptide display GBoVl variants. Huh7 cells were incubated with transduction mix comprising GBoVl peptide insertion variants with 9mer peptide inserts (A) or llmer peptide inserts (B) at an MOI (multiplicity of infection) of 3 x 105. Cells were imaged with FACS to determine the percentage of GFP positive cells. The numbers on top of the column correspond to percentages of GFP-positive cells.

[0051] Figure 7: In vitro transduction of pHAE cells with peptide display GBoVl variant with 9mer insertion at VR3.2 at an MOI (multiplicity of infection) of 5 x 104. A: fluorescence microscopy images of pHAE cells 168h post transduction. B: Percentage of GFP-positive cells, as determined by flow cytometry, 168 hours post-transduction.

[0052] DETAILED DESCRIPTION OF THE INVENTION

[0053] Definitions

[0054] Before the invention is described in detail with respect to some of its preferred embodiments, the following general definitions are provided.

[0055] 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. 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.

[0056] 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.

[0057] For the purposes of the present invention, the term “obtained” is considered to be a preferred embodiment of the term “obtainable”. If hereinafter e.g. a compound is defined to be obtainable from a specific source, this is also to be understood to disclose a compound which is obtained from this source.

[0058] 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%.

[0059] 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.

[0060] 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-stranded DNA (ssDNA). Parvoviridae a e 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 (e.g. bocavirus), copiparvovirus, dependoparvovirus (e.g. adeno-associate virus), erythroparvovirus (e.g. B19 virus), protoparvovirus (e.g. canine parvovirus, feline parvovirus), and tetraparvovirus.

[0061] 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 forms, 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.

[0062] Adeno-associated viruses (AAV) are non-pathogenic, helper-dependent members of the parvovirus 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 anti-sense 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., an immunogenic polypeptide 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.

[0063] 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 forms, 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.

[0064] “Recombinant” bocavirus capsid protein as used herein refers to a bocavirus capsid protein that has been modified in a way that results in a protein not found in nature. The term recombinant refers to the use of molecular biology methods to combine nucleic acid or protein sequences from two or more different sources. In the context of this specification, recombinant bocavirus capsid proteins are capsid proteins that have been modified to comprise exogenous peptides.

[0065] 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 AAV2 and a genome containing 5' and 3' ITR sequences from the same AAV2 serotype.

[0066] There are four known serotypes of human bocavirus (HBoV), HBoVl, HBoV2, HBoV3, and HBoV4. However, included in bocavirus are serotypes derived from other non-human mammals such as swine bocavirus or gorilla bocavirus (GBoV).

[0067] 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. 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. 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 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.

[0068] 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. The amino acid numbering is according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 amino acid sequence as defined in SEQ ID NO: 1.

[0069] 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.

[0070] 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. 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. "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.

[0071] 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 virus-like 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.

[0072] 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”.

[0073] "Gene expression" or "expression" refers to the process of gene transcription, translation, and post-translational modification.

[0074] A "helper virus" for AAV and bocavirus refers to a virus that allows the AAV / bocavirus to be replicated and packaged by a mammalian cell. A variety of suitable helper viruses 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.

[0075] 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.

[0076] "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. Host cells are typically isolated cells and / or cultured cells.

[0077] A "plasmid", “plasmid vector” 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.

[0078] 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.

[0079] "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).

[0080] 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 recombinant 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.

[0081] 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 excipients, which enhance the effectiveness of the formulation.

[0082] 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.).

[0083] 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.

[0084] 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 %-100% 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.

[0085] 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.

[0086] “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.

[0087] 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.

[0088] 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 recombinant bocavirus capsid protein of the invention.

[0089] Recombinant Capsid Proteins 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 cap open reading frame. All VPs share a common C-terminal VP3 sequence. The capsid proteins form an icosaedral 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. Thus the term “viral protein”, abbreviated “VP”, comprises VP1, VP2 and VP3.

[0090] The inventors have shown that bocavirus capsids tolerate the insertion of short peptides of a length of at least 4 amino acids into VP3. The insertion within VP3 ensures that the peptides are exposed at the surface of the capsid shell. Due to the nature of the VP proteins, the inserted peptides are also present within VP1 and VP2, which both share the common C-terminal VP3 sequence.

[0091] The inventors have identified eight insertion sites that can tolerate peptide insertion of different sizes while retaining the ability to transduce cells. The eight insertion sites disclosed herein are located immediately after an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 selected from the group consisting of amino acid S209 of VR I, amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, amino acid G408 of VR IV, amino acid T461 of VR V, amino acid T521 of VR VIII, and amino acid T636 in VR IX.

[0092] The insertion sites can also be described by the amino acid number corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 of their two flanking amino acids: VR I: 209 / 210; VR III: 332 / 333, 333 / 334, 334 / 335; VR IV: 408 / 409; VR V: 461 / 462; VR VIII: 521 / 522; VR IX: 636 / 637. The corresponding amino acids residues according to the numbering of human bocavirus 1 (H BoVl) VP1 as defined in in SEQ ID NO: 63 of their two flanking amino acids: VR I: 211 / 212; VR III: 334 / 335, 335 / 336, 336 / 337; VR IV: 408 / 409; VR V: 461 / 462; VR VIII: 521 / 522; VR IX: 636 / 637. A nucleic acid sequence encoding HBoVl VP1 is set forth in SEQ ID NO: 64.

[0093] In one particular embodiment, the invention relates to a recombinant bocavirus capsid protein, comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight heterologous peptide inserts of at least 4 amino acids.

[0094] In one embodiment, the recombinant bocavirus capsid protein comprises a peptide insert of at least 4 amino acid at each of the insertion sites located immediately after an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 consisting of amino acid S209 of VR I, amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, amino acid G408 of VR IV, amino acid T461 of VR V, amino acid T521 of VR VIII, and amino acid T636 in VR IX.

[0095] In one embodiment, the recombinant bocavirus capsid protein comprises a peptide insert of at least 4 amino acid at one, two, three, four, five, six, seven, or eight of the insertion sites located immediately after an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 consisting of amino acid S209 of VR I, amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, amino acid G408 of VR IV, amino acid T461 of VR V, amino acid T521 of VR VIII, and amino acid T636 in VR IX. In one embodiment, the insertion site is located immediately following an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 selected from the group consisting of amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, amino acid G408 of VR IV, and amino acid T461 of VR V.

[0096] In one embodiment, the recombinant bocavirus capsid protein comprises a peptide insert of at least 4 amino acid at each of the insertion sites located immediately after an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 consisting of amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, amino acid G408 of VR IV, and amino acid T461 of VR V.

[0097] In one embodiment, the recombinant bocavirus capsid protein comprises a peptide insert of at least 4 amino acid at one, two, three, four or five of the insertion sites located immediately after an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 consisting of amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, amino acid G408 of VR IV, and amino acid T461 of VR V.

[0098] In another embodiment, the insertion site is located immediately following an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 selected from the group consisting of amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, and amino acid G408 of VR IV.

[0099] In one embodiment, the recombinant bocavirus capsid protein comprises a peptide insert of at least 4 amino acid at each of the insertion sites located immediately after an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 consisting of amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, and amino acid G408 of VR IV.

[0100] In one embodiment, the recombinant bocavirus capsid protein comprises peptide inserts of at least 4 amino acid at one, two, three or four of the insertion sites located immediately after an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 consisting of amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III and amino acid G408 of VR IV.

[0101] In one embodiment, the insertion site is located within VR III of VP3.

[0102] Hence, in one embodiment, the insertion site is located immediately following an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 selected from the group consisting of amino acid D332 of VR III; amino acid G333 of VR III; or amino acid T334 of VR III.

[0103] In one embodiment, the recombinant bocavirus capsid protein comprises peptide inserts of at least 4 amino acid at one, two or three of the insertion sites located immediately after an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 consisting of amino acid D332 of VR III, amino acid G333 of VR III, and amino acid T334 of VR III.

[0104] In one embodiment, the recombinant bocavirus capsid protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21. The capsid polypeptide and the peptide are encoded by a contiguous open reading frame and the peptide is included in the structure of the capsid polypeptide during protein biosynthesis. Hence, the capsid proteins including the inserted peptides are expressed from a contiguous nucleic acid sequence and translated into a contiguous polypeptide.

[0105] In one embodiment, the recombinant bocavirus capsid protein comprises an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 and 38.

[0106] Peptide Insert

[0107] The peptide insert may be flanked by amino acid linkers or one or more flanking amino acids.

[0108] In one embodiment, the peptide insert comprises flanking amino acids of at least one amino acid on each side. In one embodiment, the peptide insert comprises flanking amino acids of one amino acid on the N-terminal side and on the C-terminal side, respectively. In another embodiment, the peptide insert comprises flanking amino acids of two, three, four or five amino acids on the N-terminal side and on the C-terminal side, respectively.

[0109] In another embodiment, the peptide insert comprises flanking amino acids of one amino acid on the N-terminal side and two amino acids on the C-terminal side. In another embodiment, the peptide insert comprises flanking amino acids of two amino acid on the N-terminal side and three amino acids on the C-terminal side. In another embodiment, the peptide insert comprises flanking amino acids of up to four amino acid on the N-terminal side and up to four amino acids on the C-terminal side, wherein the flanking amino acids do not have to be the same number on each side.

[0110] In another embodiment, the peptide insert comprises flanking amino acids of one amino acid on the C-terminal side and two amino acids on the N-terminal side. In another embodiment, the peptide insert comprises flanking amino acids of two amino acid on the C-terminal side and three amino acids on the N-terminal side. In another embodiment, the peptide insert comprises flanking amino acids of three amino acid on the C-terminal side and four amino acids on the N-terminal side.

[0111] Hence, in one embodiment, the peptide insert additionally comprises a small amino acid, preferably independently selected from G and A, at the N-terminus and / or at the C- terminus.

[0112] In one embodiment, the at least one flanking amino acid is selected from the group consisting of alanine and glycine.

[0113] In one embodiment, the peptide insert comprises flanking amino acids of one glycine on the N-terminal side and one alanine on the C-terminal side.

[0114] In one embodiment, the peptide insert comprises flanking amino acids of one, two, three or four alanines on the N-terminal side and one, two, three or four alanines on the C-terminal side.

[0115] In another embodiment, the peptide insert does not comprise flanking amino acids.

[0116] In principle, any peptide can be inserted in the insertion sites provided by the invention. The peptide may for example be a targeting peptide, i.e. a peptide which mediates the cell specificity of the recombinant bocavirus or bocavirus-like particle for a targeted cell by binding to the respective target cell. Hence, in one embodiment, the peptide insert is a targeting peptide. In one embodiment, the peptide insert comprises a binding epitope.

[0117] In one embodiment, the peptide insert does not replace any of the sequence of VP3.

[0118] Peptide libraries useful for peptide display in Parvoviridae have been described previously, for example in AAV (see Borner et al. 2019, Molecular Therapy 28(4):1016-1032). The peptide insertion sites described herein can be used with any peptide that is suitable for peptide display in Parvoviridae. For example, the peptide insertion sites described herein can be used with any peptide that is suitable for peptide display in AAV. In one embodiment, the peptide insert has a length of at least 4 amino acids. This size was chosen to ensure provision of a peptide epitope that can, for example, bind or interact with other proteins or cell components.

[0119] In one embodiment, the peptide insert has a length of at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 amino acids.

[0120] In one embodiment, the length of the peptide insert is between 4 and 20 amino acids. In another embodiment, the length of the peptide insert is between 5 and 20 amino acids. In yet another embodiment, the length of the peptide insert is between 5 and 15 amino acids. In yet another embodiment, the length of the peptide insert is between 7 and 15 amino acids. In yet another embodiment, the length of the peptide insert is between 7 and 11 amino acids. In another embodiment, the length of the peptide insert is between 9 and 11 amino acids.

[0121] Typically, the peptide insert comprises at most 11 amino acids.

[0122] In one embodiment, the peptide insert comprises an RGD motif or an NXXRXXX motif, wherein each X is independently selected from any naturally occurring amino acid.

[0123] In one particular embodiment, the peptide insert is selected from the group consisting of CDCRGDCFC and NYSRGVD.

[0124] The recombinant 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 recombinant 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 recombinant 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 recombinant 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 recombinant 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.

[0125] 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.

[0126] In one embodiment, the recombinant 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.

[0127] In another embodiment, the recombinant 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.

[0128] Nucleic acids, plasmids and host cells

[0129] The invention also provides a plasmid comprising any one of the isolated nucleic acid molecules of the invention.

[0130] 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.

[0131] Delivery of virus or virus-like particle comprising the recombinant 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.

[0132] The invention also provides a host cell comprising a recombinant 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.

[0133] In one embodiment, the host cell is a mammalian cell. In another embodiment, the host cell is a non-human mammalian cell.

[0134] Compositions and medical uses

[0135] A virus or virus-like particle comprising the recombinant 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. 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 recombinant 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.

[0136] Alternatively, a polynucleotide may be provided to the cell by a virus or virus-like particle comprising the recombinant 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.

[0137] 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 recombinant 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 recombinant 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. The introduction of a virus or virus-like particle comprising the recombinant 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.

[0138] In particular, for delivery of a virus or virus-like particle comprising the recombinant 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 viruslike particle comprising the recombinant 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.

[0139] The recombinant 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.

[0140] For purposes of intramuscular injection, solutions in an excipient 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.

[0141] 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 recombinant 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.

[0142] 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.

[0143] 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.

[0144] In some embodiments of the invention, a sample is tested for the recombinant bocavirus capsid protein by determining whether the recombinant 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 recombinant bocavirus capsid protein to a specific primer or probe, or testing for binding of the recombinant bocavirus capsid protein to a specific binding member. Detection of the presence of the recombinant bocavirus capsid protein of the invention or the nucleic acid of the invention in the sample indicates that the sample is positive for the recombinant bocavirus capsid protein.

[0145] 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 recombinant bocavirus capsid protein polypeptide to a specific binding member may employ e.g. immunofluorescence (IF), immunochromatography, or an enzyme immunoassay (EIA, ELISA).

[0146] The cargo packaged within the recombinant 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.

[0147] 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 viral genome is produced that comprises the transgene. In one embodiment, the transgene is a polynucleotide encoding a therapeutically relevant protein.

[0148] The transgene is integrated by recombinant techniques into or in place of the genomic coding region (i.e., in place of the 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. 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. The ITRs may flank a transgene within the plasmid, and the transgene may be transferred from the plasmid to the virus during packaging, forming the virus genome.

[0149] 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 recombinant 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.

[0150] The recombinant bocavirus capsid protein of the invention is especially suitable for gene therapy, or treatment of a genetic disease. Because the recombinant bocavirus capsid protein has an improved packaging capacity in comparison to conventionally gene therapy vectors, such as AAV virus vectors, the recombinant 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 4.8 kilobases.

[0151] Hence, in one aspect, the invention relates to the recombinant 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.

[0152] In one further aspect, the invention relates to the recombinant 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.

[0153] 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. 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.

[0154] 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

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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

[0159] 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.

[0160] In one embodiment, the disease-causing gene has a length of at least 1 kilobases, at least

[0161] 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. 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.

[0162] 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.

[0163] 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. The gene of interest is a wild-type, i.e. not disease-associated, version of a defective gene or disease-causing gene.

[0164] 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.

[0165] In another embodiment, the invention relates to the use of a recombinant 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.

[0166] In another embodiment, the invention relates to the use of a recombinant 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.

[0167] 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. In one embodiment, the disease is a genetic disease.

[0168] In one embodiment, the invention relates to a method of treatment of a genetic disease, comprising administering the recombinant bocavirus capsid protein, the recombinant bocavirus or bocavirus-like particle, the isolated nucleic acid, the plasmid vector, the host cell, or the composition according to the invention to a patient in need thereof.

[0169] Further Embodiments

[0170] The invention is further described by the following embodiments:

[0171] 1. A recombinant bocavirus capsid protein, comprising at least one heterologous peptide insert of at least 4 amino acids in length within viral protein 3 (VP3).

[0172] 2. The recombinant bocavirus capsid protein of item 1, wherein the peptide insert is inserted at an insertion site located within a variable region (VR) of VP3.

[0173] 3. The recombinant bocavirus capsid protein of any one of items 1 or 2, wherein the insertion site is located within a VR selected from the group consisting of VR I, VR III, VR IV, VR V, VR VIII and VR IX of VP3.

[0174] 4. The recombinant bocavirus capsid protein of any one of items 1 to 3, wherein the insertion site is located immediately following an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 selected from the group consisting of amino acid S209 of VR I, amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, amino acid G408 of VR IV, amino acid T461 of VR V, amino acid T521 of VR VIII, and amino acid T636 in VR IX.

[0175] 5. The recombinant bocavirus capsid protein of any one of items 1 or 2, wherein the insertion site is located within a VR selected from the group consisting VR III, VR IV and VR V of VP3.

[0176] 6. The recombinant bocavirus capsid protein of item 5, wherein the insertion site is located immediately following an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 selected from the group consisting of amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, amino acid G408 of VR IV, and amino acid T461 of VR V.

[0177] 7. The recombinant bocavirus capsid protein of any one of items 1 or 2, wherein the insertion site is located within a VR selected from the group consisting VR III and VR IV of VP3.

[0178] 8. The recombinant bocavirus capsid protein of item 7, wherein the insertion site is located immediately following an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 selected from the group consisting of amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, and amino acid G408 of VR IV.

[0179] 9. The recombinant bocavirus capsid protein of any one of items 1 or 2, wherein the insertion site is located within VR III of VP3.

[0180] 10. The recombinant bocavirus capsid protein of item 9, wherein the insertion site is located immediately following an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 selected from the group consisting of amino acid D332 of VR III; amino acid G333 of VR III; or amino acid T334 of VR III.

[0181] Examples

[0182] Example 1: GBoV capsid engineering - 9-mers and 11-mers peptides insertion mutants

[0183] GBoVl peptide display strategy

[0184] GBoVl variants that display model peptides on the surface using different insertion sites have been generated and subjected to manufacturability tests. Viable variants were tested for in vitro transduction efficacy resulting in a feasibility ranking of the insertion sites. Reporter-encoding vectors were produced and their performance in two cell types, Huh7, and primary human liver cells, was tested.

[0185] Eight preferred insertion sites were selected based on these results: VR I: 209 / 210; VR III: 332 / 333, 333 / 334, 334 / 335; VR IV: 408 / 409, VR V: 461 / 462; VR VIII: 521 / 522; VR IX: 636 / 637. VR refers to the number of the variable region, followed by the number of the amino acids flanking the insertion site. The amino acid numbering follows the numbering of GBoVl VP3 (SEQ ID NO: 1). VP3 is also comprised in the transcripts of VP2 and VP1. All three capsid proteins are encoded by the same mRNA and are produced by differential splicing and translation from three different start codons. The full length VP1 has 671 amino acids with an 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 VP3 and a VP1 / VP2 shared domain of 39 amino acids.

[0186] Screening was with two peptides. Peptides were selected based on literature, with Pl comprising a RGD motif: CDCRGDCFC (SEQ ID NO: 2) and P2 comprising a NXXRXXX motif: NYSRGVD (SEQ ID NO: 4). RGD is a consensus sequence for integrin binding (Shi and Bartlett 2003, Mol Ther, 7(4):515-525). NXXRXXX peptides showed the broadest improvement in transduction and expression of AAV based on data evaluation of Borner and colleagues (Borner et al. 2020, Mol Ther 28(4):1016-1032).

[0187] Two flanking amino acids were added to each peptide to maintain flexibility and promote efficient display of the peptide epitope on the surface of the bocavirus capsid, one N- terminal Glycine (G) and one C-terminal Alanine (A).

[0188] Hence, the full inserts were Pl: GCDCRGDCFCA (SEQ I D NO: 3) and P2: GNYSRGVDA (SEQ ID NO: 5).

[0189] Figure 3 shows a schematic overview of the recombinant bocavirus capsid proteins tested.

[0190] Cloning strategy for peptide insertions

[0191] For cloning, a total of six VRs were selected for peptide insertion cloning, namely VR1, VR3, VR5, VR8, and VR9. A schematic overview of the cloning strategy is depicted in Figure 2. To synthesize master templates containing peptide insertions Pl and P2 in all VRs, we employed a synthetic approach. For insertion sites 332 / 333 and 334 / 335 in VR3, separate DNA templates were synthesized. The design of primers for each VR on the master template, along with corresponding primers for the backbone plasmid linearization, was performed according to the NEBuilder® strategy, with overlaps of 15-20 nucleotides (See Table 1). Subsequently, -200 bp fragments containing the peptide insertion and the linearized backbone plasmid with matching overlaps were excised from the gel after electrophoresis. The fragments were then purified utilizing the Promega Wizard® SV Gel and PGR Clean-Up System and assembled with NEBuilder® DNA Assembly master mix. VR8 insertion plasmids were constructed using a combination of NEBuilder® strategy and restriction enzyme cloning with Mscl and Acc65l. Finally, the resulting plasmids were transformed into competent bacteria and plated on selective media, followed by screening of colonies using sequencing to confirm the presence of the peptide insertion.

[0192] Table 1 : Primer Sequences used for cloning of peptide insertion constructs. In vitro cell assay in Huh7 cells GBoVl variants that display model peptides (9mer and llmer peptides) on the surface using each of the eight potential insertion sites were also generated using the 5-plasmid system described in Example 2. These mutants were produced in the same expression system described in Example 2 of HEK293 suspension cell line in shaker flasks. Initially, a small scale to prove manufacturability potential (formation of DNase resistant particles) was tested, followed by a production in a larger scale, sufficient to receive enough vector material for transduction potency testing. The evaluation was performed in a cell-based in vitro assay and assessed influence of different insertion sites on transduction in vitro.

[0193] 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).

[0194] 5 x 104cells 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 1E4 to 2E5 based on the formula vector prep [pL] = (cell number*MOI) / (vector genomes / pL)*replicates*1.2 and filled with growth medium to 150pL*replicates*1.2-vector prep [pL] . 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.

[0195] 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). Virus copy number (VCN) and mRNA levels were determined by qPCR for selected candidates (data not shown).

[0196] GBoVl variants (9mer and llmer insertion mutants) were successfully produced and tested for in vitro biopotency. Selected variants demonstrated biopotency in the Huh7 cell line (Figure 6).

[0197] In vitro cell assay in pHAE cells

[0198] GBoVl variant with 9mer insertion at VR3.2 was also teste in an in vitro transduction assay of primary human airway epithelial (pHAE) cells. Cells were transfected at an MOI (multiplicity of infection) of 5 x 104. The percentage of GFP-positive cells was determined by flow cytometry 168 hours post-transduction. The results are depicted in Figure 7. The VR3.2 peptide display variant demonstrated biopotency in the pHAE cell line.

[0199] Exemplary embodiments of recombinant bocavirus capsid proteins of the invention are provided in Table 2 and the Sequence Listing provided with the application. Table 2: SEQ ID NOs of recombinant bocavirus capsid proteins used in the Examples

[0200] Example 2 - 3D cell culture-based Bocavirus production platform

[0201] The aim was to establish an expression platform architecture for the efficient production of recombinant bocavirus capsids. An Adeno-associated virus 2 (AAV2) ITR-based genomic payload is used as an exemplary transgene. Generation of these vectors takes place by introduction of specially designed plasmid DNA by transient transfection into HEK293 cells cultivated in serum-free suspension system (3D).

[0202] Capsid serotypes of Gorilla (GBoV) origin have been selected for the design of prototype vectors. The initial expression system layout has been adapted from Yan et al. 2019 (Yan et al. 2019 Human Gene Therapy 30(5): 556-570) by splitting of the capsid viral protein reading frames for VP1 / VP2 and VP2 / VP3 into separate plasmids to balance VP1:VP2 / VP3 expression in NPl-independent expression systems.

[0203] Gorilla Bocavirus serotype 1 VP1, VP2 and VP3 open reading frame DNA sequences were synthesized using a codon-adaptive (CAI) index as close to 100% of preferential Homo sapiens codon-usage as possible. Cryptic reading frames and splice-sites were removed. Finally, these reading frames were provided as split expression systems with ORFs for VP1 / VP2 (VP1A3) and VP2 / VP3 separated (see Figure 1). Sequences were provided as expression cassettes using the CMV promoter / enhancer combination in conjunction with a 5’ human erythropoietin (EPO) intron to enhance expression analogous to Yan et al. 2019, Human Gene Therapy 30(5): 556-570. VP3 expression was ablated from the VP1 expression cassette by introduction of a M130L mutation, which eliminated the VP3 start codon, and expression of VP1 was enhanced by introducing a Kozak consensus ribosome-binding site (RBS) in front of the VP1 start codon. VP2 expression was modulated in the VP2 / VP3 expression cassette by introducing a wild-type AAV2 RBS and upstream sequence of the VP2 start codon. All VP expression cassettes were either combined with a plasmid DNA (pDNA) backbone containing Bovine growth hormone (BGH, pVAX systems) or Simian virus 40 (SV40, pTwist systems) poly-adenylation (polyA) signal sequences downstream of the VP reading frame.

[0204] All plasmid systems were tested in 3D culture transfection in HEK293 cells for functionality and obtainable DNAse-resistant viral particle (DRP) yields. Testing was carried out by cotransfection of each 2-plasmid system for VP1 and VP2 / VP3 expression (and combinations thereof) with an Adenovirus 5 helper plasmid (pALD-X80), an AAV2 Rep containing helper plasmid and an enhanced green fluorescent (EGFP) protein-coding AAV2 proviral genomic payload plasmid, resulting in a 5-plasmid co-transfection method (see Figure 4A). Two protocols were implemented for testing, Protocol 1 consisted preparation of the material from both cell media and cell pellet, while Protocol 2 only from cell pellet. Functionality of expression systems, formation of viral vectors and expression system performance (vector yields) were determined by quantification of viral genomes after DNAse digest of free genomic DNA (DRP quantification) and expressed as titers of viral genomes (VGs) per ml of sample.

[0205] Vector purification was performed by iodixanol density gradient centrifugation using methods established for AAV purification (Strobel et al. 2015, Human Gene Therapy Methods 26(2)). Quality Control of purified viruses with recombinant capsid proteins and wild-type controls comprised qPCR titer (downstream titer) and silver staining SDS-PAGE for VP1 / VP2 / VP3 to determine expression levels and ratios of the proteins (see Figure 4B). The SDS-PAGE showed that all recombinant variants and wild-types gave rise to comparable products. Based on the results above, 3D cell culture production runs were performed in Biostat bioreactors (1.5L scale) to evaluate the performance of the GBoV 5-plasmid system in medium scale production and to produce sufficient vector material for in vitro (EGFP payload) Obtained vector yields (i.e. upstream titers) were in the range of 3.36 x 1012to 7.6 x 1012VG / 1.5L (VG = vector genomes) culture as determined by ddPCR (droplet digital PGR). In addition to the GBoVl capsid variants, GBoVl wild-type and AAV6 wild-type were used as controls.

Claims

Claims1. A recombinant bocavirus capsid protein, comprising at least one heterologous peptide insert of at least 4 amino acids in length, wherein the peptide insert is inserted at an insertion site located within a variable region (VR) of viral protein 3 (VP3).

2. The recombinant bocavirus capsid protein of claim 1, wherein the insertion site is located within a VR selected from the group consisting of VR I, VR III, VR IV, VR V, VR VIII and VR IX of VP3.

3. The recombinant bocavirus capsid protein of any one of claims 1 or 2, wherein the insertion site is located immediately following an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) viral protein 1 (VP1) as defined in SEQ ID NO: 1 selected from the group consisting of amino acid S209 of VR I, amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, amino acid G408 of VR IV, amino acid T461 of VR V, amino acid T521 of VR VIII, and amino acid T636 in VR IX.

4. The recombinant bocavirus capsid protein of claim 1, wherein the insertion site is located within a VR selected from the group consistingVR III, VR IV and VR V of VP3, preferably wherein the insertion site is locatedimmediately following an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 selected from the group consisting of amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, amino acid G408 of VR IV, and amino acid T461 of VR V.

5. The recombinant bocavirus capsid protein of claim 1, wherein the insertion site is located within a VR selected from the group consisting VR III and VR IV of VP3, preferably wherein the insertion site is located immediately following an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 selected from the group consisting of amino acid D332 of VR III, amino acid G333 of VR III, amino acid T334 of VR III, and amino acid G408 of VR IV.

6. The recombinant bocavirus capsid protein of claim 1, wherein the insertion site is located within VR III of VP3.

7. The recombinant bocavirus capsid protein of claim 6, wherein the insertion site is located immediately following an amino acid residue corresponding to an amino acid residue according to the numbering of gorilla bocavirus 1 (GBoVl) VP1 as defined in SEQ ID NO: 1 selected from the group consisting of amino acid D332 of VR III; amino acid G333 of VR III; or amino acid T334 of VR III.

8. The recombinant bocavirus capsid protein of any one of the previous claims, wherein the recombinant bocavirus capsid protein is from a primate bocavirus, preferably human or gorilla bocavirus.

9. The recombinant bocavirus capsid protein of any one of the previous claims, wherein the peptide insert is between 4 and 20 amino acids in size.

10. A recombinant bocavirus or bocavirus-like particle comprising the recombinant bocavirus capsid protein of any one of the previous claims.

11. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the recombinant bocavirus capsid protein as defined in any one of claims 1 to 9.

12. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the recombinant bocavirus or bocavirus-like particle as defined in claim 10.

13. A plasmid vector comprising the isolated nucleic acid molecule as defined in any one of claims 11 or 12.

14. A host cell comprising the recombinant bocavirus capsid protein of any one of claims 1 to 9, the recombinant bocavirus or bocavirus-like particle of claim 10, the isolated nucleic acid of claims 11 or 12, or the plasmid vector of claim 13.

15. A composition comprising the recombinant bocavirus capsid protein of any one of claims 1 to 9, the recombinant bocavirus or bocavirus-like particle of claim 10, the isolated nucleic acid of claims 11 or 12, or the plasmid vector of claim 13, or the host cell of claim 14, and optionally one or more excipients.

16. The recombinant bocavirus capsid protein of any one of claims 1 to 9, the recombinant bocavirus or bocavirus-like particle of claim 10, the isolated nucleic acid of claims 11 or 12, or the plasmid vector of claim 13, or the host cell of claim 14 orthe composition of claim 15 for use as a medicament.

17. The recombinant bocavirus capsid protein of any one of claims 1 to 9, the recombinant bocavirus or bocavirus-like particle of claim 10, the isolated nucleic acid of claims 11 or 12, or the plasmid vector of claim 13, or the host cell of claim 14 or the composition of claim 15 for use in gene therapy.

18. The recombinant bocavirus capsid protein, the recombinant bocavirus or bocavirus- like particle, the isolated nucleic acid, the plasmid vector, the host cell, or the composition for use as defined in 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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