Compositions and Methods for the Generation of Recombinant Parvoviruses

Abstract

Expression constructs and methods for generating recombinant parvoviruses are disclosed. In some embodiments, the expression construct encodes (1) the E4 protein and the E2a protein of adenovirus, (2) parvovirus proteins necessary for the production of recombinant parvoviruses, and (3) the recombinant parvovirus genome, thus enabling the production of recombinant parvoviruses by transfecting a single expression construct into a host cell.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Application No. 63 / 341,201, filed May 12, 2022, which is incorporated herein by reference.

[0002] The present invention generally relates to compositions and methods for generating recombinant viruses. More particularly, the present invention relates to compositions for the generation of recombinant parvoviruses such as adeno-associated virus (AAV).

Background Art

[0003] Recombinant viruses such as recombinant adeno-associated virus (rAAV) have been developed as vectors by replacing all viral genes with a therapeutic transgene expression cassette while retaining the inverted terminal repeats (ITRs), which are the only cis-elements required for vector packaging and DNA replication. Early methods for rAAV production relied on plasmid systems that included 1) an AAV helper plasmid (generally containing the AAV Rep coding region and the AAV Cap coding region but lacking the AAV ITRs and thus unable to replicate or package itself), and 2) an ITR-containing plasmid (generally containing a selected transgene of interest flanked by AAV ITRs that provide viral replication and packaging functions). Both the helper plasmid and the ITR-containing plasmid with the selected gene can be introduced into cells suitable for production by transient transfection. The transfected cells can then be infected with a helper virus such as adenovirus or herpes simplex virus, which transactivates the AAV promoter present on the helper plasmid that directs transcription and translation of the AAV Rep and Cap regions. For Ad helper virus, the E1a, E1b, E2a, E4, and VA RNA genes can supply the helper functions required for rAAV production.

[0004] Infection of producer cells with helper virus to generate rAAV was effective for rAAV production. However, as a result, it was also possible to generate Ad helper virus particles that could induce an immune response from the host. In certain platforms, the viral helper genes required for AAV production can be stably transfected into a production cell line (e.g., HEK293 cells), thereby reducing the possibility of an anti-helper virus immune response by the host immune system due to trace levels of residual helper virus.

[0005] More recently, a triple plasmid transfection method has been developed. This method uses AAV serotype-specific Rep and Cap plasmids as well as a transgene-containing plasmid, but eliminates the use of helper virus infection (i.e., removes or reduces the viral coding sequences) by supplying the helper virus genes essential for the third plasmid, and thus reduces the potential anti-helper virus immune response by the host immune system. By supplying the viral helper genes on the third plasmid, helper virus production in transfected cells was significantly reduced, providing only rAAV. Multiphasmid transient transfection of adherent HEK293 cells is a commonly used method for rAAV production.

Summary of the Invention

Problems to be Solved by the Invention

[0006] In a multiphasmid system, it is important to maintain appropriate plasmid size and plasmid ratio to maximize transfection efficiency and AAV production. There is a need in the art for a simplified and efficient plasmid-based system for generating rAAV that provides improved transfection and reduced immunogenicity while maintaining optimal expression of the transgene. The present invention is based on the discovery of an administrable adenovirus region in conventional helper plasmids to provide such advantages.

Means for Solving the Problems

[0007] One aspect of the present invention relates to an expression construct for the production of recombinant parvovirus. The expression construct comprises: (1) a modified or unmodified adenovirus E4 coding region encoding E4 open reading frame 6 / 7; (2) a modified or unmodified adenovirus E2a coding region encoding the E2a protein; (3) an adenovirus VA RNA coding region encoding adenovirus VAI and VAII RNAs; (4) a parvovirus protein coding region encoding parvovirus proteins necessary for the production of recombinant parvovirus; (5) (a) an expression cassette comprising a nucleotide sequence encoding a transgene and regulatory elements operably linked to the nucleotide sequence; and (b) at least one ITR at one end of the expression cassette, the recombinant parvovirus sequence; and (6) one or more regulatory elements enabling the expression of E4 open reading frame 6 / 7, E2a protein, adenovirus VA RNA, and parvovirus proteins necessary for the production of recombinant parvovirus in a host cell.

[0008] Another aspect of the present invention relates to a polynucleotide expression construct for the production of recombinant adeno-associated virus (AAV). The expression construct comprises: (a) a modified adenovirus E4 coding region encoding adenovirus E4orf6 / 7, the modified adenovirus E4 coding region comprising (i) a partial or complete deletion of E4orf6 / 7 intron 1, or (ii) a partial or complete deletion of E4orf6 / 7 intron 2, or both (i) and (ii); (b) a modified adenovirus E2a coding region encoding the adenovirus E2a protein, the modified adenovirus E2a coding region comprising at least one deletion in E4orf6 / 7 intron 1 or E4orf6 / 7 intron 2; (c) a sequence encoding one or more adenovirus VA RNAs; and (d) one or more regulatory elements operably linked to (a), (b), and (c).

[0009] Another aspect of the invention relates to a method for generating recombinant parvovirus particles using the expression constructs of the present application. The method includes introducing the expression construct of the present application into a host cell, incubating the host cell carrying the expression construct for a desired period to generate recombinant parvovirus particles, and recovering the recombinant parvovirus particles after the incubation period. BRIEF DESCRIPTION OF THE DRAWINGS

[0010]

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DETAILED DESCRIPTION OF THE INVENTION

[0011] I. DEFINITIONS As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0012] A range can be expressed in the specification as from “about” one particular value and / or to “about” another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by use of the antecedent “about”, it is understood that the particular value forms another embodiment. It is further understood that each endpoint of a range is significant both in relation to and independent of the other endpoint. Also, there are several values disclosed in the specification, and it is understood that for each value, in addition to the value itself, its particular value as “about” is also disclosed herein. For example, if the value “10” is disclosed, “about 10” is also disclosed. Also, as would be appropriately understood by one of ordinary skill in the art, when a value is disclosed, the value “less than”, the value “greater than”, and the possible ranges between the values are also understood to be disclosed. For example, if the value “10” is disclosed, “less than 10” and “greater than 10” are also disclosed.

[0013] As used herein, the term “polynucleotide” refers to a polydeoxyribonucleotide that may include unmodified or modified DNA and may include both single-stranded and double-stranded DNA.

[0014] As used herein, the term "heterologous" means derived from an entity that is genetically different from the remainder of the entity to which it is being compared, or into which it is being introduced or incorporated. For example, a polynucleotide introduced into different cell types by genetic engineering techniques is a heterologous polynucleotide (which, when expressed, can encode a heterologous polypeptide). Similarly, a cellular sequence (e.g., a gene or a portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence with respect to the vector. As an example, when used in the context of nucleic acid sequences such as coding sequences and regulatory sequences, "heterologous" can refer to sequences that are not normally linked together and / or do not normally associate with each other under ordinary circumstances found in nature. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence having codons different from a natural gene). Similarly, a cell transformed with a construct that is not normally present in the cell is considered heterologous for the purposes of this application. Allelic variations or naturally occurring mutagenic events do not give rise to heterologous DNA as used herein.

[0015] As used herein, "coding sequence" refers to a nucleic acid sequence that "encodes" a specific protein or functional nucleotide such as siRNA. The nucleic acid sequence in a polynucleotide is transcribed (in the case of DNA) and translated into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences (in the case of mRNA). The boundaries of the coding sequence are determined by the start codon at the 5' (amino) terminus and the translation termination codon at the 3' (carboxy) terminus. The coding sequence can include, but is not limited to, cDNA derived from prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. The transcription termination sequence is usually located 3' of the coding sequence.

[0016] As used herein, the term "coding region" refers to the region within genomic DNA where the coding sequence of a gene is located. The coding region of a gene may include both the exon and intron sequences of the gene, as well as the regulatory sequences of the gene (e.g., promoters and enhancers). For example, the adenovirus E4 coding region may include nucleotides 32645 - 35835 of the Ad2 genome, and the adenovirus E2a coding region may include nucleotides 22233 - 27575 of the Ad2 genome.

[0017] As used herein, the term "modified coding region" refers to a coding region that has been modified by deletion, substitution, and / or insertion of one or more nucleotides. For example, the modified adenovirus E4 coding region may include nucleotides 32645 - 35835 of the Ad2 genome and have a deletion of one or more nucleotides in intron 1 and / or intron 2 of E4orf6 / 7, and the modified adenovirus E2a coding region may include nucleotides 22233 - 27575 of the Ad2 genome and have a deletion of one or more nucleotides in late E2a intron 1 and / or late E2a intron 2.

[0018] The term "adenovirus E4 protein" refers to a protein that (1) is produced by the adenovirus E4 region and (2) is required for AAV production in host cells. The adenovirus E4 protein may be encoded by different open reading frames in different adenoviruses. As used herein, the term "adenovirus E4 protein" includes functional homologs and functional equivalents of the adenovirus E4 protein. Examples of adenovirus E4 proteins include proteins encoded by the E4 open reading frame 6 / 7 (E4orf6 / 7) of Ad2 / 5 and its functional equivalents.

[0019] The term "adenovirus E2a protein" refers to a protein encoded by the E2a region of the adenovirus genome. The adenovirus E2a protein is a DNA-binding protein that plays a role in the elongation phase of viral strand displacement replication by unwinding the template in an ATP-independent manner. As used herein, the term "adenovirus E2a protein" includes functional homologs and functional equivalents of the adenovirus E2a protein.

[0020] As used herein, the term "adenovirus VA RNA" refers to a type of non-coding RNA found in adenoviruses that plays a role in regulating translation. Examples of VA RNA include VAI and VAII. As used herein, the term "adenovirus VA RNA" includes functional homologs and functional equivalents of the adenovirus VA RNA.

[0021] The term "adenovirus VA RNA coding region" refers to the region that encodes adenovirus VA RNA. An exemplary VA coding region includes nucleotide sequences 10426-11156 (SEQ ID NO: 16) of the Ad2 genome.

[0022] The terms "regulatory element", "regulatory sequence", and "regulatory region" are used with reference to promoters, enhancers, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), etc., which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these regulatory sequences need to be present at all times, so long as the selected coding sequence can be replicated, transcribed, and translated in an appropriate host cell.

[0023] The terms "promoter" and "promoter region" are used herein to refer to a DNA regulatory sequence to which RNA polymerase binds and initiates transcription of a downstream (3' direction) coding sequence. Further, these terms should be broadly construed to additionally encompass other regulatory elements, including enhancer regions, intron splice donors and acceptors, other 5' untranslated regions, and the like. The promoter sequence may be homologous or heterologous to the desired gene sequence. A wide variety of promoters are known, including a wide range of viral and mammalian promoters, and are available in the art for use in the present invention. Cell type-selective or tissue-specific promoters can be utilized to target or enhance the expression of a gene sequence in a particular cell population as compared to others. Suitable mammalian and viral promoters. The promoter may be constitutively active, conditionally active, or inducible, depending on the cell type.

[0024] The terms "enhancer" and "enhancer region" are used herein to refer to a polynucleotide sequence that acts at a cis position on the activity of a promoter and thus stimulates the transcription of a gene or coding sequence operably linked to this promoter. Unlike a promoter, the effect of an enhancer is independent of position and orientation, and thus an enhancer can be placed either upstream or downstream of a transcription unit, within an intron, or within a coding region. An enhancer may be located both in close proximity to a transcription unit and at a considerable distance from the promoter. It is also possible to have physical and functional overlap with a promoter. Those skilled in the art will recognize various sources (and several enhancers available as independent elements or elements cloned within a polynucleotide sequence, e.g., from the ATCC or deposited from commercial and individual sources) such as the SV40 enhancer, CMV enhancer, polyoma enhancer, adenovirus enhancer, etc., deposited in a database such as GenBank. Some promoter sequences also contain enhancer sequences such as the frequently used CMV promoter. An example of an inducible enhancer is the metallothionein enhancer that can be stimulated by glucocorticoids or heavy metals.

[0025] "Operably linked" refers to the arrangement of elements so described that the components so described are configured to perform their normal functions. Thus, a control sequence operably linked to a coding sequence can affect the expression of the coding sequence. A chimeric gene is obtained by operably linking a heterologous sequence to a promoter. The control sequences need not be contiguous with the coding sequence so long as they function to direct its expression. Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0026] For purposes of describing the relative positions of nucleotide sequences in a particular nucleic acid molecule throughout the present invention, such as when a particular nucleotide sequence is described as being "upstream," "downstream," "3'," or "5'" relative to another sequence, these modifiers should be interpreted as the relevant sequence portions in the "sense" or "coding" strand of a DNA molecule, as is conventional in the art.

[0027] The term "transgene" refers to a polynucleotide that is introduced into a cell, transcribed into RNA, and optionally, can be translated and / or expressed under appropriate conditions. A transgene confers a desired property to the cell into which it is introduced or otherwise results in a desired therapeutic or diagnostic outcome. In another aspect, it may be transcribed into a molecule that mediates RNA interference, such as miRNA, siRNA, shRNA, or guide RNA for CRISPR / Cas9-mediated targeting of mutant alleles.

[0028] The term "expression" encompasses the process by which a nucleic acid (e.g., DNA) is transcribed to produce RNA, such as mRNA, which is processed and translated into a polypeptide.

[0029] The term "gene product" (also referred to herein as "gene expression product" or "expression product") encompasses products resulting from the expression of a gene, such as mRNA transcribed from the gene and polypeptides resulting from the translation of the mRNA. It will be understood that a particular gene product may be subject to processing or modification, for example, within a cell. For example, an RNA transcript may be spliced, polyadenylated, and / or a polypeptide may be subject to co-translational or post-translational processing such as removal of a secretion signal sequence, removal of an organelle targeting sequence, or modification such as phosphorylation, fatty acylation. The term "gene product" encompasses such processed or modified forms. Genomic nucleotide sequences, cDNA sequences, mRNA sequences, and polypeptide sequences from various species including humans are known in the art and are available in publicly accessible databases such as those available from the National Center for Biotechnology Information (www.ncbi.nih.gov) or the Universal Protein Resource (www.uniprot.org). Examples of databases include, for example, GenBank, RefSeq, Gene, UniProtKB / SwissProt, UniProtKB / Trembl, and the like. Generally, sequences within the NCBI reference sequence database, such as mRNA and polypeptide sequences, may be used as the gene product sequences of the gene of interest.

[0030] As used herein, the term "modified protein" refers to a protein containing one or more modifications from a wild-type protein. Examples of modifications include, but are not limited to, substitution, deletion, and insertion of one or more amino acid residues, and post-transcriptional modifications such as glycosylation.

[0031] As used herein, the terms "transfection," "gene transfer," and "gene delivery" are used interchangeably herein to refer to methods or systems for inserting foreign nucleic acid into a host cell. Gene transfer can result in transient expression of non-integrated transferred DNA, extrachromosomal replication, and expression of the transferred replicon (e.g., episome), or integration of the transferred genetic material into the genomic DNA of the host cell. A variety of techniques for introducing one or more exogenous nucleic acid molecules into a suitable host cell are known to those of skill in the art, including chemical, electrical, and virus-mediated transfection procedures.

[0032] As used herein, the term "host cell" generally refers to a cell that functions as a recipient of exogenously introduced nucleic acid or a cell that has been transfected with exogenous nucleic acid (e.g., bacterial cell, yeast cell, insect cell, mammalian cell). It should be understood that the progeny of a single parental cell may not necessarily be exactly identical to the original parent in morphology, or in genomic or total DNA complement, due to natural, accidental, or intentional mutations. A host cell may be a cell that has been engineered to express a desired gene product (e.g., stably transformed to express one or more exogenous gene products). Such cells (E1-expressing cells) that stably express adenoviral E1a and E1b proteins.

[0033] As used herein, the term "cell line" refers to a population of cells capable of continuous or long-term growth and division in vitro. In many cases, a cell line is a clonal population derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes in karyotype can occur during the storage or transfer of such clonal populations. Thus, cells derived from the referenced cell line may not be exactly identical to the ancestral cells or culture, and the referenced cell line includes such variants.

[0034] A nucleotide or amino acid residue in a first nucleic acid or protein is "corresponding" to a residue in a second nucleic acid or protein if the two residues perform one or more corresponding functions and / or are located at corresponding positions in the first and second nucleic acids or proteins. Corresponding functions are typically the same, equivalent, or substantially equivalent functions, taking into account differences in the environment of the two nucleic acids or proteins as necessary. Residues at corresponding positions typically align with each other when the sequences of the two nucleic acids or proteins are aligned to maximize identity (allowing introduction of gaps) using a sequence alignment algorithm or a computer program such as those referred to below ("identity"), and / or when the three-dimensional structures of the proteins are superimposed and the residues overlap or occupy structurally equivalent positions, and / or when they are located in positions that form the same, equivalent, or substantially equivalent intra- and / or intermolecular contacts or bonds (e.g., hydrogen bonds). The structure may be determined experimentally, for example, by X-ray crystallography or NMR, or may be predicted, for example, using structure prediction or molecular modeling software. The alignment may be over the entire length of one or more of the aligned nucleic acid or polypeptide sequences, or over at least one protein domain (or nucleotide sequence encoding a protein domain).

[0035] The term "expression cassette" is used herein to refer to a polynucleotide containing a gene of interest in operable linkage to regulatory elements (also referred to as "regulatory sequences", "expression control elements", or "expression control sequences") that mediate the expression of the coding region in an mRNA transcript that can be translated into a protein. The expression cassette may be contained or incorporated in a plasmid, viral vector, or may exist as a nucleic acid fragment.

[0036] An "expression vector" is a specialized vector containing a gene or nucleic acid sequence having regulatory regions necessary for expression in a host cell. Examples of expression vectors include plasmids and viral vectors such as adeno-associated virus (AAV) vectors, adenovirus vectors, lentivirus vectors, and herpesvirus vectors.

[0037] As used herein, the term "viral vector" refers to a recombinant polynucleotide vector containing nucleic acid derived from a virus, which facilitates the replication and expression of an exogenously incorporated transgene sequence operably linked to suitable control elements, and one or more heterologous sequences (i.e., nucleic acid sequences not of viral origin).

[0038] The term "recombinant virus" is used herein to refer to a virus that has been genetically modified, for example, by adding or inserting a heterologous nucleic acid construct into a viral particle.

[0039] The term "parvovirus" refers to a family of animal viruses that make up the Parvoviridae. They typically have a linear single-stranded DNA (ssDNA) genome containing two genes encoding a replication initiation protein called NS1 and the proteins that form the viral capsid. The coding portion of the genome has inverted terminal repeats (ITRs) adjacent to each end that form important hairpin loops during replication. Parvovirus virions are 23 - 28 nanometers in diameter, contain a genome enclosed in an icosahedral capsid that is small and has a robust surface compared to most viruses. The parvovirus family includes three subfamilies, including adeno-associated virus (AAV), bocavirus, and protoparvovirus, and 126 species.

[0040] The term "parvovirus protein coding region" refers to the region of the parvovirus genome that encodes non-structural proteins necessary for replicating the viral genome, such as NS1, and structural proteins for the viral capsid, as recognized in the art. This region need not include all of the wild-type gene, but as long as the non-structural and structural proteins present provide at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the level of support provided by the parvovirus protein coding region encoding the corresponding wild-type non-structural and structural proteins, and provide sufficient function to enable the production of a relevant recombinant parvovirus in a suitable host cell, it may be modified, for example, by nucleotide insertion, deletion, or substitution.

[0041] The term "sequence encoding a recombinant parvovirus genome" refers to a nucleotide sequence encoding a sequence that can generate a functional recombinant parvovirus genome in a suitable host cell. The sequence encoding a recombinant parvovirus genome typically includes (1) an expression cassette comprising a sequence encoding a gene of interest (GOI), also referred to as a "transgene", and regulatory sequences operably linked to the sequence encoding the GOI, and (2) an ITR adjacent to at least one end of the expression cassette. In some embodiments, the sequence encoding a recombinant parvovirus genome includes ITRs at each end of the expression cassette.

[0042] The term "sequence encoding a recombinant AAV genome" refers to a nucleotide sequence encoding a sequence that can generate a functional recombinant AAV genome in a suitable host cell. The sequence encoding a recombinant AAV genome typically includes (1) an expression cassette comprising a sequence encoding a GOI, also referred to as a "transgene", and regulatory sequences operably linked to the sequence encoding the GOI, and (2) an ITR adjacent to at least one end of the expression cassette. In some embodiments, the sequence encoding a recombinant AAV genome includes ITRs at each end of the expression cassette.

[0043] As used herein, the term "adeno-associated virus" (AAV) in the context of the present invention includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, caprine AAV, porcine AAV, ovine AAV, and other AAV serotypes and variants currently known or later discovered.

[0044] The terms "AAV cis construct" and "AAV vector" are used interchangeably herein to refer to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences not derived from AAV) adjacent to at least one AAV ITR. Such AAV vectors are present within a host cell infected with a suitable helper virus (or expressing a suitable helper function) and, when expressing the AAV Rep gene product and the AAV Cap gene product (i.e., the AAV Rep protein and the AAV Cap protein), can be replicated and packaged into infectious virus particles. When an AAV vector is incorporated into a larger polynucleotide (e.g., another vector such as a chromosome or a plasmid used for cloning or transfection), the AAV vector can be referred to as a "provirus" that can be "rescued" by replication and encapsidation in the presence of the AAV packaging function and a suitable helper function. An AAV vector can be in any of several forms, including, but not limited to, a plasmid, complexed with a lipid, encapsulated within a liposome, or encapsulated within a virus particle, e.g., an AAV particle. It is possible to package an AAV vector into an AAV viral capsid to generate "recombinant adeno-associated virus particles (AAV particles)".

[0045] The "AAV cis construct" or "AAV vector" can be derived from any adeno-associated virus serotype, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, and AAV-11, as well as others described herein. The AAV vector can have one or more AAV wild-type genes that are wholly or partially deleted, preferably the rep gene and / or the Cap gene, while retaining the functional adjacent ITR sequences. The functional ITR sequences are necessary for the rescue, replication, and packaging of the AAV virion. Thus, an AAV cis polynucleotide or AAV vector is defined herein as including at least those sequences that are cis-required for viral replication and packaging (e.g., functional ITRs). The ITRs need not be wild-type nucleotide sequences and can be modified, for example, by nucleotide insertion, deletion, or substitution, as long as the sequence provides functional rescue, replication, and packaging.

[0046] The term "AAV vector" also includes self-complementary adeno-associated virus (scAAV) vectors. An scAAV vector is a viral vector engineered from naturally occurring AAV. scAAV is termed "self-complementary" because the coding region is designed to form an intramolecular double-stranded DNA template. The rate-limiting step during the replication of the standard AAV genome involves second-strand synthesis because the typical AAV genome is a single-stranded DNA template. However, this does not apply to the scAAV genome. Upon infection, instead of waiting for cell-mediated synthesis of the second strand, the two complementary halves of scAAV associate to form one double-stranded DNA (dsDNA) unit that is immediately ready for replication and transcription.

[0047] The terms "recombinant AAV virion", "rAAV virion", and "rAAV viral particle" are used synonymously herein to refer to an infectious replication-defective viral particle composed of a viral particle composed of at least one AAV capsid protein and a capsidated rAAV vector genome containing heterologous nucleotide sequences of interest flanking both sides of the AAV ITRs. The rAAV virion is produced in a suitable host cell containing the AAV vector, AAV helper functions, and accessory functions. A host cell containing these components can encode the AAV polypeptides necessary to package the AAV vector (containing the recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.

[0048] The term "adeno-associated virus inverted terminal repeat" or "AAV ITR" refers to a region recognized in the art found at each end of the AAV genome, which functions cis as an origin of DNA replication and as a packaging signal for the viral genome. The AAV ITR, together with the AAV rep coding region, provides for efficient excision and rescue of the nucleotide sequence located between two adjacent ITRs into the mammalian cell genome, as well as integration of the nucleotide sequence. The nucleotide sequence of the AAV ITR region is well known in the art. As used herein, "AAV ITR" need not have the wild-type nucleotide sequence shown in the previously cited references, but may be modified, for example, by nucleotide insertion, deletion, or substitution. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, etc. Further, the 5' and 3' ITRs flanking a selected nucleotide sequence within an AAV vector need not be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., enable excision and rescue of the sequence of interest from the host cell genome or vector and enable integration of the heterologous sequence into the recipient cell genome when the AAV Rep gene product is present intracellularly.

[0049] The term "Rep coding region" refers to the region recognized in the art of parvoviruses that encodes the parvovirus Rep protein. This term also includes functional homologs thereof, such as the human herpesvirus 6 (HHV-6) rep gene, which is also known to mediate AAV DNA replication, and the rep genes in other parvoviruses such as bocavirus and protoparvovirus.

[0050] The term "AAV Rep coding region" refers to the region of the AAV genome that encodes the viral replication proteins necessary to replicate the viral genome and insert the viral genome into the host genome during latent infection. As used herein, the Rep coding region may be derived from any viral serotype, such as the above-described AAV serotypes.

[0051] This region need not contain all wild-type genes, but may be modified, for example, by nucleotide insertion, deletion, or substitution, as long as the modified rep gene, when expressed in a suitable host cell, provides sufficient integration function. A modified Rep gene is considered to encode a "functional variant" of the wild-type Rep protein if it supports rAAV production at a level of at least 25%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90%, or at least 95% of the level supported by the wild-type rep gene product.

[0052] The term "long form of Rep" refers to the Rep 78 gene product and the Rep 68 gene product of the AAV Rep coding region, including their functional homologs. The long form of Rep is usually expressed under the direction of the AAV p5 promoter.

[0053] The phrase "short form of Rep" refers to the Rep 52 gene product and the Rep 40 gene product of the AAV Rep coding region, including their functional homologs. The short form of Rep is expressed under the direction of the AAV p19 promoter.

[0054] The term "Cap coding region" refers to the region of the parvovirus genome that encodes the viral coat proteins necessary to package the viral genome, as recognized in the art.

[0055] The term "AAV Cap coding region" refers to the region of the AAV genome recognized in the art that encodes the viral coat proteins (e.g., VP1, VP2, and VP3) necessary for packaging the viral genome. As used herein, the AAV Cap coding region can be derived from any AAV serotype, as described above. This region need not contain all of the wild-type Cap genes, but may be modified, for example, by nucleotide insertion, deletion, or substitution, so long as the modified Cap gene provides sufficient packaging function when present in a host cell together with the AAV vector. A modified Cap gene is considered to encode a "functional variant" of the wild-type Cap protein if it supports rAAV production at a level of at least 25%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90%, or at least 95% of the level supported by the wild-type Cap gene product.

[0056] The term "AAV helper function" refers to an AAV-derived coding sequence that can be expressed to provide an AAV gene product that functions in trans for productive AAV replication. Thus, the AAV helper function includes the Rep region and the Cap region. The Rep expression product has been shown to have a number of functions including, inter alia, recognition, binding, and nicking of the AAV origin of DNA replication, DNA helicase activity, and regulation of transcription from AAV (or other heterologous) promoters. The Cap expression product provides the necessary packaging function. The AAV helper function is used herein to complement AAV functions in trans that are lacking in the AAV vector.

[0057] The term "AAV transfer construct" or "AAV helper construct" refers to a nucleic acid molecule that contains a nucleotide sequence that provides AAV functions deleted from an AAV vector used to generate a transduction vector for delivering a nucleotide sequence of interest. AAV transfer constructs are generally used to provide transient expression of the AAV Rep and / or AAV Cap genes to complement the defective AAV functions required for lytic AAV replication, but the transfer constructs lack AAV ITRs and cannot replicate or package themselves. AAV transfer constructs can be in the form of plasmids, minicircles, phages, transposons, cosmids, viruses, or virions. A number of AAV transfer constructs, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode both Rep and Cap expression products, have been described. See, for example, Samulski et al. (1989) J. Virology 63, 3822-3828; McCarty et al. (1991) J. Virology 65, 2936-2945. A number of other vectors encoding Rep expression products and / or Cap expression products have also been described. See, for example, U.S. Patent No. 5,139,941.

[0058] As used herein, the term "adenovirus" has the same meaning as an adenovirus vector and refers to a member of the family Adenoviridae. The family Adenoviridae includes all animal adenoviruses of the genus Mastadenovirus. Specifically, human adenoviruses include Subgenera A-F and their individual serotypes. Subgenera A-F include human adenoviruses 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Ad11A and Ad11P), 12, 13, 14, 15, 16, 17, 18, 19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 51.

[0059] The term "helper function" refers to non-AAV-derived viral and / or cellular functions on which AAV depends for its replication. Thus, this term encompasses the DNA, RNA, and proteins necessary for AAV replication, including portions involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products, and AAV capsid assembly. Viral-based helper functions can be derived from any of a number of known helper viruses such as adenovirus, herpesviruses (other than herpes simplex virus type 1), and vaccinia virus.

[0060] Helper functions derived from adenovirus have been widely studied, and numerous adenovirus genes involved in helper functions have been identified and partially characterized. Specifically, the early adenovirus E1A, E1B 55K, E2A, E4, and VA RNA gene regions are thought to be involved in accessory processes.

[0061] A "functional homolog" or "functional equivalent" of a given adenovirus nucleotide region includes a similar region from a heterologous adenovirus serotype, a nucleotide region from another viral or cellular source, and a polynucleotide that is recombinantly produced or chemically synthesized to function in a similar manner as the reference nucleotide region to achieve a desired result. Thus, a functional homolog of the adenovirus VA RNA gene region or the adenovirus E2A gene region includes derivatives and analogs of such gene regions that contain any single or multiple nucleotide base additions, substitutions, and / or deletions that occur within such gene regions, so long as the homolog retains its native helper function to support AAV virion production at a detectable level above background.

[0062] The term "percent identical" is used herein with reference to comparisons between nucleic acid or amino acid sequences. Nucleic acid and amino acid sequences are often compared using a computer program such as the National Library of Medicine BLAST alignment program.

[0063] II. Compositions and Kits One aspect of the present invention relates to an expression construct for the production of recombinant parvoviruses such as rAAV. The expression construct of the present invention can encode and express an adenovirus protein that provides an auxiliary function for the production of recombinant parvoviruses in host cells.

[0064] In some embodiments, the expression construct of the present invention can provide adenovirus helper functions such as adenovirus E4, E2, VA RNA, and / or E1 functions, and can be used in a triple plasmid system for the production of recombinant parvoviruses.

[0065] In some embodiments, the expression construct of the present invention includes (1) a sequence encoding an adenovirus gene that provides an adenovirus helper function, and (2) a sequence encoding parvovirus non-structural and structural proteins such as AAV Rep and Cap proteins, or a sequence encoding a recombinant parvovirus genome, and can be used in a double plasmid system for the production of recombinant parvoviruses.

[0066] In some embodiments, the expression construct of the present invention includes (1) a sequence encoding an adenovirus gene that provides an adenovirus helper function, (2) a sequence encoding parvovirus non-structural and structural proteins such as AAV Rep and Cap proteins, and (3) a sequence encoding a recombinant parvovirus genome, and can be used in a single plasmid system for the production of recombinant parvoviruses. Table 1 shows some exemplary expression constructs for producing rAAV.

[0067] [Table 1]

[0068] In some embodiments, the expression construct of the present invention also encodes the functional adenovirus E1a protein and / or E1b protein. In some embodiments, the expression construct of the present invention does not encode the functional adenovirus E1a protein and / or E1b protein.

[0069] Expression construct of the triple plasmid parvovirus production system In some embodiments, the expression construct for a preset use is designed to be used in a triple plasmid parvovirus production system. FIG. 7 shows an exemplary construct of a triple plasmid AAV production system. The expression construct includes a modified adenovirus E4 coding region or a modified adenovirus E2a coding region and can express one or more adenovirus E4 proteins or adenovirus E2a proteins required for AAV production in host cells.

[0070] In some embodiments, the expression construct includes both a modified adenovirus E4 coding region and a modified adenovirus E2a coding region, expresses one or more adenovirus E4 proteins and adenovirus E2a proteins, and can provide the adenovirus helper functions required for the production of recombinant parvovirus in suitable host cells.

[0071] In some embodiments, the expression construct includes a modified adenovirus E4 coding region, a modified adenovirus E2a coding region, and a sequence encoding one or more adenovirus-associated RNAs (VA RNAs), expresses the encoded adenovirus proteins and RNAs, and can provide the adenovirus helper functions required for the production of recombinant parvovirus in suitable host cells. In some embodiments, the expression construct does not encode the functional adenovirus E1a protein and / or E1b protein, and the host cell provides the helper functions of E1a and / or E1b.

[0072] In some embodiments, the expression construct includes a modified adenovirus E4 coding region, a modified adenovirus E2a coding region, a sequence encoding one or more adenovirus-associated RNAs (VA RNAs), and an AAV Rep coding region, and is capable of expressing the encoded adenovirus proteins, VA RNAs, and AAV Rep proteins to produce recombinant AAVs in a suitable host cell.

[0073] Modified adenovirus E4 coding region The modified adenovirus E4 coding region encodes a functional E4 protein capable of supporting parvovirus replication in host cells. In some embodiments, the functional E4 protein may include only the amino acids encoded by adenovirus E4 open reading frames 6 and 7 (E4 orfs6 / 7), and only the amino acids encoded by E4 orfs6 / 7 are required for the activity necessary to support parvovirus replication. In some embodiments, the functional E4 protein includes a polypeptide sequence encoded by all or a substantial portion of E4 orfs6 / 7 and does not include a polypeptide sequence encoded by all or a part of E4 orfs1-4 and the E4 34K protein.

[0074] In some embodiments, the modified adenovirus E4 coding region includes one or more deletions. In some embodiments, the modified adenovirus E4 coding region includes a partial or complete deletion of E4 orf6 / 7 intron 1.

[0075] In some embodiments, the modified adenovirus E4 coding region includes a partial deletion of E4 orf6 / 7 intron 1, with up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the E4 orf6 / 7 intron 1 sequence deleted.

[0076] In some embodiments, the modified adenovirus E4 coding region comprises a partial or complete deletion of E4orf6 / 7 intron 2. In some embodiments, the modified adenovirus E4 coding region comprises a partial deletion of E4orf6 / 7 intron 2, with up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the E4orf6 / 7 intron 2 sequence deleted.

[0077] In some embodiments, the modified adenovirus E4 coding region comprises a partial or complete deletion of E4orf6 / 7 intron 1 and a partial or complete deletion of E4orf6 / 7 intron 2.

[0078] In some embodiments, the modified adenovirus E4 coding region has a total size of 400 - 500, 400 - 600, 400 - 800, 400 - 1000, 400 - 1200, 400 - 1500, 400 - 2000, 500 - 600, 500 - 800, 500 - 1000, 500 - 1200, 500 - 1500, or 500 - 2000 bp and encodes a functional E4 protein that can provide the E4 functions required for parvovirus production in non-E4 expressing host cells.

[0079] In some embodiments, the modified adenovirus E4 coding region comprises a sequence corresponding to nucleotides 32645 - 35835 of the Ad2 genome, having (a) a partial or complete deletion of E4orf6 / 7 intron 1, (b) a partial or complete deletion of E4orf6 / 7 intron 2, or both (a) and (b).

[0080] In some embodiments, the modified adenovirus E4 coding region comprises a sequence corresponding to nucleotides 32645-35835 of the Ad2 genome, with a deletion of the complete sequence of E4orf6 / 7 intron 2 (711 bp, SEQ ID NO: 2) and a deletion of the complete sequence of E4orf6 / 7 intron 1 (1275 bp, SEQ ID NO: 1). In some embodiments, the modified adenovirus E4orf6 / 7 coding region comprises a codon-optimized nucleotide sequence (SEQ ID NO: 7) encoding the adenovirus E4orf6 / 7 protein (SEQ ID NO: 8).

[0081] Modified adenovirus E2a coding region In some embodiments, the modified adenovirus E2a coding region comprises (a) a partial or complete deletion of early E2a intron 1 or late E2a intron 1, (b) a partial or complete deletion of E2a intron 2, or both (a) and (b).

[0082] In some embodiments, the modified adenovirus E2a coding region comprises a partial deletion of late E2a intron 1 and / or late E2a intron 2, with up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of late E2a intron 1 and / or late E2a intron 2 deleted.

[0083] In some embodiments, the modified adenovirus E2a coding region has a total size of 1600-3500, 1600-3000, 1600-2500, 1600-2000, 1600-1800, or 1600-1700 bp and encodes a functional E2a protein capable of providing the E2a function required for parvovirus production in non-E2a expressing host cells.

[0084] In some embodiments, the modified adenovirus E2a coding region comprises a sequence corresponding to nucleotides 22233-27575 of the Ad2 genome that has (a) a partial or complete deletion of early E2a intron 1 or late E2a intron 1, (b) a partial or complete deletion of E2a intron 2, or both (a) and (b). In some embodiments, the modified adenovirus E2a coding region comprises a sequence corresponding to nucleotides 22233-27575 of the Ad2 genome that has a 1897 bp deletion (SEQ ID NO: 3) encompassing the complete sequences of late E2a intron 1 and late E2a intron 2. In some embodiments, the modified adenovirus E2a coding region comprises a partial deletion of late E2a intron 1 and / or late E2a intron 2, with up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of SEQ ID NO: 3 deleted.

[0085] In some embodiments, the modified adenovirus E2a coding region comprises (a) a partial or complete deletion following the sequence encoding the codon-optimized adenovirus E2a protein E2a intron 1, (b) a partial or complete deletion of late E2a intron 2, and (c) a codon-modified nucleotide sequence encoding the adenovirus E2a protein.

[0086] Adenovirus VA RNA coding region In some embodiments, the expression construct further comprises an adenovirus VA RNA coding region encoding one or more adenovirus VA RNAs. In some embodiments, the expression construct comprises an adenovirus VA RNA coding region encoding adenovirus VAI RNA and / or VAII RNA. In some embodiments, the VA RNA coding region encodes functional VA RNA I and / or VA RNAII that can provide the VA RNA functions required for parvovirus production in non-VA RNA expressing host cells. In some embodiments, the expression construct comprises SEQ ID NO: 16.

[0087] As used herein and hereinafter, a "functional" adenoviral E4 protein, adenoviral E2a protein, or adenoviral VA RNA I or VA RNA II can promote the production of recombinant parvoviruses such as bocavirus. Determining whether a given VA RNA I and II, E2A protein or E4 protein is functional is within the ability of one of ordinary skill in the art. In one embodiment, an adenoviral E4 protein, adenoviral E2a protein, or adenoviral VA RNA I or VA RNA II is at least 25%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90% or at least 95% of the level supported by the corresponding wild-type protein or RNA within the same triple plasmid system, double plasmid system or single plasmid system. When it supports parvovirus production at a level, it is considered "functional".

[0088] Adenoviruses and nucleic acid fragments thereof for use in the compositions and methods of the present invention include all human adenoviruses of the family Adenoviridae, including members of the Mastadenovirus genus. To date, over 51 human serotypes of adenoviruses have been identified. The adenovirus may be of serotype A, B, C, D, E, or F. Human adenoviruses include, but are not limited to, serotype 1 (Ad1), serotype 2 (Ad2), serotype 3 (Ad3), serotype 4 (Ad4), serotype 5 (Ad5), serotype 6 (Ad6), serotype 7 (Ad7), serotype 8 (Ad8), serotype 9 (Ad9), serotype 10 (Ad10), serotype 11 (Ad11), serotype 12 (Ad12), serotype 13 (Ad13), serotype 14 (Ad14), serotype 15 (Ad15), serotype 16 (Ad16), serotype 17 (Ad17), serotype 18 (Ad18), serotype 19 (Ad19), serotype 19a (Ad19a), serotype 19p (Ad19p), serotype 20 (Ad20), serotype 21 (Ad21), serotype 22 (Ad22), serotype 23 (Ad23), serotype 24 (Ad24), serotype 25 (Ad25), serotype 26 (Ad26), serotype 27 (Ad27), serotype 28 (Ad28), serotype 29 (Ad29), serotype 30 (Ad30), serotype 31 (Ad31), serotype 32 (Ad32), serotype 33 (Ad33), serotype 34 (Ad34), serotype 35 (Ad35), serotype 36 (Ad36), serotype 37 (Ad37), serotype 38 (Ad38), serotype 39 (Ad39), serotype 40 (Ad40), serotype 41 (Ad41), serotype 42 (Ad42), serotype 43 (Ad43), serotype 44 (Ad44), serotype 45 (Ad45), serotype 46 (Ad46), serotype 47 (Ad47), serotype 48 (Ad48), serotype 49 (Ad49), serotype 50 (Ad50), serotype 51 (Ad51), or combinations thereof. In certain embodiments, the adenovirus is serotype 5 (Ad5).

[0089] Regulatory element In some embodiments, the expression construct of the present invention includes one or more regulatory elements that control the expression of the encoded adenoviral protein and / or RNA. Exemplary promoters include, for example, viral promoters, mammalian promoters, composite promoters, cell type- or tissue-specific promoters, and inducible / suppressible promoters.

[0090] Examples of viral promoters include, for example, the SV40 early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), the adenovirus E4 promoter, the adenovirus e2a promoter, the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, for example, the CMV immediate early promoter region (CMV-IE), the SFFV promoter, and the Rous sarcoma virus (RSV) promoter.

[0091] Examples of mammalian promoters include, for example, promoters for expressing EF1α, ubiquitin (e.g., ubiquitin B or C), globin, actin, phosphoglycerate kinase (PGK), NSE (neuron-specific enolase), synapsin or NeuN, and composite promoters such as the CAG promoter (a combination of the CMV early enhancer element and the chicken β-actin promoter).

[0092] The promoter may be of human origin or of other species including mice. In some embodiments, a human promoter may be used. In some embodiments, a promoter that normally directs transcription by eukaryotic RNA polymerase II (a "pol II promoter") or a functional variant thereof is used. In some embodiments, a promoter that normally directs transcription by a eukaryotic RNA polymerase I promoter, for example, a promoter for the transcription of ribosomal RNA (other than 5S rRNA) or a functional variant thereof may be used. In some embodiments, a promoter that normally directs transcription by eukaryotic RNA polymerase III (a "pol III promoter"), for example, (U6, H1, 7SK or tRNA promoter or a functional variant thereof) may be used.

[0093] In some embodiments, the modified adenovirus E4 coding region in the expression construct of the present invention is operably linked to regulatory elements such as a promoter, an enhancer, or both. In some embodiments, the modified adenovirus E4 coding region is operably linked to the adenovirus E4 promoter. In some embodiments, the modified adenovirus E4 coding region is operably linked to the adenovirus E4 promoter comprising SEQ ID NO: 22.

[0094] In some embodiments, the modified adenovirus E2a coding region in the expression construct of the present invention is operably linked to regulatory elements such as a promoter, an enhancer, or both. In some embodiments, the modified adenovirus E2a coding region is operably linked to the adenovirus E2a promoter. In some embodiments, the modified adenovirus E2a coding region is operably linked to the adenovirus E2a promoter comprising SEQ ID NO: 23.

[0095] Codon-optimized coding sequence In some embodiments, the expression construct of the present invention comprises one or more polynucleotides encoding one or more codon-optimized proteins for expression in, for example, human, mammalian, or primate cells, such as HuH7, HEK293T, or CHO cells. The polynucleotide encoding the protein of the present invention can be codon-optimized to improve activity, stability, or expression in the host cell without altering the encoded amino acid sequence. Codon optimization replaces the codons present in the polynucleotide sequence with preferred codons encoding the same amino acid, for example, codons preferred for mammalian expression. Thus, the amino acid sequence is not modified during the process. Codon optimization can be performed using gene optimization software. The codon-optimized nucleotide sequence is translated and aligned with the original protein sequence to ensure that no change occurs in the amino acid sequence. Methods of codon optimization are known in the art and are described, for example, in U.S. Patent Application Publication No. 2008 / 0194511 and U.S. Patent No. 6,114,148.

[0096] In some embodiments, the expression construct of the present invention comprises (a) a modified adenovirus E4 coding region comprising a codon-modified E4 coding sequence, or (b) a modified adenovirus E2a coding region comprising a codon-modified E2a coding sequence.

[0097] In some embodiments, the expression construct of the present invention comprises (a) a modified adenovirus E4 coding region comprising a codon-modified E4 coding sequence, and (b) a modified adenovirus E2a coding region comprising a codon-modified E2a coding sequence.

[0098] In some embodiments, the expression construct of the present invention comprises a modified adenovirus E4 coding region comprising a codon-modified nucleotide sequence (SEQ ID NO: 7). In some embodiments, the expression construct of the present application comprises a modified adenovirus E2a coding region comprising a codon-modified nucleotide sequence (SEQ ID NO: 4).

[0099] Additional elements In some embodiments, the expression construct of the present invention further comprises a sequence encoding adenovirus E1a and E1b proteins.

[0100] In some embodiments, the expression construct further comprises one or more additional elements such as a plasmid origin of replication sequence and a selection marker. Examples of selection markers include, but are not limited to, an ampicillin resistance gene, a neomycin resistance gene, and a kanamycin resistance gene.

[0101] In some embodiments, the expression construct designed for the triple plasmid parvovirus production system has a size of less than 13 kb, 12 kb, 11 kb, 10 kb, 9 kb, 8 kb, 7 kb, 6 kb, 5 kb, 4 kb, 3 kb or 2 kb. In some embodiments, the expression construct has a size in the range of 2 - 11 kb, 2 - 4 kb, 2 - 6 kb, 2 - 8 kb, 2 - 10 kb, 4 - 6 kb, 4 - 8 kb, 4 - 10 kb, 4 - 11 kb, 6 - 8 kb, 6 - 10 kb, 6 - 11 kb, 8 - 10 kb, 8 - 11 kb or 10 - 11 kb.

[0102] In some embodiments, the expression construct designed for the triple plasmid parvovirus production system comprises a modified adenovirus E4 coding region, a modified adenovirus E2a coding region, a sequence encoding one or more adenovirus-associated RNAs (VA RNAs), a sequence encoding an AAV Rep protein, and has a size of less than 13 kb, 12 kb, 11 kb, 10 kb, 9 kb, 8 kb, 7 kb, 6 kb, 5 kb, or 4 kb. In some embodiments, the expression construct has a size in the range of 4 - 13 kb, 4 - 11 kb, 4 - 9 kb, 4 - 7 kb, 7 - 13 kb, 7 - 11 kb, 7 - 9 kb, 9 - 13 kb, 9 - 11 kb, or 11 - 13 kb.

[0103] Preferred embodiments of the expression construct for the triple plasmid parvovirus production system In some embodiments, the expression construct is a plasmid for triple plasmid AAV production. The plasmid includes a modified adenovirus E4 coding region comprising SEQ ID NO: 17 or SEQ ID NO: 18, a modified adenovirus E2a coding region comprising SEQ ID NO: 4, a sequence encoding an adenovirus VAI RNA coding region comprising SEQ ID NO: 16, and a regulatory region comprising SEQ ID NO: 19, and the plasmid has a size in the range of 6 - 7 kb, 6 - 8 kb, 6 - 9 kb, 7 - 8 kb, 7 - 9 kb, and 8 - 9 kb. In some embodiments, the plasmid has a size of 7 - 9 kb.

[0104] Expression construct of the dual plasmid parvovirus production system In some embodiments, the expression construct for a preset use is designed to be used in a dual plasmid parvovirus production system. FIG. 7 shows two exemplary dual plasmid systems for AAV production.

[0105] Constructs encoding adenovirus proteins and parvovirus proteins In some embodiments, the expression construct for dual plasmid parvovirus production includes a parvovirus protein coding region in addition to one or more of the elements described for the expression construct for the triple plasmid parvovirus production system. In some embodiments, the parvovirus coding region includes a sequence encompassing the AAV Rep coding region and a sequence encompassing the AAV Cap coding region.

[0106] In some embodiments, the expression construct for dual plasmid parvovirus production is constructed for AAV production and includes (1) a modified adenovirus E4 coding region encoding E4orf6 / 7, (2) a modified adenovirus E2a coding region encoding the E2a protein, (3) an adenovirus VA RNA coding region, (4) an AAV Rep coding region, (5) an AAV Cap coding region, and (6) one or more regulatory elements that enable the expression of E4orf6 / 7, the E2a protein, adenovirus VA RNA, the AAV Rep protein, and the AAV Cap protein in a host cell. As shown in FIG. 7, such an expression construct may be used in combination with another expression vector encoding a recombinant AAV genome (e.g., a transgene expression cassette flanked by at least one ITR on one side) for the production of recombinant AAV in a host cell. In some embodiments, the AAV Rep coding region is operably linked to one or more regulatory elements such as the AAV P5 promoter or the AAV P5I promoter. In some embodiments, the AAV Rep coding region is operably linked to one or more regulatory elements such as the AAV P19 promoter. In some embodiments, the regulatory element includes the P5I promoter (SEQ ID NO: 15). In some embodiments, the expression construct for dual plasmid AAV production further includes a sequence encoding the adenovirus E1a protein and / or the E1b protein. In some embodiments, the expression construct for dual plasmid AAV production does not include a sequence encoding the adenovirus E1a or E1b protein. In some embodiments, the expression construct has a size of less than 15 kb, 14 kb, 13 kb, 12 kb, 11 kb, 10 kb, 9 kb, 8 kb, or 7 kb. In some embodiments, the expression construct has a size in the range of 6-15 kb, 6-13 kb, 6-11 kb, 6-9 kb, 9-15 kb, 9-13 kb, 9-11 kb, 11-15 kb, 11-13 kb, or 13-15 kb.

[0107] Constructs encoding adenovirus proteins and recombinant parvovirus genomes In some embodiments, the expression construct for the dual plasmid parvovirus production system comprises: (1) a modified adenovirus E4 coding region encoding E4 open reading frame 6 / 7; (2) a modified adenovirus E2a coding region encoding the E2a protein; (3) a sequence encoding adenovirus VA RNA; (4) a sequence encoding a recombinant parvovirus genome (e.g., a transgene expression cassette flanked by at least one ITR on one side); and (5) one or more regulatory elements that enable the expression of E4 open reading frame 6 / 7, the E2a protein, and adenovirus VA RNA in a host cell.

[0108] In some embodiments, an expression construct designed for a dual plasmid AAV production system includes (1) a modified adenovirus E4 coding region encoding E4 open reading frame 6 / 7, (2) a modified adenovirus E2a coding region encoding the E2a protein, (3) a sequence encoding adenovirus VA RNA, (4) a sequence encoding a recombinant AAV genome (e.g., a transgene expression cassette flanked by two ITRs), and (5) one or more regulatory elements enabling the expression of E4 open reading frame 6 / 7, the E2a protein, and adenovirus VA RNA in a host cell. As shown in Figure 7, such an expression construct may be used in combination with another expression vector encoding AAV Rep and Cap proteins for the production of recombinant AAV in a host cell. In some embodiments, an expression construct for dual plasmid AAV production further includes a sequence encoding adenovirus E1a and E1b proteins. In some embodiments, the expression construct has a size of less than 16 kb, 15 kb, 14 kb, 13 kb, 12 kb, 11 kb, 10 kb, 9 kb, 8 kb, 7 kb, 6 kb, 5 kb, or 4 kb. In some embodiments, the expression construct has a size in the range of 3 - 16 kb, 3 - 14 kb, 3 - 12 kb, 3 - 10 kb, 3 - 8 kb, 3 - 6 kb, 6 - 16 kb, 6 - 14 kb, 6 - 12 kb, 6 - 10 kb, 6 - 8 kb, 8 - 16 kb, 8 - 14 kb, 8 - 12 kb, 8 - 10 kb, 10 - 16 kb, 10 - 14 kb, 10 - 12 kb, 12 - 16 kb, 12 - 14 kb, or 14 - 16 kb.

[0109] Preferred Embodiments of Expression Constructs for Dual Plasmid AAV Production Systems In some embodiments, the expression construct for the dual plasmid parvovirus production system is a plasmid for AAV production. The plasmid comprises a modified adenovirus E4 coding region comprising SEQ ID NO: 17 or SEQ ID NO: 18, a modified adenovirus E2a coding region comprising SEQ ID NO: 4, a sequence encoding an adenovirus VAI RNA coding region comprising SEQ ID NO: 16, a regulatory region comprising SEQ ID NO: 19, a sequence comprising SEQ ID NO: 20 and a sequence encoding an AAV Rep protein, and a sequence comprising SEQ ID NO: 21 and a sequence encoding an AAV Cap protein, and the plasmid has a size in the range of 12-14 kb.

[0110] In some embodiments, the expression construct for the dual plasmid parvovirus production system is a plasmid for AAV production. The plasmid comprises a modified adenovirus E4 coding region comprising SEQ ID NO: 17 or SEQ ID NO: 18, a modified adenovirus E2a coding region comprising SEQ ID NO: 4, a sequence encoding an adenovirus VAI RNA coding region comprising SEQ ID NO: 16, a regulatory region comprising SEQ ID NO: 19, a sequence encoding a recombinant AAV genome, the recombinant AAV genome comprising a transgene expression cassette comprising a transgene of interest operably linked to one or more regulatory elements, and two AAV ITRs adjacent to the transgene expression cassette, and the plasmid has a size in the range of 10-14 kb.

[0111] Expression construct for a single plasmid parvovirus production system In some embodiments, an expression construct for a preset use is designed to be used in a single plasmid parvovirus production system. The expression construct includes any or all of the modified adenovirus coding region, parvovirus protein coding region, and an expression cassette having a GOI operably linked to regulatory elements as described above, and a sequence encoding a recombinant parvovirus genome including at least one ITR at one end of the expression cassette. The expression construct enables the production of recombinant parvovirus by transfecting a single plasmid into a host cell. In some embodiments, the expression construct does not provide the E1a and E1b functions of adenovirus and needs to be used for parvovirus production in host cells that result in endogenous expression of adenovirus E1a and E1b proteins. In some embodiments, the expression construct can also provide the adenovirus E1a and E1b functions and can be used for parvovirus production in host cells that do not result in endogenous expression of the adenovirus E1a and E1b proteins.

[0112] Figure 7 provides an exemplary embodiment of an expression construct of a single plasmid AAV production system. In some embodiments, the expression construct includes (1) a modified adenovirus E4 coding region encoding E4 open reading frame 6 / 7, (2) a modified adenovirus E2a coding region encoding the E2a protein, (3) an adenovirus VA RNA coding region, (4) an AAV Rep coding region, (5) an AAV Cap coding region, (6) an expression cassette having a nucleotide sequence encoding a transgene and regulatory elements operably linked to the nucleotide sequence, and at least one ITR at one end of the expression cassette, a sequence encoding a recombinant AAV genome, and (7) one or more regulatory elements enabling the expression of E4 open reading frame 6 / 7, E2a protein, adenovirus VA RNA, AAV Rep protein, and AAV Cap protein in a host cell. In some embodiments, the expression cassette within the recombinant AAV genome is adjacent to the AAV ITRs at each end.

[0113] In some embodiments, an expression construct for a preset use comprises: (1) a modified adenovirus E4 coding region encoding E4 open reading frame 6 / 7; (2) a modified adenovirus E2a coding region encoding an E2a protein; (3) an adenovirus VA RNA coding region; (4) a parvovirus protein coding region; (5) a recombinant parvovirus cloning region; and (6) one or more regulatory elements enabling the expression of E4 open reading frame 6 / 7, E2a protein, adenovirus VA RNA, and parvovirus protein in a host cell.

[0114] In some embodiments, the recombinant parvovirus cloning region comprises a cloning site for insertion of a recombinant parvovirus sequence that includes: (1) an expression cassette containing a gene of interest (GOI) operably linked to a regulatory element; and (2) at least one ITR on one side of the expression cassette. In some embodiments, the recombinant parvovirus sequence includes two ITRs, one at each end of the expression cassette.

[0115] In some embodiments, the recombinant parvovirus cloning region comprises: (1) a cloning site for insertion of a sequence containing an expression cassette with a GOI operably linked to a regulatory element; and (2) at least one ITR on one side of the cloning site. In some embodiments, the recombinant parvovirus sequence includes two ITRs, one on each side of the cloning site.

[0116] In some embodiments, the recombinant parvovirus cloning region comprises: (1) a cloning site for insertion of a GOI and a regulatory element in the vicinity of the cloning site, and an expression cassette capable of being operably linked to the GOI when the GOI is inserted into the cloning site; and (2) at least one ITR on one side of the expression cassette. In some embodiments, the recombinant parvovirus sequence includes two ITRs, one on each side of the expression cassette.

[0117] In some embodiments, the modified adenovirus E4 coding region encodes E4orf6 / 7 with a partial or complete deletion of E4orf6 / 7 intron 1. In some embodiments, the modified adenovirus E4 coding region encodes E4orf6 / 7 with a partial or complete deletion of E4orf6 / 7 intron 2. In some embodiments, the modified adenovirus E4 coding region encodes E4orf6 / 7 with partial or complete deletions of both E4orf6 / 7 intron 1 and E4orf6 / 7 intron 2.

[0118] In some embodiments, the modified adenovirus E4 coding region has a total size of 400 - 500, 400 - 600, 400 - 800, 400 - 1000, 400 - 1200, 400 - 1500, 400 - 2000, 500 - 600, 500 - 800, 500 - 1000, 500 - 1200, 500 - 1500, or 500 - 2000 bp and encodes a functional E4 protein capable of providing the E4 functions required for parvovirus production in non-E4 expressing host cells.

[0119] In some embodiments, the modified adenovirus E4 coding region comprises a sequence corresponding to nucleotides 32645 - 35835 of the Ad2 genome, having (a) a partial or complete deletion of E4orf6 / 7 intron 1, (b) a partial or complete deletion of E4orf6 / 7 intron 2, or both (a) and (b).

[0120] In some embodiments, the modified adenovirus E4 coding region comprises a sequence corresponding to nucleotides 32645 - 35835 of the Ad2 genome, having a deletion of the complete sequence of E4orf6 / 7 intron 2 (711 bp, SEQ ID NO: 2) and a deletion of the complete sequence of E4orf6 / 7 intron 1 (1275 bp, SEQ ID NO: 1).

[0121] In some embodiments, the modified adenovirus E4 coding region comprises a partial deletion of E4orf6 / 7 intron 1 with up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of SEQ ID NO: 1 deleted, and / or a partial deletion of E4orf6 / 7 intron 2 with up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of SEQ ID NO: 2 deleted.

[0122] In some embodiments, the modified adenovirus E4orf6 / 7 coding region comprises a codon-optimized nucleotide sequence (SEQ ID NO: 7) encoding the adenovirus E4orf6 / 7 protein (SEQ ID NO: 8).

[0123] In some embodiments, the modified adenovirus E4 coding region encodes Ad2 E4orf6 / 7. In some embodiments, the modified adenovirus E4 coding region comprises SEQ ID NO: 17. In some embodiments, the modified adenovirus E4 coding region comprises SEQ ID NO: 18.

[0124] In some embodiments, the modified adenovirus E2a coding region comprises a codon-modified or unmodified sequence encoding the adenovirus E2a protein, having a partial or complete deletion of the early E2a intron 1.

[0125] In some embodiments, the modified adenovirus E2a coding region comprises a codon-modified or unmodified sequence encoding the adenovirus E2a protein, having a partial or complete deletion of the late E2a intron 1.

[0126] In some embodiments, the modified adenovirus E2a coding region comprises a codon-modified or unmodified sequence encoding the adenovirus E2a protein, having a partial or complete deletion of the E2a intron 2.

[0127] In some embodiments, the modified adenovirus E2a coding region comprises a codon-modified or unmodified sequence encoding an adenovirus E2a protein having a partial or complete deletion of early E2a intron 1 and a partial or complete deletion of E2a intron 2.

[0128] In some embodiments, the modified adenovirus E2a coding region comprises a codon-modified or unmodified sequence encoding an adenovirus E2a protein having a partial or complete deletion of late E2a intron 1 and a partial or complete deletion of E2a intron 2.

[0129] In some embodiments, the modified adenovirus E2a coding region comprises a partial deletion of late E2a intron 1 and / or late E2a intron 2, with up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of late E2a intron 1 and / or late E2a intron 2 deleted.

[0130] In some embodiments, the modified adenovirus E2a coding region has a total size of 1600 - 3500, 1600 - 3000, 1600 - 2500, 1600 - 2000, 1600 - 1800, or 1600 - 1700 bp and encodes a functional E2a protein capable of providing the E2a function required for parvovirus production in non-E2a expressing host cells.

[0131] In some embodiments, the modified adenovirus E2a coding region comprises a sequence corresponding to nucleotides 22233-27575 of the Ad2 genome having (a) a partial or complete deletion of early E2a intron 1 or late E2a intron 1, (b) a partial or complete deletion of E2a intron 2, or both (a) and (b). In some embodiments, the modified adenovirus E2a coding region comprises a sequence corresponding to nucleotides 22233-27575 of the Ad2 genome having a 1897 bp deletion (SEQ ID NO: 3) encompassing the complete sequences of late E2a intron 1 and late E2a intron 2. In some embodiments, the modified adenovirus E2a coding region comprises a partial deletion of late E2a intron 1 and / or late E2a intron 2, with up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of SEQ ID NO: 3 deleted.

[0132] In some embodiments, the modified adenovirus E2a coding region comprises (a) a partial or complete deletion following the sequence encoding the codon-optimized adenovirus E2a protein E2a intron 1, (b) a partial or complete deletion of late E2a intron 2, and (c) a codon-modified nucleotide sequence encoding the adenovirus E2a protein.

[0133] In some embodiments, the modified adenovirus E2a coding region comprises SEQ ID NO: 4.

[0134] In some embodiments, the adenovirus VA RNA coding region comprises a sequence encoding adenovirus VA1, adenovirus VAII, or both. In some embodiments, the adenovirus VA RNA coding region comprises a sequence encoding Ad2 VA1, Ad2 VAII, or both. In some embodiments, the adenovirus VA RNA coding region comprises SEQ ID NO: 16.

[0135] In some embodiments, the AAV Rep coding region comprises SEQ ID NO: 20.

[0136] In some embodiments, the AAV Cap coding region comprises SEQ ID NO: 21.

[0137] In some embodiments, the one or more regulatory elements include an adenovirus E4 promoter operably linked to a modified adenovirus E4 coding region, an adenovirus E2a promoter operably linked to a modified adenovirus E2a coding region, and a modified AAV P5 promoter operably linked to a sequence encoding AAV Rep and Cap proteins. In some embodiments, the adenovirus E4 promoter comprises SEQ ID NO: 22. In some embodiments, the adenovirus E2a promoter comprises SEQ ID NO: 23. In some embodiments, the modified AAV P5 promoter comprises SEQ ID NO: 16.

[0138] In some embodiments, the expression construct further comprises a nucleotide sequence encoding an adenovirus E1a protein and / or a nucleotide sequence encoding an adenovirus E1b protein.

[0139] In some embodiments, the expression construct designed for a single plasmid parvovirus production system has a size of less than 20 kb, 19 kb, 18 kb, 16 kb, 15 kb, 14 kb, 13 kb, 12 kb, 11 kb, 10 kb, 9 kb, 8 kb, or 7 kb.

[0140] In some embodiments, the expression construct designed for a single plasmid parvovirus production system has a size in the range of 7 - 25 kb, 7 - 20 kb, 7 - 18 kb, 7 - 16 kb, 7 - 14 kb, 7 - 12 kb, 7 - 10 kb, 10 - 25 kb, 10 - 20 kb, 10 - 18 kb, 10 - 16 kb, 10 - 14 kb, 10 - 12 kb, 12 - 25 kb, 12 - 20 kb, 12 - 18 kb, 12 - 16 kb, 12 - 14 kb, 14 - 25 kb, 14 - 20 kb, 14 - 18 kb, 14 - 16 kb, 16 - 25 kb, 16 - 20 kb, 16 - 18 kb, 18 - 25 kb, 18 - 20 kb, or 20 - 25 kb.

[0141] In some embodiments, the expression construct is a plasmid having a total size of 10 - 15 kb, 10 - 20 kb, 10 - 25 kb, 15 - 20 kb, 15 - 25 kb, or 20 - 25 kb.

[0142] Preferred embodiments of an expression construct for a single plasmid AAV production system In some embodiments, the expression construct is a plasmid for single plasmid AAV production, and comprises a modified adenovirus E4 coding region containing SEQ ID NO: 17 or SEQ ID NO: 18, a modified adenovirus E2a coding region containing SEQ ID NO: 4, a sequence encoding an adenovirus VAI RNA coding region containing SEQ ID NO: 16, a regulatory region containing SEQ ID NO: 19, an AAV Rep coding region containing SEQ ID NO: 20, an AAV Cap coding region containing SEQ ID NO: 21, and a sequence encoding a recombinant AAV genome, and the expression construct has a size in the range of 7 - 19 kb.

[0143] In another embodiment, the present invention provides one or more cells each containing one or more of the expression constructs described herein. In another embodiment, the present application provides one or more cell lines stably transformed with one or more of the expression constructs described herein.

[0144] In another aspect, the present invention provides a kit for generating recombinant AAV particles. In one embodiment, the kit comprises a plasmid containing the expression construct of the present application.

[0145] In certain embodiments, the kit further comprises instructions for use, such as instructions for use in the methods described herein. In some embodiments, the kit further comprises one or more tubes or other types of containers (e.g., Eppendorf tubes) for cell lysates and / or one or more tubes or other types of containers for generated waste.

[0146] III. Method for generating recombinant parvovirus Another aspect of the invention relates to a method for generating recombinant parvovirus particles using the expression constructs of the present application. The method includes introducing the expression construct of the present application into a host cell that provides the helper functions necessary for parvovirus production, incubating the host cell carrying the expression cassette for a desired period to generate recombinant parvovirus particles, and recovering the recombinant parvovirus particles after the incubation period. In some embodiments, the parvovirus is AAV.

[0147] In some embodiments, the method includes: (1) introducing into a host cell: (A) an expression construct of a triple plasmid parvovirus production system, (B) a parvovirus trans construct capable of expressing parvovirus proteins necessary for the production of recombinant parvovirus, and (C) a parvovirus cis construct containing a sequence encoding a recombinant parvovirus genome; (2) incubating the host cell carrying the three constructs for a desired period to generate recombinant parvovirus particles; and (3) recovering the recombinant parvovirus particles after the incubation period. In some embodiments, constructs A, B, and C are co-transfected into the host cell at an A:B:C molar ratio of 1-10:1-10:1-10. In some embodiments, constructs A, B, and C are co-transfected into the host cell at an A:B:C molar ratio of 1:1:1. This method is often referred to as the triple transfection method. In some embodiments, the parvovirus is AAV and the trans construct includes the AAV Rep coding region and the AAV Cap coding region.

[0148] In some embodiments, the method comprises: (1) introducing into a host cell: (A) an expression construct (Construct A) of a dual plasmid parvovirus production system, comprising a modified adenovirus E4 coding region encoding E4orf6 / 7, a modified adenovirus E2a coding region encoding E2a protein, an adenovirus VA RNA coding region, a parvovirus protein coding region, and one or more regulatory elements enabling the expression of adenovirus E4orf6 / 7, E2a, VA RNA, and parvovirus proteins; and (B) an expression construct (Construct B) comprising a sequence encoding a recombinant parvovirus genome; (2) incubating the host cell carrying the constructs for a desired period to generate recombinant parvovirus particles; and (3) recovering the recombinant parvovirus particles after the incubation period. In some embodiments, the recombinant parvovirus is rAAV. In some embodiments, Constructs A and B are co-transfected into the host cell at an A:B molar ratio in the range of 1:10 to 10:1. In some embodiments, Constructs A and B are co-transfected into the host cell at an A:B molar ratio of 1:1, 1:2, 1:3, 2:1, or 3:1.

[0149] In some embodiments, the method comprises: (1) introducing into a host cell: (A) an expression construct (Construct A) of a dual plasmid parvovirus production system, which contains a modified adenovirus E4 coding region encoding E4orf6 / 7, a modified adenovirus E2a coding region encoding E2a protein, an adenovirus VA RNA coding region, a sequence encoding a recombinant parvovirus genome, and one or more regulatory elements enabling the expression of adenovirus E4orf6 / 7, E2a, and VA RNA; and (B) an expression construct (Construct B) containing a parvovirus protein coding region; (2) incubating the host cell carrying the constructs for a desired period to generate recombinant parvovirus particles; and (3) recovering the recombinant parvovirus particles after the incubation period. In some embodiments, the recombinant parvovirus is rAAV. In some embodiments, Constructs A and B are co-transfected into the host cell at an A:B molar ratio in the range of 1:10 to 10:1. In some embodiments, Constructs A and B are co-transfected into the host cell at an A:B molar ratio of 1:1, 1:2, 1:3, 2:1, or 3:1.

[0150] In some embodiments, the method comprises: (1) introducing an expression construct of a single plasmid parvovirus production system into a host cell; (2) incubating the host cell carrying the expression cassette for a desired period to generate recombinant parvovirus particles; and (3) recovering the recombinant parvovirus particles after the incubation period. In some embodiments, the expression construct is a plasmid for generating recombinant AAV. In some embodiments, the plasmid does not encode adenovirus E1a and E1b proteins, and the host cell endogenously expresses adenovirus E1a and E1b proteins.

[0151] Host cells for generating parvovirus particles can contain the expression construct of the present invention, for example, in the form of an episomal plasmid. The expression construct of the present invention may also be stably integrated into the host cell genome. Furthermore, the host cell can constitute an expression system for generating adenovirus proteins such as E1a and E1b, which are necessary for parvovirus production. Examples of host cells include, but are not limited to, microorganisms, yeast cells, insect cells, and animal cells. In some embodiments, the host cell is a mammalian host cell such as human HuH7 and HEK293 cells, Chinese hamster ovary cells (“CHO”), and baby hamster kidney (“BHK”) cells. Mammalian cells suitable for carrying out the present application include, inter alia, COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573), CHOP, HuH7, HEK293, and NS-1 cells. The term “host cell” includes the progeny of the original transfected cell. It is understood that the progeny of a single parental cell may not necessarily be identical to the original parent in morphology, or in genomic or total DNA complement due to natural, accidental, or intentional mutations. A “mammalian host cell” is a cell originally derived from a mammalian or its progeny cells.

[0152] In one embodiment, the host cell expresses Ad E1a and E1b proteins. In certain preferred embodiments, the host cell is HEK-293 cells that express adenovirus E1a and E1b.

[0153] In some embodiments, the expression construct of the present application and other expression constructs (if any) are CaPO 4Transfection is carried out in HEK293 cells (available from ATCC®) via media transfection, polymer molecules such as lipids or polyethyleneimine (PEI). The HEK293 cells are then incubated for at least 60 hours to allow for the production of recombinant parvovirus particles.

[0154] Without further elaboration, it is believed that those skilled in the art can make maximum use of the present disclosure based on the above description. Accordingly, the following specific embodiments should be construed as merely exemplary and not limiting the remainder of the present disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter referred to herein.

[0155] IV. Kit Another aspect of the present invention relates to a kit for producing recombinant parvovirus. In some embodiments, the kit comprises an expression construct of the present invention and instructions for the use of the expression construct.

[0156] In some embodiments, the expression construct comprises: (1) a modified adenovirus E4 coding region encoding E4 open reading frame 6 / 7; (2) a modified adenovirus E2a coding region encoding the E2a protein; (3) an adenovirus VA RNA coding region; (4) a parvovirus protein coding region; (5) a recombinant parvovirus cloning region; and (6) one or more regulatory elements that enable the expression of E4 open reading frame 6 / 7, the E2a protein, adenovirus VA RNA, and parvovirus protein in a host cell. In some embodiments, the recombinant parvovirus cloning region comprises: (1) an expression cassette comprising a gene of interest (GOI) operably linked to a regulatory element, and (2) a cloning site for insertion of a recombinant parvovirus sequence comprising at least one ITR on one side of the expression cassette. In some embodiments, the recombinant parvovirus sequence comprises two ITRs, one at each end of the expression cassette. In some embodiments, the recombinant parvovirus cloning region comprises: (1) a cloning site for insertion of a sequence comprising an expression cassette comprising a GOI operably linked to a regulatory element, and (2) at least one ITR on one side of the cloning site. In some embodiments, the recombinant parvovirus sequence comprises two ITRs, one on each side of the cloning site. In some embodiments, the recombinant parvovirus cloning region comprises: (1) a cloning site for insertion of a GOI and a regulatory element in the vicinity of the cloning site, an expression cassette that can be operably linked to the GOI when the GOI is inserted into the cloning site, and (2) at least one ITR on one side of the expression cassette. In some embodiments, the recombinant parvovirus sequence comprises two ITRs, one on each side of the expression cassette.

[0157] In some embodiments, the expression construct comprises: (1) a modified adenovirus E4 coding region encoding E4 open reading frame 6 / 7; (2) a modified adenovirus E2a coding region encoding the E2a protein; (3) an adenovirus VA RNA coding region; (4) an AAV Rep coding region; (5) an AAV Cap coding region; (6) an expression cassette having a nucleotide sequence encoding a transgene and regulatory elements operably linked to the nucleotide sequence; and at least one ITR at one end of the expression cassette, a sequence encoding a recombinant AAV genome, and (7) one or more regulatory elements that enable the expression of E4 open reading frame 6 / 7, E2a protein, adenovirus VA RNA, AAV Rep protein, and AAV Cap protein in a host cell. In some embodiments, the expression cassette within the recombinant AAV genome is adjacent to the AAV ITRs at each end.

[0158] In some embodiments, the kit further comprises reagents for transfection of the expression construct.

Example

[0159] Example 1: Construction of AAV Helper Constructs To examine the dispersibility of the AD sequences for packaging AAV vectors, a series of AD-based deletion mutants were constructed to reduce the plasmid size and increase the helper function for generating recombinant AAV virus particles. Figure 1 shows a series of expression cassettes regarding the arrangement of the Ad2 coding region and surrounding deletions relative to the adenovirus (Ad2) nucleotide sequence coordinates.

[0160] Figure 2 shows an enlarged map showing the arrangement of the Ad E4orf and surrounding deletions shown in Figure 1 relative to the Ad2 genome sequence.

[0161] Figure 3 shows an enlarged map showing the Ad2 E2a promoter and Ad2 mRNA expressed early and late in infection, including the surrounding deletions shown in Figure 1 relative to the Ad genome sequence.

[0162] Figure 4 shows an exemplary helper plasmid, mini-pHelper plasmid (SEQ ID NO: 6), which includes an embodiment of the expression cassette of the present invention. The plasmid contains the mini-pHelper Ad helper gene arrangement shown in Figure 1. The mini-pHelper backbone plasmid includes a codon-optimized Ad E2a coding region sequence (SEQ ID NO: 4) encoding the wild-type Ad E2a amino acid sequence shown in SEQ ID NO: 5.

[0163] Figure 5 shows another exemplary helper plasmid, mini-pHelper-1.0 plasmid (SEQ ID NO: 10), which contains another embodiment of the expression cassette of the present invention. The plasmid contains the mini-pHelper Ad helper gene arrangement shown in Figure 1.

[0164] Example 2: Identification of Adenovirus Regions Dispensable for AAV Production Figure 6 shows the effect of deletions or combinations of deletions in Figure 1 on the ability of an expression cassette to fully provide helper functions for adeno-associated virus (AAV) packaging. The production titer of rAAV was obtained using a triple plasmid transfection system including an ITR-containing plasmid (such as pAAV-CAG-EGFP), an AAV helper plasmid (such as pRep2-AAV1, pRep2-AAV2, or pRep2-AAV9), and an Ad helper plasmid Ad having the Ad helper gene arrangement (such as pHelper, pHelper-dE4Orf6 / 7 intron, mini-pHelper1.0, etc.) shown in Figure 1 that provides the necessary adenovirus functions. All AAV vectors were produced in HEK293T cells. The Y-axis shows the fold increase in using different new pHelpers compared to the original pHelper.

[0165] Example 3: Construction of mini-helper Plasmids for AAV Library Production Figure 7 shows a schematic diagram comparing the original triple plasmid transfection system with the mini-pHelper-based production system. Green - Ad gene, blue - AAV gene, pink - transgene element. In the original triple plasmid system, the total mass of the three plasmids (pHelper, pRep&Cap, GOI) is 24.1 - 28.1 kb. In the mini-phelper plasmid system, the total mass of the three plasmids was reduced to 20.9 - 24.9 kb. There are two different versions of the double plasmid system (V1, the AAV gene (Rep and Cap) was inserted into mini-pHelper; V2, the transgene element was inserted into mini-pHelper). The total mass of V1 and / or V2 is 17.9 - 21.9 kb. In one plasmid system, both the AAV gene and the transgene element were inserted into mini-pHelper. The total mass of the plasmid is 13.9 - 17.9 kb.

[0166] Construction of mini-pHelper-AAV2 (SEQ ID NO: 10): The mini-pHelper1.0 plasmid was digested with ClaI. The AAV helper genes (Rep and Cap) were PCR amplified and assembled into the ClaI site.

[0167] Construction of mini-pHelper-CMV-EGFP (SEQ ID NO: 11): The mini-pHelper1.0 plasmid (SEQ ID NO: 9) was digested with PmeI and used as a backbone. The insert ITR-CMV-EGFP-ITR cassette was digested from pAAV-CMV-EGFP with SbfI. The backbone and the insert were ligated with T4 DNA ligase.

[0168] Construction of pAAVone-AAV2-CMV-EGFP (SEQ ID NO: 12): The mini-pHelper-AAV2 plasmid was digested with PmeI and used as a backbone. The insert ITR-CMV-EGFP-ITR cassette was digested from pAAV-CMV-EGFP with SbfI. The backbone and the insert were ligated with T4 DNA ligase.

[0169] Figure 8 shows the packaging efficiency of the mini-helper-based AAV production system in adherent HEK 293T cells. The production titer of rAAV was obtained in HEK 293T cells 72 hours after transfection. In the triple plasmid transfection system, a fixed amount of polyethyleneimine (PEI) (1.8 μl / well) was used to transfect HEK 293T cells with three plasmids (pHelper or mini-phelper-1.0): pRep2-AAV2: pAAV-CMV-EGFP) with a total DNA mass of 0.6 μg / well at four different molecular ratios. For the double plasmid system, three different molecular ratios were evaluated. In the single plasmid system, different amounts of plasmid were transfected with a fixed amount of PEI.

[0170] Figure 9 is a diagram showing the packaging efficiency of the mini-helper-based AAV production system in suspension-cultured HEK 293T cells. The production titer of rAAV was obtained 72 hours after transfection. In the triple plasmid transfection system, the ratio of the three plasmids was 1:1:1, and the total DNA was 0.75 μg / 10 6 cells. In the double plasmid transfection system, the ratio of the two plasmids was 1:1, and the total DNA was 0.75 μg / 10 6 cells. In the single plasmid system, different amounts of plasmid were transfected at a fixed PEI:DNA ratio of 2.

[0171] Figure 10 shows the packaging efficiency of the mini-helper-based double plasmid V2 system for different AAV serotypes. In the double plasmid V2 system, the transgene element was inserted into mini-pHelper. AAV production system in suspension-cultured HEK 293T cells. The production titer of rAAV was obtained 72 hours after transfection. In the triple plasmid transfection system, the molecular ratio of the three plasmids was 1:1:1, and the total DNA was the total DNA mass of 0.6 μg / well. In the double plasmid transfection system, the molecular ratio of the two plasmids was 1:1, and the total DNA was the total DNA mass of 0.6 ug / well.

[0172] Example 4: Use of mini-helper for AAV library generation Figure 11 shows the use of the mini-helper for AAV library generation. The productive titer of rAAV was obtained in HEK 293T cells 72 hours after transfection. The pAAV-library plasmid was used at 200 copies / cell. pHelper and pRep, mini-pHelper-1.0 and mini-pHelper-Rep (SEQ ID NO: 13) were used at 10 5 copies / cell. PEI was used at 1.5 μg / well. The mini-pHelper-Rep:mini-pHelper-AAV2 plasmid was digested with SwaI and SnaBI to remove the Cap gene.

[0173] Figure 12 shows the exemplary helper plasmid pAAVone-AAV2-CMV-EGFP (SEQ ID NO: 12) containing another embodiment of the expression cassette of the present invention. The plasmid contains the mini-pHelper Ad helper gene arrangement shown in Figure 1, the AAVRep and Cap genes, and the sequence of the recombinant AAV genome (ITR-CMV-EGFP-PolyA-ITR).

[0174] Figure 13 shows the sequences of the wild-type AAV2 P5 promoter P5 (P5-AAV2, SEQ ID NO: 14) and the modified AAV P5 promoter P5 (P5I, SEQ ID NO: 15), and their effects on the packaging efficiency of the AAVone system. The total DNA is a total DNA mass of 0.6 μg / well. The productive titer of rAAV was obtained 72 hours after transfection.

[0175] Figure 14 is a list of the sequences described herein.

[0176] In this specification, incorporated by reference is the sequence listing xml file named 2037-004US.xml, created on February 27, 2023, with a size of 107 kB, and filed on March 10, 2023.

[0177] The foregoing description is intended to teach those skilled in the art how to practice the invention. This specification is not intended to detail all obvious modifications and variations thereof that will be apparent to those skilled in the art upon reading the description. However, all such obvious modifications and variations are intended to be included within the scope of the invention as defined by the following claims. Any patents or patent publications cited herein are hereby expressly incorporated by reference in their entirety. The claims are intended to cover the claimed components and steps in any order that is effective for achieving the intended purpose thereof, unless the context specifically indicates the contrary.

Claims

1. 1. A polynucleotide expression construct for the generation of a recombinant parvovirus, comprising: (1) a modified adenovirus E4 coding region; and (2) a modified adenoviral E2a coding region; and (3) an adenovirus VA RNA coding region; and (4) a parvovirus protein coding region encoding a parvovirus protein required for producing the recombinant parvovirus; and (5) a recombinant parvovirus sequence comprising: (i) an expression cassette comprising a nucleotide sequence encoding a transgene and a regulatory element operably linked to the nucleotide sequence; and (ii) at least one ITR at one end of the expression cassette; (6) one or more regulatory elements that enable expression of (1) to (4) in a host cell; A polynucleotide expression construct comprising:

2. The expression construct of claim 1, wherein the modified adenoviral E4 coding region comprises (i) a partial or complete deletion of E4orf6 / 7 intron 1, or (ii) a partial or complete deletion of E4orf6 / 7 intron 2, or both (i) and (ii).

3. 2. The expression construct of claim 1, wherein the modified adenoviral E4 coding region comprises a sequence corresponding to nucleotide sequence 32645-35835 of the Ad2 genome with (i) a partial or complete deletion of E4orf6 / 7 intron 2 having the sequence of SEQ ID NO:2, or (ii) a partial or complete deletion of E4orf6 / 7 intron 1 having the sequence of SEQ ID NO:1, or (i) and (ii).

4. The expression construct of claim 1, wherein the modified adenovirus E2a coding region comprises (i) a partial or complete deletion of early E2a intron 1 or late E2a intron 1, (ii) a partial or complete deletion of E2a intron 2, or both (i) and (ii).

5. 2. The expression construct of claim 1, wherein the modified adenoviral E2a coding region comprises a sequence corresponding to nucleotide sequence 22233-27575 of the Ad2 genome, with (i) a partial or complete deletion of early E2a intron 1 or late E2a intron 1, (ii) a partial or complete deletion of E2a intron 2, or both (i) and (ii).

6. 2. The expression construct of claim 1, wherein the recombinant parvovirus is a recombinant adeno-associated virus (rAAV) and the parvovirus protein coding region comprises an AAV Rep coding region and an AAV Cap coding region.

7. 7. The expression construct of claim 6, wherein the AAV Rep coding region comprises an AAV Rep coding sequence operably linked to an AAV P5I promoter.

8. (1) SEQ ID NO: 17 or SEQ ID NO: 18, (2) SEQ ID NO: 4, (3) SEQ ID NO: 16, (4) SEQ ID NO: 19, (5) SEQ ID NO: 20, and (6) The expression construct of claim 1, comprising SEQ ID NO:

21.

9. 10. The expression construct of claim 1, having a size of less than 20 kilobases.

10. 1. A one-plasmid production method for producing a recombinant parvovirus, comprising: introducing a single plasmid into a host cell, said single plasmid being the expression construct of claim 1; incubating the host cells harboring the expression construct for a desired period of time to produce recombinant parvovirus particles; and recovering the recombinant parvovirus particles after an incubation period.

11. 1. A polynucleotide expression construct for the generation of a recombinant parvovirus, comprising: (a) a modified adenoviral E4 coding region comprising (i) a partial or complete deletion of E4orf6 / 7 intron 1, or (ii) a partial or complete deletion of E4orf6 / 7 intron 2, or both (i) and (ii); (b) a modified adenoviral E2a coding region encoding an adenoviral E2a protein, the modified adenoviral E2a coding region comprising (i) a partial or complete deletion of early E2a intron 1 or late E2a intron 1, (ii) a partial or complete deletion of E2a intron 2, or both (i) and (ii); (c) a sequence encoding one or more adenovirus VA RNAs; and (d) one or more regulatory elements operably linked to (a), (b), and (c).

12. 12. The expression construct of claim 11, wherein the modified adenoviral E4 coding region comprises a sequence corresponding to nucleotide sequence 32645-35835 of the Ad2 genome with (i) a partial or complete deletion of E4orf6 / 7 intron 2 having the sequence of SEQ ID NO:2, (ii) a partial or complete deletion of E4orf6 / 7 intron 1 having the sequence of SEQ ID NO:1, or both (i) and (ii).

13. 12. The expression construct of claim 11, wherein the modified adenoviral E4 coding region comprises a modified adenoviral E4orf6 / 7 coding region comprising the codon-optimized nucleotide sequence of SEQ ID NO:

7.

14. 12. The expression construct of claim 11, wherein the modified adenoviral E2a coding region comprises a sequence corresponding to nucleotide sequence 22233-27575 of the Ad2 genome, with (i) a partial or complete deletion of early E2a intron 1 or late E2a intron 1, (ii) a partial or complete deletion of E2a intron 2, or both (i) and (ii).

15. 12. The expression construct of claim 11, wherein the modified adenovirus E2a coding region comprises a sequence corresponding to nucleotide sequence 22233-27575 of the Ad2 genome, with an 1897 bp deletion of SEQ ID NO:3 encompassing the complete sequence of late E2a intron 1 and E2a intron 2.

16. The expression construct of claim 11, comprising (1) SEQ ID NO: 17 or SEQ ID NO: 18, (2) SEQ ID NO: 4, (3) SEQ ID NO: 16, and (4) SEQ ID NO:

19.

17. 12. The expression construct of claim 11, which consists of less than 9 kilobases.

18. 12. The expression construct of claim 11, further comprising an AAV Rep coding region and an AAV Cap coding region.

19. 19. The expression construct of claim 18, comprising SEQ ID NO:20 and SEQ ID NO:

21.

20. 19. The expression construct of claim 18, wherein the AAV Rep coding region and / or the AAV Cap coding region is operably linked to an AAV P5I promoter comprising SEQ ID NO:

15.

21. an expression cassette comprising a sequence encoding a gene of interest and a regulatory element operably linked to the sequence encoding the gene of interest; 12. The expression construct of claim 11, further comprising a recombinant AAV genome comprising two AAV ITRs flanking the expression cassette.

22. 1. A method for producing a recombinant AAV, comprising: Introducing into a host cell the expression construct of claim 11, an AAV trans-plasmid encoding AAV Rep and Cap proteins, and an AAV cis-plasmid encoding a recombinant AAV genome; incubating the host cells harboring the expression construct, the AAV trans-plasmid, and the AAV cis-plasmid for a desired period of time to produce recombinant AAV particles; and recovering the recombinant AAV particles after an incubation period.

23. 1. A method for producing a recombinant AAV, comprising: introducing the expression construct of claim 18 and an AAV cis plasmid encoding a recombinant AAV genome into a host cell; incubating the host cells harboring the expression construct and the AAV cis plasmid for a desired period of time to produce recombinant AAV particles; and recovering the recombinant AAV particles after an incubation period.

24. 1. A method for producing a recombinant AAV, comprising: Introducing the expression construct of claim 21 and an AAV trans-plasmid encoding AAV Rep and Cap proteins into a host cell; incubating the host cells harboring the expression construct and the AAV trans-plasmid for a desired period of time to produce recombinant AAV particles; and recovering the recombinant AAV particles after an incubation period.

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