Compositions and methods for producing recombinant parvoviruses

Abstract

Expression constructs and methods for producing recombinant parvoviruses are disclosed. In some embodiments, the expression constructs encode: (1) adenoviral E4 and E2a proteins; (2) parvovirus protein necessary for the production of the recombinant parvovirus; and (3) a recombinant parvovirus genome, allowing the recombinant parvovirus to be produced by transfecting a host cell with a single expression construct.

Description

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

[0002] The present application generally relates to compositions and methods for producing recombinant viruses. More specifically, the present application relates to compositions for producing recombinant parvoviruses such as adeno-associated viruses (AAV). Background Art

[0003] Recombinant viruses such as recombinant adeno-associated virus (rAAV) have been developed as vectors by replacing all viral genes with therapeutic transgene expression cassettes while retaining the only cis-element ITR required for vector packaging and DNA replication. Early rAAV production methods relied on plasmid systems, including: 1) AAV helper plasmids (generally covering AAV Rep and Cap coding regions, but lacking AAV ITRs, so they cannot replicate or package themselves); and 2) ITR-containing plasmids (generally covering the selected transgene of interest flanked by AAV ITRs that provide viral replication and packaging functions). By transient transfection, both the helper plasmid and the ITR-containing plasmid carrying the selected gene can be introduced into suitable cells for production. 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, thereby directing the transcription and translation of the AAV Rep and Cap regions. In the Ad helper virus, E1a, E1b, E2a, E4 and VA RNA genes can provide the auxiliary functions necessary for rAAV production.

[0004] Infection of producer cells with helper virus to generate rAAV is effective for producing rAAV. However, as a consequence, the helper virus also produces Ad helper virus particles that may cause host immune responses. In some platforms, the viral helper genes necessary for AAV production can be stably transfected into production cell lines (e.g., HEK293 cells), thereby reducing the possibility that the host immune system will produce an anti-helper virus immune response to trace levels of residual helper virus.

[0005] A three-plasmid transfection approach has recently been developed. This approach uses AAV serotype-specific Rep and Cap plasmids and a transgene-containing plasmid, but avoids the use of helper virus infection by providing the necessary helper virus genes on a third plasmid (i.e., removing or reducing viral coding sequences), thereby reducing potential anti-helper virus immune responses by the host immune system. Providing viral helper genes on a third plasmid greatly reduces the production of helper virus in transfected cells, providing only rAAV. Multi-plasmid transient transfection of adherent HEK293 cells is a common method for rAAV production.

[0006] In a multi-plasmid system, it is important to maintain appropriate plasmid size and plasmid ratios for maximizing AAV production and transfection efficiency. There is a need in the art to provide a simplified and efficient plasmid-based rAAV production system that improves transfection and reduces immunogenicity while still retaining optimal expression of the transgene. The present application provides such benefits based on the discovery of adenoviral regions that can be omitted in traditional helper plasmids. Summary of the invention

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

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

[0009] Another aspect of the present application relates to a method for producing recombinant parvovirus particles using the expression construct of the present application. The method comprises the following steps: introducing the expression construct of the present application into a host cell; incubating the host cell carrying the expression construct for a desired period of time to produce recombinant parvovirus particles; and collecting the recombinant parvovirus particles after the incubation period of time. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Shown is a diagram depicting the arrangement of adenovirus (Ad2) coding regions relative to various deletions in a series of exemplary expression cassettes of the present application. CoE2A: Codon-optimized E2A coding sequence.

[0011] Figure 2 Figure 2 depicts the Ad2 E4 coding region, the arrangement of the Ad E4orf in the coding region, and Figure 1 The missing extension diagram depicted in .

[0012] Figure 3 Figure 2 depicts the Ad2 E2a coding region, the Ad2 E2A promoter, and Ad2 mRNA expressed early and late in infection, as well as Figure 1 The missing extension diagram depicted in .

[0013] Figure 4 Shows that it contains Figure 1 Plasmid constructs with corresponding arrangements of Ad helper genes, i.e., mini-pHelper, are shown.

[0014] Figure 5 Shows that it contains Figure 1 The plasmid construct corresponding to the arrangement of Ad helper genes is shown, namely mini-pHelper-1.0.

[0015] Figure 6 Shows Figure 1 Shown are the effects of various deletions and their combinations on the ability of the expression cassette to adequately provide AAV packaging helper functions.

[0016] Figure 7 It is a comprehensive schematic diagram of the three-plasmid, two-plasmid and single-plasmid virus production systems based on mini-pHelper.

[0017] Figure 8 Shown is the packaging efficiency of the mini-pHelper-based AAV production system in adherent HEK 293T cells.

[0018] Fig. 9 Shown is the packaging efficiency of the mini-pHelper-based AAV production system in HEK 293T cells cultured in suspension.

[0019] Fig.10 The packaging efficiency of the mini-pHelper-based two-plasmid V2 system for different AAV serotypes is shown.

[0020] Fig.11 The use of the mini-pHelper for AAV library production is shown.

[0021] Fig.12 An exemplary plasmid construct for a single-plasmid AAV production system, pAAVone-AAV2-CMV-EGFP, is shown.

[0022] Fig.13 It is shown that the modified P5 promoter (P5I) increases AAV yield compared to the wild-type AAV2 P5 promoter.

[0023] Fig.14 A list of nucleotide and amino acid sequences described in this application is shown. DETAILED DESCRIPTION

[0024] I. Definitions

[0025] As used in this specification and the appended claims, the singular forms "a," "an," and "the" generally include plural referents as well, unless the content clearly dictates otherwise.

[0026] The "range" mentioned herein may be expressed as "from about a certain specific value and / or to about another specific value". In this case, "from a certain specific value and / or to another specific value" serves as another embodiment. Similarly, when the antecedent "about" is used to indicate an approximate value, the specific value should be considered to form another embodiment. It should be further understood that the endpoints of each range are meaningful whether related to or independent of the other endpoint. It should also be understood that the specific values ​​disclosed herein include both the values ​​themselves and the approximate ranges of these values, which are expressed herein as "about". For example, when the value "10" is disclosed, "about 10" is also included. It should also be understood that when a value is disclosed as "less than or equal to" a certain value, it also includes "greater than or equal to" a certain value and the possible range between the values, as appropriately understood by those skilled in the art. For example, when the value "10" is disclosed, "less than or equal to 10" and "greater than or equal to 10" are also disclosed.

[0027] As used herein, the term "polynucleotide" refers to polydeoxyribonucleotides and may include unmodified DNA or modified DNA, and may be single-stranded or double-stranded DNA.

[0028] Herein, the term "heterologous" means an entity derived from a different genotype, relative to its comparison object or the rest of the target entity introduced or incorporated therein. For example, a polynucleotide introduced into different cell types by genetic engineering techniques is a heterologous polynucleotide (and can encode a heterologous polypeptide when expressed). Similarly, a cell sequence (e.g., a gene or its fragment) incorporated into a viral vector is a heterologous nucleotide sequence relative to a vector. As an example, when used in the context of nucleic acid sequences such as coding sequences and control sequences, "heterologous" can refer to sequences that are not usually combined together, and / or sequences that are not usually associated with each other under the common conditions present in nature. Another example of a heterologous coding sequence is a construct (e.g., having a synthetic sequence different from a natural gene codon) in which the coding sequence itself does not exist in nature. Similarly, for the purposes of this application, cells transformed using constructs that are not usually present in cells are considered to be heterologous. Herein, allelic variation or naturally occurring mutation events do not produce heterologous DNA.

[0029] Herein, "coding sequence" refers to a nucleic acid sequence that "encodes" a specific protein or a functional nucleotide such as siRNA. Under the control of appropriate regulatory sequences, the nucleic acid sequence in a polynucleotide is transcribed in vitro or in vivo (in the case of DNA) and translated (in the case of mRNA) into a polypeptide. The scope of the coding sequence is defined by the start codon at the 5' (amino) end and the translation stop codon at the 3' (carboxyl) end. The coding sequence may include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. The transcription termination sequence is usually located at the 3' of the coding sequence.

[0030] Herein, "coding region" refers to a region of genomic DNA that contains a gene coding sequence. 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 nucleotide sequence 32645-35835 of the Ad2 genome, while the adenovirus E2a coding region may include nucleotide sequence 22233-27575 of the Ad2 genome.

[0031] Herein, "modified coding region" refers to a coding region modified by deletion, substitution and / or insertion of one or more nucleotides. For example, the modified adenovirus E4 coding region may include the nucleotide sequence 32645-35835 of the Ad2 genome, but contains a deletion of one or more nucleotides in E4orf6 / 7 intron 1 and / or intron 2, and the modified adenovirus E2a coding region may include the nucleotide sequence 22233-27575 of the Ad2 genome, but contains a deletion of one or more nucleotides in late E2a intron 1 and / or intron 2.

[0032] The term "adenovirus E4 protein" refers to a protein that (1) is produced by the adenovirus E4 region and (2) is required for the production of AAV in host cells. The adenovirus E4 protein can be encoded by different open reading frames in different adenoviruses. Herein, the term "adenovirus E4 protein" encompasses functional homologs and functional equivalents of the adenovirus E4 protein. For example, the adenovirus E4 protein can be a protein encoded by the E4 open reading frame 6 / 7 (E4orf6 / 7) of Ad2 / 5 and its functional equivalents.

[0033] 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 chain displacement replication by unwinding the template in an ATP-independent manner. Herein, the term "adenovirus E2a protein" encompasses functional homologs and functional equivalents of the adenovirus E2a protein.

[0034] Herein, the term "adenovirus VA RNA" refers to a class of non-coding RNAs found in adenoviruses that are involved in translational regulation. VA RNA includes VAI and VAII. Herein, the term "adenovirus VA RNA" encompasses functional homologs and functional equivalents of adenovirus VA RNA.

[0035] The term "adenovirus VA RNA coding region" refers to the region encoding adenovirus VA RNA. A typical VA coding region includes nucleotide sequence 10426-11156 of the Ad2 genome (SEQ ID NO: 16).

[0036] The terms "regulatory elements," "regulatory sequences," and "regulatory regions" are used to describe promoters, enhancers, polyadenylation signals, transcription termination sequences, upstream regulatory domains, replication origins, internal ribosome entry sites ("IRES"), etc., which together provide for replication, transcription, and translation of coding sequences in recipient cells. Not all of these regulatory sequences must be present at the same time, as long as the selected coding sequence can be replicated, transcribed, and translated in appropriate host cells.

[0037] Herein, the terms "promoter" and "promoter region" refer to DNA regulatory sequences that bind to RNA polymerase and initiate transcription of downstream (3' direction) coding sequences. Further, these terms should be broadly understood to also cover other regulatory elements, including enhancer regions, intron splicing donors and acceptors, other 5' untranslated regions, etc. The promoter sequence may be homologous or heterologous to the target gene sequence. In the field of this application, there are various known promoters available for use, including a variety of viral and mammalian promoters. Cell type selective or tissue-specific promoters can be used for targeted editing or enhancement of gene sequence expression in a specific cell population relative to other cell populations. For example, suitable mammalian and viral promoters. Depending on the cell type, the promoter may exhibit constitutive activity, conditional activity, or inducible activity.

[0038] As used herein, the terms "enhancer" and "enhancer region" refer to polynucleotide sequences that stimulate transcription of a gene or coding sequence functionally connected to the promoter by regulating promoter activity in a cis position. Unlike promoters, the effect of enhancers is not affected by position and direction, so enhancers can be positioned in front of or behind the transcription unit, in introns, or even in the coding region. Enhancers can be positioned in the immediate vicinity of the transcription unit, or they can be kept at a significant distance from the promoter. Enhancers may also overlap with promoters physically and functionally. Those skilled in the art are familiar with various enhancers from different sources (and deposited in databases such as GenBank, for example, SV40 enhancers, CMV enhancers, polyoma enhancers, adenovirus enhancers), which can be used as independent elements or elements cloned in polynucleotide sequences (for example, deposited in ATCC or from commercial and personal sources). Many promoter sequences also contain enhancer sequences, such as the commonly used CMV promoter. For example, an inducible enhancer is a metallothionein enhancer that can be induced by glucocorticoids or heavy metals.

[0039] "Operably linked" refers to an arrangement of elements in which the described components are configured to perform their usual functions. Thus, a control sequence operably linked to a coding sequence is capable of effecting expression of the coding sequence. Operable linkage of a heterologous sequence to a promoter produces a chimeric gene. The control sequence need not be immediately adjacent to the coding sequence, as long as it is able to function to direct expression. Thus, for example, there may be intervening sequences that are untranslated but transcribed between a promoter sequence and a coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence.

[0040] For purposes of describing the relative position of a nucleotide sequence in a particular nucleic acid molecule throughout this application, for example, when a particular nucleotide sequence is described as being located "upstream," "downstream," "3'," or "5'" relative to another sequence, these modifiers should be understood to refer to the relevant sequence portion of the "sense" strand or "coding" strand of the DNA molecule, as is conventional practice in the art.

[0041] The term "transgene" refers to a polynucleotide that is introduced into a cell and can be transcribed into RNA, and selectively translated and / or expressed under appropriate conditions. The introduced transgene confers the desired properties to its target cell, or otherwise contributes to the desired therapeutic or diagnostic outcome. On the other hand, the transgene can be transcribed into a molecule that mediates RNA interference (such as miRNA, siRNA, shRNA) or a guide RNA for targeted editing of mutant alleles mediated by CRISPR / Cas9.

[0042] The term "expression" refers to the process by which nucleic acid (eg, DNA) is transcribed to produce RNA (eg, mRNA), which is subsequently processed and translated into a polypeptide.

[0043] The term "gene product" (also referred to herein as "gene expression product" or "expression product") encompasses products produced by gene expression, such as mRNA transcribed from a gene and polypeptides produced by translation of the mRNA. It should be understood that certain gene products may be processed or modified, for example, in a cell. For example, before translation of the mRNA, the RNA transcript may be spliced, polyadenylated, etc., and / or the polypeptide may be co-translationally or post-translationally processed, such as removing a secretion signal sequence, removing an organelle targeting sequence, or undergoing modifications such as phosphorylation or fatty acylation. The term "gene product" encompasses all of the above-mentioned processing or modification forms. Genomic nucleotide sequences, cDNA sequences, mRNA sequences, and polypeptide sequences of a variety of species, including humans, are known in the art and are available in publicly accessible databases such as the National Center for Biotechnology Information (NCBI, website: www.ncbi.nih.gov) or the UniProt database (Universal Protein Resource, website: www.uniprot.org). Databases include, for example, GenBank, RefSeq, Gene, UniProtKB / SwissProt, UniProtKB / Trembl, etc. Generally speaking, sequences in the NCBI reference sequence database (such as mRNA and polypeptide sequences) can be used as the gene product sequence of the target gene.

[0044] Herein, the term "modified protein" refers to a protein obtained by one or more modifications of a wild-type protein, such as, but not limited to, substitution, deletion and insertion of one or more amino acid residues, and post-transcriptional modifications, such as glycosylation.

[0045] Herein, the terms "transfection", "gene transfer" and "gene delivery" are used interchangeably herein and refer to a method or system for inserting exogenous nucleic acids into host cells. Gene transfer can result in transient expression of unintegrated transferred DNA, extrachromosomal replication, and expression of transfer replicons (e.g., episomes), or integration of the transferred genetic material into the genomic DNA of the host cell. Various techniques for introducing one or more exogenous nucleic acid molecules into suitable host cells are known to those of ordinary skill in the art, including chemical transfection, electrotransfection, and virus-mediated transfection methods.

[0046] As used herein, the term "host cell" generally refers to a cell that acts as a receptor for exogenously introduced nucleic acids or has been transfected with exogenous nucleic acids (e.g., bacterial cells, yeast cells, insect cells, mammalian cells). It should be understood that the progeny of a single parent cell may not be completely identical to the original parent in morphology, genome, or total DNA complement due to natural, accidental, or artificial mutations. A host cell may be a cell that has been genetically engineered to express a gene product of interest (e.g., to express one or more exogenous gene products after stable transformation). For example, it is a cell that stably expresses adenovirus E1a and E1b proteins (i.e., E1 expressing cells).

[0047] Herein, the term "cell line" refers to a cell population that is capable of sustained proliferation or long-term growth and division in vitro. Typically, a cell line is a clonal population derived from a single progenitor cell. It is further known in the art that spontaneous or induced karyotype changes may occur during storage or transfer of such clonal populations. Therefore, cells derived from the above cell lines may not be completely identical to the progenitor cells or original cultures, and the cell lines described herein include such variants.

[0048] If the nucleotide or amino acid residue in the first nucleic acid or protein performs one or more corresponding functions with the residue in the second nucleic acid or protein, and / or is in the corresponding position in the first and second nucleic acids or proteins, the two residues are considered to "correspond". Considering the environmental differences of the two nucleic acids or proteins, the corresponding functions are usually the same, equivalent or substantially equivalent functions. The residues in corresponding positions are usually aligned with each other in the following situations: using a sequence alignment algorithm or a computer program (for example, see the "identity" section below) to achieve maximum identity (allowing the introduction of vacancies) when aligning two nucleic acid or protein sequences, and / or the position is such that these residues overlap each other or occupy structurally equivalent positions when superimposing the three-dimensional structure of the protein, and / or form the same, equivalent or substantially equivalent intramolecular and / or intermolecular contacts or bonds (such as hydrogen bonds). The above structure can be determined, for example, by X-ray crystallography or NMR experiments, or predicted, for example, using structural prediction or molecular modeling software. The comparison can cover the entire length of one or more of the compared nucleic acid or polypeptide sequences, or cover at least one protein domain (or nucleotide sequence encoding a protein domain).

[0049] The term "expression cassette" is used herein to refer to a polynucleotide containing a gene of interest, which is operably linked to regulatory elements (also referred to as "regulatory sequences", "expression control elements" or "expression control sequences"), thereby mediating the expression of the coding region therein in an mRNA transcript that can be translated into a protein. The expression cassette can be included in or incorporated into a plasmid, a viral vector, or can exist as a nucleic acid fragment.

[0050] "Expression vector" refers to a special vector that contains a gene or nucleic acid sequence and its necessary regulatory regions required for expression in a host cell. Expression vectors include, for example, plasmids and viral vectors, such as adeno-associated virus (AAV) vectors, adenovirus vectors, lentivirus vectors, herpes virus vectors, etc.

[0051] As used herein, the term "viral vector" refers to a recombinant polynucleotide vector comprising nucleic acids of viral origin containing sequences that promote the replication and expression of exogenously incorporated transgene sequences operably linked to appropriate control elements, and one or more heterologous sequences (i.e., nucleic acid sequences of non-viral origin).

[0052] The term "recombinant virus" is used herein to refer to a virus that has been genetically altered, for example, by the addition or insertion of a heterologous nucleic acid construct into the viral particle.

[0053] The term "parvovirus" refers to a class of animal viruses that make up the Parvoviridae family. Parvoviruses have a linear single-stranded DNA (ssDNA) genome, typically containing two genes that encode a replication initiation protein (i.e., NS1) and a component protein of the viral capsid. The coding portion of the genome is flanked at each end by inverted terminal repeats (ITRs) that form important hairpin loops during replication. Compared to most viruses, parvovirus virions are smaller, with a diameter of 23-28 nanometers, and their genomes are encapsulated in an icosahedral capsid with a concave-convex surface. The Parvoviridae family is divided into three subfamilies, containing 126 species, including adeno-associated virus (AAV), bocavirus, and protoparvovirus.

[0054] The term "parvovirus protein coding region" refers to a region known in the art in the parvovirus genome that encodes nonstructural proteins (such as NS1) required for viral genome replication and structural proteins of the viral capsid. The region does not necessarily include all wild-type genes, but can be altered, for example, by insertion, deletion or substitution of nucleotides, provided that the existing nonstructural and structural proteins provide sufficient function to allow the production of the corresponding recombinant parvovirus in a suitable host cell at the following levels, the level of which reaches 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 supported by the parvovirus protein coding region encoding the corresponding wild-type nonstructural and structural proteins.

[0055] The term "sequence encoding a recombinant parvoviral genome" refers to a nucleotide sequence that encodes a sequence capable of producing a functional recombinant parvoviral genome in a suitable host cell. The sequence encoding the recombinant parvoviral genome generally comprises: (1) an expression cassette comprising a sequence encoding a gene of interest (GOI, also referred to as a "transgene") and a regulatory sequence operably linked to the sequence encoding the GOI; and (2) an ITR located at at least one end of the expression cassette. In some embodiments, the sequence encoding the recombinant parvoviral genome comprises an ITR located at each end of the expression cassette.

[0056] The term "sequence encoding a recombinant AAV genome" refers to a nucleotide sequence that encodes a sequence capable of producing a functional recombinant AAV genome in a suitable host cell. The sequence encoding the recombinant AAV genome generally comprises: (1) an expression cassette comprising a sequence encoding a GOI (also referred to as a "transgene") and a regulatory sequence operably linked to the sequence encoding the GOI; and (2) an ITR located at at least one end of the expression cassette. In some embodiments, the sequence encoding the recombinant AAV genome comprises an ITR located at each end of the expression cassette.

[0057] In the context of this application, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 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 any other AAV serotypes and variants now known or later discovered.

[0058] The terms "AAV cis construct" and "AAV vector" are used interchangeably herein and refer to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences of non-AAV origin) flanked by at least one AAV ITR. If the host cell in which such an AAV vector is located has been infected with a suitable helper virus (or is expressing a suitable helper function) and is expressing AAV rep and Cap gene products (i.e., AAV Rep and Cap proteins), it can be replicated and packaged into infectious viral particles. When the AAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in other vectors such as plasmids for cloning or transfection), the AAV vector can be referred to as a "pro-vector" and can be "recovered" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. The AAV vector can take any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, or encapsulated in viral particles (e.g., AAV particles). The AAV vector can be packaged into an AAV viral capsid to generate a "recombinant adeno-associated viral particle (AAV particle)".

[0059] "AAV cis constructs" or "AAV vectors" 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 other adeno-associated virus serotypes described herein. The AAV vector may have one or more AAV wild-type genes that are completely or partially deleted, preferably the rep and / or Cap genes are deleted, but functional flanking ITR sequences are retained. Functional ITR sequences are essential for the recovery, replication and packaging of AAV virus particles. Therefore, AAV cis polynucleotides or AAV vectors are defined herein as including at least the cis sequences (e.g., functional ITRs) required for replication and packaging of the virus. ITRs are not necessarily wild-type nucleotide sequences and can be altered by, for example, insertion, deletion or substitution of nucleotides, as long as the sequence provides functional recovery, replication and packaging.

[0060] The term "AAV vector" also includes self-complementary adeno-associated virus (scAAV) vectors. scAAV vectors are viral vectors that are genetically modified from naturally occurring AAV. scAAV is called "self-complementary" because its coding region has been designed to form an intramolecular double-stranded DNA template. Since the typical AAV genome is a single-stranded DNA template, the rate-limiting step during the replication of the standard AAV genome involves second-strand synthesis. However, this is not the case with the scAAV genome. After infection, instead of waiting for cell-mediated second-strand synthesis, two complementary scAAV half molecules will combine to form a double-stranded DNA (dsDNA) unit that can be replicated and transcribed immediately.

[0061] The terms "recombinant AAV virion", "rAAV virion" and "rAAV virus particle" are used interchangeably herein and refer to an infectious, replication-defective virus particle, which consists of at least one AAV capsid protein and an encapsulated rAAV vector genome comprising a heterologous nucleotide sequence of interest flanked by AAV ITRs. rAAV virus particles are produced in suitable host cells comprising an AAV vector, AAV helper functions and accessory functions. Host cells containing these components are capable of encoding AAV polypeptides, which are required for packaging the AAV vector (containing the recombinant nucleotide sequence of interest) into infectious recombinant virus particle particles for subsequent gene delivery.

[0062] The term "adeno-associated virus inverted terminal repeat sequence" or "AAV ITR" refers to regions located at each end of the AAV genome known in the art, which serve as the starting point of DNA replication in cis and as a packaging signal for the viral genome. The AAV ITR works together with the AAV rep coding region to achieve efficient excision and recovery, and integrates the nucleotide sequence inserted between the two flanking ITRs into the mammalian cell genome. The nucleotide sequences of the AAV ITR region are well known in the art. Herein, "AAV ITR" is not required to have the wild-type nucleotide sequence depicted in the previously cited references, but can be changed, for example, by insertion, deletion or substitution of nucleotides. In addition, the AAV ITR can 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. Furthermore, the 5' and 3' ITRs flanking the selected nucleotide sequence in the AAV vector need not be identical, nor need they be derived from the same AAV serotype or isolate, as long as they function as intended, i.e., allowing excision and recovery of the sequence of interest from the host cell genome or vector, and allowing integration of the heterologous sequence into the recipient cell genome when the AAV Rep gene product is present in the cell.

[0063] The term "Rep coding region" refers to a region of a parvovirus known in the art to encode a parvovirus Rep protein. This term also includes functional homologs thereof, such as the human herpesvirus 6 (HHV-6) rep gene known to mediate AAV DNA replication, as well as rep genes in other parvoviruses such as bocavirus and proparvovirus.

[0064] The term "AAV Rep coding region" refers to a region of the AAV genome known in the art to encode viral replication proteins that are essential for inserting the viral genome into the host genome during replication of the viral genome and latent infection. Herein, the Rep coding region can be derived from any viral serotype, including the AAV serotypes described above.

[0065] The region need not include all of the wild-type gene, but may be altered, for example, by insertion, deletion or substitution of nucleotides, as long as the altered existing rep gene provides adequate integration function when expressed in a suitable host cell. An altered Rep gene will be considered a "functional variant" encoding a wild-type Rep protein if the functional variant supports production of rAAV 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.

[0066] The term "long form of Rep" refers to the Rep 78 and Rep 68 gene products of the AAV Rep coding region, including their functional homologs. The long form of Rep is usually expressed by the AAV p5 promoter.

[0067] The phrase "short form of Rep" refers to the Rep 52 and Rep 40 gene products of the AAV Rep coding region, including their functional homologs. The short form of Rep is expressed by the AAV p19 promoter.

[0068] The term "Cap coding region" refers to the region of the parvoviral genome known in the art to encode viral capsid proteins, which are required for packaging of the viral genome.

[0069] The term "AAV Cap coding region" refers to a region known in the art in the AAV genome that encodes viral capsid proteins (e.g., VP1, VP2, and VP3) that are required for viral genome packaging. Herein, the AAV Cap coding region can be derived from any AAV serotype as described above. When present in a host cell together with an AAV vector, the region does not need to include all wild-type Cap genes, but can be altered by, for example, insertion, deletion, or substitution of nucleotides, as long as the altered Cap gene provides sufficient packaging function. If the functional variant supports the production of rAAV 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, the altered Cap gene will be considered to encode a "functional variant" of the wild-type Cap protein.

[0070] The term "AAV helper function" refers to a coding sequence derived from AAV that can be expressed to provide AAV gene products, which in turn promote efficient AAV replication in trans. Therefore, AAV helper functions include the Rep region and the Cap region. Studies have shown that the Rep expression product has many functions, including: recognition, binding and cleavage of the AAV origin of DNA replication; DNA helicase activity; and transcriptional regulation from the AAV (or other heterologous) promoter. The Cap expression product provides the necessary packaging function. AAV helper functions are used herein to supplement the trans-AAV functions missing in AAV vectors.

[0071] The term "AAV trans-construct" or "AAV helper construct" refers to a nucleic acid molecule that includes a nucleotide sequence that provides AAV functions that have been deleted in an AAV vector that will be used to produce a transduction vector that delivers a nucleotide sequence of interest. AAV trans-constructs are typically used to provide transient expression of AAV Rep and / or Cap genes to complement the missing AAV functions required for lytic AAV replication; however, trans-constructs lack AAV ITRs and are neither able to replicate nor self-package. AAV trans-constructs can exist in the form of plasmids, minicircles, phages, transposons, cosmids, viruses, or virions. Many AAV trans-constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode both Rep and Cap expression products. See, for example, Samulski et al. (1989) Journal of Virology, Vol. 63, pp. 3822-3828; McCarty et al. (1991) Journal of Virology, Vol. 65, pp. 2936-2945. In addition, many other vectors encoding Rep and / or Cap expression products have been described. See, for example, U.S. Pat. No. 5,139,941.

[0072] Herein, the term "adenovirus" has the same meaning as an adenoviral vector and refers to a member of the Adenoviridae family. The Adenoviridae family includes all animal adenoviruses of the genus Mastadenovirus. Specifically, human adenoviruses include subclasses AF and their respective serotypes. Subclasses AF 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.

[0073] The term "accessory function" refers to non-AAV-derived viral and / or cellular functions that AAV replication depends on. Thus, the term encompasses DNA, RNA, and proteins required for AAV replication, including those involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, AAV DNA replication, Cap expression product synthesis, and AAV capsid assembly. Virus-based accessory functions can be derived from any known helper virus, such as adenovirus, herpes virus (excluding herpes simplex virus type 1), and vaccinia virus.

[0074] Adenovirus-derived accessory functions have been extensively studied, and many adenovirus genes associated with accessory functions have been identified and partially characterized. Specifically, early adenovirus E1A, E1B 55K, E2A, E4, and VA RNA gene regions are believed to be involved in the accessory process.

[0075] "Functional homologs" or "functional equivalents" of a given adenoviral nucleotide region include similar regions derived from heterologous adenoviral serotypes, nucleotide regions derived from another virus or derived from a cellular source, and recombinantly produced or chemically synthesized polynucleotides that function in a manner similar to the reference nucleotide region to achieve the desired result. Thus, functional homologs of the adenoviral VA RNA gene region or the adenoviral E2A gene region encompass derivatives and analogs of such gene regions, including any addition, substitution and / or deletion of single or multiple nucleotide bases occurring within the gene region, provided that the homolog is still capable of providing its inherent accessory functions to support AAV virus particle production at a level that is detectable above background levels.

[0076] The term "percent identity" is used herein to describe comparisons between nucleic acid or amino acid sequences. Comparisons of nucleic acid and amino acid sequences are often performed using computer programs, such as the BLAST alignment program in the National Library of Medicine.

[0077] II. Compositions and Kits

[0078] One aspect of the present application relates to an expression construct for producing a recombinant parvovirus (eg, rAAV). The expression construct of the present application encodes and is capable of expressing adenovirus proteins to provide the accessory functions required for producing a recombinant parvovirus in a host cell.

[0079] In some embodiments, the expression constructs of the present application are capable of providing adenovirus helper functions, such as adenovirus E4, E2, VA RNA and / or E1 functions, and can be used in a three-plasmid system for recombinant parvovirus production.

[0080] In some embodiments, the expression construct of the present application comprises: (1) a sequence encoding an adenovirus gene that provides adenovirus helper function; and (2) a sequence encoding parvovirus non-structural proteins and structural proteins (such as AAV Rep protein and Cap protein), or a sequence encoding a recombinant parvovirus genome, and can be used in a dual-plasmid system for recombinant parvovirus production.

[0081] In some embodiments, the expression construct of the present application comprises: (1) a sequence encoding an adenoviral gene that provides adenoviral helper function; (2) a sequence encoding parvovirus nonstructural proteins and structural proteins (such as AAV Rep protein and Cap protein); and (3) a sequence encoding a recombinant parvovirus genome, and can be used in a single plasmid system for recombinant parvovirus production. Table 1 shows some exemplary expression constructs for rAAV production.

[0082] Table 1: Exemplary expression constructs for AAV production.

[0083]

[0084] In some embodiments, the expression construct of the present application further encodes functional adenovirus E1a and / or E1b proteins. In some embodiments, the expression construct of the present application does not encode functional adenovirus E1a and / or E1b proteins.

[0085] Expression constructs for the three-plasmid parvovirus production system

[0086] In some embodiments, the expression constructs of the present application are designed for use in a three-plasmid parvoviral production system. Figure 7Exemplary constructs for a three-plasmid AAV production system are shown. The expression constructs contain a modified adenoviral E4 coding region or a modified adenoviral E2a coding region and are capable of expressing one or more adenoviral E4 proteins or adenoviral E2a proteins that are required for AAV production in a host cell.

[0087] In some embodiments, the expression construct comprises both a modified adenoviral E4 coding region and a modified adenoviral E2a coding region and is capable of expressing one or more adenoviral E4 proteins and adenoviral E2a proteins to provide the necessary adenoviral helper functions for production of the recombinant parvovirus in a suitable host cell.

[0088] In some embodiments, the expression construct comprises a modified adenoviral E4 coding region, a modified adenoviral E2a coding region, and sequences encoding one or more adenovirus-associated RNAs (VA RNAs), and is capable of expressing the encoded adenoviral proteins and RNAs to provide the necessary adenoviral helper functions for the production of recombinant parvovirus in a suitable host cell. In some embodiments, the expression construct does not encode functional adenoviral E1a and / or E1b proteins, and the host cell provides E1a and / or E1b helper functions.

[0089] In some embodiments, the expression construct comprises a modified adenoviral E4 coding region, a modified adenoviral 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 adenoviral proteins, VA RNAs, and AAV Rep proteins for production of recombinant AAV in a suitable host cell.

[0090] Modified adenovirus E4 coding region

[0091] The modified adenovirus E4 coding region encodes a functional E4 protein capable of supporting parvovirus replication in a host cell. In some embodiments, the functional E4 protein may only contain amino acids encoded by adenovirus E4 open reading frames 6 and 7 (E4orf6 / 7), because only the amino acids encoded by E4orf6 / 7 are required for activities necessary to support parvovirus replication. In some embodiments, the functional E4 protein comprises a polypeptide sequence encoded by all or most of E4orf6 / 7, and does not contain a polypeptide sequence encoded by all or part of E4orf 1-4 and E4 34K proteins.

[0092] In some embodiments, the modified adenoviral E4 coding region comprises one or more deletions. In some embodiments, the modified adenoviral E4 coding region comprises a partial or complete deletion of E4orf6 / 7 intron 1.

[0093] In some embodiments, the modified adenoviral E4 coding region comprises a partial deletion of E4orf6 / 7 intron 1, wherein up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the E4orf6 / 7 intron 1 sequence is deleted.

[0094] In some embodiments, the modified adenoviral E4 coding region comprises a partial or complete deletion of E4orf6 / 7 intron 2. In some embodiments, the modified adenoviral E4 coding region comprises a partial deletion of E4orf6 / 7 intron 2, wherein up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the E4orf6 / 7 intron 2 sequence is deleted.

[0095] In some embodiments, the modified adenoviral 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.

[0096] In some embodiments, the total size of the modified adenoviral E4 coding region is 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 is capable of providing the E4 functions required for production of parvovirus in a non-E4 expressing host cell.

[0097] In some embodiments, the modified adenovirus E4 coding region comprises a sequence corresponding to nucleotide sequence 32645-35835 of the Ad2 genome, comprising: (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).

[0098] In some embodiments, the modified adenoviral E4 coding region comprises a sequence corresponding to nucleotide sequence 32645-35835 of the Ad2 genome, which lacks the entire sequence of E4orf6 / 7 intron 2 (711 bp, SEQ ID NO: 2) and lacks the entire sequence of E4orf6 / 7 intron 1 (1275 bp, SEQ ID NO: 1). In some embodiments, the modified adenoviral E4orf6 / 7 coding region comprises a codon-optimized nucleotide sequence (SEQ ID NO: 7) encoding an adenoviral E4orf6 / 7 protein (SEQ ID NO: 8).

[0099] Modified adenovirus E2a coding region

[0100] In some embodiments, the modified adenoviral E2a coding region comprises: (a) a partial or complete deletion of early E2a or late E2a intron 1; (b) a partial or complete deletion of E2a intron 2; or both (a) and (b).

[0101] In some embodiments, the modified adenoviral E2a coding region comprises a partial deletion of late E2a intron 1 and / or late E2a intron 2, wherein at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of late E2a intron 1 and / or late E2a intron 2 is deleted.

[0102] In some embodiments, the total size of the modified adenoviral E2a coding region is 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 functions required for parvovirus production in a non-E2a expressing host cell.

[0103] In some embodiments, the modified adenoviral E2a coding region comprises a sequence corresponding to nucleotide sequence 22233-27575 of the Ad2 genome, the sequence comprising: (a) a partial or complete deletion of early E2a 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 adenoviral E2a coding region comprises a sequence corresponding to nucleotide sequence 22233-27575 of the Ad2 genome, the sequence having a 1897 bp deletion (SEQ ID NO: 3), the deletion encompassing the entire sequence of late E2a intron 1 and late E2a intron 2. In some embodiments, the modified adenoviral E2a coding region comprises a partial deletion of late E2a intron 1 and / or late E2a intron 2, wherein at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of SEQ ID NO: 3 is deleted.

[0104] In some embodiments, the modified adenoviral E2a coding region comprises: (a) (codon-optimized adenoviral E2a protein coding sequence) a partial or complete deletion of late E2a intron 1; (b) a partial or complete deletion of late E2a intron 2; and (c) a codon-modified nucleotide sequence encoding an adenoviral E2a protein.

[0105] Adenovirus VA RNA coding region

[0106] In some embodiments, the expression construct further comprises an adenoviral VA RNA coding region encoding one or more adenoviral VA RNAs. In some embodiments, the expression construct comprises an adenoviral VA RNA coding region encoding an adenoviral VAI RNA and / or VAII RNA. In some embodiments, the VA RNA coding region encodes functional VA RNA I and / or VA RNAII, which are capable of providing the VA RNA function required for producing parvovirus in a non-VA RNA expressing host cell. In some embodiments, the expression construct comprises SEQ ID NO: 16.

[0107] Here and hereinafter, a "functional" adenoviral E4 protein, adenoviral E2a protein, or adenoviral VA RNA I or VA RNA II is capable of promoting the production of a recombinant parvovirus, such as a bocavirus. It is within the capabilities of a skilled artisan to determine whether a given VA RNA I and II, E2A protein, or E4 protein is functional. In one embodiment, an adenoviral E4 protein, adenoviral E2a protein, or adenoviral VA RNA I or VA RNA II is considered "functional" if it is capable of supporting parvovirus production at a level that 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 in the same three-plasmid, two-plasmid, or single-plasmid system.

[0108] Adenoviruses and nucleic acid fragments thereof for use in the compositions and methods of the present application include all human adenoviruses of the family Adenoviridae, including members of the genus Adenovirus of mammals. To date, more than fifty-one human adenovirus serotypes have been identified. Adenoviruses may belong to serogroups A, B, C, D, E, or F. Human adenoviruses may be serotype 1 (Ad 1), 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 a combination thereof, but are not limited to these examples. In certain embodiments, the adenovirus is serotype 5 (Ad5).

[0109] Regulatory elements

[0110] In some embodiments, the expression construct of the present application comprises one or more regulatory elements that control the expression of the encoded adenoviral protein and / or RNA. For example, exemplary promoters include viral promoters, mammalian promoters, composite promoters, cell type-specific or tissue-specific promoters, and inducible / repressible promoters.

[0111] For example, viral promoters include SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), adenovirus E4 promoter, adenovirus e2a promoter, herpes simplex virus (HSV) promoter, CMV immediate early promoter region (CMV-IE) and other cytomegalovirus (CMV) promoters, SFFV promoters and Rous Sarcoma Virus (RSV) promoters.

[0112] For example, mammalian promoters include promoters for expression of EF1α, ubiquitin (e.g., ubiquitin B or C), globin, actin, phosphoglycerate kinase (PGK), NSE (neuron-specific enolase), synapsin, or NeuN, as well as composite promoters such as the CAG promoter (a combination of the CMV early enhancer element and the chicken β-actin promoter).

[0113] The promoter can be human, or from other species, including mice. In some embodiments, a human promoter can be used. In some embodiments, a promoter ("pol II promoter") or a functional variant thereof that is usually mediated by eukaryotic RNA polymerase II is used. In some embodiments, a promoter that is usually mediated by eukaryotic RNA polymerase I, such as a promoter for transcribing ribosomal RNA (except 5S rRNA), or a functional variant thereof can be used. In some embodiments, a promoter that is usually mediated by eukaryotic RNA polymerase III ("pol III promoter", such as U6, H1, 7SK or tRNA promoter, or a functional variant thereof can be used.

[0114] In some embodiments, the modified adenovirus E4 coding region in the expression construct of the present application is operably linked to a regulatory element (such as a promoter, an enhancer, or both). In some embodiments, the modified adenovirus E4 coding region is operably linked to an adenovirus E4 promoter. In some embodiments, the modified adenovirus E4 coding region is operably linked to an adenovirus E4 promoter comprising SEQ ID NO: 22.

[0115] In some embodiments, the modified adenoviral E2a coding region in the expression construct of the present application is operably linked to a regulatory element (such as a promoter, an enhancer, or both). In some embodiments, the modified adenoviral E2a coding region is operably linked to an adenoviral E2a promoter. In some embodiments, the modified adenoviral E2a coding region is operably linked to an adenoviral E2a promoter comprising SEQ ID NO: 23.

[0116] Codon-optimized coding sequence

[0117] In certain embodiments, the expression construct of the present application includes one or more polynucleotides, and the one or more polynucleotides encode one or more codon-optimized proteins for expression in, for example, humans, mammals or primate cells (such as HuH7, HEK293T or CHO cells). Codon optimization can be performed on the polynucleotides encoding the protein of the present application to improve activity, stability or expression in host cells without changing the encoded amino acid sequence. Codon optimization replaces the codons present in the polynucleotide sequence with preferred codons encoding the same amino acids, for example, preferably codons for mammalian expression. Therefore, the amino acid sequence will not change in the process. Codon optimization can be performed using gene optimization software. The codon-optimized nucleotide sequence is translated and compared with the original protein sequence to ensure that the amino acid sequence does not change. Codon optimization methods are known in the art and are described, for example, in U.S. Application Publication No. 2008 / 0194511 and U.S. Patent No. 6,114,148.

[0118] In some embodiments, the expression construct of the present application comprises (a) a modified adenoviral E4 coding region comprising a codon-modified E4 coding sequence, or (b) a modified adenoviral E2a coding region comprising a codon-modified E2a coding sequence.

[0119] In some embodiments, the expression construct of the present application comprises (a) a modified adenoviral E4 coding region comprising a codon-modified E4 coding sequence, and (b) a modified adenoviral E2a coding region comprising a codon-modified E2a coding sequence.

[0120] In some embodiments, the expression construct of the present application 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).

[0121] Other components

[0122] In some embodiments, the expression construct of the present application further comprises sequences encoding adenovirus E1a and E1b proteins.

[0123] In some embodiments, the expression construct further comprises one or more other elements, such as a plasmid replication initiation sequence and a selection marker. Examples of selection markers include, but are not limited to, ampicillin resistance genes, neomycin resistance genes, and kanamycin resistance genes.

[0124] In some embodiments, the size of the expression construct designed for the three-plasmid parvovirus production system is 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 size of the expression construct is 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.

[0125] In some embodiments, the expression construct designed for the three-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 is less than 13 kb, 12 kb, 11 kb, 10 kb, 9 kb, 8 kb, 7 kb, 6 kb, 5 kb, or 4 kb in size. In some embodiments, the size of the expression construct is 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.

[0126] Preferred embodiments of expression constructs for use in the three-plasmid parvoviral production system

[0127] In some embodiments, the expression construct is a plasmid for three-plasmid 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), and a regulatory region (comprising SEQ ID NO: 19), wherein the size of the plasmid is 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 size of the plasmid is 7-9 kb.

[0128] Expression constructs for dual-plasmid parvovirus production systems

[0129] In some embodiments, the expression constructs of the present application are designed for use in a dual-plasmid parvoviral production system. Figure 7 Two exemplary two-plasmid systems for AAV production are shown.

[0130] Constructs encoding adenoviral and parvoviral proteins

[0131] In some embodiments, the expression construct for dual-plasmid parvovirus production comprises not only one or more of the elements described in the expression construct for the three-plasmid parvovirus production system, but also comprises a parvovirus protein coding region. In some embodiments, the parvovirus coding region comprises a sequence encompassing the AAV Rep coding region and a sequence encompassing the AAV Cap coding region.

[0132] In some embodiments, the expression construct for dual-plasmid parvovirus production is constructed for AAV production and comprises: (1) a modified adenovirus E4 coding region encoding E4orf6 / 7; (2) a modified adenovirus E2a coding region encoding 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 allow expression of E4orf6 / 7, E2a protein, adenovirus VA RNA, AAV Rep protein, and AAV Cap protein in a host cell. Figure 7 As shown, such expression constructs can be used in combination with another expression vector encoding a recombinant AAV genome (e.g., a transgenic expression cassette with at least one ITR connected to one side) for producing recombinant AAV in a host cell. In some embodiments, the AAV Rep coding region is operably connected to one or more regulatory elements (e.g., AAV P5 promoter or AAV P5I promoter). In some embodiments, the AAV Rep coding region is operably connected to one or more regulatory elements (e.g., AAV P19 promoter). In some embodiments, the regulatory element comprises a P5I promoter (SEQ ID NO: 15). In some embodiments, the expression construct for dual-plasmid AAV production further comprises a sequence encoding adenovirus E1a and / or E1b protein. In some embodiments, the expression construct for generating dual-plasmid AAV does not comprise a sequence encoding adenovirus E1a and / or E1b protein. In some embodiments, the size of the expression construct is less than 15kb, 14kb, 13kb, 12kb, 11kb, 10kb, 9kb, 8kb, or 7kb. In some embodiments, the size of the expression construct ranges from 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.

[0133] Constructs encoding adenoviral proteins and recombinant parvoviral genomes

[0134] In some embodiments, the expression construct for the dual-plasmid parvovirus production system comprises: (1) a modified adenoviral E4 coding region encoding E4 open reading frame 6 / 7; (2) a modified adenoviral E2a coding region encoding E2a protein; (3) a sequence encoding adenoviral VA RNA; (4) a sequence encoding a recombinant parvovirus genome (e.g., a transgenic expression cassette with at least one ITR linked to one side); and (5) one or more regulatory elements that allow expression of E4 open reading frame 6 / 7, E2a protein, and adenoviral VA RNA in a host cell.

[0135] In some embodiments, an expression construct designed for a dual-plasmid AAV production system comprises: (1) a modified adenoviral E4 coding region encoding E4 open reading frame 6 / 7; (2) a modified adenoviral E2a coding region encoding E2a protein; (3) a sequence encoding adenoviral VA RNA; (4) a sequence encoding a recombinant AAV genome (e.g., a transgenic expression cassette flanked by two ITRs); and (5) one or more regulatory elements that allow expression of E4 open reading frame 6 / 7, E2a protein, and adenoviral VA RNA in a host cell. Figure 7 As shown, such expression constructs can be used in combination with another expression vector encoding AAV Rep and Cap proteins for producing recombinant AAV in host cells. In some embodiments, the expression construct for dual-plasmid AAV production further comprises sequences encoding adenovirus E1a and E1b proteins. In some embodiments, the size of the expression construct is 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 size of the expression construct ranges from 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.

[0136] Preferred embodiments of expression constructs for dual-plasmid AAV production systems

[0137] 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 an AAV Rep protein (comprising SEQ ID NO: 20), and a sequence encoding an AAV Cap protein (comprising SEQ ID NO: 21), wherein the size of the plasmid is in the range of 12-14 kb.

[0138] 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), and a sequence encoding a recombinant AAV genome, wherein the recombinant AAV genome comprises a transgene expression cassette (comprising a transgene of interest operably linked to one or more regulatory elements) and two AAV ITRs respectively located on either side of the transgene expression cassette, wherein the size of the plasmid is in the range of 10-14 kb.

[0139] Expression constructs for single-plasmid parvovirus production systems

[0140] In some embodiments, the expression construct of the present application is designed for a single plasmid parvovirus production system. The expression construct contains any or all of the aforementioned modified adenovirus coding region, parvovirus protein coding region, and a sequence encoding a recombinant parvovirus genome, wherein the recombinant parvovirus genome comprises an expression cassette having a GOI operably connected to a regulatory element and at least one ITR located at one end of the expression cassette. The expression construct allows the production of recombinant parvovirus by transfecting a host cell with a single plasmid. In some embodiments, the expression construct does not provide adenovirus E1a and E1b functions, and is required for producing parvovirus in a host cell that provides endogenous expression of adenovirus E1a and E1b proteins. In some embodiments, the expression construct also provides adenovirus E1a and E1b functions, and can be used to produce parvovirus in a host cell that does not provide endogenous expression of adenovirus E1a and E1b proteins.

[0141] Figure 7Exemplary embodiments of expression constructs for use in a single plasmid AAV production system are provided. 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 E2a protein; (3) an adenovirus VA RNA coding region; (4) an AAV Rep coding region; (5) an AAV Cap coding region; (6) a sequence encoding a recombinant AAV genome, the sequence comprising an expression cassette and at least one ITR located at one end of the expression cassette, the expression cassette having a nucleotide sequence encoding a transgene and a regulatory element operably linked to the nucleotide sequence; and (7) one or more regulatory elements that allow 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 in the recombinant AAV genome is flanked by AAV ITRs on each end.

[0142] In some embodiments, the expression construct of the present application comprises: (1) a modified adenovirus E4 coding region encoding E4 open reading frame 6 / 7; (2) a modified adenovirus E2a coding region encoding 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 allow expression of E4 open reading frame 6 / 7, E2a protein, adenovirus VA RNA and parvovirus protein in a host cell.

[0143] In some embodiments, the recombinant parvoviral cloning region comprises a cloning site for inserting a recombinant parvoviral sequence comprising: (1) an expression cassette comprising a gene of interest (GOI) operably linked to a regulatory element; and (2) at least one ITR located on one side of the expression cassette. In some embodiments, the recombinant parvoviral sequence comprises two ITRs, one at each end of the expression cassette.

[0144] In some embodiments, the recombinant parvoviral cloning region comprises: (1) a cloning site for inserting a sequence comprising an expression cassette comprising a gene of interest (GOI) operably linked to regulatory elements; and (2) at least one ITR on either side of the cloning site. In some embodiments, the recombinant parvoviral sequence comprises two ITRs, one on each side of the cloning site.

[0145] In some embodiments, the recombinant parvoviral cloning region comprises: (1) an expression cassette comprising a cloning site for inserting a GOI and a regulatory element proximal to the cloning site and capable of being operably linked to the GOI when the GOI is inserted at the cloning site; and (2) at least one ITR located on one side of the expression cassette. In some embodiments, the recombinant parvoviral sequence comprises two ITRs, one on each side of the expression cassette.

[0146] In some embodiments, the modified adenoviral E4 coding region encodes E4orf6 / 7, and E4orf6 / 7 intron 1 is partially or completely deleted. In some embodiments, the modified adenoviral E4 coding region encodes E4orf6 / 7, and E4orf6 / 7 intron 2 is partially or completely deleted. In some embodiments, the modified adenoviral E4 coding region encodes E4orf6 / 7, and both E4orf6 / 7 intron 1 and E4orf6 / 7 intron 2 are partially or completely deleted.

[0147] In some embodiments, the total size of the modified adenoviral E4 coding region is 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 is capable of providing the E4 functions required for production of parvovirus in a non-E4 expressing host cell.

[0148] In some embodiments, the modified adenovirus E4 coding region comprises a sequence corresponding to nucleotide sequence 32645-35835 of the Ad2 genome, wherein: (a) E4orf6 / 7 intron 1 is partially or completely deleted; (b) E4orf6 / 7 intron 2 is partially or completely deleted; or both (a) and (b).

[0149] In some embodiments, the modified adenovirus E4 coding region comprises a sequence corresponding to nucleotide sequence 32645-35835 of the Ad2 genome, wherein the entire sequence of E4orf6 / 7 intron 2 is deleted (711 bp, SEQ ID NO: 2) and the entire sequence of E4orf6 / 7 intron 1 is deleted (1275 bp, SEQ ID NO: 1).

[0150] In some embodiments, the modified adenoviral E4 coding region comprises: a partial deletion of E4orf6 / 7 intron 1, wherein at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of SEQ ID NO: 1 is deleted; and / or a partial deletion of E4orf6 / 7 intron 2, wherein at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of SEQ ID NO: 2 is deleted.

[0151] In some embodiments, the modified adenoviral E4orf6 / 7 coding region comprises a codon-optimized nucleotide sequence (SEQ ID NO: 7) encoding an adenoviral E4orf6 / 7 protein (SEQ ID NO: 8).

[0152] In some embodiments, the modified adenoviral E4 coding region encodes Ad2 E4orf6 / 7. In some embodiments, the modified adenoviral E4 coding region comprises SEQ ID NO: 17. In some embodiments, the modified adenoviral E4 coding region comprises SEQ ID NO: 18.

[0153] In some embodiments, the modified adenoviral E2a coding region contains a codon-modified or unmodified sequence encoding an adenoviral E2a protein in which the early E2a intron 1 is partially or completely deleted.

[0154] In some embodiments, the modified adenoviral E2a coding region contains a codon-modified or unmodified sequence encoding an adenoviral E2a protein in which the late E2a intron 1 is partially or completely deleted.

[0155] In some embodiments, the modified adenoviral E2a coding region contains a codon-modified or unmodified sequence encoding an adenoviral E2a protein, wherein E2a intron 2 is partially or completely deleted.

[0156] In some embodiments, the modified adenoviral E2a coding region contains a codon-modified or unmodified sequence encoding an adenoviral E2a protein in which early E2a intron 1 is partially or completely deleted and E2a intron 2 is partially or completely deleted.

[0157] In some embodiments, the modified adenoviral E2a coding region contains a codon-modified or unmodified sequence encoding an adenoviral E2a protein in which late E2a intron 1 is partially or completely deleted and E2a intron 2 is partially or completely deleted.

[0158] In some embodiments, the modified adenoviral E2a coding region comprises a partial deletion of late E2a intron 1 and / or late E2a intron 2, wherein at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of late E2a intron 1 and / or late E2a intron 2 is deleted.

[0159] In some embodiments, the total size of the modified adenoviral E2a coding region is 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 functions required for parvovirus production in a non-E2a expressing host cell.

[0160] In some embodiments, the modified adenoviral E2a coding region comprises a sequence corresponding to nucleotide sequence 22233-27575 of the Ad2 genome, wherein: (a) early E2a or late E2a intron 1 is partially or completely deleted; (b) E2a intron 2 is partially or completely deleted; or both (a) and (b). In some embodiments, the modified adenoviral E2a coding region comprises a sequence corresponding to nucleotide sequence 22233-27575 of the Ad2 genome, the sequence having a 1897 bp deletion (SEQ ID NO: 3), the deletion encompassing the entire sequence of late E2a intron 1 and late E2a intron 2. In some embodiments, the modified adenoviral E2a coding region comprises a partial deletion of late E2a intron 1 and / or late E2a intron 2, wherein at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of SEQ ID NO: 3 is deleted.

[0161] In some embodiments, the modified adenoviral E2a coding region comprises: (a) (codon-optimized adenoviral E2a protein coding sequence) a partial or complete deletion of late E2a intron 1; (b) a partial or complete deletion of late E2a intron 2; and (c) a codon-modified nucleotide sequence encoding an adenoviral E2a protein.

[0162] In some embodiments, the modified adenovirus E2a coding region comprises SEQ ID NO:4.

[0163] In some embodiments, the adenovirus VA RNA coding region comprises a sequence encoding adenovirus VAI, adenovirus VAII, or both. In some embodiments, the adenovirus VA RNA coding region comprises a sequence encoding Ad2 VAI, Ad2 VAII, or both. In some embodiments, the adenovirus VA RNA coding region comprises SEQ ID NO: 16.

[0164] In some embodiments, the AAV Rep coding region comprises SEQ ID NO:20.

[0165] In some embodiments, the AAV Cap coding region comprises SEQ ID NO:21.

[0166] In some embodiments, the one or more regulatory elements comprise an adenoviral E4 promoter operably linked to a modified adenoviral E4 coding region, an adenoviral E2a promoter operably linked to a modified adenoviral E2a coding region, and a modified AAV P5 promoter operably linked to sequences encoding AAV Rep and Cap proteins. In some embodiments, the adenoviral E4 promoter comprises SEQ ID NO: 22. In some embodiments, the adenoviral E2a promoter comprises SEQ ID NO: 23. In some embodiments, the modified AAV P5 promoter comprises SEQ ID NO: 16.

[0167] In some embodiments, the expression construct further comprises a nucleotide sequence encoding an adenovirus Ela protein and / or a nucleotide sequence encoding an adenovirus Elb protein.

[0168] In some embodiments, the size of an expression construct designed for use in a single plasmid parvoviral production system is 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.

[0169] In some embodiments, the size of the expression construct designed for use in the single plasmid parvovirus production system ranges from 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.

[0170] 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.

[0171] Preferred embodiments of expression constructs for single-plasmid AAV production systems

[0172] In some embodiments, the expression construct is a plasmid for producing a single-plasmid AAV and 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), an AAV Rep coding region (comprising SEQ ID NO: 20), an AAV Cap coding region (comprising SEQ ID NO: 21), and a sequence encoding a recombinant AAV genome, wherein the size of the expression construct is in the range of 7-19 kb.

[0173] In another embodiment, the present application provides one or more cells, each containing one or more expression constructs described herein. In another embodiment, the present application provides one or more cell lines, each cell line is stably transformed, comprising one or more expression constructs described herein.

[0174] On the other hand, the present application provides a kit for producing recombinant AAV particles. In one embodiment, the kit comprises a plasmid, and the plasmid comprises the expression construct of the present application.

[0175] In a specific embodiment, the kit also includes instructions for use, such as instructions for use of the methods described herein. In some embodiments, the kit further includes 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.

[0176] III. Methods for producing recombinant parvoviruses

[0177] Another aspect of the present application relates to a method for producing recombinant parvovirus particles using the expression construct of the present application. The method comprises the following steps: introducing the expression construct of the present application into a host cell that provides the necessary auxiliary functions for parvovirus production; incubating the host cell carrying the expression cassette for a desired period of time to produce recombinant parvovirus particles; and collecting the recombinant parvovirus particles after the incubation period. In some embodiments, the parvovirus is AAV.

[0178] In some embodiments, the method comprises the following steps: (1) introducing the following constructs into a host cell: (A) an expression construct of a three-plasmid parvovirus production system; (B) a parvovirus trans construct capable of expressing parvovirus proteins necessary for producing recombinant parvovirus; and (C) a parvovirus cis construct containing a sequence encoding a recombinant parvovirus genome; (2) incubating the host cells carrying the three constructs for a desired period of time to produce recombinant parvovirus particles; and (3) harvesting 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 generally referred to as a triple transfection method. In some embodiments, the parvovirus is AAV, and the trans construct comprises an AAV Rep coding region and an AAV Cap coding region.

[0179] In some embodiments, the method comprises the following steps: (1) introducing the following constructs into a host cell: (A) an expression construct of a dual plasmid parvovirus production system (Construct A), the expression construct containing a modified adenoviral E4 coding region encoding E4orf6 / 7, a modified adenoviral E2a coding region encoding E2a protein, an adenoviral VA RNA coding region, a parvovirus protein coding region, and one or more regulatory elements that allow expression of adenoviral E4orf6 / 7, E2a, VA RNA, and parvovirus proteins; and (B) an expression construct containing a sequence encoding a recombinant parvovirus genome (Construct B); (2) incubating the host cell carrying the construct for a desired period of time to produce recombinant parvovirus particles; and (3) harvesting the recombinant parvovirus particles after the incubation period. In certain 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 ranging from 1:10 to 10:1. In some embodiments, constructs A and B are co-transfected into the host cell at a molar ratio of A:B of 1:1, 1:2, 1:3, 2:1, or 3:1.

[0180] In some embodiments, the method comprises the following steps: (1) introducing the following constructs into a host cell: (A) an expression construct of a dual plasmid parvovirus production system (construct A), the expression construct containing 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 that allow expression of adenovirus E4orf6 / 7, E2a, VA RNA; and (B) an expression construct containing a parvovirus protein coding region (construct B); (2) incubating the host cell carrying the construct for a desired period of time to produce recombinant parvovirus particles; and (3) collecting 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 ranging from 1:10 to 10:1. In some embodiments, constructs A and B are co-transfected into the host cell at a molar ratio of A:B of 1:1, 1:2, 1:3, 2:1, or 3:1.

[0181] In some embodiments, the method comprises the following steps: (1) introducing an expression construct of a single plasmid parvoviral production system into a host cell; (2) incubating the host cell carrying the expression cassette for a desired period of time to produce recombinant parvoviral particles; and (3) harvesting the recombinant parvoviral particles after the incubation period. In some embodiments, the expression construct is a plasmid for producing recombinant AAV. In some embodiments, the plasmid does not encode adenoviral E1a and E1b proteins, and the host cell endogenously expresses adenoviral E1a and E1b proteins.

[0182] The host cell for producing the parvovirus particles may contain the expression construct of the present application, for example, in the form of an exon plasmid. The expression construct of the present application may also be stably integrated into the host cell genome. Further, the host cell may constitute an expression system for producing adenovirus proteins (such as E1a and E1b), which are required for producing the parvovirus. Examples of host cells include, but are not limited to, microorganisms, yeast cells, insect cells, and animal cells. In certain 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 implementing the present application include: 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 cell that has been transfected. It should be understood that the progeny of a single parent cell may not be exactly the same as the original parent in morphology, genome or total DNA complement due to natural, accidental or artificial mutations. A "mammalian host cell" is a cell originally derived from a mammal or a progeny cell thereof.

[0183] In one embodiment, the host cell expresses Ad E1a and E1b proteins. In certain preferred embodiments, the host cell is a HEK-293 cell expressing adenovirus E1a and E1b.

[0184] In some embodiments, the expression constructs of the present application and other expression constructs (if any) are transfected into HEK293 cells (available from HEK293 cells were then incubated for at least 60 hours to allow for the production of recombinant parvoviral particles.

[0185] Without further elaboration, it is believed that those skilled in the art can make full use of the present disclosure based on the above description. Therefore, the following specific examples should be considered as illustrative only and should not limit the rest of the present disclosure in any way. All documents cited herein are incorporated herein by reference for the purpose or subject reference.

[0186] IV. Kit

[0187] Another aspect of the present application relates to a kit for producing a recombinant parvovirus. In some embodiments, the kit comprises the expression construct of the present application and instructions for using the expression construct.

[0188] In some embodiments, the expression construct comprises: (1) a modified adenoviral E4 coding region encoding E4 open reading frame 6 / 7; (2) a modified adenoviral E2a coding region encoding E2a protein; (3) an adenoviral VA RNA coding region; (4) a parvoviral protein coding region; (5) a recombinant parvoviral cloning region; and (6) one or more regulatory elements that allow expression of E4 open reading frame 6 / 7, E2a protein, adenoviral VA RNA, and parvoviral protein in a host cell. In some embodiments, the recombinant parvoviral cloning region comprises a cloning site for inserting a recombinant parvoviral sequence comprising: (1) an expression cassette comprising a gene of interest (GOI) operably linked to regulatory elements; and (2) at least one ITR located on one side of the expression cassette. In some embodiments, the recombinant parvoviral sequence comprises two ITRs, one at each end of the expression cassette. In some embodiments, a recombinant parvoviral cloning region comprises: (1) a cloning site for inserting a sequence comprising an expression cassette comprising a gene of interest (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 parvoviral sequence comprises two ITRs, one on each side of the cloning site. In some embodiments, a recombinant parvoviral cloning region comprises: (1) an expression cassette comprising a cloning site for inserting a GOI and a regulatory element proximal to the cloning site and capable of being operably linked to the GOI when the GOI is inserted at the cloning site; and (2) at least one ITR on one side of the expression cassette. In some embodiments, the recombinant parvoviral sequence comprises two ITRs, one on each side of the expression cassette.

[0189] 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 E2a protein; (3) an adenovirus VA RNA coding region; (4) an AAV Rep coding region; (5) an AAV Cap coding region; (6) a sequence encoding a recombinant AAV genome, the sequence comprising an expression cassette and at least one ITR located at one end of the expression cassette, the expression cassette having a nucleotide sequence encoding a transgene and a regulatory element operably linked to the nucleotide sequence; and (7) one or more regulatory elements that allow 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 in the recombinant AAV genome is flanked on each end by an AAV ITR.

[0190] In some embodiments, the kit further comprises reagents for transfecting the expression construct.

[0191] Example

[0192] Example 1: Construction of AAV helper constructs

[0193] To investigate the omission of Ad sequences for AAV vector packaging, a series of Ad-based deletion mutants were constructed to reduce the size of the plasmid and increase the helper functions for the production of recombinant AAV viral particles. Figure 1 Depicted are a series of expression cassette arrangements for adenoviral (Ad2) coding regions and surrounding deletions relative to the coordinates of the Ad2 nucleotide sequence.

[0194] Figure 2 Shows Figure 1 Expanded diagram of the arrangement of the Ad E4orf and surrounding deletion relative to the Ad2 genomic sequence depicted in FIG.

[0195] Figure 3 An expanded diagram depicting the expression of the Ad2 E2a promoter and Ad2 mRNA during early and late stages of infection is shown, including Figure 1 The surrounding deletions are depicted relative to the Ad genome sequence.

[0196] Figure 4 An exemplary helper plasmid containing an embodiment of the expression cassette of the present application, i.e., the mini-pHelper plasmid (SEQ ID NO: 6), is shown. The plasmid contains Figure 1 The mini-pHelper Ad helper gene arrangement is shown. The mini-pHelper backbone plasmid includes a codon-optimized Ad E2a coding region sequence (SEQ ID NO:4), which encodes the wild-type Ad E2a amino acid sequence shown in SEQ ID NO:5.

[0197] Figure 5 Another exemplary helper plasmid containing another embodiment of the expression cassette of the present application, mini-pHelper-1.0 plasmid (SEQ ID NO: 10) is shown. The plasmid contains Figure 1 The mini-pHelper Ad helper gene arrangement is shown.

[0198] Example 2: Identification of adenoviral regions that can be omitted in AAV production

[0199] Figure 6 Shows Figure 1The effect of deletions or deletion combinations on the ability of the expression cassette to adequately provide the helper functions required for adeno-associated virus (AAV) packaging. rAAV production titers were obtained using a three-plasmid transfection system that included: a plasmid containing ITRs (e.g., pAAV-CAG-EGFP), an AAV helper plasmid (e.g., pRep2-AAV1, pRep2-AAV2, or pRep2-AAV9), and a plasmid with Figure 1 Ad helper plasmids (such as pHelper, pHelper-dE4Orf6 / 7 intron, mini-pHelper1.0, etc.) with the indicated Ad helper gene arrangement provide the necessary adenoviral functions. All AAV vectors were produced in HEK 293T cells. The Y axis shows the fold change using different novel pHelper compared to the original pHelper.

[0200] Example 3: Construction of mini-pHelper plasmid for AAV library production

[0201] Figure 7 A schematic diagram comparing the original three-plasmid transfection system and the mini-pHelper-based production system is shown. Green represents Ad genes, blue represents AAV genes, and pink represents transgenic elements. In the original three-plasmid system, the total mass of the three plasmids (pHelper, pRep&Cap, GOI) is between 24.1-28.1kb. In the mini-phelper plasmid system, the total mass of the three plasmids is reduced to 20.9-24.9kb. There are two different versions of the double-plasmid system: in the V1 version, AAV genes (Rep and Cap) are inserted into mini-pHelper; in the V2 version, transgenic elements are inserted into mini-pHelper. The total mass of V1 and / or V2 is 17.9-21.9kb. In the single-plasmid system, both AAV genes and transgenic elements are inserted into mini-pHelper. The total mass of the plasmid is 13.9-17.9kb.

[0202] Construction of mini-pHelper-AAV2 (SEQ ID NO: 10): Mini-pHelper1.0 plasmid was digested with ClaI, and AAV helper genes (Rep and Cap) were PCR amplified and assembled into the ClaI site.

[0203] Construction of mini-pHelper-CMV-EGFP (SEQ ID NO: 11): Mini-pHelper1.0 plasmid (SEQ ID NO: 9) was digested with PmeI and used as the backbone. The insert, i.e., ITR-CMV-EGFP-ITR box, was isolated from pAAV-CMV-EGFP using SbfI. The backbone was connected to the insert using T4 DNA ligase.

[0204] Construction of pAAVone-AAV2-CMV-EGFP (SEQ ID NO: 12): Mini-pHelper-AAV2 plasmid was digested with Pmel and used as backbone. The insert, ITR-CMV-EGFP-ITR cassette, was isolated from pAAV-CMV-EGFP using SbfI. The backbone was ligated to the insert using T4 DNA ligase.

[0205] Figure 8 The packaging efficiency of the mini-pHelper-based AAV production system in adherent HEK 293T cells is shown. The production titer of rAAV was obtained in HEK 293T cells 72 hours after transfection. In the three-plasmid transfection system, three plasmids with a total DNA mass of 0.6 μg / well at 4 different molecular ratios, namely (pHelper or mini-phelper-1.0): pRep2-AAV2: pAAV-CMV-EGFP, were transfected into HEK 293T cells using a fixed amount of polyethyleneimine (PEI) (1.8 μl / well). In the two-plasmid system, 3 different molecular ratios were evaluated. In the single-plasmid system, different amounts of plasmids were transfected using a fixed amount of PEI.

[0206] Fig. 9 The packaging efficiency of the mini-pHelper-based AAV production system in suspension cultured HEK 293T cells is shown. The production titer of rAAV was obtained 72 hours after transfection. In the three-plasmid transfection system, the ratio of the three plasmids was 1:1:1, and the total DNA was 0.75 μg / 10 6 In the dual plasmid transfection system, the ratio of the two plasmids was 1:1, and the total DNA was 0.75 μg / 10 6 In the single plasmid system, different amounts of plasmid were transfected with a fixed PEI:DNA ratio of 2:1.

[0207] Fig.10The packaging efficiency of the mini-pHelper-based dual-plasmid V2 system for different AAV serotypes is shown. In the dual-plasmid V2 system, the transgenic element is inserted into the mini-pHelper. For example, an AAV production system in HEK 293T cells cultured in suspension. 72 hours after transfection, the production titer of rAAV was obtained. In the three-plasmid transfection system, the molecular ratio of the three plasmids was 1:1:1, and the total DNA was a total DNA mass of 0.6 μg / well. In the dual-plasmid transfection system, the molecular ratio of the two plasmids was 1:1, and the total DNA was a total DNA mass of 0.6 μg / well.

[0208] Example 4: Use of mini-pHelper for AAV library production

[0209] Fig.11 The use of mini-pHelper for AAV library production is shown. rAAV production titers were obtained in HEK 293T cells 72 hours after transfection. 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 as 10 5 Copy number / cell. PEI was used as 1.5 μg / well. Mini-pHelper-Rep: Mini-pHelper-AAV2 plasmid was digested with SwaI and SnaBI to remove the Cap gene.

[0210] Fig.12 An exemplary helper plasmid pAAVone-AAV2-CMV-EGFP (SEQ ID NO: 12) containing another embodiment of the expression cassette of the present application is shown. The plasmid contains Figure 1 The arrangement of the mini-pHelper Ad helper genes, AAV Rep and Cap genes, and the sequence of the recombinant AAV genome (ITR-CMV-EGFP-PolyA-ITR) are shown.

[0211] Fig.13 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) are shown, as well as their effects on the packaging efficiency of the AAVone system. The total DNA mass was 0.6 μg / well. The production titer of rAAV was obtained 72 hours after transfection.

[0212] Fig.14 A sequence listing of the sequences described in this application is shown.

[0213] The sequence listing xml file named 2037-004US.xml filed on March 10, 2023 is incorporated herein by reference, the file was created on February 27, 2023, and the file size is 107kB.

[0214] The above description is intended to teach those of ordinary skill in the art to implement the present application. The present application does not intend to exhaustively list all obvious modifications and variations that are obvious to those skilled in the art after reading the specification. However, all such obvious modifications and variations are deemed to be included in the scope of protection of the present application as defined by the appended claims. Any patent or patent document mentioned herein is expressly incorporated by reference into the present application in its entirety. Unless the context clearly indicates otherwise, the claims are intended to cover the claimed components and steps in any order that effectively achieves the intended purpose.

Claims

1. A polynucleotide expression construct for recombinant parvovirus production, the polynucleotide expression construct comprising: (1) a modified adenovirus E4 coding region, wherein the modified adenovirus E4 coding region encodes E4 open reading frame 6 / 7; (2) a modified adenovirus E2a coding region, wherein the modified adenovirus E2a coding region encodes an E2a protein; (3) an adenovirus VA RNA coding region, wherein the adenovirus VA RNA coding region encodes adenovirus VAI and VAII RNA; (4) a parvovirus protein coding region, wherein the parvovirus protein coding region encodes a parvovirus protein necessary for the production of the recombinant parvovirus; (5) A recombinant parvovirus sequence comprising: (1) an expression cassette comprising a nucleotide sequence encoding a transgene and a regulatory element operably linked to the nucleotide sequence; as well as (2) at least one ITR located at one end of the expression cassette; as well as (6) one or more regulatory elements, which allow the expression of the E4 open reading frame 6 / 7, the E2a protein, the adenovirus VA RNA and the parvovirus proteins necessary for the production of the recombinant parvovirus in the host cell.

2. The expression construct according to claim 1, wherein The modified adenovirus 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. The expression construct according to claim 2, wherein The modified adenovirus E4 coding region comprises a sequence corresponding to nucleotide sequence 32645-35835 of the Ad2 genome, wherein the sequence has a partial or complete deletion of E4orf6 / 7 intron 2 and a partial or complete deletion of E4orf6 / 7 intron 1, wherein the E4orf6 / 7 intron 2 has a sequence of SEQ ID NO: 2, and wherein the E4orf6 / 7 intron 1 has a sequence of SEQ ID NO:

1.

4. The expression construct according to any one of claims 1 to 3, wherein The modified adenoviral E2a coding region comprises: (i) a partial or complete deletion of early E2a or late E2a intron 1; (ii) a partial or complete deletion of E2a intron 2; or both (i) and (ii).

5. The expression construct according to any one of claims 1 to 4, wherein The modified adenovirus E2a coding region comprises a sequence corresponding to nucleotide sequence 22233-27575 of the Ad2 genome, wherein the sequence has: (i) a partial or complete deletion of early E2a or late E2a intron 1; (ii) a partial or complete deletion of E2a intron 2; or both (i) and (ii).

6. The expression construct according to any one of claims 1 to 5, wherein The recombinant parvovirus is rAAV, and wherein the parvovirus protein coding region comprises an AAV Rep coding region encoding an AAV Rep protein and an AAV Cap coding region encoding an AAV Cap protein.

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

8. An expression construct according to any one of claims 1 to 7, comprising: (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) SEQ ID NO:

21.

9. The expression construct according to any one of claims 1 to 8, which is less than 20 kilobases in size.

10. A method for producing a recombinant parvovirus, the method comprising the following steps: introducing an expression construct according to any one of claims 1 to 9 into a host cell; incubating the host cells carrying the expression construct for a desired period of time to produce recombinant parvoviral particles; and The recombinant parvoviral particles are harvested after the incubation period.

11. A polynucleotide expression construct for producing a recombinant parvovirus, the polynucleotide expression construct comprising: (a) a modified adenovirus E4 coding region encoding adenovirus E4orf6 / 7, wherein: The modified adenovirus 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); (b) a modified adenovirus E2a coding region encoding an adenovirus E2a protein, wherein the modified adenovirus E2a coding region comprises at least one deletion in E4orf6 / 7 intron 1 or E4orf6 / 7 intron 2; (c) sequences encoding one or more adenoviral VA RNAs; and (d) one or more regulatory elements operably linked to (a), (b) and (c).

12. The expression construct according to claim 11, wherein The modified adenovirus E4 coding region comprises a sequence corresponding to nucleotide sequence 32645-35835 of the Ad2 genome, wherein the sequence has a partial or complete deletion of E4orf6 / 7 intron 2 and a partial or complete deletion of E4orf6 / 7 intron 1, wherein the E4orf6 / 7 intron 2 has a sequence of SEQ ID NO: 2, and wherein the E4orf6 / 7 intron 1 has a sequence of SEQ ID NO:

1.

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

7.

14. The expression construct according to any one of claims 11 to 13, wherein The modified adenoviral E2a coding region comprises: (i) a partial or complete deletion of early E2a or late E2a intron 1; (ii) a partial or complete deletion of E2a intron 2; or both (i) and (ii).

15. The expression construct according to any one of claims 11 to 14, wherein The modified adenovirus E2a coding region comprises a sequence corresponding to nucleotide sequence 22233-27575 of the Ad2 genome, wherein the sequence has: (i) a partial or complete deletion of early E2a or late E2a intron 1; (ii) a partial or complete deletion of E2a intron 2; or both (i) and (ii).

16. The expression construct according to any one of claims 11 to 15, wherein The modified adenovirus E2a coding region comprises a sequence corresponding to nucleotide sequence 22233-27575 of the Ad2 genome, wherein the sequence has a 1897 bp deletion in SEQ ID NO: 3, and the deletion covers the complete sequence of late E2a intron 1 and E2a intron 2.

17. The expression construct according to any one of claims 11 to 16, wherein The modified adenovirus E2a coding region comprises a codon-optimized nucleotide sequence of SEQ ID NO:

4.

18. 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.

19. The expression construct according to any one of claims 11 to 18, wherein The total length of the expression construct is less than 9 kilobases.

20. The expression construct according to any one of claims 11 to 19, wherein The expression construct further comprises an AAV Rep coding region and an AAV Cap coding region.

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

21.

22. The expression construct according to claim 20 or 21, wherein The AAV Rep coding region and / or the AAVCap coding region are operably linked to the AAV P5I promoter.

23. The expression construct according to claim 22, wherein The AAV P5I promoter comprises SEQ ID NO:

16.

24. An expression construct according to any one of claims 11 to 19, wherein The expression construct further comprises a recombinant AAV genome, wherein the recombinant AAV genome comprises: An expression cassette comprising a sequence encoding a target gene and a regulatory element operably linked to the sequence encoding the target gene; and Two AAV ITRs flank the expression cassette.

25. A method for producing a recombinant AAV, the method comprising the steps of: introducing into a host cell an expression construct according to any one of claims 11 to 19, an AAV trans plasmid encoding an AAV Rep protein and an AAV Cap protein, and an AAV cis plasmid encoding a recombinant AAV genome; incubating the host cells carrying the expression construct, the AAV trans plasmid, and the AAV cis plasmid for a desired period of time to produce recombinant AAV particles; and After the incubation period the recombinant AAV particles are harvested.

26. A method for producing a recombinant AAV, the method comprising the steps of: introducing an expression construct according to any one of claims 20 to 23 and an AAV cis plasmid encoding a recombinant AAV genome into a host cell; incubating the host cells carrying the expression construct and the AAV cis plasmid for a desired period of time to produce recombinant AAV particles; and After the incubation period the recombinant AAV particles are harvested.

27. A method for producing a recombinant AAV, the method comprising the steps of: introducing the expression construct according to claim 24 and an AAV trans plasmid encoding an AAV Rep protein and an AAV Cap protein into a host cell; incubating the host cells carrying the expression construct and the AAV trans plasmid for a desired period of time to produce recombinant AAV particles; and After the incubation period the recombinant AAV particles are harvested.

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