Nucleic acid constructs for VA RNA transcription

Abstract

Herein, we report novel adenovirus VA RNA nucleic acids in which the wild-type type 2 polymerase III promoter has been removed and a U6-snRNA promoter or an inducible promoter has been added.

Description

Technical Field

[0001] This specification reports novel DNA constructs and methods of using them. Using the novel DNA constructs according to the present invention, adenoviral VA RNA can be transcribed in an AAV particle-producing cell line. The novel VA RNA nucleic acid comprises a VA RNA nucleic acid operably linked to an exogenous promoter.

Background Art

[0002] Background of the Invention Gene therapy, in a broad sense, refers to the therapeutic administration of genetic material to modify gene expression in living cells and thereby change their biological properties. After decades of research, gene therapy has advanced to the market and is expected to become increasingly important. Generally, gene therapy can be divided into either an in vivo or an ex vivo approach.

[0003] Today, most in vivo therapies rely on DNA delivery via recombinant adeno-associated virus (rAAV) vectors. AAV is a small, naturally occurring, non-pathogenic parvovirus, composed of a non-enveloped icosahedral capsid. It contains a linear single-stranded DNA genome of approximately 4.7 kb. The genome of the wild-type AAV vector contains two genes, rep and cap, flanked by a reverse-end repeat (ITR). The ITR is required in cis for viral replication and packaging. The rep gene encodes four distinct proteins, whose expression is driven by two alternative promoters, P5 and P19. Furthermore, alternative splicing generates different morphologies. The rep proteins have multiple functions, such as DNA binding, endonuclease, and helicase activity. They play roles in gene regulation, site-specific integration, excision, replication, and packaging. The cap gene encodes three capsid proteins and one aggregate activator protein. The differential expression of these proteins is achieved by alternative splicing and alternative start codon use, and is driven by a single promoter P40 located in the coding region of the rep gene.

[0004] In engineered therapeutic rAAV vectors, the viral genes remain adjacent to the viral ITR but are replaced by a transgene expression cassette encoding the target gene under the control of a selected promoter. Unlike wild-type viruses, engineered rAAV vectors do not undergo site-specific integration into the host genome and remain primarily in the episome within the nucleus of transduced cells.

[0005] AAV does not replicate itself, but requires the function of helper genes. These are naturally provided by co-infecting helper viruses, such as adenoviruses or herpes simplex viruses. For example, five adenovirus genes, namely E1A, E1B, E2A, E4, and VA, are known to be essential for AAV replication. In contrast to other protein-coding helper genes, VA is a small RNA gene.

[0006] To produce an rAAV vector, DNA containing a transgene adjacent to the ITR is introduced into a packaging host cell line that also contains the rep gene, the cap gene, and the necessary helper genes. There are many methods for introducing these three DNA elements into cells, and methods for combining them on different DNA plasmids (e.g., Robert, MA, et al. Biotechnol. J. 12 (2017) 1600193 (Non-Patent Literature 1)).

[0007] Two common manufacturing methods are widely used. In the triple transfection method, HEK293 cells already expressing adenoviruses E1A and E1B are transiently and simultaneously transfected with adenovirus helper plasmids (pHELPER) carrying E2A, E4, and VA, a plasmid containing rep / cap, and a plasmid containing the rAAV transgene. Alternatively, the rep / cap gene and the viral helper gene can be combined on a single large plasmid (double transfection method). The second method involves infection of insect cells (Sf9) with two baculoviruses, one carrying the rAAV genome and the other carrying rep and cap. In this system, the helper function is provided by the baculovirus vector itself. Similarly, herpes simplex virus is used in combination with HEK293 cells or BHK cells. More recently, Mietzsch et al. (Hum.Gene Ther. 25 (2014) 212-222 (Non-Patent Literature 2); Hum.Gene Ther. Methods 28 (2017) 15-22 (Non-Patent Literature 3)) manipulated Sf9 cells in which rep and cap genes were stably integrated into the genome. In these cells, a single baculovirus containing the rAAV transgene was sufficient to produce an rAAV vector. Clark et al. (Hum.Gene Ther. 6 (1995) 1329-1341) (Non-Patent Literature 4) created a HeLa cell line in which the rep / cap gene and the rAAV transgene were integrated into its genome. Transfecting the cells with wild-type adenovirus induced rAAV vector production, resulting in the production of a mixed stock of rAAV vector and adenovirus.

[0008] No mammalian cell lines in which helper genes are stably integrated into their genome have been described to date. The expression of rep and viral helper genes is toxic to cells and needs to be strictly controlled (see, for example, Qiao, C., et al., J. Virol. 76 (2002) 1904-1913 (Non-Patent Literature 5)).

[0009] In the case of rep genes, such regulation is achieved by introducing an intron into the rep gene containing a polyadenylation site adjacent to the LoxP site. After introducing Cre recombinase using recombinant adenovirus, the polyadenylation site is removed, and the intron is removed by splicing (see, for example, Yuan, Z., et al., Hum. Gene Ther. 22 (2011) 613-624 (Non-Patent Literature 6); Qiao, C., et al. (see above)).

[0010] International Publication No. 97 / 9441 (EP 0 850 313 B1) (Patent Document 1) describes a method for producing recombinant adeno-associated virus (AAV), comprising: (1) (a) an AAV helper plasmid comprising nucleic acids encoding AAV rep and cap proteins; (b) an adenovirus helper plasmid comprising essential adenovirus helper genes, wherein the essential adenovirus helper genes present in the plasmid are selected from the group consisting of E1A, E1B, E2A, E4, E4ORF6, E4ORF6 / 7, VA RNA and combinations thereof; (c) an AAV plasmid comprising first and second AAV reverse terminal repeat sequences (ITRs), wherein the first and second AAV ITR reported a method comprising (1) culturing a composition containing cells transiently transfected with an AAV plasmid, wherein the AAV plasmid is adjacent to the DNA encoding the target polypeptide and the DNA is operably linked to a promoter DNA, in the absence of adenovirus particles; and (2) purifying the recombinant AAV produced therefrom.

[0011] International Publication No. 2001 / 36615 (EP 1 230 354 B1) (Patent Document 2) reported a permanent amniotic fluid cell line containing at least one nucleic acid that results in the expression of gene products of the adenovirus E1A and E1B regions.

[0012] International Publication No. 2004 / 29219 (Patent Document 3) reported vectors and methods for controlling the temporal and spatial expression of shRNA constructs in cells and organisms. Such vectors may be retroviral vectors, such as lentiviral vectors. In preferred embodiments, shRNA expression is regulated by an RNA polymerase III promoter. Such promoters are known to result in efficient silencing. Essentially any polIII promoter can be used, but preferred examples include the human U6 snRNA promoter, the mouse U6 snRNA promoter, the human and mouse H1 RNA promoter, and the human tRNA-val promoter.

[0013] Ventura, A. et al. reported Cre-lox-regulated conditional RNA interference from a transgene (Proc. Natl. Acad. Sci. USA 101(2004)10380-10385 (Non-Patent Literature 7)). The authors constructed two lentiviral vectors for conditional Cre-lox-regulated RNA interference. One vector allows for conditional activation, while the other allows for conditional inactivation of short hairpin RNA (shRNA) expression. The former is based on a strategy of modifying the mouse U6 promoter by including a hybrid between the LoxP site and the TATA box.

[0014] Kawabe, Y. et al. reported a gene integration system for antibody production using recombinant Chinese hamster ovary (CHO) cells (Cytotechnol. 64 (2012) 267-279 (Non-Patent Literature 8)). Exchange cassettes adjacent to wild-type and mutant LoxP sites were integrated into the chromosomes of CHO cells to establish recipient founder cells. Subsequently, a donor plasmid was prepared containing a marker antibody expression cassette adjacent to the matching pair of LoxP sites, and also including an internal non-paired LoxP site between the expression cassette for the selection marker and the antibody expression cassette. The donor plasmid and Cre recombinase expression plasmid were co-transfected into founder CHO cells to induce RMCE in the CHO genome, resulting in site-specific integration of the antibody gene that restores the original wild-type LoxP site and generates an inactive double mutant LoxP site that is no longer involved in RMCE. The RMCE procedure was repeated to increase the copy number of the incorporated gene, and at each step, the expression cassette of the selected marker present in the cell was excised and removed.

[0015] U.S. Patent Application Publication 2013 / 58871 (Patent Document 4) reports the generation of a Cre recombinase-mediated switchable inverted plasmid by using two head-to-head oriented mutant LoxP sites (Lox66 and Lox71). In the presence of Cre recombinase, the genes adjacent to the two mutant LoxP sites are inverted, forming one LoxP and one double mutant LoxP site. Since the double mutant LoxP site exhibits very low affinity for Cre recombinase, the preferred one-step inversion is nearly irreversible, allowing the gene to be stably switched "on" and "off" as desired. Leakage of expression in the absence of Cre recombinase was minimized by eliminating sequences containing a pseudo-TATA box and start codon on the side of the floxed gene.

[0016] Crawford, Y., et al. (Biotechnol. Prog. 29 (2013) 1307-1315 (Non-Patent Literature 9)) reported that they rapidly identified reliable hosts for targeted cell line development through limited genome screening by combining phiC31 integrase technology and CRE-Lox technology.

[0017] International Publication No. 2016 / 57800 (Patent Document 5) reported a TGG or DRG promoter operably linked to Cre recombinase and a LOX-stop-LOX-inducible RNA polymerase III promoter operably linked to inhibitory RNA. In vivo, the authors found that a single T-to-C mutation at position 4 of the central spacer region in the distal (3')LoxP site completely inhibited recombination in two conditional mouse models.

[0018] International Publication No. 2019 / 126634 (Patent Document 6) reported targeted integration (TI) host cells suitable for recombinant protein expression, as well as methods for producing and using said TI host cells. [Prior art documents] [Patent Documents]

[0019] [Patent Document 1] International Publication No. 97 / 9441 (EP 0 850 313 B1) [Patent Document 2] International Publication No. 2001 / 36615 (EP 1 230 354 B1) [Patent Document 3] International Publication No. 2004 / 29219 [Patent Document 4] U.S. Patent Application Publication No. 2013 / 58871 [Patent Document 5] International Publication No. 2016 / 57800 [Patent Document 6] International Publication No. 2019 / 126634 [Non-patent literature]

[0020] [Non-Patent Document 1] Robert, M. A., et al. Biotechnol. J. 12 (2017) 1600193 [Non-Patent Document 2] Mietzsch et al. (Hum. Gene Ther. 25 (2014) 212 - 222) [Non-Patent Document 3] Mietzsch et al. (Hum. Gene Ther. Methods 28 (2017) 15 - 22) [Non-Patent Document 4] Clark et al. (Hum. Gene Ther. 6 (1995) 1329 - 1341) [Non-Patent Document 5] Qiao, C., et al., J. Virol. 76 (2002) 1904 - 1913 [Non-Patent Document 6] Yuan, Z., et al., Hum. Gene Ther. 22 (2011) 613 - 624 [Non-Patent Document 7] Ventura, A. et al. (Proc. Natl. Acad. Sci. USA 101 (2004) 10380 - 10385) [Non-Patent Document 8] Kawabe, Y. et al. (Cytotechnol. 64 (2012) 267 - 279) [Non-Patent Document 9] Crawford, Y., et al. (Biotechnol. Prog. 29 (2013) 1307 - 1315) [Summary of the Invention]

[0021] This specification reports a novel deoxyribonucleic acid containing adenovirus VA RNA and a method of using the same. The novel deoxyribonucleic acid according to the present invention is useful for the production of recombinant adeno-associated virus particles.

[0022] Accordingly, one aspect of the present invention is adenovirus VA RNA nucleic acid. In the adenovirus VA RNA nucleic acid reported herein, the VA RNA coding sequence is operably ligated at its 5' end to a variant 2 polymerase III promoter, or a variant 3 polymerase III promoter or its variant, such as the U6-snRNA promoter, or a polymerase II promoter.

[0023] In the adenovirus VA RNA nucleic acid according to the present invention, the VA RNA coding sequence is operably ligated to the U6-snRNA promoter at its 5' end.

[0024] In the adenovirus VA RNA nucleic acid according to the present invention, the VA RNA coding sequence is operably linked to an inducible promoter at its 5' end.

[0025] In one preferred embodiment, the adenovirus VA RNA coding sequence has the sequence of SEQ ID NO: 38.

[0026] In all aspects and in one embodiment of the embodiments, the adenovirus VA RNA nucleic acid includes a precise transcription start site located 3' to the promoter. In one embodiment, the precise transcription start site includes at least six 5' terminal nucleotides of the adenovirus VA RNAI gene, oriented 5' to 3', including a transcription start site (TSS) (to prevent bypass of the subsequent polymerase III (pol III) terminator) and a functional polymerase III terminator (to prevent transcription from the constitutively active upstream promoter).

[0027] In all aspects and in one embodiment of the embodiments, the adenovirus VA RNA nucleic acid contains a polymerase III terminator at its 3' end.

[0028] In all aspects and in one embodiment of the embodiments, all elements of the adenovirus VA RNA nucleic acid are arranged in an operablely linked configuration.

[0029] In all aspects and in one embodiment of the embodiments, the adenovirus VA RNA nucleic acid is functional.

[0030] While not bound by this theory, it is assumed that improved control of adenovirus VA RNA transcription and the resulting AAV particle production can be achieved using the nucleic acids according to the present invention.

[0031] Another aspect of the present invention is a packaging cell line for rAAV particle production in which the rep / cap gene and the adenovirus helper gene are (stably) integrated into the genome, and the adenovirus VA RNA nucleic acid contains the adenovirus VA RNA nucleic acid according to the present invention.

[0032] In one preferred embodiment of this configuration, the rAAV plasmid containing the ITR and the transgene is also incorporated into the genome of the packaging cell. This transforms the packaging cell line into an rAAV vector and particle-producing cell line. Similarly, in certain embodiments, the rAAV plasmid / genome is introduced transiently.

[0033] After recombination, the cells of the producing cell line are genetically homogeneous and express all the genes necessary for rAAV replication and packaging at the correct stoichiometric levels (in contrast, with triple or double transfection, some cells may receive suboptimal doses of one or the other plasmid). Therefore, without being bound by this theory, stable rAAV vector / particle packaging or producing cells may result in higher product quality compared to transient packaging or producing cells.

[0034] One independent aspect of the present invention is DNA (molecule), -Adenovirus VA RNA nucleic acid according to the present invention, - The first DNA element, -Optionally, a second DNA element and, -Optionally, a third DNA element, -Optionally, rep or / and cap open reading frame and It is DNA (a molecule) that contains [this].

[0035] In one dependent embodiment of this aspect, - The first DNA element includes an E1A open reading frame and an E1B open reading frame; - The second DNA element, if present, includes an E2A open reading frame and an E4 or E4 or E4 or E6 open reading frame. The reverse is also true.

[0036] One independent aspect of the present invention is a mammalian or insect cell containing adenovirus VA RNA nucleic acid or DNA (elements) according to the present invention.

[0037] One independent aspect of the present invention is a method for producing recombinant adeno-associated virus (rAAV) vectors or particles, - The process of culturing / proliferating cells according to the present invention (under conditions suitable for cell division), - A step of recovering rAAV vectors or particles from cells or culture medium. This method includes [something].

[0038] A further independent aspect of the present invention is an adenovirus VA RNA nucleic acid or DNA (molecule) according to the present invention for producing recombinant adeno-associated virus vectors or particles.

[0039] One independent aspect of the present invention is an adenovirus VA RNA nucleic acid in which the wild-type type 2 polymerase III promoter is inactivated / deleted / removed and a U6-snRNA promoter is added. In one embodiment, a more precise transcription start site is added.

[0040] One independent aspect of the present invention is a method for generating / producing recombinant adeno-associated virus (rAAV) vectors or particles, - To generate / provide mammalian suspension-growth cells that are either stably integrated into the genome or transiently present and include the following: - A transgene expression cassette placed between two AAV ITRs; - Open reading frames encoding adenovirus E1A, E1B, E2A, E4 or E4orf6 proteins and adenovirus VA RNA nucleic acids according to the present invention; - Open reading frame encoding adeno-associated Rep / Cap protein; - Growing / culturing mammalian cells (under conditions that allow cell division); and - Isolating rAAV vectors or particles from cells or culture media, and thereby producing rAAV vectors or particles. This method includes [something]. [Invention 1001] From 5' to 3', Human U6 RNA promoter and The adenovirus VA RNA coding sequence of sequence number 38 and Adenovirus VA RNA nucleic acid, including... [Invention 1002] From 5' to 3', Inducible promoters and The adenovirus VA RNA coding sequence of sequence number 38 and Adenovirus VA RNA nucleic acid, including... [Invention 1003] Adenovirus VA RNA nucleic acid of the present invention 1001 or 1002, E1A open reading frame and E1B open reading frame, or E2A open reading frame and E4 or E4 or E4 or E6 open reading frame, rep open reading frame and cap open reading frame DNA elements and DNA, including [Invention 1004] Mammalian cells or insect cells containing adenovirus VA RNA nucleic acid of Invention 1001 or 1002 or DNA of Invention 1003. [Invention 1005] A method for producing recombinant adeno-associated virus particles, A transgene expression cassette placed between two AAV ITRs, Open reading frames encoding adenovirus E1A, E1B, E2A, E4, or E4 or Ef6 proteins. Adenovirus VA RNA nucleic acid of the present invention 1001 or 1002, Open reading frame encoding adeno-associated Rep / Cap protein To provide mammalian cells that proliferate in suspension, The aforementioned mammalian cells are cultured, The process involves isolating rAAV particles from the aforementioned cells or culture medium, and thereby producing rAAV particles. Methods that include... [Modes for carrying out the invention]

[0041] Detailed description of the embodiments of the invention This specification reports novel nucleic acids and DNA elements, as well as methods for using them. The nucleic acids according to the present invention are useful for recombinant production of AAV particles. The present invention provides novel adenovirus VA RNA nucleic acids using the intentional arrangement of promoters and coding sequences.

[0042] definition Useful methods and techniques for carrying out the present invention are described, for example, in Ausubel, FM (ed.), Current Protocols in Molecular Biology, Vols. I-III (1997); Glover, ND, and Hames, BD, ed., DNA Cloning: A Practical Approach, Vols. I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture - a practical approach, IRL Press Limited (1986); Watson, JD, et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); and Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., NY (1987).

[0043] Recombinant DNA technology enables the creation of nucleic acid derivatives. Such derivatives can be modified at individual or several nucleotide positions, for example, by substitution, alteration, exchange, deletion, or insertion. Modification or derivatization can be carried out, for example, by site-directed mutagenesis. Such modifications can be easily performed by those skilled in the art (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual (1999), Cold Spring Harbor Laboratory Press, New York, USA; Hames, BD, and Higgins, SG, Nucleic Acid Hybridization: A Practical Approach (1985), IRL Press, Oxford, England).

[0044] Deoxyribonucleic acid includes a coding strand and a non-coding strand. The terms "5'-" and "3'-" refer to positions on the coding strand, as used herein.

[0045] The term "3'-adjacent sequence" refers to a sequence located at the 3' end (downstream, below) of a base sequence.

[0046] The term "5'-adjacent sequence" refers to a sequence located at the 5' end (downstream, below) of a base sequence.

[0047] When used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple referents unless the context makes otherwise obvious. Thus, for example, a reference to “a cell” includes multiple such cells and their equivalents known to those skilled in the art, and so on. Similarly, the terms “a” (or “an”), “one or more,” and “at least one” may also be used synonymously herein. It should also be noted that the terms “comprising,” “including,” and “having” may also be used synonymously.

[0048] The term "AAV helper function" refers to AAV-derived coding sequences (proteins) that can be expressed to provide trans-functioning AAV gene products and AAV particles for AAV replication and packaging. Therefore, AAV helper functions include rep and cap, as well as AAV open reading frames (ORFs), including others such as AAP for specific AAV serotypes. Rep gene expression products have been shown to have many functions, including, among others, recognition, binding, and nicking of AAV origins in DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other xenogeneic) promoters. Cap gene expression products (capsids) provide the necessary packaging functions. AAV helper functions are used to complement trans-AAV functions that are missing from the AAV vector genome.

[0049] The term "approximately" means a range of ±20% of the number that follows. In one embodiment, the term "approximately" means a range of ±10% of the number that follows. In one embodiment, the term "approximately" means a range of ±5% of the number that follows.

[0050] The term "comprising" also includes the term "consisting of".

[0051] The terms "empty capsid" and "empty particle" refer to AAV particles that possess an AAV protein shell but lack the nucleic acid that encodes a protein or is transcribed into a target transcript adjacent to the AAV ITR, i.e., the entire or partial vector. Therefore, empty capsids do not function to transfer the nucleic acid that encodes a protein or is transcribed into a target transcript into a host cell.

[0052] The term "endogenous" refers to something that arises naturally within a cell; something that is naturally produced by a cell. Similarly, an endogenous locus / intracellular locus is a locus of genes that arises naturally within a cell.

[0053] As used herein, the term “exogenous” means that a nucleotide sequence does not originate from a particular cell and is introduced into such cell by DNA delivery methods, such as transfection, electroporation, or transformation by a viral vector. Therefore, an exogenous nucleotide sequence is an artificial sequence, which may arise, for example, from a combination of subsequences of different origins (e.g., a combination of a recombinase recognition sequence with an SV40 promoter and a green fluorescent protein coding sequence is an artificial nucleic acid), or from a partial deletion or mutation of nucleic acid bases in a sequence (e.g., a sequence or cDNA that codes only for the extracellular domain of a membrane-bound receptor). The term “endogenous” means a nucleotide sequence that originates from a cell. An “exogenous” nucleotide sequence may have an “endogenous” counterpart that has the same base composition but is becoming an “exogenous” sequence through introduction into a cell, for example, via recombinant DNA technology.

[0054] As used herein, the term “adjacent” means that the first nucleotide sequence is located at either the 5' or 3' end, or both ends, of the second nucleotide sequence. The adjacent nucleotide sequence may be adjacent to the second nucleotide sequence or at a predetermined distance therefrom. There are no specific restrictions on the length of the adjacent nucleotide sequence other than the practical requirements. For example, the adjacent sequence may be a few base pairs or several thousand base pairs. The term “adjacent nucleotide sequence” means the sequence segments of nucleic acid before and after the sequence to be inserted (=target sequence).

[0055] The term "gene locus" refers to the location of a gene on a chromosome, that is, the location of a gene within the genome, i.e., the gene location.

[0056] An "isolated" composition is one that has been separated from one or more components of its natural environment. In some embodiments, the composition is purified to a purity of 95% or more than 99% as measured, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis, CE-SDS) or chromatography (e.g., size exclusion chromatography or ion exchange or reversed-phase HPLC). For an overview of methods for evaluating antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0057] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from one or more components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally found inside cells containing nucleic acid molecules, but the nucleic acid molecules are located outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0058] An "isolated" polypeptide or antibody means a polypeptide molecule or antibody molecule that has been isolated from one or more components of its natural environment.

[0059] The term "integration site" refers to a nucleic acid sequence within the cellular genome where an exogenous nucleotide sequence is inserted. In certain embodiments, an integration site is located between two adjacent nucleotides in the cellular genome. In certain embodiments, an integration site includes a stretch of nucleotides. In certain embodiments, an integration site is located within a specific gene locus in the genome of a mammalian cell. In certain embodiments, an integration site is located within an endogenous gene in a mammalian cell.

[0060] The term "LoxP site" refers to a 34 bp nucleotide sequence consisting of two 13 bp palindromic sequences (reverse repeats) at the ends (ATAACTTCGTATA (SEQ ID NO: 01) and TATACGAAGTTAT (SEQ ID NO: 02)) and a central 8 bp core (asymmetric) spacer sequence. The spacer sequence determines the orientation of the LoxP site. Depending on the relative orientation and position of the two LoxP sites, the intervening DNA is either cut (LoxP sites oriented in the same direction) or inverted (LoxP sites oriented in opposite directions). The term "floxed" refers to a DNA sequence located between two LoxP sites. When two floxed sequences exist, i.e., a target floxed sequence in the genome and a floxed sequence in the donor nucleic acid, both sequences can be exchanged with each other. This is called "recombinase-mediated cassette exchange."

[0061] Exemplary LoxP sites are shown in the table below. TIFF0007857714000001.tif58128

[0062] The term “mammalian cell containing an exogenous nucleotide sequence” encompasses cells into which one or more exogenous nucleic acids have been introduced, including offspring of such cells. These can serve as a starting point for further genetic modification. Therefore, the term “mammalian cell containing an exogenous nucleotide sequence” encompasses cells containing an exogenous nucleotide sequence integrated into a single site within a locus of the genome of the mammalian cell, the exogenous nucleotide sequence comprising at least one first and at least one second recombination recognition site (these recombination recognition sites are distinct) adjacent to at least one first choice marker. In a particular embodiment, a mammalian cell containing an exogenous nucleotide sequence is a cell containing an exogenous nucleotide sequence integrated into a single site within a locus of the genome of the cell, the exogenous nucleotide sequence comprising a first recombination recognition site and a second recombination recognition site adjacent to at least one first choice marker, and a third recombination recognition site located between the first and second recombination recognition sites, all of which are distinct.

[0063] Both "mammalian cells containing exogenous nucleotide sequences" and "recombinant cells" are "transfected cells." This term includes both primary transfected cells and their offspring, regardless of the number of passages. Offspring may contain mutations, for example, but may not have the exact same nucleic acid content as the parent cells. Offspring of mutants that have the same function or biological activity as the initially transfected cells are included.

[0064] The term "nucleic acid encoding AAV packaging proteins" generally refers to one or more nucleic acid molecules containing nucleotide sequences that provide the AAV functionality deleted from an AAV vector, used to produce transduction-eligible recombinant AAV particles. Nucleic acids encoding AAV packaging proteins are commonly used to provide expression of AAV rep and / or cap genes to complement the missing AAV functionality required for AAV replication. However, nucleic acid constructs lack AAV ITR and cannot replicate or package. Nucleic acids encoding AAV packaging proteins can be in the form of plasmids, phages, transposons, cosmids, viruses, or particles. Many nucleic acid constructs, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode both rep and cap gene expression products, have been described (see, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945). Several plasmids encoding rep and / or cap gene expression products have been described (e.g., U.S. Patent No. 5,139,941 and U.S. Patent No. 6,376,237). Any one of these nucleic acids encoding AAV packaging proteins may contain a DNA element or nucleic acid according to the present invention.

[0065] The term "nucleic acid encoding a helper protein" generally refers to one or more nucleic acid molecules containing a nucleotide sequence that encodes a protein providing adenovirus helper function. Plasmids having nucleic acids encoding helper proteins can be transfected into suitable cells, and as a result, the plasmid can support AAV particle production in said cells. Any one of these nucleic acids encoding helper proteins may include DNA elements or nucleic acids according to the present invention. Infectious viral particles present in nature, such as adenovirus, herpesvirus, or vaccinia virus particles, are specifically excluded from this term.

[0066] As used herein, the term “operably linked” means the close arrangement of two or more components that are related in a way that enables them to function in the desired manner. For example, if a promoter and / or enhancer plays a role in regulating the transcription of a coding sequence / open reading frame / gene, then the promoter and / or enhancer is operably linked to the coding sequence / open reading frame / gene. In certain embodiments, “operably linked” DNA sequences are contiguous. In certain embodiments, for example, when it is necessary to link the coding regions of two proteins, such as one secretory leader and one polypeptide, these sequences are contiguous and reside in the same reading frame. In certain embodiments, an operably linked promoter may be located upstream of the coding sequence / open reading frame / gene and adjacent to the coding sequence. In certain embodiments, for example, with respect to an enhancer sequence that regulates the expression of a coding sequence / open reading frame / gene, the two components may not be adjacent but may be operably linked. If an enhancer increases the transcription of a coding sequence / open reading frame / gene, the enhancer is operably ligated to the coding sequence / open reading frame / gene. An operably ligated enhancer may be located upstream, within, or downstream of the coding sequence / open reading frame / gene, and may be located at a considerable distance from the promoter of the coding sequence / open reading frame / gene.

[0067] The term "packaging protein" refers to non-AAV-derived viral and / or cellular functions on which AAV replication depends. Therefore, this term encompasses the capture of proteins and RNA necessary for AAV replication, including regions involved in AAV gene transcription, step-specific AAV mRNA splicing, AAV DNA replication, Cap expression product synthesis, and activation of AAV capsid assembly. Virus-based accessory functions may originate from any known helper viruses, such as adenoviruses, herpesviruses (other than type I herpes simplex virus), and vaccinia viruses.

[0068] As used herein, “AAV packaging protein” refers to an AAV-derived sequence that functions in trans for the production of AAV replication. Thus, AAV packaging proteins are encoded by the major AAV open reading frame (ORF), rep, and cap. The rep protein has been shown to have many functions, including, among others, recognition, binding, and nicking of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The cap (capsid) protein provides the necessary packaging function. AAV packaging proteins are used herein to complement the trans AAV function missing from the AAV vector.

[0069] A "plasmid" is typically a form of nucleic acid or polynucleotide having additional elements for plasmid expression (e.g., transcription, replication, etc.) or proliferation (replication). Plasmids as used herein can also be used to reference such nucleic acid or polynucleotide sequences. Thus, in all embodiments, the compositions and methods of the present invention are applicable to nucleic acids, polynucleotides, and plasmids, for example, to produce cells that produce viral (e.g., AAV) vectors, to produce viral (e.g., AAV) particles, to produce cell culture media containing viral (e.g., AAV) particles, etc.

[0070] As used herein, the term “recombinant cell” refers to a cell that has undergone final genetic modification, for example, a cell that produces the AAV particle of interest and can be used to produce the AAV particle of interest on any scale. For example, a “mammalian cell containing an exogenous nucleotide sequence” that has been subjected to recombinase-mediated cassette exchange (RMCE) so that the coding sequence of the polypeptide of interest has been introduced into the genome of the host cell is a “recombinant cell.” This cell can still undergo further RMCE reactions, but this is not the purpose.

[0071] A "recombinant AAV vector" is obtained from the wild-type genome of a virus (e.g., AAV) by using molecular biological methods to remove the wild-type genome and replacing it with a non-natural nucleic acid, such as a nucleic acid transcribed into a transcript or a protein-coding nucleic acid. Typically, for AAV, one or both of the reverse-end repeat (ITR) sequences of the wild-type AAV genome are retained in the recombinant AAV vector. A "recombinant" AAV vector is distinguished from a wild-type viral AAV genome because all or part of the viral genome is replaced with non-natural (i.e., heterologous) sequences with respect to the viral genome nucleic acid. Therefore, the incorporation of non-natural sequences defines a viral vector (e.g., AAV) as a "recombinant" vector, which in the case of AAV can be called an "rAAV vector".

[0072] Recombinant vectors (e.g., AAVs) are packaged for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo, and may be referred to herein as “particles.” When a recombinant vector sequence is encapsulated or packaged within an AAV particle, the particle may also be referred to as “rAAV.” Such particles contain proteins that encapsulate or package the vector genome. Specific examples include viral envelope proteins, and in the case of AAV, capsid proteins, e.g., AAV VP1, VP2, and VP3.

[0073] A "recombination recognition site" (RRS) is a nucleotide sequence that is recognized by a recombinase and is necessary and sufficient for a recombinase-mediated recombination event. Using RRSs, the location within a nucleotide sequence where a recombination event is expected to occur can be determined.

[0074] As used herein, the term “selection marker” means a gene that enables cells possessing that gene to be positively or negatively specific selected in the presence of a corresponding selective agent. For example, but not limited to, a selection marker may enable host cells transformed with that selection marker gene to be positively selected in the presence of each selective agent (under selective culture conditions); untransformed host cells are considered unable to proliferate or survive under selective culture conditions. Selection markers may be positive, negative, or bifunctional. A positive selection marker may enable the selection of cells possessing the marker, while a negative selection marker may enable the selective exclusion of cells possessing the marker. Selection markers can confer drug resistance in host cells or compensate for metabolic or catabolic defects. In prokaryotic cells, in particular, genes conferring resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol may be used. Useful resistance genes as selection markers in eukaryotic cells include, but are not limited to, those of aminoglycoside phosphotransferases (APHs) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthase (GS), asparagine synthase, tryptophan synthase (indole), and histidinol dehydrogenase (histidinol D)), as well as genes encoding resistance to puromycin, blasticidine, bleomycin, phleomycin, chloramphenicol, zeosin, and mycophenolic acid. Further marker genes are described in International Publication Nos. 92 / 08796 and 94 / 28143.

[0075] Beyond facilitating selection in the presence of a corresponding selector, the selection marker may, alternatively, be a molecule not normally present in cells, such as green fluorescent protein (GFP), high-sensitivity GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), high-sensitivity YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire. For example, cells expressing such molecules can be distinguished from cells that do not contain this gene based on the detection or absence of fluorescence emitted by the encoded polypeptide, respectively.

[0076] As used herein, the term “serotype” refers to a distinction based on serologically distinct AAV capsids. Serological specificity is determined based on the absence of cross-reactivity between antibodies against a given AAV compared to other AAVs. Such differences in cross-reactivity are typically due to differences in capsid protein sequences / antigenic determinants (e.g., differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). An AAV variant containing a capsid variant differs from the reference or other AAV serotype by at least one nucleotide or amino acid residue, even though it may not be serologically distinguishable from the reference or other AAV serotypes.

[0077] Under the conventional definition, a serotype means that the virus of interest has been tested against serums specific to all existing and characterized serotypes for neutralizing activity, and no antibody neutralizing the virus of interest has been found. As more naturally occurring virus isolates are discovered and / or capsid variants are generated, they may or may not be serologically distinct from any of the currently existing serotypes. Therefore, if a new virus (e.g., AAV) has no serological differences, this new virus (e.g., AAV) is a subgroup or variant of the corresponding serotype. Often, serological testing for neutralizing activity has not yet been performed on mutant viruses with capsid sequence modifications to determine whether they are other serotypes according to the conventional definition of serotypes. Therefore, for convenience and to avoid repetition, the term “serotype” in a broad sense refers to both serologically distinct viruses (e.g., AAV) and serologically non-distinguishable viruses (e.g., AAV) that may fall within a subgroup or variant of a given serotype.

[0078] The terms "transduction" and "transfect" refer to the introduction of molecules such as nucleic acids (viral vectors, plasmids) into cells. A cell is "transduced" or "transfected" when an exogenous nucleic acid is introduced inside the cell membrane. Therefore, a "transduced cell" is a cell into which a "nucleic acid" or "polynucleotide" has been introduced, or its offspring into which an exogenous nucleic acid has been introduced. In certain embodiments, a "transduced" cell (e.g., in mammals, e.g., in cell, tissue, or organ cells) undergoes genetic changes after the incorporation of an exogenous molecule, e.g., nucleic acid (e.g., a transgene). Transduced cells can be grown to transcribe and / or express the introduced nucleic acid as a protein.

[0079] In transfected or transfected cells, nucleic acids (viral vectors, plasmids) may or may not be incorporated into the genomic nucleic acid. Once the introduced nucleic acid is incorporated into the nucleic acid (genomic DNA) of a recipient cell or organism, it can be stably maintained within the cell or organism and further passed on to or inherited by the progeny cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid may exist outside the chromosome or transiently only in the recipient cell or host organism. Several techniques are known; see, for example, Graham et al. (1973) Virology, 52:456; Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al. (1986), Basic Methods in Molecular Biology, Elsevier; and Chu et al. (1981) Gene 13:197. Using such techniques, one or more exogenous DNA segments can be introduced into suitable host cells.

[0080] The term “transgene” is used herein to conveniently refer to a nucleic acid that is intended or introduced into a cell or organism. Transgenes include any nucleic acid, for example, a gene that is transcribed into a transcript or codes for a polypeptide or protein.

[0081] The term "vector" refers to the portion of a recombinant plasmid sequence that is ultimately packaged or encapsulated, either directly or in single-stranded or RNA form, to form a viral particle (e.g., AAV). When a recombinant plasmid is used to construct or produce a recombinant viral particle, the viral particle does not contain the portion of the plasmid that does not correspond to the vector sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid is called the "plasmid backbone," and while it is crucial for plasmid cloning and amplification, which are processes necessary for replication and recombinant virus production, it is not itself packaged or encapsulated within the viral particle (e.g., AAV). Therefore, the term "vector" refers to the nucleic acid packaged or thereby encapsulated within the viral particle (e.g., AAV).

[0082] Generation of recombinant cell lines Generally, for the efficient and large-scale production of a target proteinaceous compound, such as rAAV particles or therapeutic polypeptides, cells that stably express and, if possible, secrete the proteinaceous compound are required. Such cells are called “recombinant cells” or “recombinant-producing cells.” The process for generating such recombinant cells is called “cell line development” (CLD).

[0083] In the first step, suitable host cells are transfected with the necessary nucleic acid sequence encoding the proteinaceous compound of interest. Additional transfection of helper polypeptides may be required. In the second step, cells that stably express the proteinaceous compound of interest are selected. This can be done, for example, based on the co-expression of a selection marker co-transfected with the nucleic acid sequence encoding the proteinaceous compound of interest, or it can be the expression of the proteinaceous compound itself.

[0084] The expression of a coding sequence, i.e., an open reading frame, requires additional regulatory elements such as a promoter and a polyadenylation signal (sequence). Therefore, the open reading frame is operably linked to these additional regulatory elements for transcription. This can be achieved by incorporating it into a so-called expression cassette. The minimum regulatory elements required for an expression cassette to be functional in mammalian cells are a promoter functional in the mammalian cell, located upstream (i.e., at the 5' end) of the open reading frame, and a polyadenylation signal (sequence) functional in the mammalian cell, located downstream (i.e., at the 3' end) of the open reading frame. Furthermore, a terminator sequence may be present at the 3' end of the polyadenylation signal (sequence). For expression, the promoter, open reading frame / coding region, and polyadenylation signal sequence must be arranged in an operably linked form.

[0085] Similarly, nucleic acids transcribed into non-protein-coding RNA are called "RNA genes." RNA gene expression also requires additional regulatory elements, such as promoters and transcription termination signals or polyadenylation signals (sequences). The nature and localization of such elements depend on the RNA polymerase intended to drive RNA gene expression. Therefore, RNA genes are typically incorporated into expression cassettes as well.

[0086] If the target proteinaceous compound is a heteromultimeric polypeptide composed of different (monomer) polypeptides, not only is a single expression cassette required, but one cassette is needed for each of the different polypeptides, i.e., the open reading frame / coding sequence and, if present, the RNA gene. These expression cassettes differ in at least the open reading frame / coding sequence they contain, but they may also differ in their promoters and / or polyadenylation signal sequences.

[0087] For example, if the target protein compound is a full-length antibody that is a heteromultimeric polypeptide containing two copies of a light chain and two copies of a heavy chain, then two different expression cassettes are required, one for the light chain and one for the heavy chain. For example, if the full-length antibody is a bispecific antibody, i.e., if the antibody contains two different binding sites that specifically bind to two different antigens, then each of the light chains and each of the heavy chains are also different from each other. Therefore, a bispecific full-length antibody is composed of four different polypeptides, and thus four expression cassettes are required, each containing four different open reading frames encoding the four different polypeptides.

[0088] If the target proteinaceous compound is an AAV particle composed of different (monomer) polypeptides and single-stranded DNA molecules, and further requires other cofactors for production and encapsulation, then multiple expression cassettes with different open reading frame / coding sequences are required. In this case, at least one expression cassette is needed for each of the transgenes, different polypeptides that form the capsid of the AAV vector, and VA RNA for the required helper functions. Therefore, individual expression cassettes are needed for each of the helper E1A, E1B, E2A, E4orf6, VA RNA, rep, and cap genes.

[0089] As outlined in the previous paragraph, the more complex the target proteinaceous compound, or the greater the number of additional helper polypeptides and / or RNAs required, the greater the number of different expression cassettes needed. Essentially, along with the number of expression cassettes, the size of the nucleic acid incorporated into the host cell's genome also increases. However, there is a practical upper limit to the size of transferable nucleic acids, in the range of approximately 15 kbp (kilobase pairs). Beyond this limit, handling and processing efficiency decreases significantly. This problem can be addressed by using two or more distinct nucleic acids, where different expression cassettes are assigned to different nucleic acids, and each nucleic acid contains only a portion of the expression cassette.

[0090] For cell line development, random integration (RI) of nucleic acids carrying expression cassettes for target protein compounds can be used. Generally, by using RI, nucleic acids or fragments thereof are randomly integrated into the genome of host cells.

[0091] Alternatively, targeted integration (TI) can be used for CLDs in radioisotopes. In TI CLDs, one or more nucleic acids containing different expression cassettes are introduced into a predetermined locus in the host cell's genome.

[0092] In TI, either homologous recombination or recombinase-mediated cassette exchange (RMCE) can be used to integrate nucleic acids containing their respective expression cassettes into specific loci in the genome of TI host cells.

[0093] In a particular embodiment, a method is provided for the targeted incorporation of a single deoxyribonucleic acid into the genome of a (host) mammalian cell (i.e., a method for producing recombinant mammalian cells), the method comprising subsequently a nucleic acid encoding a proteinogenic compound, the method comprising the following steps: a) A step of providing a mammalian cell comprising an exogenous nucleotide sequence incorporated into a defined (optionally, single) site within a gene locus of the genome of the mammalian cell, wherein the exogenous nucleotide sequence comprises first and second recombinant sequences adjacent to at least one first selection marker, so that all recombinant sequences are different and / or incompatible (i.e., they do not result in cross-exchange reactions); b) A step of introducing deoxyribonucleic acid containing two different recombinant sequences and 1 to 8 expression cassettes into mammalian cells provided in a), The deoxyribonucleic acid is directed from 5' to 3', - The first recombinant sequence, -1 to 8 expression cassettes, one of which encodes a second choice marker, - Second recombinant sequence and Includes, A step of introducing deoxyribonucleic acid, wherein the first and second recombinant sequences of the deoxyribonucleic acid are matched with the first and second recombinant sequences on the incorporated exogenous nucleotide sequence; c) Optionally, introduce or activate the mammalian cells obtained in step b) with the first and second recombinant sequences and a functional recombinase (by exchanging a portion of the exogenous nucleotide sequence between the first and second recombinant sequences with a portion of the deoxyribonucleic acid between the first and second recombinant sequences, thereby incorporating the latter into the genome of the mammalian cells); d) optionally including the step of selecting cells that express the second selection marker and produce a protein compound encoded by the introduced deoxyribonucleic acid, This process produces recombinant mammalian cells containing nucleic acids encoding proteinaceous compounds, thereby producing the proteinaceous compounds.

[0094] In a particular embodiment, a method is provided for the simultaneous targeted incorporation of two deoxyribonucleic acids into the genome of a (host) mammalian cell (i.e., a method for producing recombinant mammalian cells), comprising nucleic acids encoding a proteinogenic compound, wherein the proteinogenic compound is optionally expressed, the method comprising the following steps: a) Providing a mammalian cell comprising an exogenous nucleotide sequence incorporated into a defined (optionally, single) site within a gene locus of the mammalian cell genome, wherein the exogenous nucleotide sequence comprises first and second recombinant sequences adjacent to at least one first selection marker, and a third recombinant sequence located between the first and second recombinant sequences, and all recombinant sequences are different and / or incompatible (i.e., they do not result in cross-exchange reactions); b) A step of introducing two deoxyribonucleic acid compositions, each containing three different recombinant sequences and 1 to 8 expression cassettes, into the cells provided in a), The first deoxyribonucleic acid is in the 5' to 3' direction, - The first recombinant sequence, -One or more (preferably up to four in one embodiment) expression cassettes, -The 5' end portion of the expression cassette encoding one second choice marker, - The first copy of the third recombinant sequence and Includes, and The second deoxyribonucleic acid is located in the 5' to 3' direction. - The second copy of the third recombinant sequence, - The 3' end portion of the expression cassette encoding one second choice marker, -One or more (preferably up to four in one embodiment) expression cassettes, - Second recombinant sequence and Includes, The first to third recombinant sequences of the first and second deoxyribonucleic acids match the first to third recombinant sequences of the incorporated exogenous nucleotide sequence. The 5' and 3' ends of an expression cassette encoding a second choice marker combine to form a functional expression cassette for that second choice marker. c) Optionally, introduce or activate the mammalian cells obtained in step b) with the first, second, and third recombinant sequences and functional recombinases (by exchanging a portion of the exogenous nucleotide sequence between the first and third sequences and a portion between the third and second recombinant sequences with a portion of the deoxyribonucleic acid between the first and third sequences and the third and second recombinant sequences, thereby incorporating the latter into the genome of the mammalian cells); d) optionally includes the step of selecting cells that express a second selection marker and optionally produce a protein product encoded by the introduced deoxyribonucleic acid, This produces recombinant mammalian cells containing nucleic acids encoding the aforementioned proteinaceous compound.

[0095] To increase the selective pressure, the first selection marker is a negative selection marker, such as, in one embodiment, a thymidine kinase derived from herpes simplex virus (making cells sensitive to thymidine analogs such as 5-iodo-2'-fluoro-2'-deoxy-1-β-D-arabino-flonosyluracil (FIAU) or ganciclovir) or a diphtheria toxin fragment A derived from Corynebacterium diphtheria (causing toxicity by inhibiting protein synthesis; for example, by phosphoglycerate kinase promoter (PGK)-driven expression of the diphtheria toxin A fragment gene). The negative selection marker is removed during exchange with the introduced deoxyribonucleic acid. This makes it possible to distinguish between correct targeted integration and incorrect random integration.

[0096] In all aspects and in one embodiment of the embodiments, each expression cassette comprises a promoter, an open reading frame / coding sequence or RNA gene and a polyadenylation signal sequence and / or a terminator sequence, in the 5' to 3' direction. In one embodiment, the open reading frame encodes a polypeptide, and the expression cassette comprises a polyadenylation signal sequence with or without an additional terminator sequence. In one embodiment, the expression cassette comprises an RNA gene, the promoter is a polII promoter, and a polyadenylation signal sequence or polyU terminator is present. See, for example, Song et al. Biochemical and Biophysical Research Communications 323(2004) 573-578. In one embodiment, the expression cassette comprises an RNA nucleic acid, the promoter is a polIII promoter and a polyU terminator sequence.

[0097] In all aspects and in one embodiment of the embodiments, the open reading frame encodes a polypeptide, the promoter is a human CMV promoter with or without intron A, the polyadenylation signal sequence is a bGH (bovine growth hormone) polyA signal sequence, and the terminator is an hGT (human gastrin terminator).

[0098] In all aspects and in one embodiment of the embodiments, the promoter is a human CMV promoter having intron A, the polyadenylation signal sequence is a bGH polyadenylation signal sequence, and the terminator is hGT, except for the RNA nucleic acid expression cassette and the expression cassette for the selection marker, for the selection marker, the promoter is an SV40 promoter, the polyadenylation signal sequence is an SV40 polyadenylation signal sequence, and there is no terminator; for the RNA nucleic acid, the promoter is a type 3 polymerase III promoter such as a variant 2 polymerase III promoter or a U6-snRNA promoter, and the terminator is a polymerase II or III terminator.

[0099] In one embodiment of all the embodiments and models described herein, the human CMV promoter has the sequence of SEQ ID NO: 13. In one embodiment, the human CMV promoter has the sequence of SEQ ID NO: 14. In one embodiment, the human CMV promoter has the sequence of SEQ ID NO: 15.

[0100] In one embodiment of all the embodiments and models described above, the BGH polyadenylated signal sequence is sequence number 16.

[0101] In one embodiment of all the embodiments and models described above, hGT has the sequence of sequence number 17.

[0102] In one embodiment of all the embodiments and models described herein, the SV40 promoter has the sequence of Sequence ID No. 18.

[0103] In one embodiment of all the embodiments and models described above, the SV40 polyadenylated signal sequence is sequence number 19.

[0104] It should be noted that the present invention does not include permanent human cell lines comprising nucleic acid sequences for adenovirus gene functions E1A and E1B, and nucleic acid sequences for SV40 large T antigen or Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1).

[0105] Homologous recombination In certain embodiments, targeted integration is mediated by homologous recombination.

[0106] Targeted integration by homologous recombination is an established technique in this field. For example, homologous recombination has been used for over 30 years to introduce site-specific gene modifications in mouse embryonic stem cells (Doetschman, T., et al., Nature 330 (1987) 576-578; Thomas, K. and Capecchi, MR, Cell 51 (1987) 503-512; Thompson, S., et al., Cell 56 (1989) 313-321; Zijlstra, M., et al., Nature 342 (1989) 435-438; Bouabe, H. and Okkenhaug, K., Meth. Mol. Biol. 1064 (2013) 315-336).

[0107] In the case of homologous recombination for targeted integration, the recombinant sequence is homologous to the exogenous nucleic acid sequence and is called a "homologous arm." In this case, the deoxyribonucleic acid introduced into the host cell contains, as the first recombinant sequence, a sequence homologous to the exogenous nucleic acid sequence (i.e., the landing site) at sequence 5' (upstream), and as the second recombinant sequence, a sequence homologous to the exogenous nucleic acid sequence at sequence 3' (downstream). Generally, the frequency of targeted integration increases with the length and isogenicity of the homologous arm. Ideally, the homologous arm is derived from genomic DNA prepared from each host cell.

[0108] Nuclease In certain embodiments, targeted integration is achieved by homologous recombination mediated by site-specific nucleases.

[0109] In one embodiment, the site-specific nuclease is selected from zinc finger nucleases (ZFNs), activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-related protein 9 nuclease (Cas9) systems.

[0110] Nuclease-coding genes can be delivered to cells via plasmid DNA, viral vectors, or in vitro transcribed mRNA. Transfection of plasmid DNA or mRNA can be performed by electroporation or cationic lipid-based reagents. Integrase-deficient lentiviral vectors can be used to deliver nucleases to transfection-resistant cell types. AAV vectors can also be used for nuclease delivery.

[0111] Recombinase Recombination systems such as Cre / LoxP or Flp / FRT can be used for the exchange of partial nucleic acid sequences between different nucleic acid molecules, the excision of nucleic acid fragments from nucleic acid molecules, or the inversion of parts within nucleic acid molecules. The results of the action of the recombinase can be permanent using a single on / off event, or they can be defined but limited in duration and can be regulated to a defined specific cell type or tissue.

[0112] FLp recombinase The Flp / FRT site-specific recombination system involves sequence recombination between flippase-recognition target (FRT) sites by the recombinase flippase (Flp). The flippase is derived from Saccharomyces cerevisiae. The sequence of Flp is available, for example, from UniProt P03870. The 34 bp FRT site has the sequence GAAGTTCCTATTCtctagaaaGAATAGGAACTTC (SEQ ID NO: 20; lowercase letters indicate the central spacer sequence), and the Flp recombinase binds to the inverted 13 bp repeat of GAAGTTCCTATTC (forward SEQ ID NO: 21; reverse SEQ ID NO: 22) adjacent to the 8 bp central spacer sequence.

[0113] Exemplary FRT sites are shown in the table below (Branda and Dymecki, Dev. Cell 6(2004)7-28). TIFF0007857714000002.tif22128

[0114] Cre recombinase The Cre / LoxP site-directed recombination system is widely used in many biological experimental systems. Cre recombinase is a 38 kDa site-directed DNA recombinase that recognizes a 34 bp LoxP sequence. Cre recombinase is derived from bacteriophage P1 and belongs to the tyrosine family of site-directed recombinases. Cre recombinase can mediate both intramolecular and intermolecular recombination between LoxP sequences. A canonical LoxP sequence consists of two 13 bp reverse repeats flanked by an 8 bp non-palindromic spacer sequence. Cre recombinase binds to the 13 bp repeats, thereby mediating recombination within the 8 bp spacer sequence. Cre / LoxP-mediated recombination occurs with high efficiency and does not require other host factors. If the two LoxP sequences are located on the same nucleotide sequence and in the same orientation, Cre recombinase-mediated recombination will cleave the DNA sequence located between the two LoxP sequences as a covalently closed ring. When two LoxP sequences are located on the same nucleotide sequence in opposite / reverse orientations to each other, Cre recombinase-mediated recombination reverses the orientation of the DNA sequence located between the two LoxP sequences. When the two LoxP sequences are on two different DNA molecules, and one of the DNA molecules is circular, Cre recombinase-mediated recombination results in the integration of the circular DNA sequence.

[0115] Cre recombinase can be introduced into or activated within cells by any known method. For example, liposome-based gene delivery (WO 93 / 24640; Mannino and Gould-Fogerite, BioTechniques 6(1988)682-691; US ​​5,279,833; WO 91 / 06309; Feigner et al., Proc.Natl.Acad.Sci.USA 84(9871)7413-7414), or viral vectors, such as papillomavirus, retroviral vectors, and adeno-associated virus vectors (e.g., Berns et al., Ann.NY Acad.Sci.772(1995)95-104; Ali et al., Gene Ther.1(1994)367-384; Haddada et al. al.,Curr.Top.Microbiol.Immunol.199(1995)297-306;Buchscher et al.,J.Virol.66(1992)2731-2739;Johann et al.,J.Virol.66(1992)1635-1640;Sommerfelt et al. al.,Virol.176(1990)58-59;Wilson et al.,J.Virol.63(1989)2374-2378;Miller et al.,J.Virol.65(1991)2220-2224;WO 94 / 26877;Rosenburg and Fauci in Fundamental Immunology,Third Edition Paul (ed.) Raven Press, Ltd., New York (1993) and references therein; West et al.,Virology 160(1987)38-47;US 4,797,368;WO 93 / 24641;Kotin,Human Gene Therapy 5(1994)793-801;Muzyczka,J.Clin.Invest.94(1994)1351;US 5,173,414;Tratschin et al. al.,Mol.Cell.Biol.5(1985)3251-3260;Tratschin et al.,Mol.Cell.Biol.4 (1984) 2072-2081; Hermonat and Muzyczka, Proc. Natl. Acad. Sci. USA 81 (1984) 6466-6470; Samulski et al., J. Virol. 63 (1989) 3822-3828). .

[0116] For example, recombinant AAV vectors of serotype 2 expressing Cre recombinase have been described by Li, X., et al. (PLOS ONE 7(2012)e50063) and Scammell, E., et al. (J. Neurosci. 23(2003)5762-5770). Using this rAAV-Cre, we were able to induce very complete recombination of the target LoxP site. For delivery based on rAAV vectors, see also Muzyczka, Curr. Top. Microbiol. Immunol. 158(1992)97-129; US 4,797,368; WO 91 / 18088; Samulski, Current Opinion in Genetic and Development 3(1993)74-80.

[0117] For example, a Cre recombinase expression plasmid can be used.

[0118] For example, mRNA encoding Cre recombinase can be used.

[0119] Numerous functional LoxP sites are known, such as Lox511, Lox66, Lox11, Lox76, Lox75, Lox43, and Lox44 (see, for example, Hoess, R., et al., Nucl. Acids Res. 14 (1986) 2287-2300; Albert, H., et al., Plant J. 7 (1995) 649-659).

[0120] For example, when Cre recombinase is used, the sequences to be exchanged are defined by the locations of two LoxP sites in the genome and the donor nucleic acid. These LoxP sites are recognized by Cre recombinase. Nothing more is needed; i.e., ATP, etc., is not required.

[0121] The Cre / LoxP system functions in various types of cells, including those of mammals, plants, bacteria, and yeast.

[0122] Targeted integration using recombinase In certain embodiments, targeted integration is performed by a recombinase-mediated cassette exchange reaction (RMCE).

[0123] RMCE is an enzymatic process in which the sequence of an integration site in the genome is exchanged with that of a donor nucleic acid. Any recombinase, such as Cre recombinase, Flp recombinase, Bxb1-integrase, pSR1-recombinase, or φC31-integrase, can be used in this process.

[0124] One specific TI method is dual recombinase-mediated cassette exchange (dual RMCE).

[0125] Dual RMCE is a method for producing recombinant mammalian cells containing deoxyribonucleic acid encoding a target proteinaceous compound by recombinase-mediated introduction of two nucleic acid sequences into the host cell genome at a single locus. After integration, the two nucleic acid sequences are operably linked to each other.

[0126] For example, but not limited to, the incorporated exogenous nucleotide sequence, i.e., the TI landing site, may contain two recombination recognition sites (RRS), and the (donor) nucleic acid sequence may contain two RRS that match the RRS on the incorporated exogenous nucleotide sequence. Such a single plasmid RMCE strategy allows for the introduction of multiple open reading frames by incorporating an appropriate number of expression cassettes in each sequence between pairs of RRS.

[0127] For example, but not limited to, the incorporated exogenous nucleotide sequence, i.e., the TI landing site, may include three recombination recognition sites (RRS), for example, a sequence in which a third RRS ("RRS3") is located between the first RRS ("RRS1") and the second RRS ("RRS2"), with the first (donor) nucleic acid containing two RRSs matching the first and third RRS on the incorporated exogenous nucleotide sequence, and the second (donor) nucleic acid containing two RRSs matching the third and second RRS on the incorporated exogenous nucleotide sequence. Such a dual RMCE strategy enables the introduction of multiple genes by incorporating an appropriate number of expression cassettes in each sequence between each pair of RRSs.

[0128] Furthermore, a two-plasmid RMCE also requires two selection markers. One selection marker expression cassette is divided into two parts. The first (front) nucleic acid may contain the promoter, followed by the translation start codon and the RRS3 sequence. The second (back) nucleic acid correspondingly contains the RRS3 sequence fused to the N-terminus of the selection marker coding sequence minus the translation start codon (e.g., ATG). Additional nucleotides may need to be inserted between the RRS3 site and the selection marker coding sequence to ensure in-frame translation from the fusion gene, i.e., a functional linkage. Only when both nucleic acids (front and back) are precisely inserted can a complete expression cassette of the selection marker be assembled, and thus resistance to each selector is conferred to the cells.

[0129] Both single-plasmidal RMCEs and dual-plasmidal RMCEs enable the integration of one or more donor DNA molecules into predetermined locations in the mammalian cell genome by precisely exchanging DNA sequences present on the donor DNA with DNA sequences in the mammalian cell genome where the integration site is located. These DNA sequences are characterized by i) at least one selection marker, or a “split selection marker” as in certain two-plasmidal RMCEs, and / or ii) at least one exogenous gene of interest, and two heterospecific RRSs adjacent to it.

[0130] RMCE involves a recombinase-catalyzed double recombination crossover event between two heterologous RRSs within a target genomic locus and a donor DNA molecule. It is designed to introduce a combined copy of the DNA sequence, derived from a dual front and back nucleic acid, into a predetermined locus in the mammalian cell genome. The RMCE procedure can be repeated using multiple DNA sequences.

[0131] In certain embodiments, targeted integration is achieved by dual RMCE, in which two different DNA sequences are both integrated into predetermined sites in the genome of a TI-suitable mammalian cell, and each DNA sequence includes at least one expression cassette encoding a portion of the target protein compound and / or two heterospecific RRSs adjacent to at least one select marker or a portion thereof. In certain embodiments, targeted integration is achieved by multiple RMCEs, in which DNA sequences derived from multiple nucleic acids are all integrated into predetermined sites in the genome of a TI-suitable mammalian cell, and each DNA sequence includes at least one expression cassette encoding a portion of the target protein compound and / or two heterospecific RRSs adjacent to at least one select marker or a portion thereof. In certain embodiments, the select marker may be partially encoded in a first nucleic acid (front) and partially encoded in a second nucleic acid (back), and as a result, expression of the select marker is possible only when both nucleic acids are precisely integrated by dual RMCE.

[0132] For single-RMCE and dual-RMCE, the methods for targeted integration of donor nucleic acids into the genome of recipient / target cells, as well as the methods for simultaneous targeted integration of two donor nucleic acids into the genome of recipient / target cells as outlined above, include an additional step of introducing / activating a recombinase.

[0133] Therefore, in one embodiment, the recombinant sequence is a recombinant recognition sequence, and the method is as follows: c) i) b) Simultaneously with the introduction of deoxyribonucleic acid; or ii) Subsequently, sequentially, The process further includes the step of introducing or activating a recombinase, The recombinases recognize the recombination recognition sequences of the first and second deoxyribonucleic acids (and optionally, one or more recombinases perform recombinase-mediated cassette exchange).

[0134] In certain embodiments, the RRS is selected from the group consisting of LoxP, L3, 2L, LoxFas, Lox511, Lox2272, Lox2372, Lox5171, Loxm2, Lox71, Lox66, FRT, F3, F5, Bxb1 attP, Bxb1 attB, φC31 attP, and φC31 attB sequences. If multiple RRSs must exist, the selection of each sequence depends on the other, insofar as non-identical RRSs are selected.

[0135] In certain embodiments, RRS can be recognized by Cre recombinase. In certain embodiments, RRS can be recognized by Flp recombinase. In certain embodiments, RRS can be recognized by Bxb1 integrase. In certain embodiments, RRS can be recognized by φC31 integrase. In certain embodiments, RRS can be recognized by pSR1 recombinase.

[0136] In certain embodiments, if the RRS is a LoxP site, the cell requires Cre recombinase to perform recombination.

[0137] In certain embodiments, if the RRS is the FRT site, the cell requires Flp recombinase to perform recombination.

[0138] In certain embodiments where the RRS is a Bxb1 attP site or a Bxb1 attB site, the cells require Bxb1 integrase to undergo recombination.

[0139] In certain embodiments, if the RRS is a φC31 attP site or a φC31 attB site, the cell requires φC31 integrase to undergo recombination.

[0140] In certain embodiments, if RRS is the recognition site for pSR1-recombinase in Zygosaccharomyces rouxii, the cell requires pSR1-recombinase to undergo recombination.

[0141] Recombinase-coding genes can be delivered to cells as DNA, via viral vectors, or as mRNA. DNA or mRNA transfection can be carried out by electroporation or cationic lipid-based reagents. Integrase-deficient lentiviral vectors can be used to deliver recombinase to transfection-resistant cell types. AAV vectors can also be used for recombinase delivery. Recombinase proteins can also be introduced via non-vesicles.

[0142] In all aspects and in one embodiment of the embodiments, the recombinase is introduced into the cell as mRNA.

[0143] In all aspects and in one embodiment of the embodiments, the recombinase is introduced into the host cell as DNA. In one embodiment, the DNA is a sequence encoding the recombinase contained in an expression cassette.

[0144] In all aspects and in one embodiment of the embodiments, the recombinase is Cre recombinase, which is introduced into cells as Cre recombinase-coding mRNA encoding a polypeptide having the amino acid sequence of SEQ ID NO: 26.

[0145] In all aspects and in one embodiment, Cre recombinase mRNA encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 26 and further comprising a nuclear localization sequence at its N-terminus, C-terminus, or both. In one embodiment, Cre recombinase mRNA encodes a polypeptide having the amino acid sequence of SEQ ID NO: 26 and further comprising 1 to 5 nuclear localization sequences at its N-terminus, C-terminus, or both, independently of each other.

[0146] In all embodiments and one embodiment of the embodiments, the mRNA encoding Cre recombinase comprises the nucleotide sequence of SEQ ID NO: 27 or a variant thereof having a different codon frequency. In all embodiments and one embodiment of the embodiments, the mRNA encoding Cre recombinase comprises the nucleotide sequence of SEQ ID NO: 27 or a variant thereof having a different codon frequency, and further comprises a further nucleic acid encoding a nuclear localization sequence at its 5' end, 3' end, or both. In all embodiments and one embodiment of the embodiments, the mRNA encoding Cre recombinase comprises the nucleotide sequence of SEQ ID NO: 27 or a variant thereof having a different codon frequency, and further comprises 1 to 5 nucleic acids encoding a nuclear localization sequence independently at its 5' end, 3' end, or both.

[0147] In certain embodiments, the LoxP sequence is a wild-type LoxP sequence. In certain embodiments, the LoxP sequence is a mutant LoxP sequence. The mutant LoxP sequence was developed to enhance the efficiency of Cre recombinase-mediated integration or substitution. In certain embodiments, the mutant LoxP sequence is selected from the group consisting of L3, 2L, LoxFas, Lox511, Lox2272, Lox2372, Lox5171, Loxm2, Lox71, and Lox66 sequences. For example, the Lox71 sequence has a 5bp mutation in the left 13bp repeat. The Lox66 sequence has a 5bp mutation in the right 13bp repeat. Both wild-type and mutant LoxP sequences can mediate Cre recombinase-dependent recombination.

[0148] The term "matching RRS" indicates that recombination occurs between two matching RRS. In a particular embodiment, the two matching RRS are the same. In a particular embodiment, both RRS are wild-type LoxP sequences. In a particular embodiment, both RRS are mutant LoxP sequences. In a particular embodiment, both RRS are wild-type FRT sequences. In a particular embodiment, both RRS are mutant FRT sequences. In a particular embodiment, the two matching RRS are different sequences but can be recognized by the same recombinase. In a particular embodiment, the first matching RRS is the Lox71 sequence and the second matching RRS is the Lox66 sequence. In a particular embodiment, the first matching RRS is the Bxb1 attP sequence and the second matching RRS is the Bxb1 attB sequence. In a particular embodiment, the first matching RRS is the φC31 attB sequence and the second matching RRS is the φC31 attB sequence.

[0149] In all aspects and in one embodiment of the embodiments, the recombinant recognition sites in the dual RMCE are L3, 2L, and LoxFas. In one embodiment, L3 includes the sequence of sequence number 04 as a spacer sequence, 2L includes the sequence of sequence number 05 as a spacer sequence, and LoxFas includes the sequence having the sequence of sequence number 06 as a spacer sequence. In one embodiment, the first recombinant recognition site is L3, the second recombinant recognition site is 2L, and the third recombinant recognition site is LoxFas.

[0150] In all aspects and in one embodiment of the embodiments, the expression cassette encoding the selection marker is located partially 5' and partially 3' relative to a third recombinant recognition site, wherein the 5' portion of the expression cassette includes a promoter and a translation initiation codon, and the 3' portion of the expression cassette includes a coding sequence without a translation initiation codon and a polyA signal sequence.

[0151] In all aspects and in one embodiment of the embodiments, the 5' portion of the expression cassette encoding the selection marker includes a promoter sequence operably linked to a translation start codon, thereby the promoter sequence is adjacent upstream to a second, third, or fourth expression cassette (i.e., located downstream of the second, third, or fourth expression cassette), the start codon is adjacent downstream to a third recombinant recognition sequence (i.e., located upstream of the third recombinant recognition sequence), and the 3' portion of the expression cassette encoding the selection marker includes a nucleic acid encoding a selection marker lacking a translation start codon, adjacent upstream to a third recombinant recognition sequence, adjacent downstream to a poly(A) signal sequence, and then adjacent to a third, fourth, or fifth expression cassette, respectively.

[0152] Any known or future mammalian cells suitable for targeted integration, including the exogenous nucleic acids described herein ("landing sites"), can be used in the present invention.

[0153] In all aspects and one preferred embodiment, the mammalian cell containing an exogenous nucleotide sequence integrated into a single site within a gene locus of the mammalian cell genome is a hamster cell or a human cell, and in one embodiment is a CHO cell.

[0154] Exemplary mammalian cells suitable for use in the present invention, which contain an exogenous nucleotide sequence integrated into a single site within a locus of their genome, are CHO cells, HEK293 cells, or Per.C6 cells that have a landing site (= an exogenous nucleotide sequence integrated into a single site within a locus of the genome of a mammalian cell) containing three heterospecific LoxP sites for Cre recombinase-mediated cassette exchange. In one embodiment, these heterospecific LoxP sites are L3, LoxFas, and 2L (see, for example, Lanza et al., Biotechnol. J. 7 (2012) 898-908; Wong et al., Nucleic Acids Res. 33 (2005) e147), where L3 and 2L are adjacent to the 5' and 3' ends of the landing site, respectively, or vice versa, and LoxFas is located between the L3 and 2L sites. In all aspects and in certain embodiments of the embodiments, the landing site further includes a bisistronic unit that links the expression of a selection marker via IRES to the expression of green fluorescent protein (GFP), thereby stabilizing the landing site by positive selection and enabling the selection of samples in which the site is absent after transfection and Cre recombinase-mediated recombination (negative selection). An exemplary GFP has the sequence of SEQ ID NO: 28.

[0155] Such a configuration of the landing site, as outlined in the previous paragraph, allows for the simultaneous integration of two nucleic acids from different plasmids: a so-called front nucleic acid with L3 and LoxFas sites, and a back nucleic acid with LoxFas and 2L sites. Functional elements of the selection marker gene, distinct from those present in the landing site, are distributed between the two nucleic acids: the promoter and translation start codon are located on the front nucleic acid, while the coding region and poly(A) signaling pathway are located on the back nucleic acid. Only correct Cre recombinase-mediated integration of both nucleic acids induces resistance to their respective selectors.

[0156] Generally, mammalian cells suitable for TI are mammalian cells that contain an exogenous nucleotide sequence integrated into a gene locus in their genome, the exogenous nucleotide sequence comprising first and second recombination recognition sites adjacent to at least one first selection marker, and a third recombination recognition site located between the first and second recombination recognition sites, all of which are distinct. The exogenous nucleotide sequence is called the “landing site”.

[0157] The subject matter disclosed in this invention uses mammalian cells suitable for TI with an exogenous nucleotide sequence. In certain embodiments, the mammalian cells suitable for TI contain an exogenous nucleotide sequence that is integrated into an integration site in the genome of the mammalian cell. Such mammalian cells suitable for TI may also be referred to as "TI host cells."

[0158] In all aspects and in specific embodiments of the embodiments, suitable mammalian cells for TI are hamster cells, human cells, rat cells, or mouse cells, including the landing site. In specific embodiments, suitable mammalian cells for TI are Chinese hamster ovary (CHO) cells, CHO K1 cells, CHO K1SV cells, CHO DG44 cells, CHO DUKXB-11 cells, CHO K1S cells, CHO K1M cells, human cells, HEK293 cells, or Per.C6 cells, including their respective landing sites.

[0159] In all aspects and in specific embodiments of the embodiments, a mammalian cell suitable for TI comprises an incorporated exogenous nucleotide sequence, the exogenous nucleotide sequence comprising one or more recombination recognition sites (RRS). In specific embodiments, the exogenous nucleotide sequence comprises at least two RRSs. The RRSs can be recognized by recombinases, such as Cre recombinase, Flp recombinase, Bxb1 integrase, or φC31 integrase. The RRSs may be selected from the group consisting of LoxP sites, L3 sites, 2L sites, LoxFas sites, Lox511 sites, Lox2272 sites, Lox2372 sites, Lox5171 sites, Loxm2 sites, Lox71 sites, Lox66 sites, FRT sites, F3 sites, F5 sites, Bxb1 attP sites, Bxb1 attB sites, φC31 attP sites, and φC31 attB sites.

[0160] In all aspects and in one embodiment of the embodiments, the selection marker is independently selected from the group consisting of genes encoding resistance to aminoglycoside phosphotransferases (APHs) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthase (GS), asparagine synthase, tryptophan synthase (indole), histidinol dehydrogenase (histidinol D), and puromycin, blasticidine, bleomycin, phleomycin, chloramphenicol, zeosin, and mycophenolic acid. Furthermore, the selection marker may be a fluorescent protein selected from the group consisting of green fluorescent protein (GFP), high-sensitivity GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), high-sensitivity YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald6, CyPet, mCFPm, Cerulean, and T-Sapphire.

[0161] An exogenous nucleotide sequence is a nucleotide sequence that does not originate from a specific cell but can be introduced into the cell by a DNA delivery method, such as transfection, transduction, electroporation, or transformation. In all aspects and in specific embodiments of the embodiments, a mammalian cell suitable for TI contains at least one exogenous nucleotide sequence integrated into many integration sites in the genome of the mammalian cell. In specific embodiments, the exogenous nucleotide sequence is integrated into an integration site within a specific gene locus in the genome of the mammalian cell.

[0162] In all aspects and in certain embodiments of the embodiments, the incorporated exogenous nucleotide sequence includes one or more recombination recognition sites (RRSs) that can be recognized by a recombinase. In certain embodiments, the incorporated exogenous nucleotide sequence includes at least two RRSs. In certain embodiments, the incorporated exogenous nucleotide sequence includes three RRSs, with the third RRS located between the first and second RRSs. In certain embodiments, the first and second RRSs are identical, and the third RRS is different from either the first or second RRS. In certain embodiments, all three RRSs are different. In certain embodiments, the RRS can be independently selected from the group consisting of LoxP site, L3 site, 2L site, LoxFas site, Lox511 site, Lox2272 site, Lox2372 site, Lox5171 site, Loxm2 site, Lox71 site, Lox66 site, FRT site, F3 site, F5 site, Bxb1 attP site, Bxb1 attB site, φC31 attP site, and φC31 attB site.

[0163] In all aspects and in specific embodiments of the embodiments, the incorporated exogenous nucleotide sequence includes at least one selection marker. In specific embodiments, the incorporated exogenous nucleotide sequence includes first, second, and third RRSs, and at least one selection marker. In specific embodiments, the selection marker is located between the first RRS and the second RRS. In specific embodiments, the two RRSs are adjacent to at least one selection marker. That is, the first RRS is located 5' (upstream) of the selection marker, and the second RRS is located 3' (downstream) of the selection marker. In specific embodiments, the first RRS is adjacent to the 5' end of the selection marker, and the second RRS is adjacent to the 3' end of the selection marker.

[0164] In all aspects and in specific embodiments of the embodiments, the selection marker is located between a first and a second RRS, and these two adjacent RRSs are distinct from each other. In specific embodiments, the first adjacent RRS is the L3 sequence, and the second adjacent RRS is the 2L sequence. In specific embodiments, the L3 sequence (sequenced) is located on the 5' side of the selection marker, and the 2L sequence is located on the 3' side of the selection marker.

[0165] In all aspects and in certain embodiments of the embodiments, the first adjacent RRS is a LoxP sequence having a wild-type reverse repeat, and the second adjacent RRS is a LoxP sequence having one mutated reverse repeat. In a particular embodiment, the first adjacent RRS is a LoxP sequence having a first mutated reverse repeat, and the second adjacent RRS is a LoxP sequence having a second mutated reverse repeat that is the same as or different from the first mutated reverse repeat. In a particular embodiment, the first adjacent RRS is a LoxP sequence having a wild-type reverse repeat, and the third RRS is a LoxP sequence having one mutated reverse repeat. In a particular embodiment, the second adjacent RRS is a LoxP sequence having a wild-type reverse repeat, and the third RRS is a LoxP sequence having one mutated reverse repeat. In certain embodiments, the first adjacent RRS is a LoxP sequence having a first mutated reverse repeat, and the third RRS is a LoxP sequence having a second mutated reverse repeat. In all aspects and in certain embodiments of the embodiments, the second adjacent RRS is a LoxP sequence having a first mutated reverse repeat, and the third RRS is a LoxP sequence having a second mutated reverse repeat.

[0166] In all aspects and in specific embodiments of the embodiments, the first adjacent RRS is a wild-type FRT sequence, and the second adjacent RRS is a mutant FRT sequence. In specific embodiments, the first adjacent RRS is a first mutant FRT sequence, and the second adjacent RRS is a second mutant FRT sequence.

[0167] In all aspects and in specific embodiments of the embodiments, the first adjacent RRS is a Bxb1 attP sequence, and the second adjacent RRS is a Bxb1 attB sequence.

[0168] In all aspects and in specific embodiments of the embodiments, the first adjacent RRS is a φC31 attP sequence, and the second adjacent RRS is a φC31 attB sequence.

[0169] In all aspects and in specific embodiments of the embodiments, the incorporated exogenous nucleotide sequence includes first and second selection markers, where two RRSs are adjacent, and the first selection marker is distinct from the second selection marker. In specific embodiments, both of the two selection markers are independently selected from the group consisting of glutamine synthase selection markers, thymidine kinase selection markers, HYG selection markers, and puromycin resistance selection markers. In specific embodiments, the incorporated exogenous nucleotide sequence includes a thymidine kinase selection marker and a HYG selection marker. In specific embodiments, the first selection marker is an aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418). The second selection marker is selected from the group consisting of genes encoding resistance to APH, dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthase (GS), asparagine synthase, tryptophan synthase (indole), histidinol dehydrogenase (histidinol D), and puromycin, blasticidine, bleomycin, phleomycin, chloramphenicol, zeosin, and mycophenolic acid. The second selection marker is selected from the group consisting of GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire fluorescent proteins. In certain embodiments, the first selection marker is a glutamine synthase selection marker, and the second selection marker is a GFP fluorescent protein. In certain embodiments, the two RRSs adjacent to both selection markers are different.

[0170] In all aspects and in specific embodiments of the embodiments, the selection marker is operably linked to a promoter sequence. In specific embodiments, the selection marker is operably linked to the SV40 promoter. In specific embodiments, the selection marker is operably linked to the human cytomegalovirus (CMV) promoter.

[0171] Regardless of the method used for introducing donor deoxyribonucleic acid, successfully transfected cells can be selected based on the introduced second selection marker.

[0172] It should be noted that when the DNA element, DNA molecule, or VA RNA nucleic acid according to the present invention is used in combination with a recombinase-mediated cassette exchange reaction, different recombinases are used for RMCE and RMCI.

[0173] For example, in the DNA element, DNA molecule, or VA RNA according to the present invention, the Cre / LoxP system is used for recombinase-mediated cassette exchange (RMCE), and the Flp / FRT system is used for recombinase-mediated cassette inversion (RMCI). Similarly, in the DNA element, DNA molecule, or VA RNA according to the present invention, the Flp / FRT system is used for recombinase-mediated cassette exchange (RMCE), and the Cre / LoxP system is used for recombinase-mediated cassette inversion (RMCI).

[0174] Adeno-associated virus vector For a general review of the helper functions of AAV and adenoviruses or herpesviruses, see Berns and Bohensky, Advances in Virus Research, Academic Press, 32(1987)243-306. The AAV genome is described in Srivastava et al., J. Virol., 45(1983)555-564. U.S. Patent No. 4,797,368 describes design considerations for constructing recombinant AAV vectors (see also International Publication No. 93 / 24641). Further references describing AAV vectors are West et al., Virol. 160(1987)38-47; Kotin, Hum. Gene Ther. 5(1994)793-801; and Muzyczka J. Clin. Invest. 94(1994)1351. Construction of recombinant AAV vectors as described in U.S. Patent No. 5,173,414; Lebkowski et al., Mol. Cell. Biol. 8 (1988) 3988-3996; Tratschin et al., Mol. Cell. Biol. 5 (1985) 3251-3260; Tratschin et al., Mol. Cell. Biol. 4 (1994) 2072-2081; Hermonat and Muzyczka Proc. Natl. Acad. Sci. USA 81 (1984) 6466-6470; Samulski et al. J. Virol. 63 (1989) 3822-3828.

[0175] Adeno-associated viruses (AAVs) are replication-deficient parvoviruses. They can only replicate in cells where specific viral function is provided by co-infecting helper viruses such as adenoviruses, herpesviruses, and, in some cases, poxviruses such as vaccinia. Nevertheless, AAVs can replicate in substantially any cell line of human, monkey, or rodent origin, provided that appropriate helper viral function is present.

[0176] If the helper virus gene is absent, AAV establishes an incubation period in its host cell. Its genome is integrated into a specific site on chromosome 19 [(Chr)19(q13.4)] called adeno-associated virus integration site 1 (AAVS1). For certain serotypes such as AAV-2, other integration sites have been found, such as AAVS2 on chromosome 5 [(Chr)5(p13.3)] and AAVS3 on chromosome 3 [(Chr)3(p24.3)].

[0177] AAV is classified into different serotypes. These are assigned based on parameters such as hemagglutination, tumorigenicity, and DNA sequence homology. To date, more than 10 different serotypes and more than 100 sequences have been identified, corresponding to different clades of AAV.

[0178] The type and symmetry of the capsid protein determine the tissue tropism of each AAV. For example, AAV-2, AAV-4, and AAV-5 are specific to the retina; AAV-2, AAV-5, AAV-8, AAV-9, and AAVrh-10 are specific to the brain; AAV-1, AAV-2, AAV-6, AAV-8, and AAV-9 are specific to cardiac tissue; AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and AAV-10 are specific to the liver; and AAV-1, AAV-2, AAV-5, and AAV-9 are specific to the lungs.

[0179] Pseudotyping involves the cross-packaging of AAV genomes across different serotypes, meaning the genome is packaged with capsid proteins of different origins.

[0180] The wild-type AAV genome is approximately 4.7 kb in size. The AAV genome further contains two duplicate genes called rep and cap, which contain multiple open reading frames (see, e.g., Srivastava et al., J. Viral., 45(1983) 555-564; Hermonat et al., J. Viral. 51(1984) 329-339; Tratschin et al., J. Viral., 51(1984) 611-619). The open reading frame encoding the Rep protein provides four proteins of different sizes called Rep78, Rep68, Rep52, and Rep40. These are involved in AAV replication, rescue, and integration. The open reading frame encoding the Cap protein provides four proteins called VP1, VP2, VP3, and AAP. VP1, VP2, and VP3 are part of the proteinaceous capsid of the AAV particle. The combined open reading frames of rep and cap are flanked at their 5' and 3' ends by so-called reverse terminal repeats (ITRs). For replication, AAV requires, in addition to the Rep and Cap proteins, the products of adenovirus genes E1A, E1B, E4orf6, E2A, and VA, or corresponding factors from another helper virus.

[0181] For example, in the case of serotype 2 (AAV-2) AAV, each ITR is 145 nucleotides long and flanks a coding region of approximately 4470 nucleotides. Of the 145 nucleotides in the ITR, 125 nucleotides have a palindromic sequence and can form a T-shaped hairpin structure. This structure functions as a primer during viral replication. The remaining 20 unpaired nucleotides are shown as the D sequence.

[0182] The AAV genome has three transcription promoters, P5, P19, and P40, for the expression of rep and cap genes (Laughlin et al., Proc. Natl. Acad. Sci. USA 76(1979) 5567-5571).

[0183] ITR sequences must be cis relative to the coding region. ITRs provide functional origins of replication (ori), signals necessary for integration into the target cell genome, and efficient excision and rescue from host cell chromosomes or recombinant plasmids. ITRs further contain origin-of-replication elements such as Rep protein binding sites (RBS) and terminal dissociation sites (TRS). It has been found that ITRs themselves can function as transcription promoters in AAV vectors (Flotte et al., J. Biol. Chem. 268 (1993) 3781-3790; Flotte et al., Proc. Natl. Acad. Sci. USA 93 (1993) 10163-10167).

[0184] The replication of the viral single-stranded DNA genome and the formation of the capsid both require the trans transformation of the rep gene product and the cap gene product, respectively.

[0185] The rep locus contains two internal promoters called P5 and P19. It contains open reading frames for four proteins. Promoter P5 is operably ligated to nucleic acid sequences that provide a non-splicing 4.2kb mRNA encoding the Rep protein Rep78 (a chromatin niccasse for cell cycle arrest) and a splicing 3.9kb mRNA encoding the Rep protein Rep68 (a site-specific endonuclease). Promoter P19 is operably ligated to nucleic acid sequences that provide a non-splicing mRNA encoding the Rep protein Rep52 and a splicing 3.3kb mRNA encoding the Rep protein Rep40 (a DNA helicase for accumulation and packaging).

[0186] The two larger Rep proteins, Rep78 and Rep68, appear to be essential for AAV double-stranded DNA replication, while the smaller Rep proteins, Rep52 and Rep40, seem to be essential for the accumulation of single-stranded DNA in offspring (Chejanovsky & Carter, Virology 173(1989)120-128).

[0187] The larger Rep proteins, Rep68 and Rep78, can specifically bind to the hairpin conformation of the AAV ITR. They exhibit predetermined enzymatic activity required to degrade replication at the AAV terminus. Expression of Rep78 or Rep68 may be sufficient for infectious particle formation (Holscher, C., et al. J. Virol. 68(1994) 7169-7177 and 69(1995) 6880-6885).

[0188] All Rep proteins, mainly Rep78 and Rep68, are thought to exhibit regulatory activity such as induction and repression of AAV genes and inhibitory effects on cell proliferation (Tratschin et al., Mol.Cell.Biol.6(1986)2884-2894; Labow et al., Mol.Cell.Biol.,7(1987)1320-1325; Khleif et al., Virology,181(1991)738-741).

[0189] Recombinant overexpression of Rep78 results in a phenotype characterized by reduced cell proliferation due to induced DNA damage. This causes host cells to arrest in the S phase, thereby promoting latent viral infection (Berthet, C., et al., Proc. Natl. Acad. Sci. USA 102(2005)13634-13639).

[0190] Tratschin et al. reported that the P5 promoter is negatively autoregulated by Rep78 or Rep68 (Tratschin et al., Mol.Cell.Biol.6(1986)2884-2894). Due to the toxic effects of Rep protein expression, very low expression has been reported in certain cell lines after stable integration of AAV (see, for example, Mendelson et al., Virol.166(1988)154-165).

[0191] The cap locus contains a single promoter called P40. Promoter P40 is operably linked to nucleic acid sequences providing 2.6kb mRNA encoding the Cap proteins VP1 (87kDa, unspliced ​​mRNA transcript), VP2 (72kDa from spliced ​​mRNA transcript), and VP3 (61kDa from alternative start codon) through alternative splicing and the use of alternative start codons. VP1-VP3 constitute the building blocks of the viral capsid. The capsid has the function of binding to cell surface receptors and enabling intracellular transport of the virus. VP3 accounts for approximately 90% of the total viral particle protein. Nevertheless, all three proteins are essential for effective capsid production.

[0192] Inactivation of all three capsid proteins VP1-VP3 has been reported to prevent the accumulation of single-stranded progeny AAV DNA. Mutations at the amino terminus of VP1 ("lipid-negative" or "inf-negative") still allow the assembly of single-stranded DNA into the viral particle, thereby significantly reducing the infectivity titer.

[0193] The AAP open reading frame encodes the assembly activation protein (AAP). It is approximately 22 kDa in size and transports the native VP protein to the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein-coding sequence.

[0194] Each AAV particle contains only a single-stranded DNA molecule. This can be either a "positive" or "negative" strand. AAV virus particles containing DNA molecules are infectious. Inside an infected cell, the parental infectious single strand is converted to a double strand and then amplified. Amplification results in a large pool of double-stranded DNA molecules from which single strands are substituted and packaged into a capsid.

[0195] Adeno-associated virus (AAV) vectors can transduce both dividing and quiescent cells. Transgenes introduced into target cells using AAV vectors are thought to be expressed for extended periods. One drawback of using AAV vectors is the limited size of the transgenes that can be introduced into cells.

[0196] Carter et al. demonstrated that the entire rep and cap open reading frames can be deleted and replaced with the transgene (Carter, BJ, "Handbook of Parvoviruses", ed. by P. Tijssen, CRC Press, pp. 155-168 (1990)). Furthermore, it has been reported that the ITR must be maintained to preserve the functions of replicating, rescuing, packaging, and integrating the transgene into the target cell genome.

[0197] When cells containing each viral helper gene are transduced by an AAV vector, or vice versa, when cells containing the incorporated AAV provirus are transduced by an appropriate helper virus, the AAV provirus is activated and the lytic infection cycle is restarted (Clark, KR, et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, RJ, Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0198] E1A is the first viral helper gene expressed after adenovirus DNA enters the cell nucleus. The E1A gene encodes 12S and 13S proteins based on the same E1A mRNA through alternative splicing. Expression of 12S and 13S proteins leads to the activation of other viral functions E1B, E2, E3, and E4. Furthermore, expression of 12S and 13S proteins pushes the cell into the S phase of the cell cycle. If only E1A-derived proteins are expressed, the cell dies (apoptosis).

[0199] E1B is the second viral helper gene that is expressed. It is activated by E1A-derived proteins 12S and 13S. E1B gene-derived mRNA can be spliced ​​in two different ways, resulting in a first 55kDa transcript and a second 19kDa transcript. The E1B 55kDa protein is involved in regulating the cell cycle, preventing the transport of cellular mRNA in the later stages of infection, and preventing E1A-induced apoptosis. The E1B 19kDa protein is involved in preventing E1A-induced apoptosis in cells.

[0200] The E2 gene codes for different proteins. The E2A transcript codes for single-strand binding proteins (SSBPs), which are essential for AAV replication.

[0201] The E4 gene also codes for several proteins. The 34kDa protein derived from the E4 gene (E4orf6), along with the E1B 55kDa protein, prevents the accumulation of cellular mRNA in the cytoplasm, but also promotes the transport of viral RNA from the cell nucleus to the cytoplasm.

[0202] Generally, different complementary plasmids are simultaneously transfected into host cells to produce recombinant AAV particles. One plasmid contains a transgene sandwiched between two cis-acting AAV ITRs. The open reading frames of the missing AAV elements, namely Rep and Cap proteins, necessary for replication and subsequent packaging of the progeny recombinant genome are trans-contained on the second plasmid. Overexpression of the Rep protein results in an inhibitory effect on cell proliferation (Li, J., et al., J. Virol. 71 (1997) 5236-5243). Furthermore, a third plasmid containing helper virus genes, namely adenovirus-derived E1, E4 or F6, E2A, and VA, is required for AAV replication.

[0203] To reduce the number of plasmids required, the Rep, Cap, and adenovirus helper genes may be combined on a single plasmid.

[0204] Alternatively, the host cells may already be stably expressing the E1 gene product. Such cells are HEK293 cells. The human embryonic kidney clone, designated as 293, was created in 1977 by incorporating adenovirus DNA into human embryonic kidney cells (HEK cells) (Graham, FL, et al., J. Gen. Virol. 36(1977) 59-74). The HEK293 cell line contains base pairs 1-4344 of the adenovirus serotype 5 genome. This includes the E1A and E1B genes as well as the adenovirus packaging signal (Louis, N., et al., Virology 233(1997) 423-429).

[0205] When using HEK293 cells, the missing E2A, E4orf6, and VA genes can be introduced by co-infection with adenovirus or by co-transfection with E2A, E4orf6, and VA expression plasmids (e.g., Samulski, RJ, et al., J. Virol. 63 (1989) 3822-3828; Allen, JM, et al., J. Virol. 71 (1997) 6816-6822; Tamayose, K., et al., Hum. Gene Ther. 7 (1996) 507-513; Flotte, TR, et al., Gene Ther. 2 (1995) 29-37; Conway, JE, et al., J. Virol. 71 (1997) 8780-8789; Chiorini, JA, et al., Hum. Gene Ther. 71 (1997) 8780-8789; Chiorini, JA, et al., Hum. Gene Ther. Ther.6(1995)1531-1541;Ferrari,FK,et al.,J.Virol.70(1996)3227-3234;Salvetti,A.,et al.,Hum.Gene Ther.9(1998)695-706;Xiao,X.,et al., J. Virol. 72 (1998) 2224-2232; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Zhang, X., et al., Hum. Gene Ther. 10 (1999) 2527-2537). Alternatively, adenovirus / AAV or herpes simplex virus / AAV hybrid vectors can be used (see, for example, Conway, JE, et al., J. Virol. 71 (1997) 8780-8789; Johnston, KM, et al., Hum. Gene Ther. 8 (1997) 359-370; Thrasher, AJ, et al., Gene Ther. 2 (1995) 481-485; Fisher, JK, et al., Hum. Gene Ther. 7 (1996) 2079-2087; Johnston, KM, et al., Hum. Gene Ther. 8 (1997) 359-370).

[0206] Therefore, cell lines into which the rep gene is incorporated and expressed tend to grow slowly or express the Rep protein at very low levels.

[0207] A major safety concern is contamination of rAAV particle preparations with reproducible adenovirus (RCA). RCA is produced when the vector genome and adenovirus DNA integrated into the host cell are recombined during viral replication via homologous recombination (Lochmueller, H., et al., Hum. Gene Ther. 5 (1994) 1485-1491; Hehir KM, et al., J. Virol. 70 (1996) 8459-8467). Therefore, HEK293 cells are not suitable for producing adenovirus vectors for pharmaceutical use.

[0208] To restrict the activity of a transgene to a specific tissue, i.e., to restrict the integration site, the transgene can be operably linked to an inducible promoter or a tissue-specific promoter (see, for example, Yang, Y., et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0209] To date, the main difficulty in rAAV particle production has been the inefficient packaging of rAAV vectors, resulting in low titers. Packaging has been difficult for several reasons, including: - If wild-type AAV genomes are present, their preferred capsid formation; -Difficulty in generating sufficient complementary functions, such as those provided by wild-type rep and cap genes, due to inhibitory effects associated with the rep gene product; - Limited efficiency of simultaneous transfection of plasmid constructs.

[0210] All of these problems stem from the biological properties of Rep proteins. In particular, the inhibitory (cell proliferation suppression and cytotoxic) properties of Rep proteins, as well as their ability to reverse the immortalization phenotype of cultured cells, are problematic. Furthermore, Rep proteins downregulate their own expression when the widely used AAV P5 promoter is used (see, for example, Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894).

[0211] Exemplary compounds and compositions according to the present invention This specification reports novel nucleic acids and methods for using them. The novel nucleic acids according to the present invention are useful for the production of recombinant adeno-associated virus particles.

[0212] Accordingly, one aspect of the present invention is a novel adenovirus VA RNA nucleic acid. In the VA RNA nucleic acid according to the present invention, the VA RNA coding sequence includes, or is ligated to at its 5' end, a variant 2 polymerase III promoter, or a variant 3 polymerase III promoter, or a variant 3 polymerase III promoter, for example, in one preferred embodiment, a U6-snRNA promoter, or a polymerase II promoter. In certain embodiments, the VA RNA nucleic acid further includes a precise transcription start site located at 3' of the promoter and 5' of the VA RNA coding sequence. In certain embodiments, the VA RNA nucleic acid further includes a polymerase III terminator at its 3' end. In certain embodiments, the precise transcription start site includes at least six 5' terminal nucleotides of the adenovirus VA RNA I gene, in the 5'-to-3' direction, including a transcription start site (TSS) (to prevent bypass of a subsequent polymerase III (poly III) terminator) and a functional polymerase III terminator (to prevent transcription from a constitutively active upstream promoter). In all aspects and in specific embodiments of the embodiments, all elements of the adenovirus VA RNA nucleic acid (in) according to the present invention are arranged in an operablely linked configuration.

[0213] To further enhance the advantageous effects of the adenovirus VA RNA nucleic acid according to the present invention, the promoter used can also be selected to be activatable, particularly in the case of the polymerase II promoter. Thus, transcription of the VA RNA coding sequence can only be turned on by further specific promoter activation. This results in improved control of VA RNA coding sequence transcription on the one hand, and on the other hand, the possibility of turning transcription off again. The combination of adenovirus VA RNA nucleic acid and an inductive promoter according to the present invention can further suppress the potential leakage of the inductive promoter when used in isolation. Inductive systems such as the Tet on / off system are known in the art.

[0214] The subject matter of this disclosure provides not only methods for the preparation of recombinant mammalian rAAV packaging or cell lines, and optionally for nucleic acids suitable for inducible transcription of VA RNA, but also methods for the stable large-scale production of rAAV particles. Similarly, recombinant stable mammalian rAAV-producing cells with high productivity of rAAV particles can be obtained.

[0215] Therefore, in all aspects and in specific embodiments of the embodiments, the promoter is an inductive promoter. In specific embodiments, the inductive promoter is selected from the group of inductive promoters consisting of a tetracycline-controlled promoter, a kmate-controlled promoter, a FKBP12-mTOR-controlled promoter, a rapamycin-controlled promoter, a FKCsA-controlled promoter, an abscisic acid-controlled promoter, a tamoxifen-controlled promoter, and a riboswitch-controlled promoter (a heterodimer of FKCsA = FK506 and cyclosporine A).

[0216] For a review of inductive promoters, see, for example, Kallunki, T., et al., Cells 8(2019)796.

[0217] In all aspects and in specific embodiments of the embodiments, the promoter is a repressive promoter. In specific embodiments, the repressive promoter is selected from the group of repressive promoters, including tetracycline-controlled promoters, GAL4 / UAS-controlled promoters, and LexA / lexAop-controlled promoters.

[0218] Recombinant AAV particles The generation of recombinant AAV particles requires the expression of Rep and Cap proteins, helper proteins E1A, E1B, E2A, and E4orf6, and adenovirus VA RNA in a single mammalian cell. Helper proteins E1A, E1B, E2A, and E4orf6 can be expressed using any promoter shown by Matsushita et al. (Gene Ther. 5(1998) 938-945), particularly the CMV IE promoter. Therefore, any promoter can be used below.

[0219] DNA according to the present invention, including E1A, E1B, E2A, and E4orf6 open reading frames One independent aspect of the invention is DNA (molecule), -Adenovirus VA RNA nucleic acid according to the present invention, - The first DNA element, and -Optionally, rep or / and cap open reading frame and It is DNA (a molecule) that contains [this].

[0220] In one dependent embodiment, - The first DNA element includes an E1A open reading frame and an E1B open reading frame; or - The first DNA element includes an E2A open reading frame and an E4 or E4 or E4 or f6 open reading frame; or - The first DNA element is the Rep protein open reading frame and the Cap protein open reading frame Includes.

[0221] One independent aspect of the present invention is a mammalian or insect cell containing adenovirus VA RNA nucleic acid or DNA (elements) according to the present invention.

[0222] One independent aspect of the present invention is a method for producing recombinant adeno-associated virus (rAAV) particles, - The process of culturing / proliferating cells according to the present invention (under conditions suitable for cell division), - A step of recovering rAAV particles from cells or culture medium and This method includes [something].

[0223] Therefore, one independent aspect of the present invention is an adenovirus VA RNA nucleic acid or DNA (molecule) according to the present invention for producing recombinant adeno-associated virus particles.

[0224] One independent aspect of the present invention is a method for generating / producing recombinant adeno-associated virus (rAAV) particles, - To provide mammalian suspension-growing cells that are either stably incorporated or transiently present and include the following: - A transgene expression cassette placed between two AAV ITRs; - Open reading frames encoding adenovirus E1A, E1B, E2A, E4 or E4orf6 proteins and adenovirus VA RNA nucleic acids according to the present invention; - Open reading frames encoding adeno-associated Rep and Cap proteins; - To grow / culture mammalian cells (under conditions suitable for cell division); and - Isolating rAAV particles from cells or culture medium, and thereby producing rAAV particles. This method includes [something].

[0225] In all aspects and in specific embodiments of the embodiments, each open reading frame is located within the expression cassette, i.e., operably coupled to the promoter and the polyadenylation signal sequence and / or transcription termination element.

[0226] The coding sequences for E1A and E1B (open reading frames) are derived in all aspects and specific embodiments from human adenoviruses, for example, particularly human adenovirus serotype 2 or 5. An exemplary sequence for human Ad5 (adenovirus serotype 5) can be found in GenBank entry X02996, and an exemplary sequence for human Ad2 can be found in GenBank entry AC_000007. In all aspects and specific embodiments, nucleotides 505-3522 include nucleic acid sequences encoding human adenovirus serotype 5 E1A and E1B. Plasmid pSTK146, reported in EP 1 230 354 B1, and plasmids pGS119 and pGS122, reported in International Publication 2007 / 056994, can also be used as sources for E1A and E1B open reading frames.

[0227] DNA according to the present invention, including Rep and Cap open reading frames With the exception of the P5 promoter, the promoters driving open reading-frame expression of rep and cap are located within the Rep polypeptide coding sequence.

[0228] One independent aspect of the invention is DNA (molecule), -Adenovirus VA RNA nucleic acid according to the present invention, - The first DNA element, and -Optionally, a DNA (molecule) containing one or more or all of the E1A, E1B, E2, E4, and E4 or f6 reading frames.

[0229] In certain embodiments of all aspects and embodiments, the first DNA element comprises a rep open reading frame and / or a cap open reading frame.

[0230] In certain embodiments of all aspects and embodiments, the first DNA element comprises an open reading frame encoding one, two, three, or four different Rep proteins.

[0231] In certain embodiments of all aspects and embodiments, the first DNA element comprises one rep open reading frame containing a coding sequence that encodes either only the Rep78 protein or only the Rep68 protein but not both, with the internal P40 promoter inactivated and splice donor and acceptor sites removed.

[0232] In certain embodiments of all aspects and embodiments, the rep open reading frame is operably linked at its 5' end to the adeno-associated virus promoter P5 or a functional fragment or variant thereof.

[0233] In certain embodiments of all aspects and embodiments, the first DNA element comprises two rep open reading frames. The first rep open reading frame contains a coding sequence that encodes either only the Rep78 protein or only the Rep68 protein but not both, with the internal P40 promoter inactivated and splice donor and acceptor sites removed. The second rep open reading frame contains a coding sequence encoding the Rep52 / Rep40 protein.

[0234] In all aspects and in certain embodiments of the embodiments, the first DNA element comprises two rep open reading frames, the first rep open reading frame comprising a coding sequence that encodes either the Rep78 protein only or the Rep68 protein only, but not both, with the internal P40 promoter inactivated and the splice donor and acceptor sites removed, and the second rep open reading frame comprising a coding sequence that encodes the Rep52 protein.

[0235] In all aspects and in specific embodiments of the embodiments, the first DNA element comprises two rep open reading frames, the first rep open reading frame comprising a coding sequence encoding either the Rep78 protein only or the Rep68 protein only, but not both, with the internal P40 promoter inactivated and the splice donor and acceptor sites removed, and the second rep open reading frame comprising a coding sequence encoding the Rep52 / Rep40 protein and the Cap protein, which includes a common polyadenylation signal.

[0236] In all aspects and in specific embodiments of the embodiments, the first rep open reading frame is operably linked at its 5' end to the adeno-associated virus promoter P5 or a functional fragment thereof or a variant thereof.

[0237] In all aspects and in specific embodiments of the embodiments, the second rep open reading frame is operably linked at its 5' end to the adeno-associated virus promoter P19 or a functional fragment thereof or a variant thereof.

[0238] One independent aspect of the present invention is a mammalian or insect cell containing adenovirus VA RNA nucleic acid or DNA (elements) according to the present invention.

[0239] One independent aspect of the present invention is a method for producing recombinant adeno-associated virus (rAAV) particles, - The process of culturing / proliferating cells according to the present invention (under conditions suitable for cell division), - A step of recovering rAAV particles from cells or culture medium and This method includes [something].

[0240] Therefore, one independent aspect of the present invention is an adenovirus VA RNA nucleic acid or DNA (molecule) according to the present invention for producing recombinant adeno-associated virus particles.

[0241] One aspect of the present invention is a method for generating / producing recombinant adeno-associated virus (rAAV) particles, - To provide mammalian suspension-growing cells that are either stably incorporated or transiently present and include the following: - A transgene expression cassette placed between two AAV ITRs; - Open reading frames encoding adenovirus E1A, E1B, E2A, E4 or E4orf6 proteins and adenovirus VA RNA nucleic acids according to the present invention; - Open reading frame encoding adeno-associated Rep / Cap protein; - To grow / culture mammalian cells (under conditions suitable for cell division); and - Isolating rAAV particles from cells or culture medium, and thereby producing rAAV particles. This method includes [something].

[0242] In all aspects and in specific embodiments of the embodiments, each open reading frame is located within the expression cassette, i.e., operably coupled to the promoter and the polyadenylation signal sequence and / or transcription termination element.

[0243] Adenovirus VA RNA nucleic acid according to the present invention The VA RNA gene is driven by a type 2 polymerase III promoter containing two intragenetic elements, the A-box and the B-box. Snouwaert et al. (Nucl. Acids Res. 15 (1987) 8293-8303) identified a VA RNAI B-box mutant that completely disables promoter activity. These mutations are unlikely to affect VA RNAI binding to PKR and related functions (Clark, KR, et al., Hum. Gene Ther. 6 (1995) 1329-1341).

[0244] The inventors have found that, in order to enable stringent control of VA RNA transcription, it is advantageous to inactivate the wild-type type 2 polymerase III promoter of the VA RNA gene and replace it with a different promoter, such as the type 3 polymerase III promoter, for example, the U6-snRNA promoter in one preferred embodiment, or the polymerase II promoter or an inducible promoter.

[0245] Therefore, one embodiment of the present invention is an AAV adenovirus VA RNA coding sequence under the control of a polymerase III type 3 promoter. In a preferred embodiment, the polymerase III type 3 promoter is a human U6-snRNA promoter.

[0246] Therefore, one aspect of the present invention is an AAV adenovirus VA RNA coding sequence under the control of the polymerase II promoter.

[0247] The type 3 polymerase III promoter contains two extragenic elements named proximal sequence element (PSE) and TATA box. In this regard, the type 3 polymerase III promoter is similar to the polymerase II promoter that drives protein gene expression. The spacing requirements between the two elements and between the elements and the transcription start site (TSS) are very strict, and the distance is quite short. The PSE of the human U6 promoter extends from position -66 to -47, and the TATA box extends from -29 to -23. Generally, transcription starts with a G or, less preferably, an A nucleotide present within the range of these +3 and -3 distances (Goomer and Kunkel, 1992).

[0248] The adenoviral VA RNA nucleic acid according to the invention allows, inter alia, for tight transcriptional control. In certain embodiments, VA RNA nucleic acid transcription is driven by a type 3 polymerase III promoter, such as the human U6-snRNA promoter, or a polymerase II promoter or an inducible promoter.

[0249] Certain aspects of the invention are shown in Figure 2.

[0250] In certain embodiments of all aspects and embodiments, the promoter driving the transcription of the adenoviral VA RNA according to the invention is the human U6 promoter. In certain embodiments, this promoter has the sequence of SEQ ID NO: 42.

[0251] In certain embodiments of all aspects and embodiments, the promoter driving the transcription of the transcriptional adenoviral VA RNA according to the invention is the mouse U6 promoter. In certain embodiments, this promoter has the sequence of SEQ ID NO: 43.

[0252] In certain embodiments of all aspects and embodiments, the promoter driving the transcription of the adenoviral VA RNA according to the invention is the human H1 pRNA promoter. In certain embodiments, this promoter has the sequence of SEQ ID NO: 44.

[0253] In all aspects and in specific embodiments of the embodiments, the promoter that drives the transcription of adenovirus VA RNA according to the present invention is a human tRNA val promoter. In specific embodiments, this promoter has the sequence of SEQ ID NO: 44.

[0254] In all aspects and in specific embodiments of the embodiments, a precise transcription start site is introduced into the non-coding element, i.e., the regulatory element, of the adenovirus VA RNA according to the present invention.

[0255] Virus-associated RNA (VA RNA) is the non-coding RNA of adenoviruses (Ad) that regulates translation. The adenovirus genome contains two independent copies: VAI (VA RNAI) and VAII (VA RNAII). Both are transcribed by RNA polymerase III (see, e.g., Machitani, M., et al., J. Contr. Rel. 154 (2011) 285-289).

[0256] The structure, function, and evolution of adenovirus-associated RNAs were investigated using a phylogenetic approach by Ma, Y. and Mathews, MB (J. Virol. 70(1996) 5083-5099). These provided alignment and consensus VA RNA sequences based on 47 known human adenovirus serotypes. The foregoing disclosure is incorporated in its entirety by reference.

[0257] VA RNA, VAI, and VAII consist of 157 to 160 nucleotides (nt).

[0258] Depending on the serotype, adenoviruses contain one or two VA RNA genes. VA RNAI plays the role of the dominant provirus, while VA RNAII is thought to partially compensate for the absence of VA RNAI (Vachon, VKand Conn, GL, Virus Res. 212 (2016) 39-52).

[0259] VA RNA is not essential, but it plays a crucial role in efficient viral replication by overcoming the cell's antiviral mechanisms. In other words, although VA RNA is not essential for viral replication, VA RNA-deficient adenoviruses cannot replicate during the initial stages of vector generation, when only a few copies of the viral genome exist per cell, because other viral genes that likely block the cell's antiviral mechanisms are not sufficiently expressed (see Maekawa, A., et al. Nature Sci. Rep. 3 (2013) 1136).

[0260] The A-box and B-box, which constitute the internal regulatory region (or promoter) of RNA polymerase III, are experimentally defined for adenovirus serotype 2 (Ad2) VA RNA I. These are well conserved. All VA RNAs have both boxes in similar positions. B-box homology is very high. The A-box, located 34-40 nt upstream of the B-box, has slightly lower homology in some VA RNAs. The pair of complementary tetranucleotides that form part of the apical stem of the VA RNA, CCGG (SEQ ID NO: 29) and (U / C)CCGG (SEQ ID NO: 30), are fairly well conserved in VA RNA sequences. The first CCGG, including the first two bases of the B-box, is invariant. All VA RNA genes except one have sequences that are 5' half-homologous to the tRNA transcription start element, the A-box and B-box consensus sequences RRYNNARYGG (SEQ ID NO: 31) and GWTCRANNC (SEQ ID NO: 32), respectively. A-box homology in VA RNA II genes is generally weaker than that in VA RNA I genes, consistent with the finding that A-boxes are less important to VA RNA transcription than B-boxes. The ends of VA RNA coding sequences contain a chain of T residues adjacent to nucleotides C and G, typical of polymerase III termination sites. The number of thymidines varies from a minimum of 4 to more than 10, and A residues are absent at least 3nt on either side of T-rich chains (with the exception of Ad12 and Ad18, which have A residues in the middle of very long T chains) (Ma, Y. and Mathews, MB, J. Virol. 70(1996) 5083-5099).

[0261] The B-box sequences of VA RNAI and VA RNAII are known to be essential for the activity of the internal polymerase III promoter.

[0262] Maekawa, A., et al. (Nature Sci. Rep. 3 (2013) 1136) reported the efficient production of an adenovirus vector lacking the gene for virus-associated RNA that disrupts the cellular RNAi mechanism. In this study, HEK293 cells constitutively and highly expressing flipper jelly combinase were infected, and VA RNA-deficient adenoviruses were obtained by FLP recombinase-mediated excision of the VA RNA locus.

[0263] The sequence of human adenovirus 2 VA RNAI (nucleotides 10586-10810 of GenBank entry AC_000007) is shown in SEQ ID NO: 33. This is G58T / G59T / C68A (continuous residue numbering) of SEQ ID NO: 34. SEQ ID NO: 34 is also an embodiment of the present invention. The sequence of human adenovirus 5 VA RNAI (nucleotides 10579-10820 of GenBank entry AC_000008) is shown in SEQ ID NO: 35. This is the combination of human adenovirus 5 VA RNAI and VA RNAII of SEQ ID NO: 36.

[0264] Hahn, S. (Nat. Struct. Mol. Biol. 11 (2004) 394-403) and Revyakin, A., et al. (Gen. Devel. 26 (2012) 1691-1702) reported on the structure and mechanism of RNA polymerase II transcription, while Nikitina, TV and Tishchenko, LI (Mol. Biol. 39 (2005) 161-172) outlined the RNA polymerase III transcription mechanism. These are summarized below.

[0265] Transcription, or RNA synthesis on a DNA template, is carried out by DNA-dependent RNA polymerase (Pols, [EC 2.7.7.6]). In addition to RNA polymerase, further factors called basic transcription factors (GTFs) are involved. These are necessary for promoter sequence recognition, response to regulatory factors, and conformational changes required for polymerase activity during transcription.

[0266] The core promoter (the minimal DNA sequence required to identify non-regulated or basal transcription) helps position Pol in a state called the pre-start complex (PIC). In this state, Pol and GTF are all bound to the promoter, but they are not in a conformation that is active for initiating transcription.

[0267] Eukaryotic cells contain three Pols, designated as I, II, and III, which differ in their subunit composition.

[0268] Genes transcribed by a particular Pol are assigned to correspond to class I, II, or III.

[0269] Pol I transcribes pre-rRNA genes. Pol II transcribes all protein-coding genes and snRNA genes except U6 snRNA. Pol III transcribes 5S rRNA, tRNA, U6 snRNA, 7SK RNA, 7SL RNA genes; Alu repeats; some viral genes; and genes for small, stable uncoding RNAs.

[0270] Different classes of genes have different promoter structures that determine the Pol involved in the formation of basic transcription factors and PICs.

[0271] RNA polymerase II (Pol II) is responsible for the flow of genetic information from DNA to messenger RNA (mRNA) in eukaryotic cells. Studies have identified GTF-TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, which assemble with Pol II at the promoter site into the PIC, initiating transcription initiation at the basal activity level. Further regulation of transcriptional activity depends on cis-regulatory elements in the DNA template recognized by sequence-specific activators / repressors assisted by coactivators.

[0272] Sequence elements found in the Pol II core promoter include the TATA element (TATA-binding protein (TBP) binding site), BRE (TFIIB recognition element), Inr (initiation factor element), and DPE (downstream promoter element). Most promoters contain one or more of these elements, but none of them are absolutely essential for promoter function. Promoter elements are binding sites for subunits of the transcription mechanism and help to asymmetrically orient the transcription mechanism within the promoter to direct unidirectional transcription.

[0273] The core domain of TBP consists of two incomplete repeats that form a molecule that binds to DNA with an 8 bp TATA element. In a TATA-containing promoter, the formation of this protein-DNA complex is the first step in the assembly of the transcription mechanism. The TATA-like sequence is located approximately 30 bp upstream of the transcription start site.

[0274] RNA polymerase III (Pol III) has the most complex structure of all eukaryotic Pol enzymes: the enzyme consists of 17 subunits ranging from approximately 10 kDa to 160 kDa, with a total molecular weight of 600 to 680 kDa.

[0275] Class III genes transcribed by Pol III primarily contain three structurally distinct promoters located within the gene. The basic transcription factors of the Pol III mechanism are TFIIIA, TFIIIB, TFIIIC, and the small nuclear RNA-activated protein complex (SNAPc).

[0276] The assembly of PICs on different promoters of class III genes (types 1, 2, and 3) requires one or more A-boxes, B-boxes, and C-boxes; an internal regulatory region (ICR); a TATA box; and distal (DSE) and proximal (PSE) sequence elements. Type 1 genes include an A-box at +57 and a C-box at +90, relative to transcription initiation at +1. Type 2 genes include an A-box and a B-box. Type 3 genes include a DSE at -250, a PSE at -60, and a TATA box at -27, relative to transcription initiation at +1. An A-box may be present but is not required.

[0277] The recruitment of Pol III and transcription initiation in all three promoters require the action of transcription factor IIIB (TFIIIB) and are highly regulated. The TFIIIB binding site is + / - 8nt around the TATA box. Furthermore, TBP is required for transcription by all three polymerases (Han, Y., et al., Cell. Discover. 4 (2018) 40).

[0278] Regarding the three types of Pol III genes, Oler, AJ, et al. (Nat. Struct. Mol. Biol. 17 (2010) 620-628) outlined the factors required to direct Pol III to target genes and the three “types” of Pol III genes in humans, based on 1) the presence and location of cis regulatory elements, and 2) the need for specific basal or subtranscriptional factors. Briefly, 5S rRNA is the only type 1 gene and uniquely requires TFIIIA. Both type 1 and type 2 genes require TFIIIC, a basal and targeting complex that recognizes the internal A-box and B-box elements of type 2 genes, not type 1 genes. The TFIIIB complex contains TBP, which is necessary for TATA / promoter recognition and Pol III initiation. Type 2 and type 3 genes utilize alternative assemblies of TFIIIB: BRF1 (TFIIIB-related factor 1) for type 2 genes and BRF2 (TFIIIB-related factor 2) for type 3 genes. Type 3 genes lack an internal A-box or B-box, are independent of TFIIIC, and instead depend on upstream PSE and DSE as well as specific factors for targeting (OCT1, SNAPc, etc.). In particular, type 3 Pol III promoters are similar to Pol II genes in their structure, utilizing upstream regulatory elements rather than internal gene elements.

[0279] In certain embodiments, the adenovirus VA RNA nucleic acid according to the present invention includes, in the 5' direction from 5' to 3', at its 5' end (in the absence of a promoter) or between the promoter and the VA RNA coding sequence (in the presence of a promoter): - At least six 5' terminal nucleotides of the adenovirus VA RNAI gene, including the transcription start site (TSS) (to prevent bypass of the subsequent polymerase III (poly III) terminator); - Functional polymerase III terminator (to prevent transcription of VA RNA from constitutively active upstream promoters), and - Adenovirus VA RNAI sequence.

[0280] In all aspects and in certain embodiments of the embodiments, the adenovirus VA RNA nucleic acid according to the present invention further comprises a polymerase promoter operably ligated to its 5' end. In certain embodiments, the promoter is a type 2 polymerase III promoter or a variant thereof, or a type 3 polymerase III promoter or a variant thereof, or a polymerase II promoter or a variant thereof, or an inducible promoter. In one preferred embodiment of all aspects and embodiments, the promoter is a human U6-snRNA promoter.

[0281] In all aspects and embodiments of the present invention, the enumerated elements are operably connected to one another.

[0282] In all aspects and in specific embodiments of the embodiments, the adenovirus VA RNA nucleic acid according to the present invention comprises all or part of the wild-type adenovirus VA RNAI sequence of SEQ ID NO: 37 gggcactctt ccgtggtctg gtggataaat tcgcaagggt atcatggcgg acgaccgggg ttcgaacccc ggatccggcc gtccgccgtg atccatgcgg ttaccgcccg cgtgtcgaac ccaggtgtgc gacgtcagac aacgggggag cgctcctttt ggcttccttc caggcgcggc ggctgctgcg ctagcttttt t.

[0283] In all aspects and in specific embodiments of the embodiments, the adenovirus VA RNA nucleic acid according to the present invention comprises all or part of the wild-type adenovirus VA RNAI sequence having the mutations G58T, G59T and C68A (sequence numbering) of SEQ ID NO: 38: gggcactctt ccgtggtctg gtggataaat tcgcaagggt atcatggcgg acgaccgttg ttcgaacacc ggatccggcc gtccgccgtg atccatgcgg ttaccgcccg cgtgtcgaac ccaggtgtgc gacgtcagac aacgggggag cgctcctttt ggcttccttc caggcgcggc ggctgctgcg ctagcttttt t.

[0284] Figure 1 shows the alignment including the above sequence.

[0285] In a particular embodiment, the adenovirus VA RNA nucleic acid according to the present invention includes the following sequence in the 5' to 3' direction: (1) A type 2 polymerase III promoter or a variant thereof, or a type 3 polymerase III promoter or a variant thereof, in one preferred embodiment, a human U6-snRNA promoter, or a polymerase II promoter, or an inducible promoter; and (2)gggcactctt ccgtggtctg gtggataaat tcgcaagggt atcatggcgg acgaccgggg ttcgaacccc ggatccggcc gtccgccgtg atccatgcgg ttaccgcccg cgtgtcgaac ccaggtgtgc gacgtcagac aacgggggag cgctcctttt ggcttccttc caggcgcggc ggctgctgcg ctagcttttt t (SEQ ID NO: 37).

[0286] In one preferred embodiment, the adenovirus VA RNA nucleic acid according to the present invention comprises the following sequence: aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgggcac tcttccgtgg tctggtggat aaattcgcaa gggtatcatg gcggacgacc ggggttcgaa ccccggatcc ggccgtccgc cgtgatccat gcggttaccg cccgcgtgtc gaacccaggt gtgcgacgtc agacaacggg ggagcgctcc ttttggcttc cttccaggcg cggcggctgc tgcgctagct ttttt (Sequence ID 39; Figure 2).

[0287] Exemplary uses and methods including nucleic acids and DNA according to the present invention The adenovirus VA RNA nucleic acid and DNA (elements) according to the present invention can be used for the production of recombinant AAV vectors and recombinant AAV particles containing them.

[0288] Various methods known in the art for generating rAAV particles. For example, transfection using an AAV vector and an AAV helper sequence in conjunction with co-infection with one AAV helper virus (e.g., adenovirus, herpesvirus, or vacciniavirus), or transfection with recombinant AAV plasmids, AAV helper plasmids, and helper functional plasmids. Non-limiting methods for generating rAAV particles are described, for example, in U.S. Patent No. 6,001,650, U.S. Patent No. 6,004,797, International Publication No. 2017 / 096039, and International Publication No. 2018 / 226887. After recombinant rAAV particle production (i.e., particle generation in a cell culture system), rAAV particles can be obtained and purified from host cells and cell culture supernatants.

[0289] Aspects of the present invention include a method for transducing molecules such as nucleic acids (e.g., plasmids) according to the present invention into cells and the production of the respective gene products. Furthermore, when such cells are transduced with sequences such as plasmids encoding viral packaging proteins and / or helper proteins, they can produce recombinant viral particles containing nucleic acids encoding the target protein or sequences transcribed into the target transcript, at least one of which contains adenovirus VA RNA nucleic acid or DNA (elements) according to the present invention, and produce recombinant viral particles in high yield.

[0290] The present invention provides a virus (e.g., AAV) particle manufacturing platform that includes features that distinguish it from current "industry standard" virus (e.g., AAV) particle manufacturing processes by using nucleic acids or DNA (elements) according to the present invention.

[0291] When discussing nucleic acids (plasmids), a specific polynucleotide sequence or structure may be described herein in accordance with the convention of providing the sequence in the 5' to 3' direction.

[0292] More generally, cells transfected or transfected with VA RNA nucleic acid or DNA (elements) according to the present invention may be called “recombinant cells.” Such cells may be yeast cells, insect cells, or mammalian cells used as recipients of nucleic acids (plasmids) encoding packaging proteins such as AAV packaging proteins, nucleic acids (plasmids) encoding helper proteins, nucleic acids (plasmids) encoding proteins or transcribed into desired transcripts, i.e., transgenes placed between two AAV ITRs, or other transgenic nucleic acids (plasmids), at least one of which contains adenovirus VA RNA nucleic acid or DNA (elements) according to the present invention. This term includes offspring of the transfected or transfected original cells. It is understood that offspring of a single parent cell may not necessarily be morphologically or genomically or in terms of whole nucleic acid complements to the original parent due to natural, accidental, or intentional mutations.

[0293] Numerous suitable cell growth media are commercially available or can be easily manufactured to maintain cell viability or to provide cell growth and / or proliferation. Examples of such media include serum-free eukaryotic growth media, such as media for maintaining viability or media for providing growth of mammalian (e.g., human) cells. Non-limiting examples include Ham's F12 or F12K medium (Sigma-Aldrich), FreeStyle (FS) F17 medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific), and mixtures thereof. Such media may be supplemented with vitamins and / or trace minerals and / or salts and / or amino acids, such as essential amino acids for mammalian (e.g., human) cells.

[0294] Helper proteins can be provided in the form of plasmids, phages, transposons, or cosmids. In particular, it has been demonstrated that complete complementation of adenovirus genes is not required for helper function. For example, adenovirus mutants that are unable to perform DNA replication and late gene synthesis have been shown to allow AAV replication. Ito et al., J.Gen.Virol.9(1970)243; Ishibashi et al, Virology 45(1971)317.

[0295] Mutants within the E2B and E3 regions have been shown to support AAV replication, suggesting that the E2B and E3 regions are likely not involved in providing helper function. Carter et al., Virology 126(1983)505. However, adenoviruses with defects in the E1 region or deletions in the E4 region cannot support AAV replication. Therefore, in the case of adenovirus helper proteins, the E1A and E4 regions are likely required for AAV replication, either directly or indirectly (see, for example, Laughlin et al., J.Virol.41(1982)868; Janik et al., Proc.Natl.Acad.Sci.USA 78(1981)1925; Carter et al., Virology 126(1983)505). Other characteristic adenovirus variants include: E1B (Laughlin et al. (1982), previously mentioned; Janik et al. (1981), previously mentioned; Ostrove et al., Virology 104 (1980) 502); E2A (Handa et al., J.Gen.Virol.29 (1975) 239; Strauss et al., J.Virol.17 (1976) 140; Myers et al., J.Virol.35 (1980) 665; Jay et al., Proc.Natl.Acad.Sci.USA 78 (1981) 2927; Myers et al., J.Biol.Chem.256 (1981) 567); E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed., 1990); E3 (Carter et al. (1983), cited above); and E4 (Carter et al. (1983), cited above; Carter (1995)).

[0296] Studies of helper proteins provided by adenoviruses with E1B mutations have reported that the E1B 55kDa protein is required for AAV particle production, while the E1B 19kDa protein is not. Furthermore, International Publication No. 97 / 17458 and Matshushita et al. (Gene Therapy 5(1998) 938-945) described helper functional plasmids encoding various adenovirus genes. Examples of helper plasmids include the adenovirus VA RNA coding region, the adenovirus E4 ORF6 coding region, the adenovirus E2A 72kDa coding region, the adenovirus E1A coding region, and the adenovirus E1B region lacking the intact E1B 55kDa coding region (see, for example, International Publication No. 01 / 83797 pamphlet).

[0297] Accordingly, this specification provides a method for producing a recombinant AAV vector or AAV particles containing the recombinant AAV vector, which includes a nucleic acid to be transcribed into a protein-coding nucleic acid or a transcript of interest, using adenovirus VA RNA nucleic acid or DNA (elements) according to the present invention.

[0298] One aspect of the present invention is a method for producing a recombinant AAV vector comprising a nucleic acid encoding a protein or a nucleic acid to be transcribed into a transcript of interest, or AAV particles comprising the recombinant AAV vector, (i) A step of providing one or more plasmids comprising nucleic acids encoding an AAV packaging protein and / or a helper protein, wherein at least one of the plasmids comprises an adenovirus VA RNA nucleic acid or DNA (element) according to the present invention; (ii) A step of providing a plasmid comprising nucleic acid that encodes a protein of interest or is transcribed into a transcript of interest; (iii) The step of bringing one or more mammalian cells into contact with the provided plasmid; (iv) A step of further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or a step of introducing nucleic acid into cells by providing a physical means such as an electric current; (v) A step of culturing transfected cells and inducing RMCI at a certain point in time / cultivation period during culture; (vi) A step of recovering cultured cells and / or culture medium from the cultured cells to produce a cell and / or culture medium recovery product; and (vii) The process of isolating and / or purifying recombinant AAV vectors or AAV particles from cells and / or culture medium recoveries to produce recombinant AAV vectors or AAV particles containing nucleic acids that encode a protein of interest or are transcribed into a transcript of interest. This method includes [something].

[0299] One aspect of the present invention is a method for producing a recombinant AAV vector comprising a nucleic acid encoding a protein or a nucleic acid to be transcribed into a transcript of interest, or AAV particles comprising the recombinant AAV vector, (i) A step of providing one or more plasmids comprising nucleic acids encoding an AAV packaging protein and / or a helper protein, wherein at least one of the plasmids comprises an adenovirus VA RNA nucleic acid or DNA (element) according to the present invention; (ii) A step of providing a plasmid comprising nucleic acid that encodes a protein of interest or is transcribed into a transcript of interest; (iii) The step of bringing one or more mammalian cells into contact with the plasmid provided in (i); (iv) A step of further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or a step of introducing nucleic acid into cells by providing a physical means such as an electric current; (v) A step to select stably transfected cells; (vi) The step of contacting the selected cells from (v) with the provided plasmid from (ii); (vii) A step of further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or a step of introducing nucleic acid into cells by providing a physical means such as an electric current; (viii) A step of culturing transfected cells of (viii) and inducing RMCI at a certain point in time / culturing time during culture; (ix) A step of recovering cultured cells and / or culture medium from the cultured cells to produce a cell and / or culture medium recovery product; and (x) The process of isolating and / or purifying recombinant AAV vectors or AAV particles from cells and / or culture medium recoveries to produce recombinant AAV vectors or AAV particles containing nucleic acids that encode a protein of interest or are transcribed into a transcript of interest. This method includes [something].

[0300] One aspect of the present invention is a method for producing a recombinant AAV vector comprising a nucleic acid encoding a protein or a nucleic acid to be transcribed into a transcript of interest, or AAV particles comprising the recombinant AAV vector, (i) A step of providing mammalian cells comprising nucleic acids encoding an AAV packaging protein and / or nucleic acids encoding a helper protein, wherein at least one of these cells comprises an adenovirus VA RNA nucleic acid or DNA (element) according to the present invention; (ii) A step of providing a plasmid comprising nucleic acid that encodes a protein of interest or is transcribed into a transcript of interest; (iii) The step of bringing the cells from (i) into contact with the provided plasmid from (ii); (iv) A step of further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or a step of introducing nucleic acid into cells by providing a physical means such as an electric current; (v) A step to select stably transfected cells; (vi) A step of culturing cells that have been stably transfected according to (v), and inducing RMCI at a certain point in time / cultivation period during culture; (vii) A step of recovering cultured cells and / or culture medium from the cultured cells to produce a cell and / or culture medium recovery product; and (viii) The process of isolating and / or purifying recombinant AAV vectors or AAV particles from cells and / or culture medium recoveries to produce recombinant AAV vectors or AAV particles containing nucleic acids that encode a protein of interest or are transcribed into a transcript of interest. This method includes [something].

[0301] The introduction of adenovirus VA RNA nucleic acid or nucleic acid containing DNA (elements) into cells according to the present invention can be carried out by multiple methods.

[0302] A variety of methods for DNA transfer into mammalian cells have been reported in the art. All of these are useful in the methods according to the present invention. In all aspects and in specific embodiments of the embodiments, electroporation, nucleofection, or microinjection is used for nucleic acid transfer / transfection. In all aspects and in specific embodiments of the embodiments, inorganic substances (e.g., calcium phosphate / DNA coprecipitation), cationic polymers (e.g., polyethyleneimine, DEAE-dextran), or cationic lipids (lipofection) are used for nucleic acid transfer / transfection. Calcium phosphate and polyethyleneimine are the most commonly used reagents for transfection for nucleic acid transfer on a larger scale (see, for example, Baldi et al., Biotechnol. Lett. 29 (2007) 677-684), with polyethyleneimine being preferred.

[0303] In all aspects and in specific embodiments of the embodiments, the nucleic acid comprising adenovirus VA RNA nucleic acid or DNA (element) according to the present invention is provided in a composition in combination with polyethyleneimine (PEI) and optionally in combination with cells. In specific embodiments, the composition comprises a plasmid / PEI mixture having the following components: (a) one or more plasmids comprising nucleic acids encoding an AAV packaging protein and / or a helper protein, at least one of which comprises adenovirus VA RNA nucleic acid or DNA (element) according to the present invention; (b) a plasmid comprising nucleic acids encoding a protein or to be transcribed into a transcript of interest; and (c) a polyethyleneimine (PEI) solution. In specific embodiments, the plasmids are in a molar ratio range of about 1:0.01 to about 1:100 or in a molar ratio range of about 100:1 to about 1:0.01, and the mixture of components (a), (b), and (c) is optionally incubated for a period of about 10 seconds to about 4 hours.

[0304] In all aspects and in certain embodiments of the embodiment, the composition further comprises cells. In certain embodiments, the cells are in contact with a plasmid / PEI mixture of components (a), (b), and / or (c).

[0305] In all aspects and in specific embodiments of the embodiments, the composition further comprises free PEI, optionally in combination with cells. In specific embodiments, the cells are in contact with the free PEI.

[0306] In all aspects and in specific embodiments of the embodiments, the cells are in contact with a mixture of components (a), (b), and / or (c) for at least about 4 hours, or about 4 hours to about 140 hours, or about 4 hours to about 96 hours. In one preferred embodiment, the cells are in contact with a mixture of components (a), (b), and / or (c), and optionally free PEI, for at least about 4 hours.

[0307] In addition to nucleic acids comprising adenovirus VA RNA or DNA (elements) according to the present invention, the composition may further comprise plasmids. Such plasmids and cells may be in contact with free PEI. In certain embodiments, the plasmids and / or cells are in contact with free PEI for at least about 4 hours, or about 4 hours to about 140 hours, or about 4 hours to about 96 hours.

[0308] The present invention also provides a method for producing transfected cells using nucleic acids comprising adenovirus VA RNA nucleic acid or DNA (elements) according to the present invention. The method comprises the steps of: providing nucleic acids comprising adenovirus VA RNA nucleic acid or DNA (elements) according to the present invention, and optionally one or more further plasmids; providing a solution comprising polyethyleneimine (PEI); and mixing the nucleic acid and optionally the plasmid with the PEI solution to produce a nucleic acid / plasmid / PEI mixture. In certain embodiments, such a mixture is incubated for a period ranging from about 10 seconds to about 4 hours. In such a method, cells are then brought into contact with the nucleic acid / plasmid / PEI mixture to produce a nucleic acid / plasmid / PEI cell culture. Free PEI is then added to the produced nucleic acid / plasmid / PEI cell culture to produce a free PEI / nucleic acid / plasmid / PEI cell culture. The produced free PEI / nucleic acid / plasmid / PEI cell culture is then incubated for at least about 4 hours to produce transfected cells. In certain embodiments, the plasmid comprises nucleic acids that encode a protein or are transcribed into a transcript of interest.

[0309] A method for producing transfected cells that produce recombinant AAV vectors or AAV particles, comprising one or more plasmids comprising nucleic acids encoding an AAV packaging protein and / or a helper protein (at least one of which is the adenovirus VA according to the present invention). A method is further provided comprising: providing a plasmid containing RNA nucleic acid or DNA (elements); a plasmid containing nucleic acid that encodes a protein or is transcribed into a transcript of interest; providing a solution containing polyethyleneimine (PEI); mixing the aforementioned plasmid with the PEI solution to produce a plasmid / PEI mixture, wherein the plasmid is in a molar ratio range of about 1:0.01 to about 1:100 or in a molar ratio range of about 100:1 to about 1:0.01 (and optionally incubating the plasmid / PEI mixture for a period of about 10 seconds to about 4 hours); contacting cells with the plasmid / PEI mixture to produce a plasmid / PEI cell culture; adding free PEI to the produced plasmid / PEI cell culture to produce a free PEI / plasmid / PEI cell culture; and incubating the free PEI / plasmid / PEI cell culture for at least about 4 hours to produce transfected cells that produce recombinant AAV vectors or particles containing nucleic acid that encodes a protein or is transcribed into a transcript of interest.

[0310] A method for producing recombinant AAV vectors or AAV particles comprising nucleic acids encoding a protein or being transcribed into a transcript of interest, comprising: providing one or more plasmids comprising nucleic acids encoding an AAV packaging protein and / or a helper protein (at least one comprising adenovirus VA RNA nucleic acid or DNA (element) according to the present invention); providing plasmids comprising nucleic acids encoding a protein of interest or being transcribed into a transcript of interest; providing a solution comprising polyethyleneimine (PEI); mixing the plasmids with the PEI solution to produce a plasmid / PEI mixture, wherein the plasmids are in a molar ratio range of about 1:0.01 to about 1:100 or about 100:1 to about 1:0.01 (and optionally incubating the plasmid / PEI mixture for a period of about 10 seconds to about 4 hours); contacting cells with the plasmid / PEI mixture produced as described to produce a plasmid / PEI cell culture; A method is further provided comprising: adding free PEI to a rasmid / PEI cell culture to produce a free PEI / plasmid / PEI cell culture; incubating the produced plasmid / PEI cell culture or free PEI / plasmid / PEI cell culture for at least about 4 hours to produce transfected cells; recovering the produced transfected cells and / or culture medium from the produced transfected cells to produce a cell and / or culture medium recovery product; and isolating and / or purifying recombinant AAV vectors or particles from the produced cell and / or culture medium recovery product to produce recombinant AAV vectors or particles containing nucleic acids that encode a protein or are transcribed into a transcript of interest.

[0311] The method for producing recombinant AAV vectors or AAV particles using adenovirus VA RNA or DNA (elements) according to the present invention may include one or more further steps or features. Exemplary steps or features include, but are not limited to, the step of recovering the produced cultured cells and / or the step of recovering the culture medium from the produced cultured cells to produce cells and / or culture medium recoveries. Further exemplary steps or features, but are not limited to, the step of isolating and / or purifying the recombinant AAV vector or AAV particles from the cells and / or culture medium recoveries to produce recombinant AAV vectors or AAV particles containing nucleic acids that encode a protein or are transcribed into a transcript of interest.

[0312] In all aspects and in specific embodiments of the embodiments, PEI is added to the plasmid and / or cells at various points in time. In specific embodiments, free PEI is added to the cells before, simultaneously with, or after contact of the plasmid / PEI mixture with the cells.

[0313] In all aspects and in specific embodiments of the embodiments, the cells have a specific density and / or cell growth phase and / or viability when in contact with the plasmid / PEI mixture and / or free PEI. In one preferred embodiment, the cells have a density in the range of about 1 × 10⁵ cells / mL to about 1 × 10⁸ cells / mL when in contact with the plasmid / PEI mixture and / or free PEI. In specific embodiments, the viability of the cells when in contact with the plasmid / PEI mixture or free PEI is about 60% or greater than 60%, or the cells are in the logarithmic growth phase when in contact with the plasmid / PEI mixture, or the viability of the cells when in contact with the plasmid / PEI mixture or free PEI is about 90% or greater than 90%, or the cells are in the logarithmic growth phase when in contact with the plasmid / PEI mixture or free PEI.

[0314] In all aspects and in specific embodiments of the embodiments, the encoded AAV packaging protein includes AAV rep and / or AAV cap proteins. In all aspects and in specific embodiments of the embodiments, such AAV packaging protein includes AAV rep and / or AAV cap proteins of any AAV serotype.

[0315] In all aspects and in specific embodiments of the embodiments, the encoded helper proteins include adenovirus E2 and / or E4 and / or non-AAV helper proteins.

[0316] In all aspects and in specific embodiments, nucleic acids (plasmids) are used in specific amounts or ratios. In specific embodiments, the total amount of nucleic acids encoding a protein or transcribed into a transcript of interest and one or more plasmids containing nucleic acids encoding an AAV packaging protein and / or a helper protein (at least one of which is a plasmid containing adenovirus VA RNA nucleic acid or DNA (elements) according to the present invention) is in the range of about 0.1 μg to about 15 μg per mL of cells. In specific embodiments, the molar ratio of plasmids containing nucleic acids encoding a protein or transcribed into a transcript of interest to one or more plasmids containing nucleic acids encoding an AAV packaging protein and / or a helper protein (at least one of which is a plasmid containing adenovirus VA RNA nucleic acid or DNA (elements) according to the present invention) is in the range of about 1:5 to about 1:1, or in the range of about 1:1 to about 5:1.

[0317] A plasmid may contain nucleic acids on different or the same plasmid. In all aspects and in certain embodiments of the embodiments, the first plasmid contains nucleic acids encoding an AAV packaging protein, and the second plasmid contains nucleic acids encoding a helper protein. At least one of these nucleic acids further comprises adenovirus VA RNA nucleic acid or DNA (element) according to the present invention.

[0318] In all aspects and in specific embodiments of the embodiments, the molar ratio of a plasmid containing nucleic acid encoding a protein or to be transcribed into a transcript of interest to a first plasmid containing nucleic acid encoding an AAV packaging protein and a second plasmid containing nucleic acid encoding a helper protein is in the range of approximately 1 to 5:1:1, or 1:1 to 5:1, or 1:1:1 to 5 in simultaneous transfection.

[0319] In all aspects and in specific embodiments of the embodiments, the cells are eukaryotic cells. In specific embodiments, the eukaryotic cells are mammalian cells. In one preferred embodiment, the cells are HEK293 cells or CHO cells.

[0320] Culture can be carried out using conditions commonly used for eukaryotic cell culture, such as approximately 37°C, 95% humidity, and 8% CO2 by volume. Culture can be carried out in serum-containing medium or serum-free medium, in adherent culture or suspension culture. Suspension culture can be carried out in any fermentation vessel, such as a stirred tank reactor, wave reactor, shaker or spinner vessel, or so-called roller bottle. Transfection can be carried out in high-throughput and screening formats, for example, in 96 or 384-well formats.

[0321] The method according to the present invention comprises AAV particles of any serotype or variant thereof. In all aspects and in specific embodiments of the embodiments, the recombinant AAV particles comprise any of AAV serotypes 1-12, AAV VP1, VP2 and / or VP3 capsid proteins, or modified or variant AAV VP1, VP2 and / or VP3 capsid proteins, or wild-type AAV VP1, VP2 and / or VP3 capsid proteins. In all aspects and in specific embodiments of the embodiments, the AAV particles comprise an AAV serotype or an AAV pseudotype, the AAV pseudotype comprises an AAV capsid serotype different from the ITR serotype.

[0322] Methods according to the present invention that provide or include AAV vectors or particles may also include other elements. Examples of such elements include, but are not limited to, introns, expression regulatory elements, one or more adeno-associated virus (AAV) reverse-end repeat sequences (ITRs) and / or filler / stuffer polynucleotide sequences. Such elements may be present in or adjacent to a nucleic acid encoding a protein or transcribed to a transcript of interest, or expression regulatory elements may be operably ligated to a nucleic acid encoding a protein or transcribed to a transcript of interest, or AAV ITRs may be adjacent to the 5' or 3' end of a nucleic acid encoding a protein or transcribed to a transcript of interest, or filler polynucleotide sequences may be adjacent to the 5' or 3' end of a nucleic acid encoding a protein or transcribed to a transcript of interest.

[0323] Expression regulatory elements include constitutive or modulotable regulatory elements such as tissue-specific expression regulatory elements or promoters (e.g., those providing expression in the liver).

[0324] ITR may be any of the following: AAV2 or AAV6 or AAV8 or AAV9 serotype, or a combination thereof. AAV particles may contain any VP1, VP2 and / or VP3 capsid protein having 75% or more sequence identity to any of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8 or AAV rh74 VP1, VP2 and / or VP3 capsid proteins, or may contain modified or variant VP1, VP2 and / or VP3 capsid proteins selected from any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8 and AAV rh74 AAV serotypes.

[0325] After the production of recombinant virus (e.g., AAV) particles as described herein, the virus (e.g., rAAV) particles can, if desired, be purified and / or isolated from host cells using a variety of conventional methods. Such methods include column chromatography and CsCl gradients. For example, multiple column purification steps can be used, such as purification by anion exchange column, affinity column and / or cation exchange column (see, e.g., WO02 / 12455 and US2003 / 0207439). Alternatively or additionally, a CsCl gradient step can be used (see, e.g., US2012 / 0135515 and US2013 / 0072548). Furthermore, if infectious virus is used for packaging and / or expression of helper proteins, residual virus can be inactivated using a variety of methods. For example, adenovirus can be inactivated by heating at a temperature of about 60°C for, for example, 20 minutes or more. AAV is heat-stable, but helper adenoviruses are heat-unstable; therefore, this treatment effectively inactivates helper viruses.

[0326] Recombinant AAV vectors, as well as methods and uses thereof, may include any viral strain or serotype. As a non-limiting example, a recombinant AAV vector may be based on any AAV genome, e.g., AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, 2i8, or AAV rh74. Such vectors may be based on the same strain or serotype (or subgroup or variant), or they may be different from each other. As a non-limiting example, a recombinant AAV vector based on a single serotype genome may be identical to one or more of the capsid proteins packaging the vector. Furthermore, a recombinant AAV vector genome may be based on an AAV (e.g., AAV2) serotype genome different from one or more of the AAV capsid proteins packaging the vector. For example, an AAV vector genome may be based on AAV2, but at least one of the three capsid proteins may be, for example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, or AAV rh74 or a variant thereof. AAV variants include variants and chimeras of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, and AAV rh74 capsids.

[0327] In all aspects and in specific embodiments of the embodiments, adeno-associated virus (AAV) vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, and AAV rh74, as well as their variants (e.g., capsid variants, e.g., amino acid insertions, additions, substitutions, and deletions), as described, for example, in WO2013 / 158879, WO2015 / 013313 and US2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0328] AAV and AAV variants (e.g., capsid variants) and serotypes (e.g., VP1, VP2, and / or VP3 sequences) may or may not be distinguishable from other AAV serotypes, including AAV1-AAV12 (e.g., different from any of the VP1, VP2, and / or VP3 sequences of AAV1-AAV12 serotypes).

[0329] In all aspects and in specific embodiments of the embodiments, the AAV particles associated with the reference serotype have a polynucleotide, polypeptide, or a subsequence thereof that is identical to or consists of at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%) of one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV12, AAV-2i8, or AAV rh74 (e.g., an ITR sequence, or a VP1 sequence, a VP2 sequence, and / or a VP3 sequence, etc.) of the sequence.

[0330] The compositions, methods, and uses of the present invention include AAV sequences (polypeptides and nucleotides), as well as partial sequences that exhibit less than 100% sequence identity to reference AAV serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, or AAV rh74, but are distinct from and not identical to known AAV genes or proteins such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, or AAV rh74 genes or proteins. In all aspects and in specific embodiments of the embodiments, the AAV polypeptide or a subsequence thereof comprises or comprises a sequence identical to any reference AAV sequence or subsequence thereof by at least 75%, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, and up to 100% identical, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, or AAV rh74 (e.g., VP1, VP2 and / or VP3 capsids or ITRs). In certain embodiments, the AAV variant has 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions.

[0331] Recombinant AAV particles, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, or AAV rh74, as well as variants, related, hybrid, and chimeric sequences, can be constructed using recombinant techniques known to those skilled in the art to include one or more nucleic acid sequences (transgenes) adjacent to one or more functional AAV ITR sequences.

[0332] Recombinant particles (e.g., rAAV particles) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are particularly useful for administration and delivery to subjects in vivo or ex vivo. In certain embodiments, the pharmaceutical composition contains pharmaceutically acceptable carriers or excipients. Such excipients include any pharmaceuticals that do not induce an adverse immune response in the individual receiving the composition and can be administered without excessive toxicity.

[0333] Protocols for the production of adenovirus vectors are described in US5,998,205;US6,228,646;US6,093,699;US6,100,242;WO94 / 17810 and WO94 / 23744, which are incorporated herein by reference in their entirety.

[0334] Despite their pathogenicity to humans, the objective of rAAV vector production and purification systems is to implement strategies to minimize / control the generation of production-related impurities, such as wild-type / pseudowild-type AAV species (wtAAV) and AAV-encapsulated residual DNA impurities, as well as proteins, nucleic acids, and vector-related impurities.

[0335] Given that rAAV particles represent only a small fraction of biomass, they need to be purified to a level of purity suitable for use as a clinical human gene therapy product (see, for example, reports from Smith PH, et al., Mo. Therapy 7(2003)8348; Chadeuf G., et al, Mo. Therapy 12(2005)744; and CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP 2005,183989 / 2004).

[0336] As a first step, typically, cultured cells producing rAAV particles are collected, optionally combined with the recovered cell culture supernatant (medium) in which the rAAV particle-producing cells (suspension or adherent) were cultured. The collected cells and optionally the cell culture supernatant can be used as is or concentrated as needed. Furthermore, if infection is used to express helper function, residual helper viruses can be inactivated. For example, adenoviruses can be inactivated by heating them to a temperature of approximately 60°C for, for example, 20 minutes or more, which inactivates only the helper viruses because AAV is heat-stable while helper adenoviruses are heat-unstable.

[0337] The supernatant of the cells and / or recovered material is dissolved by chemical or physical means, such as detergent, microfluidization, and / or homogenization, to disrupt the cells and release rAAV particles. During or after cell lysis, a nuclease, such as benzonase, is added to degrade the contaminating DNA. Typically, the resulting lysate is clarified to remove cell debris, for example, by filtration or centrifugation, to give a clarified cell lysate. In specific examples, the lysate is filtered through a micron-diameter pore filter (e.g., a filter with a pore size of 0.1–10.0 μm, e.g., a filter with a pore size of 0.45 μm and / or 0.2 μm) to produce a clarified lysate.

[0338] The lysate (optionally clarified) contains AAV particles (including the rAAV vector and empty capsid) and production / process-related impurities, such as soluble cellular components from host cells, which may include, in particular, cellular proteins, lipids and / or nucleic acids, as well as components of the cell culture medium. The optionally clarified lysate is then subjected to a purification step to purify the AAV particles (including the rAAV vector) from impurities using chromatography. The clarified lysate may be diluted or concentrated with a suitable buffer before the first chromatography step.

[0339] After cell lysis, optional clarification, and optional dilution or concentration, rAAV particles can be purified using a series of subsequent chromatography steps.

[0340] The first chromatography step may be cation exchange chromatography or anion exchange chromatography. If the first chromatography step is cation exchange chromatography, the second chromatography step may be anion exchange chromatography or size exclusion chromatography (SEC). Thus, in all aspects and in specific embodiments of the embodiments, rAAV particle purification is performed by cation exchange chromatography, followed by purification by anion exchange chromatography.

[0341] Alternatively, if the first chromatography step is cation exchange chromatography, the second chromatography step may be size exclusion chromatography (SEC). Thus, in all aspects and in specific embodiments of the embodiments, rAAV particle purification is performed by cation exchange chromatography, followed by purification by size exclusion chromatography (SEC).

[0342] Alternatively, the first chromatography step may be affinity chromatography. If the first chromatography step is affinity chromatography, the second chromatography step may be anion exchange chromatography. Thus, in all aspects and in specific embodiments of the embodiments, rAAV particle purification is performed by affinity chromatography, followed by purification by anion exchange chromatography.

[0343] Optionally, a third chromatography step can be added to the aforementioned chromatography process. Typically, the optional third chromatography step follows cation exchange, anion exchange, size exclusion, or affinity chromatography.

[0344] Therefore, in all aspects and in specific embodiments of the embodiments, rAAV particle purification is performed by cation exchange chromatography, followed by purification by anion exchange chromatography, and then by size exclusion chromatography (SEC).

[0345] Furthermore, in all aspects and in certain embodiments of the embodiments, further rAAV particle purification is performed by cation exchange chromatography, followed by purification by size exclusion chromatography (SEC), and then by anion exchange chromatography.

[0346] In all aspects and further embodiments of the embodiments, rAAV particle purification is performed by affinity chromatography, followed by purification by anion exchange chromatography, and then by size exclusion chromatography (SEC).

[0347] In all aspects and further embodiments of the embodiments, rAAV particle purification is performed by affinity chromatography, followed by purification by size exclusion chromatography (SEC), and then by anion exchange chromatography.

[0348] Cation exchange chromatography functions to separate AAV particles from cellular and other components present in lysates and / or column eluents clarified from affinity chromatography or size exclusion chromatography. Examples of strong cation exchange resins that can bind to rAAV particles over a wide pH range include, but are not limited to, any sulfonic acid resins characterized by the presence of sulfonate functional groups, including aryl and alkyl-substituted sulfonates such as sulfopropyl or sulfoethyl resins. Typical matrices include, but are not limited to, POROS HS, POROS HS 50, POROS XS, POROS SP, and POROS S (strong cation exchangers available from Thermo Fisher Scientific, Inc., Waltham, MA, USA). Further examples include Capto S, Capto S ImpAct, and Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), as well as the commercially available DOWEX®, AMBERLITE®, and AMBERLYST® resin families from Aldrich Chemical Company (Milwaukee, WI, USA). Weak cation exchange resins include, but are not limited to, any carboxylic acid-based resins. Exemplary cation exchange resins include carboxymethyl (CM), phospho(phosphate-based), methyl sulfonate (S), and sulfopropyl (SP) resins.

[0349] Anion exchange chromatography functions to separate AAV particles from proteins, cellular components, and other components present in the lysate and / or column eluate clarified from affinity chromatography, cation exchange chromatography, or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and control the amount of empty capsids in the eluate. For example, an anion exchange column bound to rAAV particles can be washed with a solution containing a moderate concentration of NaCl (e.g., about 100–125 mM, e.g., 110–115 mM), allowing some of the empty capsids to flow through without substantially eluting the rAAV particles. Subsequently, the rAAV particles bound to the anion exchange column can be eluted with a solution containing a higher concentration of NaCl (e.g., about 130–300 mM NaCl) to produce a column eluate with a reduced or depleted amount of empty capsids and a proportionally increased amount of rAAV vector-containing rAAV particles.

[0350] Examples of anion exchange resins include, but are not limited to, those based on polyamine resins and other resins. Examples of strong anion exchange resins include, but are not limited to, those based on quaternary nitrogen atoms, including quaternary ammonium salt resins such as trialkylbenzylammonium resins. Suitable exchange chromatography materials include, but are not limited to, MACRO PREP Q (strong anion exchanger available from BioRad, Hercules, CA, USA); UNOSPHERE Q (strong anion exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS XQ (strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS SOD (weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (GE Examples include a strong anion exchanger available from Healthcare, Marlborough, MA, USA; DEAE Sepharose (a weak anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA); and Q Sepharose (a strong anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA). Further exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

[0351] The manufacturing process for purifying recombinant AAV particles intended as a product for treating human diseases should achieve the following objectives: 1) consistent particle purity, efficacy, and safety; 2) scalability of the manufacturing process; and 3) acceptable manufacturing costs.

[0352] An exemplary process for purifying recombinant AAV particles is described in International Publication No. 2019 / 006390.

[0353] The purification and production methods for recombinant adeno-associated virus particles (rAAV particles) outlined below can be scaled up to large volumes, for example, up to 5, 10, 10-20, 20-50, 50-100, 100-200 or more liters of suspension culture. The purification and production methods for recombinant adeno-associated virus particles are applicable to a wide variety of AAV serotypes / capsid variants.

[0354] In all aspects and in specific embodiments of the embodiments, the purification of rAAV particles includes the following steps: (a) A step of collecting the cell culture supernatant containing cells and / or rAAV particles to produce a recovered product; (b) Optionally, a step of concentrating the recovered material produced in step (a) to produce a concentrated recovered material; (c) A step of dissolving the recovered material produced in step (a) or the concentrated recovered material produced in step (b) to produce a dissolved product; (d) A step of processing the lysate produced in step (c) to reduce the amount of contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) Optionally, filter the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally, dilute the clarified lysate to produce a diluted clarified lysate; (f) Subjecting the nucleic acid reduction lysate from step (d), the clarified lysate from step (e), or the diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or other manufacturing / process-related impurities, and optionally diluting the column eluate to produce a diluted column eluate; (g) The column eluate or diluted column eluate generated in step (f) is subjected to anion exchange chromatography to produce a second column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or manufacturing / process-related impurities, and optionally the second column eluate is concentrated to produce a concentrated second column eluate; (h) The second column eluate or concentrated second column eluate generated in step (g) is subjected to size exclusion column chromatography (SEC) to generate a third column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or manufacturing / process-related impurities, and optionally the third column eluate is concentrated to generate a concentrated third column eluate; and (i) A step of filtering the third column eluate or concentrated third column eluate produced in step (h) to produce purified rAAV particles.

[0355] In one embodiment, steps (a) to (f) are maintained and combined with the following steps. (g) The column eluate or concentrated column eluate generated in step (f) is subjected to size exclusion column chromatography (SEC) to produce a second column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or other manufacturing / process-related impurities, and optionally diluting the second column eluate to produce a concentrated second column eluate; (h) The second column eluate or diluted second column eluate produced in step (g) is subjected to anion exchange chromatography to produce a third column eluate containing rAAV particles, thereby separating the rAAV particles from the production / process-related impurities of the protein impurities, and optionally diluting the third column eluate to produce a diluted third column eluate; and (i) A step of filtering the third column eluate or concentrated third column eluate produced in step (h) to produce purified rAAV particles.

[0356] In one embodiment, steps (a) to (g) are maintained and combined with the following steps. (h) A step of filtering the second column eluate or concentrated second column eluate produced in step (g) to produce purified rAAV particles.

[0357] In this embodiment, steps (a) to (e) are maintained and combined with the following steps. (f) The nucleic acid reduction lysate from step (d), or the clarified lysate or diluted clarified lysate produced in step (e), is subjected to AAV affinity chromatography to produce a column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or other manufacturing / process-related impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) The column eluate or concentrated column eluate generated in step (f) is subjected to size exclusion column chromatography (SEC) to produce a second column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or other manufacturing / process-related impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) optionally subject the second column eluate or diluted second column eluate produced in step (g) to anion exchange chromatography to produce a third column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or other manufacturing / process-related impurities, and optionally diluting the third column eluate to produce a diluted third column eluate; and (i) A step of filtering the second column eluate or diluted second column eluate produced in step (g), or filtering the third column eluate or concentrated third column eluate produced in step (h), thereby producing purified rAAV particles.

[0358] In all aspects and in specific embodiments of the embodiments, the concentration in step (b) and / or step (f) and / or step (g) and / or step (h) is by ultrafiltration / dialysis filtration, for example, tangential flow filtration (TFF).

[0359] In all aspects and in certain embodiments of the embodiments, the concentration in step (b) reduces the volume of the recovered cells and cell culture supernatant by about 2 to 20 times.

[0360] In all aspects and in specific embodiments of the embodiments, the concentration in step (f) and / or step (g) and / or step (h) reduces the volume of the column eluate by approximately 5 to 20 times.

[0361] In all aspects and in specific embodiments of the embodiments, the dissolution of the recovered material produced in step (a) or the concentrated recovered material produced in step (b) is by physical or chemical means. Non-limiting examples of physical means include microfluidization and homogenization. Non-limiting examples of chemical means include detergents. Detergents include nonionic detergents and ionic detergents. Non-limiting examples of nonionic surfactants include Triton X-100. Non-limiting examples of detergent concentrations are about 0.1–1.0% (v / v) or (w / v) (including both ends).

[0362] In all aspects and in specific embodiments of the embodiments, step (d) includes treatment with a nuclease to reduce the contaminating nucleic acid. Non-limiting examples of nucleases include benzonase.

[0363] In all aspects and in specific embodiments of the embodiments, the filtration of the clarified or diluted clarified solution in step (e) is performed by a filter. Non-limiting examples of filters include those having a pore size of approximately 0.1 microns to 10.0 microns (including both ends).

[0364] In all aspects and in specific embodiments of the embodiments, the dilution of the clarified solution in step (e) is with buffered phosphoric acid, acetic acid, or an aqueous Tris solution. Non-limiting examples of solution pH are about pH 4.0 to pH 7.4 (inclusive). Non-limiting examples of Tris solution pH are greater than pH 7.5, for example, about pH 8.0 to pH 9.0 (inclusive).

[0365] In all aspects and in specific embodiments of the embodiments, the dilution of the column eluate in step (f) or the second column eluate in step (g) is with buffered phosphoric acid, acetic acid, or an aqueous Tris solution. Non-limiting examples of solution pH are approximately pH 4.0 to pH 7.4 (inclusive). Non-limiting examples of Tris solution pH are greater than pH 7.5, for example, approximately pH 8.0 to pH 9.0 (inclusive).

[0366] In all aspects and in specific embodiments of the embodiments, the rAAV particles obtained from step (i) are formulated together with a surfactant to produce an rAAV particle formulation.

[0367] In all aspects and in certain embodiments of the embodiments, the anion exchange column chromatography of step (f), (g), and / or (h) includes polyethylene glycol (PEG)-modified column chromatography.

[0368] In all aspects and in specific embodiments of the embodiments, the anion exchange column chromatography of step (g) and / or (h) is washed with PEG solution before elution of rAAV particles from the column. In all aspects and in specific embodiments of the embodiments, PEG has an average molecular weight in the range of about 1,000 g / mol to 80,000 g / mol (inclusive). In all aspects and in specific embodiments of the embodiments, PEG is concentrated at a concentration of about 4% to about 10% (w / v) (inclusive).

[0369] In all aspects and in specific embodiments of the embodiments, the anion exchange column of step (g) and / or (h) is washed with an aqueous surfactant solution before elution of rAAV particles from the column.

[0370] In all aspects and in specific embodiments of the embodiments, the cation exchange column of step (f) is washed with a surfactant solution before elution of rAAV particles from the column.

[0371] In all aspects and in specific embodiments of the embodiments, the PEG solution and / or surfactant solution comprises an aqueous Tris-HCl / NaCl buffer, an aqueous phosphoric acid / NaCl buffer, or an aqueous acetic acid / NaCl buffer.

[0372] In all aspects and in specific embodiments of the embodiments, the concentration of NaCl in the buffer or solution is in the range of about 20 to 300 mM NaCl (including both ends) or about 50 to 250 mM NaCl (including both ends).

[0373] In all aspects and in specific embodiments of the embodiments, the surfactant includes cationic or anionic surfactants.

[0374] In all aspects and in specific embodiments of the embodiments, the surfactant includes a 12-carbon chain surfactant.

[0375] In all aspects and in specific embodiments of the embodiments, the surfactant comprises dodecyltrimethylammonium chloride (DTAC) or sarcosyl.

[0376] In all aspects and in specific embodiments of the embodiments, rAAV particles are eluted from the anion exchange column of steps (f), (g), and / or (h) with aqueous Tris-HCl / NaCl buffer.

[0377] In all aspects and in specific embodiments of the embodiments, the Tris-HCl / NaCl buffer solution contains 100 mM to 400 mM (inclusive) NaCl at a pH optionally in the range of approximately pH 7.5 to approximately pH 9.0 (inclusive).

[0378] In all aspects and in specific embodiments of the embodiments, the anion exchange column in steps (f), (g), and / or (h) is washed with aqueous Tris-HCl / NaCl buffer.

[0379] In all aspects and in specific embodiments of the embodiments, the NaCl concentration in the aqueous Tris-HCl / NaCl buffer is in the range of approximately 75 to 125 mM (inclusive).

[0380] In all aspects and in specific embodiments of the embodiments, the aqueous Tris-HCl / NaCl buffer has a pH of approximately 7.5 to approximately 9.0 (including both ends).

[0381] In all aspects and in certain embodiments of the embodiments, the anion exchange column in steps (f), (g), and / or (h) is washed once or more to reduce the amount of empty capsid in the second or third column eluent.

[0382] In all aspects and in specific embodiments of the embodiments, anion exchange column washing removes empty capsids from the column before and / or instead of rAAV particle elution, thereby reducing the amount of empty capsids in the second or third column eluate.

[0383] In all aspects and in specific embodiments of the embodiments, anion exchange column washing removes at least about 50% of the total empty capsid from the column before and / or instead of rAAV particle elution, thereby reducing the amount of empty capsid in the second or third column eluate by about 50%.

[0384] In all aspects and in specific embodiments of the embodiments, the NaCl concentration in the aqueous Tris-HCl / NaCl buffer is in the range of approximately 110–120 mM (inclusive).

[0385] In all aspects and in specific embodiments of the embodiments, the ratio and / or amount of eluted rAAV particles to empty capsids is controlled by the washing buffer.

[0386] In all aspects and in specific embodiments of the embodiments, rAAV particles are eluted from the cation exchange column of step (f) in aqueous phosphate / NaCl buffer or aqueous acetic acid / NaCl buffer. Non-limiting NaCl concentrations in the buffer range from about 125 to 500 mM NaCl (inclusive). Non-limiting examples of buffer pH range from about pH 5.5 to about pH 7.5 (inclusive).

[0387] In all aspects and in specific embodiments of the embodiments, the anion exchange column of steps (f), (g), and / or (h) contains a quaternary ammonium functional group such as quaternized polyethyleneimine.

[0388] In all aspects and in specific embodiments of the embodiments, the size exclusion column (SEC) of step (g) and / or (h) has a separation / fractionation range (molecular weight) of about 10,000 g / mol to about 600,000 g / mol (including both ends).

[0389] In all aspects and in specific embodiments of the embodiments, the cation exchange column of step (f) contains a functional group such as a sulfonic acid or a sulfopropyl.

[0390] In all aspects and in specific embodiments of the embodiments, the AAV affinity column includes a protein or ligand that binds to the AAV capsid protein. Non-limiting examples of proteins include antibodies that bind to the AAV capsid protein. More specific non-limiting examples include single-chain camelid antibodies that bind to the AAV capsid protein.

[0391] In all aspects and in certain embodiments of the embodiment, the method excludes the step of cesium chloride gradient ultracentrifugation.

[0392] In all aspects and in specific embodiments of the embodiments, the method recovers approximately 50-90% of the total rAAV particles from the recovered material produced in step (a) or the concentrated recovered material produced in step (b).

[0393] In all aspects and in specific embodiments of the embodiments, the method produces rAAV particles having a higher purity than rAAV particles produced or purified by single AAV affinity column purification.

[0394] In all aspects and in certain embodiments of the embodiments, steps (c) and (d) are performed substantially simultaneously.

[0395] In all aspects and in specific embodiments of the embodiments, the NaCl concentration is adjusted after step (c) but before step (f) to be in the range of about 100–400 mM NaCl (including both ends) or in the range of about 140–300 mM NaCl (including both ends).

[0396] In all aspects and in specific embodiments of the embodiments, the rAAV particles are derived from AAVs selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, and Rh74.

[0397] In all aspects and in specific embodiments of the embodiments, the rAAV particles include a capsid sequence having 70% or more sequence identity with the capsid sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh 10, Rh 74, SEQ ID NO: 75, or SEQ ID NO: 76.

[0398] In all aspects and in specific embodiments of the embodiments, the rAAV particles include an ITR sequence having 70% or more sequence identity with an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh 10, or Rh74 ITR sequence.

[0399] In all aspects and in specific embodiments of the embodiments, the cells are suspension-growth cells or adherent-growth cells.

[0400] In all aspects and in specific embodiments of the embodiments, the cells are mammalian cells. Non-limiting examples include HEK cells such as HEK-293 cells, and CHO cells such as CHO-K1 cells.

[0401] Methods for determining the infectivity titer of rAAV particles containing transgenes are known in the art (see, e.g., Zhen et al., Hum. Gene Ther. 15(2004) 709). Methods for assaying empty capsids and rAAV particles packaged with transgenes are also known (see, e.g., Grimm et al., Gene Therapy 6(1999) 1322-1330; Sommer et al., Malec. Ther. 7(2003) 122-128).

[0402] To determine the presence or amount of degraded / denatured capsids, purified rAAV particles can be subjected to SDS-polyacrylamide gel electrophoresis using any gel capable of separating the three capsid proteins, such as a gradient gel. The gel can then be run until the sample is separated, and the gel can be blotted onto a nylon or nitrocellulose membrane. An anti-AAV capsid antibody is then used as the primary antibody to bind to the denatured capsid proteins (see, e.g., Wobus et al., J.Viral. 74(2000) 9281-9293). The secondary antibody, which binds to the primary antibody, includes means for detecting the primary antibody. The amount of capsids is determined by semi-quantitatively detecting the binding between the primary and secondary antibodies. Another method is analytical HPLC using an SEC column or analytical ultracentrifuge.

[0403] In addition to the various embodiments illustrated and claimed, the subject matter of this disclosure also covers other embodiments having other combinations of the features disclosed and claimed herein. Accordingly, certain features presented herein may be combined with each other in other ways within the scope of the subject matter of this disclosure so that the subject matter of this disclosure includes any preferred combination of the features disclosed herein. The foregoing descriptions of specific embodiments of the subject matter of this disclosure are provided for illustrative and explanatory purposes only. They are not intended to be exhaustive or to limit the subject matter of this disclosure to those embodiments disclosed.

[0404] All references mentioned herein are incorporated herein by reference.

[0405] The following examples, sequences, and drawings are provided to aid in understanding the present invention, and the true scope of the invention is set forth in the appended claims. It is understood that modifications to the procedures described may be made without departing from the spirit of the invention. [Brief explanation of the drawing]

[0406] [Figure 1] Alignment of adenovirus VA RNA and adenovirus VA RNA G58T / G59T / C68A variants. [Figure 2] A scheme of an embodiment of the present invention, in which a human U6 promoter is operably linked to an adenovirus VA RNAI sequence. [Examples]

[0407] general technology 1) Recombinant DNA technology Manipulate DNA using standard methods, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989). Use molecular biological reagents according to the manufacturer's instructions.

[0408] 2) DNA and protein sequence analysis and sequence data management The EMBOSS (European Molecular Biology Open Software Suite) software package, Invitrogen's Vector NTI and Geneious Prime are used for sequence generation, mapping, analysis, annotation, and visualization.

[0409] 3) Gene and oligonucleotide synthesis The desired gene segment is prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragment is cloned into an E. coli plasmid for propagation / amplification. The DNA sequence of the subcloned gene fragment is confirmed by DNA sequencing. Alternatively, short synthetic DNA fragments are constructed by annealing chemically synthesized oligonucleotides or via PCR. Each oligonucleotide is prepared by metabion GmbH (Planegg-Martinsried, Germany).

[0410] 4) Reagents Unless otherwise specified, all commercially available chemicals, antibodies, and kits should be used as provided in accordance with the manufacturer's protocol.

[0411] 5) Culture of TI host cell lines TI CHO host cells are cultured at 37°C in a humidified incubator with 85% humidity and 5% CO2. They are cultured in a proprietary DMEM / F12-based medium containing 300 μg / ml hygromycin B and 4 μg / ml of a second selective marker. Cells are divided into 30 ml total volumes at a concentration of 0.3 x 10⁶ cells / ml every 3 or 4 days. A 125 ml non-baffled Erlenmeyer flask is used for culture. Cells are shaken at 150 rpm with a shaking amplitude of 5 cm. Cell count is determined by a Cedex HiRes Cell Counter (Roche). Cells are cultured until they reach 60 days of age.

[0412] 6) Cloning overview Cloning at the R site depends on the DNA sequence adjacent to the target gene (GOI), which is equivalent to the sequence in the next fragment. Thus, the assembly of the fragments is made possible by the overlapping of the equivalent sequences and subsequent sealing of the nicks in the assembled DNA by DNA ligase. Therefore, cloning of a single gene, especially a preliminary plasmid containing the appropriate R site, is necessary. After successful cloning of these preliminary plasmids, the target gene flanked by the R site is excised via restriction digestion with an enzyme that cuts immediately adjacent to the R site. The final step is to assemble all the DNA fragments in one step. More specifically, a 5'-exonuclease removes the 5' end of the overlapping region (R site). Then, annealing of the R site can be performed, and DNA polymerase extends the 3' end to fill the sequence gap. Finally, DNA ligase seals the nicks between nucleotides. Adding an assembly master mix containing different enzymes such as exonuclease, DNA polymerase, and ligase, and then incubating the reaction mix at 50°C, results in the assembly of a single fragment into a single plasmid. Subsequently, competent E. coli cells are transformed with the plasmid.

[0413] For some plasmids, restriction enzyme-mediated cloning strategies were employed. By selecting the appropriate restriction enzyme, the desired target gene can be excised and then inserted into another plasmid by ligation. Therefore, it is preferable to use enzymes that cut at multiple cloning sites (MCS) and select them in a smart manner to ensure that ligation of the fragments in the correct array is performed. If the plasmid and fragments have been previously cut with the same restriction enzyme, the sticky ends of the fragment and plasmid will fit together perfectly and can then be ligated with DNA ligase. After ligation, competent E. coli cells are transformed with the newly constructed plasmids.

[0414] Cloning by restricted digestion For restriction enzyme digestion of plasmids, pipette the following components together on ice. Table: Restriction Digestive Mix TIFF0007857714000003.tif31128

[0415] If using more enzymes in a single digestion, use 1 μl of each enzyme and adjust the volume by adding more or less PCR-grade water. All enzymes are selected on the premise that they are suitable for use with New England Biolabs CutSmart buffer (100% activity) and at the same incubation temperature (all 37°C).

[0416] Incubation is performed using a thermomixer or thermal cycler, allowing the sample to be incubated at a constant temperature (37°C). Do not agitate the sample during incubation. Set the incubation time to 60 minutes. Afterwards, mix the sample directly with the loading dye and load it onto an agarose electrophoresis gel, or store it on 4°C / ice for further use.

[0417] Prepare a 1% agarose gel for gel electrophoresis. Weigh 1.5 g of multipurpose agarose into a 125° Erlenmeyer flask and fill with 150 ml of TAE buffer. Heat the mixture in a microwave oven until the agarose is completely dissolved. Add 0.5 μg / ml of ethidium bromide to the agarose solution. Then pour the gel into a mold. After the agarose has solidified, place the mold in the electrophoresis chamber and fill the chamber with TAE buffer. Then load the sample. (From left) Load the appropriate DNA molecular weight marker into the first pocket, followed by the sample. Electrophoresis the gel at <130 V for approximately 60 minutes. After electrophoresis, remove the gel from the chamber and analyze it with a UV-Imager.

[0418] Cut the target band and transfer it to a 1.5 ml Eppendorf tube. For gel purification, use Qiagen's QIAquick Gel Extraction Kit according to the manufacturer's instructions. Store the DNA fragment at -20°C for further use.

[0419] For ligation, the fragments are pipetteed together in a plasmid-to-insertion molar ratio of 1:2, 1:3, or 1:5, depending on the lengths of the insertion fragment and plasmid fragment, and their correlation with each other. Use a 1:5 ratio if the fragment to be inserted into the plasmid is short. If the insertion fragment is longer, less of the insertion fragment will be used for correlation with the plasmid. Use 50 ng of plasmid for each ligation and calculate the specific amount of insertion fragment using the NEBioCalculator. Use the NEB T4DNA ligation kit for ligation. An example of a ligation mixture is shown in the following table. Table: Ligation reaction mix TIFF0007857714000004.tif36131

[0420] Begin by mixing the DNA and water, then add the buffer, and finally the enzyme, pipetting all components together on ice. Gently mix the reaction mixture by pipetting up and down, microfuse briefly, and then incubate at room temperature for 10 minutes. After incubation, inactivate the T4 ligase at 65°C for 10 minutes. Cool the sample on ice. In the final step, transform 10 beta-competent E. coli cells with 2 μl of ligation plasmid (see below).

[0421] Transformed 10-beta competent E. coli cells For transformation, thaw 10-beta competent Escherichia coli (E. coli) cells on ice. Then, pipette 2 μl of plasmid DNA directly into the cell suspension. Flick the tube and leave it on ice for 30 minutes. Then, place the cells in a 42°C thermal block and give them a strict 30-second heat shock. Immediately afterward, cool the cells on ice for 2 minutes. Add 950 μl of NEB10 beta growth medium to the cell suspension. Incubate the cells at 37°C for 1 hour with shaking. Next, pipette 50-100 μl onto a pre-warmed (37°C) LB-Amp agar plate and spread it with a disposable spatula. Incubate the plate at 37°C overnight. Only bacteria that have successfully incorporated the plasmid and possess the ampicillin resistance gene can grow on these plates. On the following day, pick a single colony and culture it in LB-Amp medium for subsequent plasmid preparation.

[0422] bacterial culture E. coli cultures are performed in LB medium (Luria Bertani abbreviation), and 100 mg / ml ampicillin is added to 1 ml / L to bring the ampicillin concentration to 0.1 mg / ml. For different plasmid preparations, a single bacterial colony is inoculated into the following amounts. Table: Culture volume of E. coli TIFF0007857714000005.tif31128

[0423] For minipreps, fill a 96-well 2ml deep-well plate with 1.5ml of LB-Amp medium per well. Pick up colonies and press them into the medium with a toothpick. Once all colonies have been collected, close the plate with an adhesive air-porous membrane. Incubate the plate in a 37°C incubator at a shaking speed of 200 rpm for 23 hours.

[0424] For minipreps, fill 15 ml tubes (ventilated and lidded) with 3.6 ml of LB-Amp medium and inoculate the bacterial colonies evenly. Do not remove the toothpicks; leave them in the tubes during incubation. Incubate the tubes at 37°C and 200 rpm for 23 hours, similar to 96-well plates.

[0425] For Maxiprep, 200 ml of LB-Amp medium is packed into a 1 L glass Erlenmeyer flask that has been autoclaved, and 1 ml of daytime bacterial culture that has been incubated for approximately 5 hours is inoculated. The Erlenmeyer flask is sealed with a paper stopper and incubated at 37°C and 200 rpm for 16 hours.

[0426] Plasmid preparation For minipreps, transfer 50 μl of bacterial suspension to a 1 ml deep-well plate. Then, centrifuge the bacterial cells in the plate at 3000 rpm at 4°C for 5 minutes. Remove the supernatant and place the plate containing the bacterial pellet into an EpMotion device. After approximately 90 minutes, perform the analysis and you can remove the eluted plasmid DNA from the EpMotion device for further use.

[0427] For minipreps, remove a 15 ml tube from the incubator and divide 3.6 ml of bacterial culture into two 2 ml Eppendorf tubes. Centrifuge the tubes in a benchtop microcentrifuge at 6,800 xg at room temperature for 3 minutes. Then, perform the miniprep using the Qiagen QIAprep Spin miniprep kit according to the manufacturer's instructions. Measure the plasmid DNA concentration with Nanodrop.

[0428] Perform Maxi-Prep using the Macherey-Nagel NucleoBond® Xtra Maxi EF kit according to the manufacturer's instructions. Measure DNA concentration with Nanodrop.

[0429] Ethanol precipitate Mix the volume of the DNA solution with 2.5 times the volume of 100% ethanol. Incubate the mixture at -20°C for 10 minutes. Next, centrifuge the DNA at 14,000 rpm at 4°C for 30 minutes. Carefully remove the supernatant and wash the pellet with 70% ethanol. Again, centrifuge the tube at 14,000 rpm at 4°C for 5 minutes. Carefully remove the supernatant by pipetting and dry the pellet. Once the ethanol has evaporated, add an appropriate amount of endotoxin-free water. Allow the DNA to redissolve in water overnight at 4°C. Take a small aliquot and measure the DNA concentration with a Nanodrop device.

[0430] Composition of the expression cassette For the expression of the open reading frame, a transcription unit containing the following functional elements is used: - Pre- and early enhancers and promoters derived from human cytomegalovirus, including intron A. - Human heavy chain immunoglobulin 5' untranslated region (5'UTR), - Nucleic acids containing each open reading frame, including the signal sequence, as needed. - Bovine growth hormone polyadenylated sequence (BGH pA), and -Optionally, a human gastrin terminator (hGT).

[0431] In addition to the expression unit / cassette containing the desired gene to be expressed, basic / standard mammalian expression plasmids are: - The origin of replication from plasmid pUC18 that enables replication of this plasmid in Escherichia coli (E. coli), and - Beta-lactamase gene that confers ampicillin resistance to E. coli Includes.

[0432] Cell culture technology Standard cell culture techniques will be used, as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J. and Yamada, KM (eds.), John Wiley & Sons, Inc.

[0433] Temporary transfection in the HEK293 series Cells containing DNA elements according to the present invention are generated by transient transfection with each plasmid (see Examples 1-4 below) using the HEK293 system (Invitrogen) according to the manufacturer's instructions. Briefly, HEK293 cells (Invitrogen), grown in suspension in serum-free FreeStyle® 293 expression medium (Invitrogen) in either a shake flask or a stirred fermenter, are transfected with each plasmid and a mix of 293fectin® or fectin (Invitrogen). HEK293 cells are placed in 600 mL of a 2 L shake flask (Corning) at a rate of 1 × 10⁶ 6 Seed at a density of cells / mL and incubated at 120 rpm with 8% CO2. Later, approximately 1.5*10 6 Transfect cells with a cell density of cells / mL with either A) 20 mL of Opti-MEM (Invitrogen) containing a total of 600 μg of plasmid DNA (1 μg / mL) or B) approximately 42 mL of a mix of 20 mL of Opti-MEM + 1.2 mL of 293 fectin or fectin (2 μL / mL). Add glucose solution during the fermentation process according to glucose consumption.

[0434] SDS-PAGE LDS sample buffer, 4x concentrate: 4g glycerol, 0.682g TRIS base, 0.666g TRIS hydrochloride, 0.8g LDS (lithium dodecyl sulfate), 0.006g EDTA (ethylenediaminetetraic acid), 0.75ml 1% w / w aqueous solution of Serva Blue G250, 0.75ml 1% w / w solution of phenol red, and water are added to make a total volume of 10ml.

[0435] Cells in the culture medium were lysed. The solution was then centrifuged to remove cell debris. Aliquots of the clarified supernatant were mixed with 1 / 4 volume (v / v) of 4 x LDS sample buffer and 1 / 10 volume (v / v) of 0.5 M 1,4-dithiothreitol (DTT). The samples were then incubated at 70°C for 10 minutes, and proteins were separated by SDS-PAGE. The NuPAGE® Pre-Cast gel system (Invitrogen Corp.) was used according to the manufacturer's instructions. In particular, 10% NuPAGE® Novex® Bis-TRIS precast gel (pH 6.4) and NuPAGE® MOPS electrophoresis buffer were used.

[0436] Western blot Transfer buffer: 39 mM glycine, 48 mM TRIS hydrochloride, 0.04% wt (w / w) SDS, and 20% v / v methanol. After SDS-PAGE, the separated polypeptides were electrophoretically transferred to a nitrocellulose filter membrane (pore size: 0.45 μm) according to Burnette's "Semidry-Blotting-Method" (Burnette, WN, Anal. Biochem. 112 (1981) 195-203).

[0437] Example 1 DNA production for adenovirus VA RNAI transcription using the human U6 promoter according to the present invention A DNA fragment containing the human U6 promoter sequence (the distance between TATA and the transcription start site and the nucleotide sequence of the U6 promoter remained unchanged; SEQ ID NO: 42) and the adenovirus serotype 2 (Ad2) VA RNAI gene (GenBank AC_000007), which includes the polymerase III terminator sequence (SEQ ID NO: 33), in the 5' to 3' direction was chemically synthesized.

[0438] This fragment is ligated with a plasmid backbone containing a puromycin selection marker to obtain a plasmid for stable transfection of mammalian cells.

[0439] Figure 2 shows the order and orientation of the elements within this DNA fragment.

[0440] Example 2 Stable embedded CHO-K1 cells adapted for growth in suspension are grown at 37°C and 5-7 volume %CO2 in 50 mL of chemically defined medium in a disposable, ventilated 125 mL shaking flask. The culture is shaken at a constant stirring speed of 140-180 rpm / min and refrigerated in fresh medium every 3-4 days for 2-3 × 10⁶ days. 5 Dilute to a cell / mL density. Determine the density and viability of the culture using a Cedex HiRes cell counter (Roche Innovates AG, Bielefeld, Germany).

[0441] For stable incorporation of nucleic acids in Example 1, suspension-grown CHO-K1 cells were prepared in a 4 × 10⁶ arrangement. 5 Seeds are seeded in fresh, chemically defined medium with a cell density of 3 × 10⁶ cells / mL. The following day, transfection is performed using the Nucleofector Kit V (Lonza, Switzerland) with a Nucleofector device according to the manufacturer's protocol. 7 Individual cells are transfected with 30 μg of linearized plasmid DNA. After transfection, the cells are seeded in 30 ml of fresh, chemically defined medium without any selective agents.

[0442] Two days after transfection, cells are seeded at a rate of 300-500 cells per well in 384-well plates containing 1 μg / mL to 10 μg / mL puromycin as a selective agent. Three weeks later, cell colonies are identified by imaging using a NYONE Plate imager (SYNENTECH GmbH, Elmshorn, Germany). Colonies are transferred to 96-well plates and analyzed for integration by PCR. Cell lines containing nucleic acids are further grown in a chemically defined medium containing puromycin and then cryopreserved.

[0443] Example 3 Production of AAV particles For the production of recombinant AAV particles, 3 × 10⁶ obtained according to Example 2 7 Each cell is transfected with 30 μg of nucleic acid, consisting of plasmid DNA providing a recombinant AAV genome (transgene, e.g., the GFP gene adjacent to the AAV ITR) and an expression cassette of helper genes and / or rep / cap genes that have not yet been integrated into the cell's genome.

[0444] One day before transfection, the cells were divided into 4 x 10 5 Seed cells in fresh medium at a density of cells / mL. The following day, transfection is performed using the Nucleofector Kit V (Lonza, Switzerland) with a Nucleofector device according to the manufacturer's protocol.

[0445] Alternatively, plasmids are sequentially and stably integrated into the host cell's genome, with the rep / cap gene being the last to be incorporated.

[0446] AAV particles are recovered from cell culture supernatant or whole cell lysate and analyzed by ELISA, quantitative PCR, and target cell transduction.

Claims

1. Adenovirus VA RNA nucleic acid, The adenovirus VA RNA is characterized by having the sequence of Sequence ID No. 38 and being operably ligated at its 5' end to a type II polymerase III promoter, a type III polymerase III promoter, or a polymerase II promoter. The aforementioned adenovirus VA RNA nucleic acid.

2. The adenovirus VA RNA according to claim 1, characterized in that the VA RNA coding sequence is operably ligated to a human U6-snRNA promoter at its 5' end.

3. The adenovirus VA RNA according to claim 1, characterized in that the VA RNA coding sequence is operably ligated to an inducible promoter at its 5' end.

4. The adenovirus VA RNA according to any one of claims 1 to 3, characterized in that the adenovirus VA RNA nucleic acid includes a precise transcription start site located on the 3' side of the promoter, and the precise transcription start site includes at least six 5' terminal nucleotides of the adenovirus VA RNA gene, which includes the transcription start site and a functional polymerase III terminator, in the 5' to 3' direction.

5. Adenovirus VA RNA according to any one of claims 1 to 4, characterized in that the adenovirus VA RNA nucleic acid contains a polymerase III terminator at its 3' end.

6. Adenovirus VA RNA according to any one of claims 1 to 5, characterized in that all elements of the adenovirus VA RNA nucleic acid are arranged in a operably linked form.

7. Packaging cells for recombinant AAV (rAAV) particle production, The cell is characterized in that the rep / cap gene and the adenovirus helper gene are stably incorporated into the genome, and the adenovirus VA RNA nucleic acid comprises the adenovirus VA RNA nucleic acid described in any one of claims 1 to 6.

8. The packaging cell according to claim 7, characterized in that the rAAV plasmid containing the ITR and the transgene is also stably incorporated into the genome of the packaging cell.

9. The packaging cell according to claim 7, characterized in that an rAAV plasmid containing an ITR and a transgene is transiently introduced into the genome of the packaging cell.

10. - Adenovirus VA RNA nucleic acid according to any one of claims 1 to 6, and one or more or all of open reading frames selected from rep, cap, E1A, E1B, E2, E4 and E4orf6 DNA containing this.

11. Mammalian cells or insect cells comprising adenovirus VA RNA according to any one of claims 1 to 6, or DNA according to claim 10.

12. The cell according to any one of claims 7 to 9 and 11, which is a HEK or CHO cell.

13. The cell according to claim 12, which is a HEK293 cell.

14. - A step of culturing the cells according to any one of claims 7 to 9 or 11 to 13 under conditions suitable for cell division; and - A step of recovering rAAV particles from cells or culture medium, A method for producing rAAV particles, characterized by containing [a certain element].

15. A method for producing rAAV particles, - A step of providing mammalian suspension-growing cells that are either stably integrated into the genome or transiently present and include the following: - A transgene expression cassette placed between two AAV ITRs; - An open reading frame encoding adenovirus E1A, E1B, E2A, E4 or E4 or f6 protein and adenovirus VA RNA nucleic acid according to any one of claims 1 to 6; - Open reading frame encoding adeno-associated Rep / Cap protein; - A step of culturing mammalian cells under conditions that enable cell division; and - A process of isolating rAAV particles from cells or culture medium and thereby producing rAAV particles. The method, including the method described above.

16. The method according to claim 15, characterized in that the cells are HEK or CHO cells.

17. The method according to claim 16, wherein the cells are HEK293 cells.

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