Composition and method for a stable human embryonic kidney-293 cell line producing adeno-associated virus

Abstract

The subject matter disclosed herein relates to a composition and a method for generating a stable human embryonic kidney-293 (HEK-293) cell line for the production of adeno-associated virus (AAV).

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 612,667 filed December 20, 2023 and U.S. Provisional Application No. 63 / 667,323 filed July 3, 2024, the entire contents of which are incorporated herein by reference.

[0003] Technology field

[0004] The subject matter disclosed herein relates to a composition and a method for generating a stable human embryonic kidney-293 (HEK-293) cell line for the production of adeno-associated virus (AAV). Background Technology

[0005] background

[0006] AAV vectors offer several advantages over other viral vectors in the field of gene therapy. In addition to their non-integrating and non-pathogenic characteristics, AAV vectors are suitable carriers for clinical application as they can induce long-term stable transplant gene expression by transfecting not only dividing cells but also non-dividing cells. Furthermore, AAV vectors can be designed to alter tissue affinity and evade host immunity, which can enable more efficient transfection of intended target tissues and the successful establishment of transplant gene expression.

[0007] Given the advantages of AAV vectors, there is a growing demand for the development of stable mammalian host cells to produce them. However, achieving production-grade virus production presents several challenges. For example, the method of producing AAV by transiently transfecting production cell lines with plasmids containing the AAV gene and genes related to adenovirus support, capsid, and replication enzyme function results in high manufacturing costs due to poor scalability and reproducibility. The toxicity of viral genes also reduces manufacturing efficiency, further increasing costs. Moreover, existing strategies for developing production cell lines involve the random integration of nucleotide sequences, which is not only a rare event but can also lead to undesirable gene expression and cell growth phenotypes (e.g., unstable cell growth). Due to the heterogeneity resulting from this random integration process, time-consuming and labor-intensive clone screening is often required to isolate cell lines exhibiting viral titers of desired levels and quality. In addition to these drawbacks, virus production using adherent cells is excessively expensive due to limited scalability options. Furthermore, many manufacturing platforms utilizing adherent cells exhibit poor performance in serum-free media, whereas the use of serum leads to viral particle contamination, increasing downstream disposal costs. Therefore, there is a demand in the field for cost-effective compositions and methods that enable more efficient and GMP-compliant viral vector production. The problem to be solved

[0008] Summary of the Invention

[0009] The subject matter disclosed herein relates in part to stable mammalian host cells suitable for AAV production. In a specific embodiment, the stable mammalian host cell comprises a first exogenous nucleic acid sequence incorporated at a target locus in the genome of the stable mammalian host cell (TI host cell) and a second exogenous nucleic acid sequence incorporated at least once in the genome of the mammalian cell. In this embodiment, the first exogenous nucleic acid sequence comprises two recombination recognition sequences (RRS) and encodes one or more of the adeno-associated virus (AAV) replication enzyme (REP), AAV capsid (CAP), adenovirus (Ad) E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2-DNA binding protein (E2DBP), or Ad virus-associated (Ad VA) gene products (e.g., proteins and / or RNA) and a first selection marker. Also in this embodiment, the second exogenous nucleic acid sequence comprises one or more of the polypeptide of interest (POI), AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA, and an inversion terminal repeat (ITR) adjacent to a coding sequence for the second selection marker.

[0010] In a specific embodiment, the present invention provides a stable mammalian host cell in which a first exogenous nucleic acid sequence encodes AAV REP, Ad E2A, Ad E4, and Ad VA. In a specific embodiment, a second exogenous nucleic acid sequence encodes AAV CAP.

[0011] In a specific embodiment, the mammalian cell comprises one to ten additional exogenous nucleic acids, each comprising an ITR adjacent to a coding sequence for a selection marker, and one or more of AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA, and each of the one to ten additional exogenous nucleic acids is incorporated into the genome of the mammalian cell at least once. In a specific embodiment, each of the one to ten additional exogenous nucleic acids comprises a different selection marker. In a specific embodiment, two or more of the one to ten additional exogenous nucleic acids comprise the same selection marker. In a specific embodiment, one or more of the one to ten additional exogenous nucleic acids further comprise a coding sequence for a polypeptide of interest (POI) within the adjacent ITR.

[0012] In certain embodiments, the stable mammalian host cell is a stable human cell (e.g., HEK293 cell line).

[0013] In certain embodiments, the integration of the first exogenous nucleic acid sequence may be facilitated by an exogenous nuclease. In certain embodiments, the exogenous nuclease may be selected from the group consisting of zinc finger nucleases (ZFN), ZFN dimers, transcription activator-like effector nucleases (TALEN), TAL effector domain fusion proteins, RNA guide DNA endonucleases, engineered meganucleases, and clustered regularly spaced short palindromic repeat (CRISPR) associated (Cas) endonucleases.

[0014] In a specific embodiment, one or more proteins encoded by the first exogenous nucleic acid sequence are inductively expressed. In a specific embodiment, one or more AAV proteins encoded by the first exogenous nucleic acid sequence are inductively expressed. In a specific embodiment, one or more proteins encoded by the second exogenous nucleic acid sequence are inductively expressed. In a specific embodiment, one or more AAV proteins encoded by the second exogenous nucleic acid sequence are inductively expressed.

[0015] In a specific embodiment, one or more proteins encoded by the first exogenous nucleic acid sequence are inductively expressed, and one or more proteins encoded by the second exogenous nucleic acid sequence are constitutively expressed. In a specific embodiment, one or more proteins encoded by the first exogenous nucleic acid sequence are inductively expressed, and one or more proteins encoded by the second exogenous nucleic acid sequence are inductively expressed. In a specific embodiment, one or more proteins encoded by the first exogenous nucleic acid sequence are constitutively expressed, and one or more proteins encoded by the second exogenous nucleic acid sequence are constitutively expressed. In a specific embodiment, one or more proteins encoded by the first exogenous nucleic acid are constitutively expressed, and one or more proteins encoded by the second exogenous nucleic acid are inductively expressed. In another embodiment, a plurality of proteins encoded by the first and / or second exogenous nucleic acid sequences are inductively expressed.

[0016] In certain embodiments, the inductively expressed protein is induced by the same inducer.

[0017] The subject matter disclosed herein also provides a method for producing a recombinant AAV of interest (rAAV). In certain embodiments, the method comprises the following steps: A) providing a stable mammalian host cell comprising a first exogenous nucleotide sequence incorporated into a target locus of the genome of the mammalian host cell, wherein the first exogenous nucleotide sequence comprises two recombinant recognition sequences (RRS) adjacent to a first selection marker; B) introducing a second exogenous nucleic acid sequence into the stable mammalian host cell, wherein the second exogenous nucleic acid sequence comprises two RRS adjacent to a sequence encoding one or more of the AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA proteins and the second selection marker; C) introducing a recombinase or a nucleic acid encoding the recombinase, wherein the recombinase recognizes the RRS; D) introducing, simultaneously with or sequentially after B) and C), one or more of AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2-DNA binding protein (E2DBP), Ad VA gene products (e.g., protein and / or RNA) or a third exogenous nucleic acid sequence encoding a polypeptide of interest and a third selection marker into the genome of a stable mammalian cell via transposon-mediated genomic integration; E) selecting a mammalian host cell that stably expresses the second and third selection markers; and F) culturing the selected mammalian host cell under conditions sufficient to produce recombinant AAV. In certain embodiments, the method may further include the step of recovering the AAV of interest from the cell culture.

[0018] In a specific embodiment, the second exogenous nucleic acid sequence encodes AAV REP, Ad E2A, Ad E4, and Ad VA. In a specific embodiment, the third exogenous nucleic acid sequence encodes AAV CAP. In a specific embodiment, the mammalian host cell is a human cell (e.g., HEK293).

[0019] In a specific embodiment, the method comprises the following steps: A) providing a stable mammalian host cell comprising a first exogenous nucleotide sequence incorporated at a target locus of the genome of a mammalian host cell, wherein the first exogenous nucleotide sequence comprises first and second RRSs adjacent to at least one first selection marker and a third RRS located between the first and second RRSs, and all RRSs are heterospecific; B) introducing into the cell provided in a) a first vector comprising two RRSs adjacent to a sequence encoding one or more of AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA proteins and a selection marker; c) introducing into the cells provided in a) a second vector comprising two RRSs that are matched with the second and third RRSs in at least one integrated exogenous nucleotide sequence and adjacent to a sequence encoding one or more of AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA proteins and a selection marker; d) introducing two or more recombinases or one or more nucleic acids encoding two or more recombinases, wherein the two or more recombinases recognize the RRSs; and E) a step of introducing additional exogenous nucleic acid sequences encoding AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2-DNA binding protein (E2DBP), Ad VA gene products (e.g., protein and / or RNA) or polypeptides of interest and selection markers into the genome of a stable mammalian host cell via transposon-mediated genomic integration, simultaneously with B), C), and D);F) a step of selecting mammalian host cells that stably express one or more of the selection markers; and G) a step of culturing the selected mammalian host cells under conditions sufficient to produce recombinant AAV. In certain embodiments, this method may further include a step of recovering the AAV of interest from the cell culture.;

[0020] In certain embodiments, the target integration of the first exogenous nucleotide sequence is facilitated by an exogenous nuclease. In certain embodiments, the exogenous nuclease may be selected from the group consisting of zinc finger nucleases (ZFN), ZFN dimers, transcription activator-like effector nucleases (TALEN), TAL effector domain fusion proteins, RNA guide DNA endonucleases, engineered meganucleases, and clustered regularly spaced short palindromic repeat (CRISPR) associated (Cas) endonucleases.

[0021] In certain embodiments, the expression of one or more proteins is controlled by a regulated promoter. In certain embodiments, the regulated promoter is selected from the group consisting of SV40 and CMV promoters.

[0022] In a specific embodiment, one or more proteins encoded by a first exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian host cell are inductively expressed. In a specific embodiment, one or more proteins encoded by a second exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian host cell are inductively expressed. In a specific embodiment, one or more proteins encoded by a third exogenous nucleic acid incorporated into a transposon-mediated genome are inductively expressed. In a specific embodiment, one or more proteins encoded by a third exogenous nucleic acid incorporated into a transposon-mediated genome are inductively expressed. In a specific embodiment, one or more proteins encoded by a first exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian host cell are inductively expressed, and one or more proteins encoded by a third exogenous nucleic acid incorporated into a transposon-mediated genome are constitutively expressed. In a specific embodiment, one or more proteins encoded by a first exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian host cell are inductively expressed, and one or more proteins encoded by a third exogenous nucleic acid incorporated into a transposon-mediated genome are inductively expressed. In a specific embodiment, one or more proteins encoded by a first exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian host cell are constitutively expressed, and one or more proteins encoded by a third exogenous nucleic acid incorporated into a transposon-mediated genome are constitutively expressed. In a specific embodiment, one or more proteins encoded by a first exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian host cell are constitutively expressed, and one or more proteins encoded by a third exogenous nucleic acid incorporated into a transposon-mediated genome are inductively expressed.

[0023] In a specific embodiment, a stable mammalian cell selected to express a third selection marker comprises 1 to 10 copies of a transposon-mediated genome-integrated third exogenous nucleic acid sequence.

[0024] In certain embodiments, a plurality of proteins encoded by the first and / or second exogenous nucleic acid are inductively expressed. In certain embodiments, the proteins that are inductively expressed are induced by the same inducer. Brief explanation of the drawing

[0025] Brief explanation of the drawing Fig. 1a-1b Is This describes an exemplary strategy for generating an integrated stable mammalian host cell pool and isolated single-cell clones (SCCs) using targeted integration (TI). Figure 1a illustrates a sequential transfection approach for TI. Figure 2a illustrates a simultaneous transfection approach for TI. Fig. 2 Is It shows an exemplary melted landing cassette encoding the GFP gene and the hygromycin-thymidine kinase (HYTK) selection marker, which are lateralized by two LoxP sites (L3 and 2L), an additional LoxP site (Loxfas), and a frt3 site. Fig. 3 silver This shows the results of comparing AAV titers obtained using HEK293 TI (targeted integration) host cells and wild-type (wt) HEK293 host cells. Figures 4a-4e Is Exemplary plasmids for generating integrated, stable host cells that produce AAV are shown. Fig. 4a shows a front TI plasmid containing a Tet inducible promoter for inducing REP gene expression, a transcriptional activator (rTA) expression cassette, and a downstream SV40 promoter. Fig. 4b shows a back TI plasmid containing a puromycin-N-acetyltransferase (PAC) gene followed by a Tet inducible promoter for inducing auxiliary gene expression. Fig. 4c shows PiggyBac plasmid #1 containing a Tet inducible promoter for inducing Cap gene expression followed by a gene of interest (GOI) sequence adjacent to an ITR sequence and a selection marker (SM), e.g., blasticidin (BSD), zeocin (ZEO), or hygromycin (HYGRO) resistance gene. Figure 4d shows PiggyBac plasmid #2 containing a Tet inducible promoter for inducing REP gene expression followed by a zeosin resistance gene (ZEO). Figure 4e shows PiggyBac plasmid #3 containing a Tet inducible promoter for inducing auxiliary gene expression followed by a zeosin resistance gene (ZEO). Figs. 5a-5c Is This illustrates an exemplary combination of PiggyBac (PB) plasmids encoding CAP and GOI, which can be transfected into TI-packaged hosts to produce producer cell lines. Fig. 5a shows a PB plasmid comprising a Tet-inducible promoter that induces CAP gene expression, followed by two inversion terminal repeats (ITRs) and a GOI expression cassette fused with a transcription activator / blasticidin (rTA-Bsd) selection marker. Fig. 5b shows a PB plasmid comprising a Tet-inducible promoter that induces CAP gene expression, followed by two inversion terminal repeats (ITRs) and two GOI expression cassettes fused with a transcription activator / zeosin (rTA-Zeo) selection marker, respectively. Figure 5c shows a PB plasmid containing a Tet inducible promoter that induces CAP gene expression, followed by two inversion terminal repeats (ITRs) and two GOI expression cassettes fused with a transcription activator / hygromycin (rTA-Hyg) selection marker, respectively. Figures 6a-6f Is This document illustrates a TI pre-packaging clone screening method including first and second round screenings. Figure 6a is a schematic diagram of the first and second round screenings. Figure 6b shows the first round screening based on clone analysis. Figure 6c shows the results of comparing clone analysis with stable TI / P pool data. Figure 6d shows the results of screening 190 clones using clone analysis. Figure 6e shows the second round screening based on transient transfection analysis. Figure 6f shows the results of screening 36 clones (identified in clone analysis) using transient transfection analysis. Fig. 7 silver This describes the process of generating a TI / P producer pool from clones identified using replication and transient transfection analysis. Fig. 8 silver We show Cap / GOI plasmids with various double- and triple-selection marker combinations used for TI / P producer pool production. Figs. 9a-9d Is This shows the results of protein expression, integrated copy number, and rAAV titer analysis in the TI / P producer pool. Figure 9a shows the GFP expression results in cells transfected with dual-selection and triple-selection plasmids. Figure 9b shows the integrated copy number of Cap / GOI in cells transfected with dual-selection and triple-selection plasmids. Figure 9c shows the rAAV titer in cells transfected with dual-selection and triple-selection plasmids. Figure 9d shows the rAAV titer using different TI pools. Figs. 10a-10d Is Exemplary plasmids and methods for generating a pre-packaging pool containing REP and helper genes using a 2-plasmid RMCE strategy are shown. Fig. 10a shows an exemplary method for generating a producer clone. Fig. 10b shows an exemplary plasmid for generating an integrated, stable host cell that produces AAV. Fig. 10c shows the results of mass spectrometry (MP) measuring the total / empty ratio of rAAV purified from a crude lysate generated from a TI / P producer clone using the methods and plasmids described in Figs. 10a and 10b. Fig. 10d shows the total / empty ratio of rAAV purified from a crude lysate generated from a trivalent plasmid transient transfection. Specific details for implementing the invention

[0026] details

[0027] The subject matter disclosed herein relates to TI host cells suitable for AAV production, wherein the TI host cells are also subjects of transposon-mediated genome integration of one or more exogenous nucleic acids, and the subject matter disclosed herein also relates to methods for producing and utilizing said combined transposon-mediated genome integration and TI host cells. In certain embodiments, the host cells, genetic constructs (e.g., vectors), compositions, and methods described herein may be utilized for the development and / or utilization of said combined transposon-mediated genome integration and TI host cells. In certain embodiments, the present invention relates to an integration ("TIP") strategy for producing stable mammalian cells for AAV production, wherein the TI and transposon-mediated genome integration steps are combined. In certain embodiments, the present invention relates to a sequential integration ("TI / P") strategy for producing stable mammalian host cells for AAV production, wherein the TI and transposon-mediated genome integration steps are performed sequentially.

[0028] For clarity of disclosure without limitation, the detailed description is divided into the following subsections:

[0029] 1. Definition

[0030] 2. AAV Vector Production

[0031] 3. Integrated area

[0032] 4. Exogenous nucleotide sequences

[0033] 5. Targeted Integration and Transposon-Mediated Genome Integration Methods

[0034] 6. Method for producing recombinant AAV

[0035] 7. Examples

[0036] 1. Definition

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art. In the event of a conflict, this specification, including definitions, shall prevail. Methods and materials similar or equivalent to those described herein may be used to carry out or test the essential parts disclosed herein, but preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and embodiments disclosed herein are merely illustrative and are not intended to be limiting.

[0038] As used herein, the terms “comprising,” “comprising,” “having,” “having,” “can,” “containing,” and variations thereof are intended as open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms “a,” “an,” and “the” include the plural forms unless otherwise specified in the context. The present invention also considers other embodiments that “comprising,” “composed of,” and “essentially composed of” the embodiments or elements presented herein, regardless of whether they are explicitly described.

[0039] When specifying numerical ranges in this specification, each intermediate number in between is explicitly considered with the same precision. For example, in the range of 6-9, 7 and 8 are considered in addition to 6 and 9, and in the range of 6.0-7.0, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly considered.

[0040] As used herein, the terms “about” or “approximately” mean within a tolerance range for a specific value as determined by a person skilled in the art, which depends in part on the method of measuring or determining said value, i.e., the limitations of the measurement system. For example, “about” may mean within 3 standard deviations or exceeding 3 standard deviations according to the practice of the art. Alternatively, “about” may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly in relation to biological systems or processes, this term may mean within one digit of the value, preferably within five times, and more preferably within two times.

[0041] As used herein, the term “selection marker” may be a gene that causes cells possessing the gene to be specifically selected or not selected when a corresponding selector is present. For example, but without limitation, a selection marker may cause host cells transformed with the selection marker gene to be positively selected when the gene is present; untransformed host cells cannot grow or survive under selection conditions. A selection marker may be positive, negative, or dual-functional. A positive selection marker causes cells possessing the marker to be selected, whereas a negative selection marker causes cells possessing the marker to be selectively eliminated. A selection marker may confer resistance to a drug or compensate for metabolic or catabolic defects in host cells. In prokaryotic cells, genes conferring resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol may be used. Resistance genes useful as selection markers in eukaryotic cells include, but are not limited to, genes for aminoglycoside phosphotransferases (APH) (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 genes encoding resistance to puromycin, blasticidin, bleomycin, pleomycin, chloramphenicol, zeocin, and mycophenolic acid. Additional marker genes are described in WO 92 / 08796 and WO 94 / 28143.

[0042] In addition to facilitating selection when a corresponding selector is present, selection markers may alternatively provide genes encoding molecules not typically present in cells, such as green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced 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. Cells possessing these genes can be distinguished from cells not possessing the corresponding genes, for example, by detecting fluorescence emitted by the encoded polypeptide.

[0043] As used herein, the term “operably linked” means that two or more components are juxtaposed and have a relationship in which they can function in an intended manner. For example, if a promoter and / or enhancer serve to regulate the transcription of a coding sequence, the promoter and / or enhancer are operably linked to the coding sequence. In certain embodiments, the “operably linked” DNA sequences are continuous and adjacent on a single chromosome. In certain embodiments, if two protein encoding regions, such as a secretory leader and a polypeptide, need to be linked, these sequences are continuous, adjacent, and within the same reading frame. In certain embodiments, the operably linked promoter may be located upstream of the coding sequence and may be adjacent to the coding sequence. In certain embodiments, for example, with respect to an enhancer sequence that regulates the expression of the coding sequence, the two components may be operably linked even if they are not adjacent. If the enhancer increases the transcription of the coding sequence, the enhancer is operably linked to the coding sequence. Operatably linked enhancers may be located upstream, internally, or downstream of the coding sequence and may be located at a significant distance from the promoter of the coding sequence. Operatably linked can be achieved through recombination methods known in the art, for example, using PCR methods and / or through ligation at a convenient restriction site. If a convenient restriction site does not exist, synthetic oligonucleotide adapters or linkers may be used according to existing convention. The internal ribosome entry site (IRES) is operably linked to the ORF in such a way that it allows translation initiation of the open translation frame (ORF) at the internal location in a 5' end-independent manner.

[0044] As used herein, the term “expression” means transcription and / or translation. In certain embodiments, the transcription level of the desired product may be determined based on the amount of the corresponding mRNA present. For example, mRNA transcribed from the gene of interest may be quantified by PCR or Northern hybridization. In certain embodiments, the protein encoded by the gene of interest may be quantified by various methods, such as ELISA, measurement of the biological activity of the protein, or assays independent of such activity, such as Western blotting or radioimmunoassay using antibodies that recognize and bind to the protein.

[0045] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced (including the offspring of such cells). Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their offspring regardless of the number of passages. Offspring may not be exactly identical to the parent cells in nucleic acid content and may contain mutations. Mutant offspring having the same function or biological activity as that selected or chosen for the original transformed cell are included herein.

[0046] The terms “nucleic acid molecule” or “polynucleotide” include all compounds and / or substances comprising polymers of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by their base sequences, said bases representing the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically indicated from 5’ to 3’. As used herein, the term nucleic acid molecule includes, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, including complementary DNA (cDNA) and genomic DNA, and hybrid polymers comprising two or more of these molecules. Nucleic acid molecules may be linear or circular. Additionally, the term nucleic acid molecule includes both sense and antisense strands, and both single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include derivatized sugars or modified nucleotide bases having phosphate backbone bonds, or chemically modified residues. Nucleic acid molecules also include DNA and RNA molecules suitable as vectors for the direct expression of proteins required for AAV production by stable mammalian host cells. These DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule so that the corresponding mRNA can be injected into an individual to generate POI in vivo (see, for example, Stadler er al, Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0047] "Isolated" nucleic acids refer to nucleic acid molecules separated from components of the natural environment. Isolated nucleic acids include nucleic acid molecules present within cells that normally contain nucleic acid molecules, but said nucleic acid molecules exist outside the chromosomes or at chromosomal locations different from natural chromosomal positions.

[0048] As used herein, the term “vector” refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to itself. This term includes vectors as self-replicating nucleic acid structures as well as vectors introduced and integrated into the host cell genome. In certain embodiments, a vector induces the expression of a nucleic acid operably linked to itself. Such a vector is referred to herein as an “expression vector.”

[0049] As used herein, the term “homologous sequence” refers to a sequence that shares significant sequence similarity determined through sequence alignment. For example, two sequences may have about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% homology. Alignment is performed through algorithms and computer programs, including but not limited to BLAST, FASTA, and HMME, which compare sequences and calculate the statistical significance of alignment based on factors such as sequence length, sequence identity and similarity, and the presence and length of sequence mismatches and gaps. Homologous sequences may refer to both DNA sequences and protein sequences.

[0050] As used herein, the term “sideways” means that the first nucleotide sequence is located at either or both ends of the 5’ or 3’ end of the second nucleotide sequence. The sideways nucleotide sequence may be adjacent to the second nucleotide sequence or within a certain distance from the second nucleotide sequence. There is no particular limitation on the length of the sideways nucleotide sequence. For example, the sideways sequence may be a few base pairs or thousands of base pairs. In a specific embodiment, the length of the adjacent nucleotide sequence may be about at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, at least 3,000 base pairs, at least 4,000 base pairs, at least 5,000 base pairs, at least 6,000 base pairs, at least 7,000 base pairs, at least 8,000 base pairs, at least 9,000 base pairs, and at least 10,000 base pairs.

[0051] As used herein, the term “exogenous” indicates that a nucleotide sequence is not derived from a host cell but is introduced into a host cell through traditional DNA delivery methods, such as transfection, electroporation, or transformation methods. The term “endogenous” means that a nucleotide sequence is derived from a host cell. An “exogenous” nucleotide sequence may have an “endogenous” counterpart that has the same base composition but is introduced into a host cell, for example, through recombinant DNA technology.

[0052] 2. AAV Vector Production

[0053] Adeno-associated virus (AAV) is a non-enveloped capsid virus containing 4.7 kb of single-stranded DNA and encodes four replication proteins (Rep; Rep78, Rep68, Rep52, Rep40), three capsid proteins (VP; VP1 / VP2 / VP3), and an assembly-activating protein (AAP). The coding region is adjacent to a pair of inversion terminal repeats (ITRs) that serve as primers for genome replication. Wild-type AAVs are generally replication-deficient and require the presence of cofactors derived from helper viruses such as adenoviruses (Ad).

[0054] E1A, E1B55K, E2A, E4orf6, E4orf6 / 7, E2DBP, and VA RNA have been identified as Ad cofactors associated with AAV replication. An exemplary method for AAV production involves the multiplasmid transfection of HEK293 cells (human cell lines expressing the Ad El gene), which allows for the efficient production of rAAV without the need for separate infection with helper viruses. However, this transfection method is inefficient, lacks scalability and reproducibility, and is expensive to manufacture. In addition to these drawbacks, the constitutive expression of E1A by HEK293 cells leads to the activation of the AAV promoter, resulting in Rep and helper protein-induced cytotoxicity, which makes it difficult to establish stable producer and packaging cell lines. Alternatively, for example, but without limitation, a regulated expression system can be used to express toxic proteins associated with AAV production, in which case the sequence(s) encoding the potentially toxic protein(s) are under the control of an inducible promoter.

[0055] 3. Integrated area

[0056] The subject disclosed herein provides a mammalian host cell suitable for the targeted integration (TI) of a nucleotide sequence required for AAV production and / or transposon-mediated genomic integration. In certain embodiments, the host cell of the present invention comprises a locus in which a first exogenous nucleic acid sequence is integrated by targeted integration. In certain embodiments, the host cell of the present invention comprises a second exogenous nucleic acid sequence integrated at least once into the genome of the host cell. In certain embodiments, the host cell of the present invention comprises one to ten additional exogenous nucleic acid sequences integrated at least once into the genome of the host cell. In certain embodiments, the integration of the second exogenous nucleic acid sequence is mediated using a transposon.

[0057] In certain embodiments, the mammalian host cell is a human host cell, for example, a human embryonic kidney 293 (HEK293) cell or a variant thereof.

[0058] In a specific embodiment, a mammalian host cell is said to be stable for AVV production if the viral titer produced in the cell is maintained at a specific level, increased, or decreased to less than 20% over 10, 20, 30, 50, 100, 200, or 300 generations. In a specific embodiment, a mammalian host cell is said to be stable for AVV production if viral production is maintained without any selection process.

[0059] The “integration site” comprises a nucleic acid sequence into which a first exogenous nucleotide sequence is inserted within the host cell genome. In certain embodiments, the integration site is located between two adjacent nucleotides on the host cell genome. In certain embodiments, the integration site comprises a stretch of nucleotides into which the exogenous nucleotide sequence can be inserted. In certain embodiments, the integration site is located within a specific locus of the TI host cell genome. In certain embodiments, the integration site is within an endogenous gene of the TI host cell.

[0060] In a specific embodiment, the first exogenous nucleotide sequence is incorporated into a safe harbor locus of the host cell genome. In a specific embodiment, the first exogenous nucleotide sequence is incorporated into a region within a specific locus of the TI host cell genome. In a specific embodiment, the host cell is a human host cell, and the first exogenous nucleic acid sequence is incorporated into an adeno-associated virus integration site.

[0061] In certain embodiments, the locus comprising the integration site of the first exogenous nucleotide sequence does not encode an open reading frame (ORF). In certain embodiments, the locus comprising the integration site of the first exogenous nucleotide sequence comprises cis action elements, e.g., a promoter and an enhancer. In certain embodiments, the locus comprising the integration site of the exogenous nucleotide sequence does not have cis action elements that enhance gene expression, e.g., a promoter and an enhancer.

[0062] In certain embodiments, the integration site and / or the nucleotide sequence adjacent to the integration site may be identified experimentally. In certain embodiments, the integration site and / or the nucleotide sequence adjacent to the integration site may be identified through a whole-genome screening approach to isolate host cells producing a selection marker and / or AAV at a desired level. In certain embodiments, the integration site and / or the nucleotide sequence adjacent to the integration site may be identified through a whole-genome screening approach after a transposase-based cassette integration event. In certain embodiments, the integration site and / or the nucleotide sequence adjacent to the integration site may be identified through brute-force random integration screening. In certain embodiments, the integration site and / or the nucleotide sequence adjacent to the integration site may be determined through conventional sequencing approaches such as next-generation sequencing (NGS) and whole-genome NGS after target locus amplification (TLA). In certain embodiments, the location of the integration site on the chromosome can be determined through conventional cell biology approaches such as fluorescence in situ hybridization (FISH) analysis.

[0063] In certain embodiments, the integration sites may be located on the same chromosome. In certain embodiments, the integration sites are located within the same chromosome by 1-1,000 nucleotides, 1,000-100,000 nucleotides, 100,000-1,000,000 nucleotides, or more than that. In certain embodiments, the integration sites are located on different chromosomes. In certain embodiments, a TI host cell containing a first exogenous nucleotide sequence in one integration site may be used to insert at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or more exogenous nucleotide sequences into the same or different integration sites.

[0064] In certain embodiments, the targeted integration of the first exogenous nucleic acid sequence is performed by recombinase-mediated cassette exchange (RMCE). The feasibility of RMCE at various sites can be evaluated by methods known in the art, e.g., viral titer measurement, capsid titer measurement, or viral production measurement. In certain embodiments, the evaluation can be performed by methods known in the art, e.g., by evaluating the titer and / or specific productivity of TI host cell cultures. Exemplary culture strategies include, but are not limited to, fed-batch shaking flask culture and fed-batch bioreactor culture. The AAV titer and specific productivity of TI host cells can be evaluated by methods known in the art, e.g., droplet digital polymerase chain reaction (ddPCR); Gyrolab® AAVX Titer, ELISA, FACS, fluorescence microvolume analysis technology (FMAT), affinity chromatography, Western blot analysis, etc.

[0065] 4. Exogenous nucleotide sequences

[0066] An exogenous nucleotide sequence is a nucleotide sequence that does not originate from a host cell but can be introduced into a host cell through traditional DNA delivery methods, such as transfection, electroporation, or transformation methods. In certain embodiments, the exogenous nucleotide sequence codes for a protein associated with AAV production. In certain embodiments, the exogenous nucleotide sequence used in the context of the present invention comprises elements such as a nucleic acid sequence encoding one or more of the adeno-associated virus (AAV) proteins REP, CAP, adenovirus (Ad) proteins E2A, Ad E4, E4orf6, E4orf6 / 7, E2DBP, or Ad VA, one or more recombination recognition sequences (RRS), and a first selection marker. In certain embodiments, a first exogenous nucleotide sequence that facilitates the introduction of additional nucleic acid sequences is referred to herein as a "landing pad." Accordingly, in certain embodiments, a TI host cell may comprise: (1) an exogenous nucleotide sequence encoding an AAV or AAV-related gene product (e.g., protein and / or RNA (e.g., Ad polypeptide and Ad VA RNA)) incorporated into a specific locus of the host cell genome via targeted integration (e.g., exogenous site-specific nuclease-mediated (e.g., CRISPR / Cas9-mediated) targeted integration); (2) an exogenous nucleotide sequence comprising one or more landing pads; and / or (3) an exogenous nucleotide sequence comprising one or more landing pads incorporated with one or more exogenous nucleotide sequences encoding an AAV or AAV-related polypeptide (e.g., Ad polypeptide).

[0067] 4.1 landing pad

[0068] In certain embodiments, the targeted integrated first exogenous nucleotide sequence comprises one or more recombination recognition sequences (RRS), said RRS may be recognized by a recombinase. In certain embodiments, the integrated first exogenous nucleotide sequence comprises at least two RRSs. In certain embodiments, the integrated first exogenous nucleotide sequence comprises two identical RRSs. In certain embodiments, the integrated first exogenous nucleotide sequence comprises two RRSs, said two RRSs being heterospecific, that is, not recognized by the same recombinase. In certain embodiments, the integrated first exogenous nucleotide sequence comprises three RRSs, said three RRSs being located between the first and second RRSs. In certain embodiments, the first and second RRSs are identical, and the third RRS is different from the first or second RRS. In certain embodiments, all three RRSs are heterospecific. In certain embodiments, the integrated first exogenous nucleotide sequence comprises four, five, six, seven, or eight RRSs. In certain embodiments, the integrated first exogenous nucleotide sequence comprises multiple RRSs. In certain embodiments, multiple two or more RRSs are identical. In certain embodiments, two or more RRSs are heterospecific. In certain embodiments, each RRS can be recognized by a distinct recombinase. In certain embodiments, some of the total RRSs are homospecific, i.e., recognized by the same recombinase, and some of the total RRSs are heterospecific, i.e., not recognized by the same recombinase.In certain embodiments, RRS or RRSs may be selected from the group consisting of LoxP sequence, LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, flippase recognition target (FRT) sequence, Bxb1 attP sequence, Bxb1 attB sequence, φC31 attP sequence and φC31 attB sequence.

[0069] In certain embodiments, a first exogenous nucleic acid sequence incorporated into the genome of a TI host cell encodes one or more of the AAV replication enzyme (REP) protein, AAV capsid (CAP) protein, adenovirus (Ad) E2A protein, Ad E4 protein, Ad E4orf6 protein, Ad E4orf6 / 7 protein, Ad E2DBP protein, or Ad VA RNA. In certain embodiments, the first exogenous nucleic acid sequence encodes the AAV REP, Ad E2A, Ad E4, and Ad VA gene products, e.g., proteins and / or RNA. In certain embodiments, the first exogenous nucleic acid sequence encodes a first selection marker adjacent to two recombination recognition sequences (RRS). In a specific embodiment, the first exogenous nucleic acid sequence encodes one or more of the adeno-associated virus (AAV) REP, AAV CAP, adenovirus (Ad) E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA gene products (e.g., protein and / or RNA), and a first selection marker adjacent to two recombination recognition sequences (RRS).

[0070] In a specific embodiment, the integrated exogenous nucleotide sequence comprises three RRSs. In a specific embodiment, the third RRS is located between the first and second RRSs. In a specific embodiment, all three RRSs are identical. In a specific embodiment, the first and second RRSs are identical, and the third RRS is different from the first or second RRS. In a specific embodiment, all three RRSs are heterospecific.

[0071] In a specific embodiment, the integrated first exogenous nucleotide sequence comprises at least one selection marker. In a specific embodiment, the integrated first exogenous nucleotide sequence comprises one RRS and at least one selection marker. In a specific embodiment, the integrated first exogenous nucleotide sequence comprises a first and a second RRS and at least one selection marker. In a specific embodiment, the selection marker is located between the first and second RRS. In a specific embodiment, two RRSs adjoin at least one selection marker, namely, the first RRS is located 5' upstream of the selection marker and the second RRS is located 3' downstream of the selection marker. In a specific embodiment, 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.

[0072] In a specific embodiment, the selection marker is located between the first and second RRS, and the two adjacent RRS are identical. In a specific embodiment, the two RRS adjacent to the first selection marker are both LoxP sequences. In a specific embodiment, the two RRS adjacent to the first selection marker are both FRT sequences. In a specific embodiment, the first selection marker is located between the first and second RRS, and the two adjacent RRS are heterospecific. In a specific embodiment, the first adjacent RRS is a LoxP L3 sequence, and the second adjacent RRS is a LoxP 2L sequence. In a specific embodiment, the LoxP L3 sequence is located at the 5' of the first selection marker, and the LoxP 2L sequence is located at the 3' of the first selection marker. In a specific embodiment, the first adjacent RRS is a wild-type FRT sequence, and the second adjacent RRS is a mutant FRT sequence. In a specific embodiment, the first side RRS is the Bxb1 attP sequence and the second side RRS is the Bxb1 attB sequence. In a specific embodiment, the first side RRS is the φC31 attP sequence and the second side RRS is the φC31 attB sequence. In a specific embodiment, the two RRSs are located in the same direction. In a specific embodiment, both RRSs are located in the forward or reverse direction. In a specific embodiment, the two RRSs are located in opposite directions.

[0073] In certain embodiments, the selection marker may be an aminoglycoside phosphotransferase (APH) (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 a gene encoding resistance to puromycin, blasticidin, bleomycin, pleomycin, chloramphenicol, zeocin, or mycophenolic acid. In certain embodiments, the selection marker may be GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet, It may be an Emerald, CyPet, mCFPm, Cerulean, or T-Sapphire marker. In certain embodiments, the selection marker may be a fusion construct comprising at least two selection markers. In certain embodiments, a gene encoding a selection marker or a fragment of a selection marker may be fused with another selection marker or a gene encoding the fragment thereof.

[0074] In certain embodiments, the integrated exogenous nucleotide sequence comprises two selection markers adjacent to two RRSs, wherein the first selection marker is different from the second selection marker. In certain embodiments, both selection markers are selected from the group consisting of a glutamine synthase selection marker, a thymidine kinase selection marker, a HYG selection marker, and a puromycin resistance selection marker. In certain embodiments, the integrated exogenous nucleotide sequence comprises a thymidine kinase selection marker and a HYG selection marker. In certain embodiments, the first selection marker is selected from the group consisting of aminoglycoside phosphotransferase (APH) (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 genes encoding resistance to puromycin, blasticidin, bleomycin, pleomycin, chloramphenicol, zeocin, and mycophenolic acid, and the second selection marker is GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, It is selected from the group consisting of YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire markers. In certain embodiments, the first selection marker is a glutamine synthase selection marker, and the second selection marker is a GFP marker. In certain embodiments, the two RRSs adjacent to the two selection markers are identical. In certain embodiments, the two RRSs adjacent to the two selection markers are different from each other.

[0075] In a specific embodiment, the selection marker is operably linked to a promoter sequence. In a specific embodiment, the selection marker is operably linked to an SV40 promoter. In a specific embodiment, the selection marker is operably linked to a Cytomegalovirus (CMV) promoter.

[0076] In a specific embodiment, the integrated exogenous nucleotide sequence comprises at least one selection marker and an IRES, wherein the IRES is operably linked to the selection marker. In a specific embodiment, the selection marker operably linked to the IRES 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 markers. In a specific embodiment, the selection marker operably linked to the IRES is the GFP marker. In a specific embodiment, the integrated exogenous nucleotide sequence comprises two selection markers adjacent to the IRES and two RRSs, wherein the IRES is operably linked to the second selection marker. In a specific embodiment, the integrated exogenous nucleotide sequence comprises three selection markers adjacent to an IRES and two RRSs, wherein the IRES is operably linked to a third selection marker. In a specific embodiment, the integrated exogenous nucleotide sequence comprises three selection markers adjacent to an IRES and two RRSs, wherein the IRES is operably linked to a third selection marker. In a specific embodiment, the third selection marker is different from the first or second selection marker. In a specific embodiment, the integrated exogenous nucleotide sequence comprises a first selection marker operably linked to a promoter and a second selection marker operably linked to an IRES. In a specific embodiment, the integrated exogenous nucleotide sequence comprises a glutamine synthase selection marker operably linked to an SV40 promoter and a GFP selection marker operably linked to an IRES.In certain embodiments, the integrated exogenous nucleotide sequence comprises a thymidine kinase selection marker and a HYG selection marker operably linked to a CMV promoter, and a GFP selection marker operably linked to an IRES.

[0077] In certain embodiments, a stable mammalian host cell comprises a second exogenous nucleic acid sequence incorporated at least once into the genome of the stable mammalian cell. In some embodiments, the integration of the second exogenous nucleic acid sequence is mediated by a transposon.

[0078] In certain embodiments, the second exogenous nucleic acid sequence comprises an ITR that adjacent to the polypeptide of interest (POI) and the gene of interest (GOI) coding sequence for the second selection marker. In certain embodiments, one or more GOIs comprise coding sequences for one or more of AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA gene products (e.g., protein and / or RNA). In certain embodiments, the mammalian cell comprises one to ten additional exogenous nucleic acids comprising one or more of AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA and an ITR that adjacent to the coding sequence for the selection marker, wherein each of the one to ten additional exogenous nucleic acids is incorporated into the genome of the mammalian cell at least once. In certain embodiments, each of the one to ten additional exogenous nucleic acids comprises a different selection marker. In certain embodiments, two or more of one to ten additional exogenous nucleic acids include the same selection marker. In certain embodiments, one or more of the additional exogenous nucleic acids may additionally include a coding sequence for the POI within the adjacent ITR.

[0079] In a specific embodiment, one or more of the one to ten additional exogenous nucleic acids further comprise a coding sequence for the gene of interest (GOI) within a lateral inversion terminal repeat.

[0080] In a specific embodiment, a stable mammalian host cell comprises one to ten additional exogenous nucleic acids, each incorporated at least once into the genome of the said stable mammalian cell. In a specific embodiment, the one to ten additional exogenous nucleic acids comprise one or more of AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA, and an inversion terminal repeat adjacent to a coding sequence for a selection marker. In a specific embodiment, the one to ten additional exogenous nucleic acids each comprise a different selection marker. In a specific embodiment, two or more of the one to ten additional exogenous nucleic acids comprise the same selection marker. In a specific embodiment, one or more of the one to ten additional exogenous nucleic acids further comprise a coding sequence for a polypeptide of interest (POI) within the adjacent inversion terminal repeat.

[0081] In a specific embodiment, the stable mammalian host cell further comprises a coding sequence for a second polypeptide of interest within a lateral inversion terminal repeat.

[0082] In certain embodiments, a stable mammalian host cell further comprises an expression cassette of a transcriptional activator (rTA) sequence, and the rTA sequence is fused to a first and / or second selection marker. In certain embodiments, the rTA is fused to the first and / or second selection marker via a 2A peptide. For example, but not limited to, the 2A peptide may be selected from P2A, T2A, E2A, and F2A. In certain embodiments, the rTA is fused to the first and / or second selection marker via a P2A peptide sequence.

[0083] In certain embodiments, the selection marker may be an aminoglycoside phosphotransferase (APH) (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 a gene encoding resistance to puromycin, blasticidin, bleomycin, pleomycin, chloramphenicol, zeocin, or mycophenolic acid. In certain embodiments, the selection marker may be GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet, It may be an Emerald, CyPet, mCFPm, Cerulean, or T-Sapphire marker. In certain embodiments, the selection marker may be a fusion construct comprising at least two selection markers. In certain embodiments, a gene encoding a selection marker or a fragment of a selection marker may be fused with another selection marker or a gene encoding the fragment thereof.

[0084] In certain embodiments, the integrated exogenous nucleotide sequence comprises two selection markers adjacent to two ITRs, wherein the first selection marker is different from the second selection marker. In certain embodiments, both selection markers are selected from the group consisting of a glutamine synthase selection marker, a thymidine kinase selection marker, a HYG selection marker, and a puromycin resistance selection marker. In certain embodiments, the integrated exogenous nucleotide sequence comprises a thymidine kinase selection marker and a HYG selection marker. In certain embodiments, the first selection marker is selected from the group consisting of aminoglycoside phosphotransferase (APH) (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 genes encoding resistance to puromycin, blasticidin, bleomycin, pleomycin, chloramphenicol, zeocin, and mycophenolic acid, and the second selection marker is GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, It is selected from the group consisting of Ypet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire markers. In certain embodiments, the first selection marker is a glutamine synthase selection marker, and the second selection marker is a GFP marker. In certain embodiments, the two ITRs adjacent to the two selection markers are identical. In certain embodiments, the two ITRs adjacent to the two selection markers are different from each other.

[0085] In a specific embodiment, the selection marker is operably linked to a promoter sequence. In a specific embodiment, the selection marker is operably linked to an SV40 promoter. In a specific embodiment, the selection marker is operably linked to a Cytomegalovirus (CMV) promoter.

[0086] In a specific embodiment, the integrated exogenous nucleotide sequence comprises at least one selection marker and an IRES, wherein the IRES is operably linked to the selection marker. In a specific embodiment, the selection marker operably linked to the IRES 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 markers. In a specific embodiment, the selection marker operably linked to the IRES is the GFP marker. In a specific embodiment, the integrated exogenous nucleotide sequence comprises two selection markers adjacent to the IRES and two ITRs, wherein the IRES is operably linked to the second selection marker. In a specific embodiment, the integrated exogenous nucleotide sequence comprises three selection markers adjacent to an IRES and two ITRs, wherein the IRES is operably linked to a third selection marker. In a specific embodiment, the integrated exogenous nucleotide sequence comprises three selection markers adjacent to an IRES and two ITRs, wherein the IRES is operably linked to a third selection marker. In a specific embodiment, the third selection marker is different from the first or second selection marker. In a specific embodiment, the integrated exogenous nucleotide sequence comprises a first selection marker operably linked to a promoter and a second selection marker operably linked to an IRES. In a specific embodiment, the integrated exogenous nucleotide sequence comprises a glutamine synthase selection marker operably linked to an SV40 promoter and a GFP selection marker operably linked to an IRES.In certain embodiments, the integrated exogenous nucleotide sequence comprises a thymidine kinase selection marker and a HYG selection marker operably linked to a CMV promoter, and a GFP selection marker operably linked to an IRES.

[0087] 5. Targeted Integration and Transposon-Mediated Genome Integration Methods

[0088] In certain embodiments, target integration (TI) may be combined with transposon-mediated genomic integration. In certain embodiments, transposon-mediated genomic integration may be performed after target integration. In certain embodiments, target integration may be performed simultaneously with transposon-mediated genomic integration. In certain embodiments, transposon-mediated genomic integration may be performed after target integration. FIGS. 1a-1b illustrate exemplary TIP and TI / P strategies for target integration and transposon-mediated integration. FIG. 2 illustrates an exemplary construct for target integration and transposon-mediated integration of one or more AAV-producing genes into the genome of a mammalian host cell.

[0089] 5.1 Target integration through recombinase-mediated recombination

[0090] A “recombination recognition sequence (RRS)” is a nucleotide sequence recognized by a recombinase that is necessary and sufficient for a recombinase-mediated recombination event. The RRS can be used to define the location in a nucleotide sequence where a recombination event occurs.

[0091] In a specific embodiment, the RRS is selected from the group consisting of LoxP sequence, LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1 attP sequence, Bxb1 attB sequence, φC31 attP sequence and φC31 attB sequence.

[0092] 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.

[0093] In a specific embodiment where the RRS is a LoxP site, the host cell requires Cre recombinase to perform recombination. In a specific embodiment where the RRS is a flippase recognition target (FRT) site, the host cell requires FLP recombinase to perform recombination. In a specific embodiment where the RRS is a Bxb1 attP or Bxb1 attB site, the host cell requires Bxb1 integrase to perform recombination. In a specific embodiment where the RRS is a φC31 attP or φC31 atB site, the host cell requires φC31 integrase to perform recombination. These recombinases can be introduced into the host cell using an expression vector containing the coding sequence of the corresponding enzyme.

[0094] The Cre-LoxP site-specific recombination system has been widely used in various biological experimental systems. Cre is a 38-kDa site-specific DNA recombinase that recognizes 34-bp LoxP sequences. Cre is derived from bacteriophage P1 and belongs to the tyrosine family of site-specific recombinases. Cre recombinase can mediate both intramolecular and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8-bp non-palindromic core region adjacent to two 13-bp inversion terminal repeats. Cre recombinase binds to the 13-bp repeats and mediates recombination within the 8-bp core region. Cre-LoxP-mediated recombination occurs with high efficiency and does not require other host factors. When two LoxP sequences are located in the same direction within the same nucleotide sequence, Cre-mediated recombination excises the DNA sequence located between the two LoxP sequences into a covalently closed circle. When two LoxP sequences are located in inversions on the same nucleotide sequence, Cre-mediated recombination reverses the orientation of the DNA sequence located between the two sequences. LoxP sequences may also be located on different chromosomes, facilitating recombination between different chromosomes. If two LoxP sequences are located on two different DNA molecules, and one of the DNA molecules is circular, Cre-mediated recombination results in the integration of the circular DNA sequence.

[0095] 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 increase the efficiency of Cre-mediated integration or substitution. In certain embodiments, the mutant LoxP sequence is selected from the group consisting of LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, and Lox66 sequence. For example, in the Lox71 sequence, 5 bp is mutated in the left 13 bp repeat. In the Lox66 sequence, 5 bp is mutated in the right 13 bp repeat. Both the wild-type and mutant LoxP sequences can mediate Cre-dependent recombination.

[0096] The FLP-FRT site-specific recombination system is similar to the Cre-Lox system. It includes a flippase (FLP) recombinase derived from a 2 μm plasmid of the yeast Saccharomyces cerevisiae. FLP also belongs to the tyrosine family of site-specific recombinases. The FRT sequence is a 34 bp sequence consisting of two 13 bp palindromic sequences adjacent to an 8 bp spacer. FLP binds to the 13 bp palindromic sequence and mediates DNA cleavage, exchange, and ligation within the 8 bp spacer. Similar to Cre recombinase, the position and orientation of the two FRT sequences determine the outcome of FLP-mediated recombination. In certain embodiments, the FRT sequence is a wild-type FRT sequence. In certain embodiments, the FRT sequence is a mutant FRT sequence. Both wild-type and mutant FRT sequences can mediate FLP-dependent recombination. In a specific embodiment, the FRT sequence is fused to a reactive receptor domain sequence, such as a tamoxifen reactive receptor domain sequence.

[0097] Bxb1 and φC31 belong to the serine recombinase family. Both are derived from bacteriophages and are utilized to facilitate site-specific integration of the phage genome into the bacterial genome by establishing lysogenicity. These integrases catalyze site-specific recombination events between short (40–60 bp) DNA substrates, known as the attP and attB sequences, which are attachment sites located on the original phage DNA and bacterial DNA, respectively. Following recombination, two new sequences, named attL and attR, are formed, each containing a half-sequence derived from attP and attB. Additionally, recombination may occur between the attL and attR sequences, allowing the integrated phage to detach from the bacterial DNA. Both integrases can catalyze recombination without the assistance of additional host factors. In the absence of cofactors, these integrases mediate unidirectional recombination between attP and attB with an efficiency exceeding 80%. Because the DNA sequences that these integrases can recognize are short and the recombination direction is unidirectional, this recombination system was developed to complement the Cre-LoxP and FRT-FLP systems, which are widely used for genetic engineering purposes.

[0098] The terms "matching RRS" and "homogeneous RRS" indicate that recombination occurs between two RRSs. In certain embodiments, the two matching RRSs are identical. In certain embodiments, both RRSs are wild-type LoxP sequences. In certain embodiments, both RRSs are mutant LoxP sequences. In certain embodiments, both RRSs are wild-type FRT sequences. In certain embodiments, both RRSs are mutant FRT sequences. In certain embodiments, the two matching RRSs have different sequences but can be recognized by the same recombinase. In certain embodiments, the first matching RRS is a Bxb1 attP sequence and the second matching RRS is a Bxb1 attB sequence. In certain embodiments, the first matching RRS is a φC31 attB sequence and the second matching RRS is a φC31 attB sequence.

[0099] In certain embodiments, the integrated exogenous nucleotide sequence comprises two RRSs, and the vector comprises two RRSs that match the two RRSs in the integrated exogenous nucleotide sequence, namely, the first RRS in the integrated exogenous nucleotide sequence matches the first RRS in the vector, and the second RRS in the integrated exogenous nucleotide sequence matches the second RRS in the vector. In certain embodiments, the first RRS in the integrated exogenous nucleotide sequence and the first RRS in the vector are identical to the second RRS in the integrated exogenous nucleotide sequence and the second RRS in the vector. A non-limiting example of this “single vector RMCE” strategy is described in FIG. 2a of PCT application PCT / US2018 / 067070 (publication number WO2019126634). In certain embodiments, the first RRS in the integrated exogenous nucleotide sequence and the first RRS in the vector are different from the second RRS in the integrated exogenous nucleotide sequence and the second RRS in the vector. In certain embodiments, the first RRS in the integrated exogenous nucleotide sequence and the first RRS in the vector are both LoxP L3 sequences, and the second RRS in the integrated exogenous nucleotide sequence and the second RRS in the vector are both LoxP 2L sequences.

[0100] In certain embodiments, a "2-vector RMCE" strategy is used. For example, but without limitation, the integrated exogenous nucleotide sequence may comprise an arrangement of three RRSs, for example, a third RRS ("RRS3") existing between a first RRS ("RRS1") and a second RRS ("RRS2"), the first vector comprises two RRSs that match the first and third RRSs in the integrated exogenous nucleotide sequence, and the second vector comprises two RRSs that match the third and second RRSs in the integrated exogenous nucleotide sequence. An example of a 2-vector RMCE strategy is illustrated in FIG. 4 of PCT application PCT / US2018 / 067070 (publication number WO2019126634). In this example, RRS1, RRS2, and RRS3 are heterospecific, for example, do not cross-react with each other. In some embodiments, one vector (front) contains RRS1 and a promoter, followed by a start codon and RRS3 (in this order). Another vector (back) contains RRS3, GOI, and RRS2 (in this order) fused to the coding sequence of a marker that lacks a start codon (ATG). To ensure in-frame translation of the fusion protein, additional nucleotides may be inserted between the RRS3 site and the selected marker sequence.

[0101] Both single-vector and two-vector RMCEs unidirectionally incorporate one or more donor DNA molecule(s) into a predetermined region of the host cell genome and enable the precise exchange of a DNA cassette present on the donor DNA with a DNA cassette on the host genome containing the integration site. The DNA cassette features at least one selection marker (however, in certain two-vector RMCE examples, a "split selection marker" may be used as described herein) and / or two heterospecific RRSs adjacent to at least one exogenous GOI. The RMCE involves a double recombination crossover event catalyzed by a recombinase between the two heterospecific RRSs and the donor DNA molecule within a target genomic locus. The RMCE is designed to introduce an AAV gene, an Ad helper gene, and / or a selection marker into a predetermined locus of the host cell genome. Unlike recombination involving only a single crossover event, RMCE can be implemented so that prokaryotic vector sequences are not introduced into the host cell genome, thereby reducing and / or preventing unwanted triggering of host immunity or defense mechanisms. The RMCE procedure can be repeated using multiple DNA cassettes.

[0102] In a specific embodiment, target integration is achieved by a single cross-recombination event, wherein one exogenous nucleotide sequence comprising one RRS adjacent to at least one exogenous gene encoding an AAV protein, an Ad helper protein, and / or at least one selection marker is integrated into a predetermined site of the host cell genome. In a specific embodiment, target integration is achieved by a single RMCE, wherein a DNA cassette comprising at least one exogenous AAV gene, an Ad helper gene, and / or at least one selection marker adjacent by two heterospecific RRSs is integrated into a predetermined site of the host cell genome. In a specific embodiment, target integration is achieved by two RMCEs, wherein two different DNA cassettes each comprising at least one exogenous AAV gene, an Ad helper gene, and / or at least one selection marker adjacent by two heterospecific RRSs are both integrated into a predetermined site of the host cell genome. In a specific embodiment, target integration is achieved by a plurality of RMCEs, wherein DNA cassettes from a plurality of vectors, each comprising at least one exogenous AAV gene, an Ad auxiliary gene, and / or at least one selection marker, which are respectively adjacent by two heterospecific RRSs, are all integrated into a predetermined site of the host cell genome. In a specific embodiment, the selection marker may be partially encoded in the first vector and partially encoded in the second vector, and the integration of the two RMCEs enables the expression of the selection marker. An example of such a system is shown in FIG. 4 of PCT application PCT / US2018 / 067070 (publication number WO2019126634).

[0103] In certain embodiments, targeted integration via recombinase-mediated recombination allows a selection marker or one or more exogenous AAV genes, an Ad auxiliary gene, and / or at least one selection marker to be incorporated into one or more predetermined integration sites of the host cell genome along with sequences from a prokaryotic vector. In certain embodiments, targeted integration via recombinase-mediated recombination allows an AAV gene, an Ad auxiliary gene, and / or a selection marker to be incorporated into one or more predetermined integration sites of the host cell genome without sequences from a prokaryotic vector.

[0104] 5.2 Target integration via homologous recombination, HDR, or NHEJ

[0105] The subject matter disclosed herein also relates to target integration mediated by homologous recombination, or by an exogenous site-specific nuclease followed by an HDR or NHEJ.

[0106] Homologous recombination is the recombination between DNA molecules that share extensive sequence homology. It can be utilized to induce error-free repair of double-stranded DNA breaks and to generate sequence variations in germ cells during meiosis. Since homologous recombination is a process in which genetic information is exchanged between two homologous DNA molecules, it does not alter the entire gene sequence on the chromosome. During homologous recombination, a nick or break is formed in the double-stranded DNA (dsDNA), followed by the single-stranded DNA ends intruding into the homologous dsDNA molecule to pair homologous sequences. After the formation of a Holiday junction through branching, the Holiday junction is finally degraded.

[0107] Double-strand breaks (DSBs) are the most severe form of DNA damage, and repairing such damage is essential for maintaining genomic integrity in all organisms. There are two main repair pathways for repairing DSBs. The first repair pathway is the Homology-Induced Repair (HDR) pathway, where homologous recombination is the most common form of HDR. Since HDR requires the presence of homologous DNA within the cell, this repair pathway is typically activated during the S and G2 phases of the cell cycle, during which newly replicated sister chromatids are utilized as homologous templates. HDR is also the primary repair pathway for repairing replication forks that have collapsed during DNA replication. HDR is considered a relatively error-free repair pathway. The second repair pathway for DSBs is Non-Homologous End Junction (NHEJ). NHEJ is a repair pathway that ligates both ends of damaged DNA without the need for a homologous DNA template.

[0108] Target integration can be promoted through exogenous site-specific nucleases followed by HDR. This is because the frequency of homologous recombination can be increased by introducing DSBs into specific target genomic sites. In certain embodiments, the exogenous nuclease may be selected from the group consisting of zinc finger nucleases (ZFN), ZFN dimers, transcription activator-like effector nucleases (TALEN), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly spaced short palindromic repeat (CRISPR) associated (Cas) endonucleases.

[0109] CRISPR / Cas and TALEN systems are two genome editing tools that offer the highest ease of construction and high efficiency. CRISPR / Cas has been identified as a bacterial immune defense mechanism against bacteriophage invasion. Cas is a nuclease that, guided by synthetic guide RNA (gRNA), binds to specific nucleotide sequences within the cell and can edit DNA within or around those sequences by inducing one or more of, for example, single-strand breaks, DSBs, and / or point mutations. TALEN is a sophisticatedly designed site-specific nuclease composed of the DNA binding domain of a TALE (transcription activator-like effector) and the catalytic domain of the restriction endonuclease FokI. By altering amino acids present in a highly variable residue region of the DNA binding domain monomer, various artificial TALENs targeting diverse nucleotide sequences can be created. Subsequently, the DNA binding domain orients the nuclease toward the target sequence to generate DSBs.

[0110] Target integration via homologous recombination or HDR presupposes the presence of a homologous sequence at the integration site. In certain embodiments, the homologous sequence is present on the vector. In certain embodiments, the homologous sequence is present on the polynucleotide.

[0111] In certain embodiments, homologous recombination is performed without any cofactors. In certain embodiments, homologous recombination is facilitated by the presence of an integrable vector. For example, in the context of the present invention, the integrable vector is an adeno-associated virus vector.

[0112] 5.3 Transposon-mediated genome integration

[0113] The subject matter disclosed herein relates to TI host cells, said host cells also being subjects of transposon-mediated genome integration of one or more exogenous nucleic acids, and the subject matter disclosed herein also relates to a method of producing and using said combined transposon-mediated genome integration and TI host cells. As set forth in this specification, in certain embodiments, target integration may be performed simultaneously with transposon-mediated genome integration. In certain embodiments, transposon-mediated genome integration may be performed after target integration. In certain embodiments, transposon-mediated genome integration may be performed before target integration.

[0114] Transposons are useful in connection with the methods described herein. Exemplary transposons that may be used include, but are not limited to, piggyBac, Sleeping Beauty, Tol2, and variations thereof;

[0115] (i) Natural insect-derived piggyBac transposable factors (Wilson et. Al., Molecular Therapy, 15(1):139-145 (2007)) can be transposable into the genome by "cut and paste" to target integration sites (e.g., TTAA) via transposases. Studies using human cells have revealed that piggyBac-mediated transposability is efficient and possesses several advantages, including non-random integration site selectivity, precise excision (i.e., no "footprint" mutations in donor plasmids and genomic DNA), high integration frequency, and the absence of overproduction inhibition. In addition to these advantages, the ability to deliver large transposable factors ranging from ~9.1 kb to ~14.3 kb without a significant reduction in efficiency makes it one of the transposons of choice for gene therapy.

[0116] (ii) Engineered Tc1 / Mariner transposon Sleeping Beauty (Ivics et al., Curr. Issues Mol. Biol. 2004, 6 (1), 43-56; Ivics et al. Cell 91:501-510 (1997)) enables the transfer of gene cargo (2kb to 10kb) at target integration sites (e.g., TA dinucleotide; random). Unlike piggyBac transposons, gene transfer by sleeping beauty transposons is characterized by a loss of transfer efficiency in larger transplant genes, predictable "footprint" mutations in donor plasmids and genomic DNA, and sensitivity to suppression of overproduction.

[0117] Tol2 (Balciunas et al., PloS Genent. Nov 10;2(11):e169 (2006)), a natural medaka hAT gene lineage transposer, and its variant miniTol2 are highly likely to be utilized for mammalian gene transfer. Similar to piggyBac, Tol2 offers several advantages, such as the absence of overproduction inhibition and the ability to carry large volumes (2kb to 10kb). Similar to Sleeping Beauty, Tol2 demonstrates random integration capabilities.

[0118] Generally, the transposon useful in the method of the present invention is transposed via a non-replicative 'cut and paste' mechanism. For example, without being bound by theory, the transposition catalyzed by the transposon useful in the method described herein may proceed by a DNA transposase recognizing two inverted terminal repeats (ITRs), cleaving the target, and consequently releasing the DNA transposon from the donor sequence (e.g., donor plasmid). Upon excision, the transposon may be cleaved at the corresponding sequence within the genome by the same transposase and incorporated into the host cell genome.

[0119] 5.4 Regulated expression of proteins involved in AAV production

[0120] Protein expression levels are often suboptimal because the expression of the encoded protein is difficult. Low expression levels of difficult-to-express proteins can be varied and difficult to identify the cause of. One possibility is that the expressed protein is toxic to the host cell. In such cases, a regulated expression system can be used to express the toxic protein, whereby the sequence of interest encoding the protein is under the control of an inducible promoter. In these systems, the expression of the difficult-to-express protein is promoted only when a small molecule, such as a regulator, for example tetracycline or its analog, doxycycline (DOX), is added to the culture. By regulating the expression of the toxic protein (e.g., Rep), the toxic effects are mitigated, and the culture is enabled to achieve desired cell growth prior to production. In certain embodiments, the regulated expression system comprises at least one protein transcribed under an operablely linked regulated promoter. In certain embodiments, the regulated expression system can be used to identify the underlying cause of low AAV production or low protein expression of a difficult-to-express polypeptide of interest (POI). In certain embodiments, the ability to selectively block protein expression in a regulated expression system can be used to elucidate the association between protein expression and observed side effects.

[0121] In certain embodiments, a regulatory expression system may be used to minimize transcriptional and cell line variability effects during the analysis of the root cause of molecules that are difficult to express. For example, but without limitation, combined transposon-mediated genome integration and protein expression in TI hosts may be induced by adding a regulator (inducer), such as doxycycline, to the culture. In certain embodiments, a regulatory expression vector enables the regulatory expression of a protein by utilizing a tetracycline regulatory promoter to express the protein.

[0122] In certain embodiments, the regulatory expression system described in the present invention can be used to successfully identify the underlying cause(s) of low protein expression compared to a control cell line. In certain embodiments, if the relative expression of a protein is found to be low in the regulatory expression cell line, the levels of intracellular accumulation and secretion of the corresponding protein can be evaluated by leveraging protein translation inhibitor treatment, e.g., Dox and cycloheximide.

[0123] As described in detail herein, regulated expression may be based on a gene switch for blocking or activating mRNA synthesis by regulatoryly coupling a transcription repressor or activator to a constitutive or minimal promoter. In certain non-limiting embodiments, repression may be achieved by binding to a repressor protein (e.g., where the protein sterically blocks transcription initiation) or by actively repressing transcription through a transcription silencer. In certain non-limiting embodiments, activation of a mammalian or viral minimal promoter without an enhancer may be achieved by regulatoryly coupling to an activating domain.

[0124] In certain embodiments, conditional coupling of a transcription repressor or activator may be achieved by using an allosteric protein that binds to a promoter in response to an external stimulus. In certain embodiments, conditional coupling of a transcription repressor or activator may be achieved by using an intracellular receptor that is released from a sequestering protein and can bind to a target promoter. In certain embodiments, conditional coupling of a transcription repressor or activator may be achieved by using a chemically induced dimerizing agent.

[0125] In certain embodiments, the allosteric protein used in the regulatory expression system of the present invention may be a protein that regulates transcriptional activity in response to culture parameters such as antibiotics, bacterial quorum sensing messengers, degradation metabolites, or temperature (e.g., low or high temperature). In certain embodiments, such a regulatory expression system may be based on a degradation metabolite when a bacterial repressor that regulates catabolic genes for alternative carbon sources is delivered to mammalian cells, for example. In certain embodiments, repression of a target promoter may be achieved by the co-mate-reactive binding of the repressor CymR. In certain embodiments, the degradation metabolite-based system may rely on the activation of a chimeric promoter by the 6-hydroxynicotine-reactive binding of the prokaryotic repressor HdnoR fused to the herpes simplex VP16 transcriptional activation domain.

[0126] In certain embodiments, the regulatory expression system of the present invention may utilize a prokaryotic-derived quorum sensing-based expression system that manages within-population and inter-population communication through a quorum sensing molecule. Such a quorum sensing molecule binds to a receptor on a target cell and modulates the receptor's affinity for a cognate promoter to induce the initiation of a specific regulon switch. In certain embodiments, the quorum sensing molecule may be an N-(3-oxo-octanoyl)-homoserine lactone, and in its presence, the TraR-p65 fusion protein activates expression from a minimal promoter fused to a TraR-specific operator sequence. In certain embodiments, the quorum sensing molecule may be butyrolactone SCB1 (racemi 2-(1'-hydroxy-6-methylheptyl)-3-(hydroxymethyl)-butanolide) in a system based on a Streptomyces coelicolor A3(2) ScbR repressor that binds to the homologous operator OscbR in the absence of SCB1. In certain embodiments, the quorum sensing molecule may be a homoserine-derived inducer used in an RTI system in which Pseudomonas aeruginosa quorum sensing repressors RhlR and LasR are fused to the SV40 T antigen nuclear localization sequence and the herpes simplex VP16 domain to activate a promoter containing a specific operator sequence (las box).

[0127] In certain embodiments, the inducing molecule that regulates an allosteric protein used in the regulatory expression system of the present invention may be, but is not limited to, cumate, isopropyl-β-D-thiogalactopyranoside (IPTG), macrolide, 6-hydroxynicotine, doxycycline, streptogramin, NADH, and tetracycline.

[0128] In certain embodiments, the intracellular receptor used in the regulatory expression system of the present invention may be a cytoplasmic or nuclear receptor. In certain embodiments, the regulatory expression system of the present invention may utilize a method of releasing transcription factors from a sequestering and repressor protein using small molecules. In certain embodiments, the regulatory expression system of the present invention may rely on steroid regulation, wherein the hormone receptor is released from HSP90 in the cytoplasm, translocates to the nucleus, and fused to a natural or artificial transcription factor capable of activating a selected promoter. In certain embodiments, a mutant receptor regulated by a synthetic steroid analog may be used to avoid interference by endogenous steroid hormones. In certain embodiments, the receptor may be an estrogen receptor variant responsive to 4-hydroxytamoxifen or a progesterone receptor variant that can be induced by RU486. In certain embodiments, a nuclear receptor-derived rosiglitazone-responsive transcription switch based on human nuclear peroxisome proliferator-activated receptor γ (PPARγ) may be used in the regulatory expression system of the present invention. In certain embodiments, the variant of the steroid-responsive receptor may be a RheoSwitch based on a modified Choristoneura fumiferana ecdysone receptor and a mouse retinoid X receptor (RXR) fused with a Gal4 DNA binding domain and a VP16 transcription activator. Where synthetic ecdysone is present, the RheoSwitch variant may bind to and activate a minimal promoter fused with several repeats of the Gal4 responsive element.

[0129] In certain embodiments, the regulatory expression system disclosed herein may utilize the chemically induced dimerization of a DNA-binding protein and a transcription activator for the activation of a minimal core promoter fused with a homologous operator. In certain embodiments, the regulatory expression system disclosed herein may utilize the rapamycin-regulated dimerization of FRB and FKBP. In this system, FRB is fused to a p65 transcription activator, and FKBP is fused to a zinc finger domain specific to a homologous operator site located upstream of a engineered minimal interleukin-12 promoter. In certain embodiments, FKBP may be mutated. In certain embodiments, the regulatory expression system disclosed herein may utilize a bacterial gyrase B subunit (GyrB), wherein GyrB is dimerized in the presence of the antibiotic coumermycin and dissociated into novobiosin.

[0130] In certain embodiments, the regulated expression system of the present invention may be used for regulated siRNA expression. In certain embodiments, the regulated siRNA expression system may be a tetracycline, a macrolide, or an OFF- and ON-type QuoRex system. In certain embodiments, the RTI system may utilize a Xenopus terminal oligopyrimidine element (TOP) that blocks translation initiation by forming a hairpin structure in the 5' non-translation region.

[0131] In certain embodiments, the regulatory expression system described in the present invention may utilize gaseous regulatory expression, for example, an acetaldehyde-induced regulation (AIR) system. The AIR system may utilize the Aspergillus nidulans AlcR transcription factor, which specifically activates a PAIR promoter assembled from an AlcR-specific operator fused to a minimal human cytomegalovirus promoter in the presence of non-toxic concentrations of gaseous or liquid acetaldehyde.

[0132] In certain embodiments, the regulated expression system of the present invention may utilize a Tet-On or Tet-Off system. In such a system, the expression of one or more GOIs may be regulated by tetracycline or doxycycline, which is an analog thereof.

[0133] In certain embodiments, the regulated expression system of the present invention may utilize a PIP-on or PIP-off system. In such a system, the expression of a protein may be regulated, for example, by pristinamycin, tetracycline and / or erythromycin.

[0134] 6. Method for producing recombinant AAV

[0135] In a specific embodiment of the present invention, a method for producing recombinant AAV (rAAV) is provided, the method comprising the following steps: (A) providing a stable mammalian host cell comprising a first exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian cell, wherein the first exogenous nucleotide sequence comprises two recombinant recognition sequences (RRS) adjacent to a first selection marker; (B) introducing a second exogenous nucleic acid sequence into the stable mammalian host cell provided in (A), wherein the second exogenous nucleic acid sequence comprises two RRSs adjacent to a sequence encoding one or more of AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA gene products (e.g., protein and / or RNA) and a second selection marker; (C) a step of introducing a recombinant enzyme or a nucleic acid encoding the recombinant enzyme, wherein the recombinant enzyme recognizes RRS; (D) a step of introducing, simultaneously with (B) and (C) or sequentially after (B) and (C), one or more of the gene products (e.g., proteins and / or RNA) of AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, Ad VA or a third exogenous nucleic acid sequence encoding a polypeptide of interest and a third selection marker into the genome of a stable mammalian cell through transposon-mediated genomic integration; a step of selecting a stable mammalian host cell that stably expresses the second and third selection markers; and a step of culturing the selected stable mammalian host cell under conditions sufficient to produce recombinant AAV.

[0136] In a specific embodiment, the method of the present invention comprises the following steps: A) providing a stable mammalian host cell comprising a first exogenous nucleotide sequence incorporated at a target locus of the genome of a mammalian host cell, wherein the first exogenous nucleotide sequence comprises first and second RRSs adjacent to at least one first selection marker and a third RRS located between the first and second RRSs, and all RRSs are heterospecific; B) introducing into the cell provided in a) a first vector comprising two RRSs adjacent to a sequence encoding one or more of AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA proteins and a selection marker; c) introducing into the cells provided in a) a second vector comprising two RRSs that are matched with the second and third RRSs in at least one integrated exogenous nucleotide sequence and adjacent to a sequence encoding one or more of AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA proteins and a selection marker; d) introducing two or more recombinases or one or more nucleic acids encoding two or more recombinases, wherein the two or more recombinases recognize the RRSs; and E) a step of introducing additional exogenous nucleic acid sequences encoding AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2-DNA binding protein (E2DBP), Ad VA gene products (e.g., protein and / or RNA) or polypeptides of interest and selection markers into the genome of a stable mammalian host cell via transposon-mediated genomic integration, simultaneously with B), C), and D);F) a step of selecting mammalian host cells that stably express one or more of the selection markers; and G) a step of culturing the selected mammalian host cells under conditions sufficient to produce recombinant AAV. In certain embodiments, this method may further include a step of recovering the AAV of interest from the cell culture.;

[0137] In a specific embodiment, a stable mammalian host cell selected to express a third selection marker comprises 1 to 10 copies of a transposon-mediated genome-integrated third exogenous nucleic acid sequence.

[0138] In a specific embodiment, the nucleic acid sequence in step (D) further comprises a coding sequence encoding the second polypeptide of interest.

[0139] In certain embodiments, the first, second, and third exogenous nucleotide sequences further comprise an expression cassette of a transcriptional activator (rTA) sequence, and the rTA sequence is fused to one or more of the first, second, and third selection markers. In certain embodiments, the rTA is fused to the first and / or second selection markers via a 2A peptide. For example, but not limited to, the 2A peptide may be selected from P2A, T2A, E2A, and F2A. In certain embodiments, the rTA is fused to the first and / or second selection markers via a P2A peptide sequence.

[0140] In a specific embodiment, the cell culture of a selected stable mammalian host cell is a suspension culture. In a specific embodiment, the cell culture of a selected stable mammalian host cell is a two-dimensional adhesion culture. In a specific embodiment, the cell culture of a selected stable mammalian host cell is a three-dimensional matrix culture. In a specific embodiment, the cell culture is a serum-free culture.

[0141] In certain embodiments, the target integration of the first exogenous nucleotide sequence is promoted by the exogenous nuclease disclosed in Section 5.2 of the present invention.

[0142] In certain embodiments, the expression of one or more proteins is controlled by a regulated promoter. For example, but not limited to, the regulated promoter is an SV40 and / or CMV promoter.

[0143] In certain embodiments, a plurality of proteins encoded by the first and / or second exogenous nucleic acid are inductively expressed. In certain embodiments, the expression of one or more proteins encoded by the first exogenous nucleotide sequence and / or the second exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian cell is inductively expressed.

[0144] In certain embodiments, the expression of one or more proteins encoded by the transposon-mediated genome-integrated third exogenous nucleic acid is inductively expressed. In certain embodiments, the expression of one or more proteins encoded by the transposon-mediated genome-integrated third exogenous nucleic acid is constitutively expressed.

[0145] In a specific embodiment, one or more proteins encoded by a first and / or second exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian cell are inductively expressed, and one or more proteins encoded by a third exogenous nucleic acid incorporated into a transposon-mediated genome are constitutively expressed. In a specific embodiment, one or more proteins encoded by a first and / or second exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian cell are inductively expressed, and one or more proteins encoded by a third exogenous nucleic acid incorporated into a transposon-mediated genome are inductively expressed. In a specific embodiment, one or more proteins encoded by a first and / or second exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian cell are constitutively expressed, and one or more proteins encoded by a third exogenous nucleic acid incorporated into a transposon-mediated genome are constitutively expressed. In certain embodiments, one or more proteins encoded by a first and / or second exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian cell are constitutively expressed, and one or more proteins encoded by a third exogenous nucleic acid incorporated into a transposon-mediated genome are inductively expressed.

[0146] In certain embodiments, the protein that is inductively expressed is induced by the same inducer. In certain embodiments, the protein that is inductively expressed is induced by two or more different inducers. Non-limiting examples of such inducers include doxycycline, tetracycline, cumate, isopropyl-β-D-thiogalactopyranoside (IPTG), macrolide, 6-hydroxynicotine, streptogramin, and NADH, or combinations thereof.

[0147] In certain embodiments, the protein may be inductively expressed using a regulatory expression system. Non-limiting examples of such inductive promoters for regulatory protein expression include Tet-On and Tet-Off systems. In such systems, the expression of one or more GOIs may be regulated by tetracycline or its analogue, doxycycline.

[0148] This specification also provides a high-throughput screening method for reducing the production time of TI / P producer cells of the present invention. In certain embodiments, such a high-throughput screening method comprises the following steps:

[0149] (A) A step of obtaining a first plurality of TI pre-packaged clones through single-cell cloning by incorporating exogenous nucleotide sequences encoding Rep and Helper genes into a TI host cell;

[0150] (B) A step of transfecting rAAV encoding the gene of interest (GOI) into the first plurality of clones;

[0151] (C) Screening the transduced clones for viral genome replication to obtain a second plurality of TI pre-packaging clones that exhibit desirable viral genome replication efficiency upon induction of Rep and Helper genes;

[0152] (D) a step of obtaining multiple producer cell clones by performing transient transfection in a second plurality of clones using an exogenous nucleotide sequence encoding the Cap gene and GOI; and

[0153] (E) A step of screening multiple producer cell clones for rAAV production upon induction of Cap, Rep and Helper genes.

[0154] In certain embodiments, one or more of the Rep, Helper, and Cap proteins used in replication analysis and / or transient transfection analysis are induced. In certain embodiments, the protein(s) that are induced are induced by the same inducer. In certain embodiments, the protein(s) that are induced are induced by different inducers. Non-limiting examples of inducers used in connection with replication and / or transient transfection analysis described herein include, but are not limited to, doxycycline, tetracycline, cumate, isopropyl-β-D-thiogalactopyranoside (IPTG), macrolide, 6-hydroxynicotine, streptogramin, and NADH, or combinations of two or more of these.

[0155] In certain embodiments, the inductively expressed protein(s) may be inductively expressed using a regulatory expression system. For example, but without limitation, one or more of the Rep, Cap and Helper genes may be induced by tetracycline.

[0156] In certain embodiments, screening of the transfected clones for viral genome replication or rAAV production is performed using PCR. In certain embodiments, this screening is performed using droplet digital PCR (ddPCR).

[0157] 7. Examples

[0158] Comparison of cell pools and single colony clones produced through sequential and simultaneous integration of AAV genes

[0159] In this study, we compare AAV titers obtained from stable mammalian host cell pools and isolated single-cell clones (SCCs) integrated in sequential (TI / P, Fig. 1a) and simultaneous transfection (TIP, Fig. 1b) ways using the targeted integration (TI) plasmid and / or piggyBac plasmid.

[0160] 7.1 Melted landing cassette

[0161] A knock-in landing cassette encoding the GFP gene and a hygromycin-thymidine kinase (HYTK) selection marker was constructed, flanked by two LoxP sites (L3 and 2L), an additional LoxP site (Loxfas), and a frt site (frt3) (Fig. 2). The GFP expression cassette is flanked by AAVS1-LA (left homologous arm) and AAVS1-RA (right homologous arm), which are the homologous arms of the AAVS1 site for targeting in HEK293 cells. To distinguish random integration, the GFP expression cassette is placed downstream of the right homologous arm.

[0162] Guide RNA sequences were designed using CRISPR Guide RNA Design software (Benchling, CA) and produced by Integrated DNA Technologies (IDT, Coralville, IA, USA). The selected gRNAs formed a complex with Hifi Crispr-cas9 nuclease for 15 minutes at room temperature. RNPs and melted landing cassettes were transfected into 10 million HEK293 cells using the Neon™ Transfection System and the Neon™ Transfection System 100 μL Kit (Thermo Fisher Scientific, Waltham, MA, USA). Transfected cells were selected at 100 μg / mL hygromycin. Once the transfected pools were recovered, genomic DNA was isolated, and genomic PCR was performed to verify that the landing cassettes had melted at the correct locations. The KI pools were subjected to SCC using the restriction dilution method at a target density of 0.6 cells per well on 384-well plates. The plates were incubated for 3 weeks under conditions of 37°C, 8% CO2, and 80% humidity. Single-cell clones were selected and expanded for characterization. HEK293 TI hosts were verified based on 1) whether the landing cassette was correctly integrated and 2) whether the TI host could perform 2-plasmid RMCE and 3-plasmid RMCE as previously described (see Fig. 2 for rAAV titer comparison with wild-type (WT) HEK293).

[0163] 7.2 Case Study I - Production of packaging cell lines

[0164] Expression Plasmid:The front TI plasmid (Fig. 4a) consists of a Tet inducible promoter that induces REP gene expression followed by a transcription activator (rTA) expression cassette. Downstream of this is the SV40 promoter. All of the aforementioned elements are lateralized by two Cre recombinase recognition sites (L3, Loxfas). The back TI plasmid (Fig. B) consists of the ORF of the puromycin-N-acetyltransferase (PAC) gene followed by a Tet inducible promoter that induces auxiliary gene expression. All of the aforementioned elements are lateralized by two Cre recombinase recognition sites (Loxfas, 2L).

[0165] The PB plasmid (Fig. 4c) consists of a Tet inducible promoter that induces CAP gene expression and a GOI GFP expression cassette followed by two inversion terminal repeats (ITRs). Downstream of this are selection markers (SMs), such as a blasticidin resistance marker (BSD), a zeocin resistance marker (ZEO), or a hygromycin resistance marker (HYGRO). All of the aforementioned elements are connected within the plasmid by two ITR sites (different from the pITRs that connect the GFP gene).

[0166] Transfection HEK293 TI host cells were seeded at a concentration of 4E+5 cells / mL 2 days prior to transfection. Two different transfection schedules were tested: a strategy of simultaneously transfecting HEK293 TI hosts with TI and PB plasmids (TIP strategy); and a strategy of transfecting HEK293 TI host cells with the TI plasmid, then recovering the TI RMCE pool and transfecting with the PB plasmid (TI / P strategy).

[0167] For TIP transfection, equimolar amounts of TI front and back plasmids and Piggybac plasmid #1 were used along with Cre recombinase and transposase expression plasmids. 30E6 cells were used for each transfection, which was performed using MaxCyte electroporation. 48 hours after transfection, the pool was transferred to selective medium.

[0168] For TI / P transfection, equal molar amounts of TI front and back plasmids were used along with the Cre recombinase plasmid. 30e6 cells were used for each transfection, performed using MaxCyte electroporation. 48 hours after transfection, the pool was transferred to selective medium. Once the RMCE pool was recovered, PiggyBac plasmid #1 and transposase plasmid were transfected into the RMCE pool cells using MaxCyte electroporation. 48 hours after transfection, the pool was transferred to selective medium.

[0169] Production and Analysis Viral genome (VG) and viral particle (VP) productivity of the transfected pool were measured using a 3-day fed-batch shake flask production assay. Cells were seeded at 2E6 cells / ml in commercial production medium in shake flasks on Day 0. Viral genetic components were induced by adding doxycycline on Day 0. Batch feed was added on Day 1. Cells were shaken at 150 rpm, 37°C, and 8% CO2 throughout the 3-day period. Cell pellet samples were harvested and processed starting on Day 3. VG was measured by GFP-specific droplet PCR, and VP was measured using the Gyrolab AAVx titer kit. Table 1 summarizes the data obtained from Case Study I.

[0170] Table 1 Quantification of TI / P and TIP pools and clones

[0171] parameters TI / P cell pool TI / P cell clone - Clone A Clone B potency 1 (10E8 VG / mL) 3.5 10.3 8.2 Total capsid 2 (10E8 VP / mL) 12 87 27.6 Total / Blank Ratio (%) 26 11.7 29.5 parameters TIP Cell Pool TIP cell clone - Clone C Clone D Clone E potency 1 (10E8 VG / mL) 0.4x 4.7 1.7 4.5 Total capsid 2 (10E8 VP / mL) 31 664 79.4 176 Total / Blank Ratio (%) 1.3 0.7 2.2 2.5 1 ddPCR (Droplet Digital Polymerase Chain Reaction); 2 Gyrolab® AAVX titer

[0172] 7.3 Case Study II

[0173] expression plasmid The front and back TI plasmids are as described in Case Study I (Figs. 4a-4b). PiggyBac plasmid #1 is as described in Case Study I (Fig. 4c). PiggyBac plasmid #2 (Fig. 4d) consists of a Tet-inducible promoter that induces REP gene expression followed by a PAC gene. All of the aforementioned elements are lateralized by two pITR sites within the plasmid. PiggyBac plasmid #3 (Fig. 4e) consists of a Tet-inducible promoter that induces auxiliary gene expression followed by a zeosin resistance gene (ZEO). All of the aforementioned elements are lateralized by two pITR sites within the plasmid.

[0174] Transfection For the HEK293 packaging cell line, HEK293 TI host cells were seeded at 4E+5 cells / mL 2 days prior to transfection. Equal molar TI front and back plasmids were used in conjunction with the Cre recombinase plasmid. 30E+6 cells were used for each transfection, which was performed by electroporation using MaxCyte. The pool was transferred to selective medium 48 hours after transfection. Once the RMCE pool was recovered, equal molar PiggyBac plasmids #1, #2, and #3 were transfected into the RMCE pool cells by electroporation using MaxCyte in conjunction with the transposase plasmid. The pool was transferred to selective medium 48 hours after transfection. For the H10 packaging cell line, H10 TI host cells were seeded at 4E+5 cells / mL 2 days prior to transfection. On the day of transfection, 30E+6 H10 cells were transfected with 12.5 μg of TI front plasmid, 12.5 μg of TI back plasmid, and 5 μg of Cre recombinase plasmid, and complexed with 60 μL of PEIpro. 48 hours after transfection, the pool was transferred to selective medium.

[0175] Selection: The TI pool was initially selected at 6E+5 cells / mL using 1.5 μg / mL of puromycin and 0.5 μM of 1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil (FIAU). The TI packaging pool was recovered when the viability was >90%.

[0176] Production and Analysis The production and analysis procedures are as described in Case Study I.

[0177] Table 2 Comparison of TI / P Pool and PiggyBac Standalone Pool

[0178] method Titer (ddPCR) 10E8 VG / mL Total / Blank Ratio (%) Case I TI / P 109 2.8 PiggyBac Solo 56 0.6 Increase in multiples 1.9x 4.6x Case II TI / P 36 ~100 PiggyBac Solo 28 53.7 Increase in multiples 1.3x 1.8x

[0179] 7.4 Case Study III - Generation of TI / P Producer Cell Lines

[0180] expression plasmidFigures 5a-5c show exemplary combinations of PiggyBac (PB) plasmids encoding CAP and GOI, which can be transfected into TI-packaged hosts to produce producer cell lines. The PiggyBac (PB) transposon donor plasmid consists of a Tet-inducible promoter that induces CAP gene expression and a GFP expression cassette followed by two AAV inversion terminal repeats (ITRs). Downstream of the Tet-inducible CAP gene is a GOI expression cassette followed by two AAV inversion terminal repeats (ITRs) (Figure 5a). Downstream of the ITR is an EF1a promoter that induces the expression of a transcription activator (rTA) and a selection marker (SM), e.g., a blasticidin resistance marker (BSD), a zeocin resistance marker (ZEO), or a hygromycin resistance marker (HYGRO). In PB plasmids, selection markers can be fused to the expression cassette of transcriptional activator (rTA) elements via sequences that selectively encode 2A peptide sequences (e.g., P2A, T2A, E2A, F2A). Some PB transposon donor plasmids additionally contain a second copy of a GOI sequence (e.g., GFP) that is lateralized by two AAV ITRs upstream of the Tet-induced CAP gene (Figs. 5b, 5c). All of the elements mentioned above are lateralized by two PB ITR sites (different from those lateralizing the GOI).

[0181] Transfection: For TI / P transfection, TI pre-packaged cells were seeded at 2E+6 cells / ml 1-3 hours prior to transfection. 46E+6 cells were transfected with PB transposase plasmid (3 μg) or PB transposase mRNA (13 μg) and PB transposon donor plasmid (27 μg) using polyethyleneimine (PEI).

[0182] SelectionTwo days after transfection, the transfected cell pool was transferred to selective medium. The inclusion of blasticidin, zeocin, and hygromycin in the selective medium was based on the combination of PB transposon donor plasmids used for transfection. The TI / P producer pool was recovered when the viability was >90%.

[0183] Production and Analysis: The productivity of the transfected pool was measured using a 5-day fed-batch shake flask production assay. Cells were seeded at 20E+6 cells / ml (17 ml) in a 125 ml shake flask on Day 0. Dox was added to activate viral gene expression, and batch feed was added on Days 1 and 4. Cells were shaken throughout the 5-day period at 150 rpm, 37°C, and 8% CO2. Starting on Day 5, cell samples were harvested and lysed, after which titers were measured using GFP-specific droplet PCR and a capsid titer kit (Gyrolab AAVx titer kit). A ViCell xR cell counter was used at all steps.

[0184] result

[0185] Table 3 silver We summarize the TI / P pool titer results obtained from TI-packaged cells transfected with a PiggyBac plasmid containing Cap and GOI sequences.

[0186] Table 3 Effect of selection markers on TI / P titer and Cap / GOI copy number

[0187] BSD screening + - - + + - + ZEO screening - + - + - + + HYGRO Selection - - + - + + + Sample ID → Analysis ↓ P1b P2z P3h P1b2z P1b3h P2z3h P1b2z3h ddPCR titer (vg / ml) 1.84E+10 3.20E+10 3.80E+10 5.60E+10 1.30E+11 1.30E+11 1.20E+11 GOI copies per cell 3.8 2.5 1.8 7.5 9.1 7 11.1 Cap copy number per cell 3.8 1.2 1.1 4.5 5.3 5 9.2

[0188] 7.5 High-throughput process for TI / P producer cell production

[0189] 7.5.1 Rapid screening

[0190] A rapid screening method was developed to significantly reduce the production of TI pre-packaged host cells and TI / P producer cells (Fig. 6a). Briefly, HEK293 TI host cells were seeded at 4E+5 cells / mL 2 days prior to transfection and transfected with a plasmid containing Rep and Helper sequences under the control of the Tet promoter. After transfection and RMCE pool retrieval, clones generated via single-cell cloning were prepared for primary round screening ("cloning analysis") at a rate of 1.5–3 x 10⁶ 6 The replication ability of each clone was evaluated by seeding and culturing them on multi-well culture plates at a density of 10 cells / mL. As shown in Fig. 6b, the clones in each well were transfected with rAAV (MOI = 100) containing the gene of interest, and viral genome replication after induction of the Rep and Helper genes was measured by droplet digital PCR (ddPCR). As shown in Fig. 6a, the clones selected based on the first round screening underwent a second round screening ("transient transfection analysis"). As shown in Fig. 6e, the clones selected based on the replication analysis were transiently transfected with a vector containing Cap and GOI sequences, and the produced rAAV particles were measured by ddPCR after induction of the Rep, Helper, and Cap genes.

[0191] result

[0192] Replication analysis

[0193] Figures 6c and 6d show a comparison of replication analysis results with stable TI / P producer pool data. Replication analysis allows for the evaluation of the replication capability of TI test clones in about 4 days. 190 clones were screened using replication analysis with both VG / mL and VG / cell as selection criteria. 36 clones identified through screening proceeded to a second round of screening (Figs. 6e-6f).

[0194] Analysis of transient transfection

[0195] result: Figures 6e and 6f show the results of a second round screening using a transfection assay that can be completed in approximately 4 days. Of the 36 identified clones, 80% demonstrated superior performance compared to the stable TI pre-packaging pool. The candidate clones identified in the cloning assay also demonstrated superior performance in the transfection assay, showing at least a threefold increase in VG titers. In conclusion, this high-throughput assay, completed in approximately 8 days, offers significant advantages over the existing method, which takes 4–5 weeks to complete.

[0196] 7.5.2 TI / P Producer Pool Production

[0197] Clones that showed high performance in replication and transient transfection analyses were tested for the insertion of Cap / GOI elements using PiggyBac transposase (Fig. 7). The productivity of the TI / P clone pool generated from each TI host was evaluated for various dual- and triple-selection marker combinations using protein expression, integrated copy number, and rAAV titer as readouts (Fig. 8).

[0198] result: 20x10 6 When seeded at 1 / ml and induced on days 1 and 4, followed by harvesting on day 5, double and triple screening yielded cells with high GFP expression % and high average fluorescence intensity (Fig. 9a), higher Cap / GOI integrated copy number (Fig. 9b), and higher rAAV titer (Fig. 9c). A similar trend was observed when using different TI pools for TI / P producer pool generation (Fig. 9d).

[0199] 7.6 Comparison of viral titers between TI / P producer clone hosts and triple transient transfection

[0200] In this study, we compare the AAV titers obtained through a method using TI / P producer clones (Figs. 10a-10b) and triple transient transfection.

[0201] As described above, two TI plasmids (front TI plasmid and back TI plasmid) were transfected into a TI host (Host 1) using 2-plasmid RMCE to generate a pre-packaging pool containing REP and helper genes. The recovered pre-packaging pool was cloned into a single cell to pre-packaging host (Host 2) ...was obtained. Then, a producer pool was generated by stably transfecting pre-packaged hosts with the GOI and Cap genes using PiggyBac transposase. The recovered producer pool was single-cell cloned to obtain producer clones.

[0202] Virus and Capsid Titer Evaluation

[0203] Clones were selected and expanded for shaking flask production. Viral titer (VG / mL) was measured by GFP-specific droplet PCR, and capsid titer (VP / mL) was measured by an AAVx-based titer assay. The total / empty ratio (%) of the crude lysate was calculated by dividing VG / mL by VP / mL. The total / empty ratio (%) of purified rAAV from crude lysates produced from the best TI / P producer clone and transient transfection was confirmed by mass spectrometry (MP). The total / empty ratio (%) measured by MP was calculated by defining the total, partial, and empty particle regions in the mass histogram based on empty particle correction mass and transplant gene size. The data presented in Table 4 and Figures 10c-10d show that when using GOIs of the same size, the best TI / P producer clone has a higher VG / mL and a slightly lower total / empty ratio (%) than the standard triple transient transfection method.

[0204] Table 4 Comparison of viral titers using TI / P producer clones and triple transient transfection

[0205] VG / mL VP / mL Total / Empty ratio of dissolved substances (%) Total / Bin ratio (%) measured by MP Host 2 clone 4.0E11 1.2E12 40.0 23.3 Triple plasmid transient transfection 5.3E10 1.5E11 36.3 31.0

Claims

Claim 1 A stable mammalian host cell comprising the following: A) a first exogenous nucleic acid sequence incorporated into a target locus of the genome of a stable mammalian host cell, (i) one or more of adeno-associated virus (AAV) REP, AAV CAP, adenovirus (Ad) E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2-DNA binding protein (E2DBP), or Ad VA gene products; and (ii) a first exogenous nucleic acid sequence comprising two recombination recognition sequences (RRS) encoding a first selection marker and adjacent to (A)(i) and A(ii); and B) a second exogenous nucleic acid sequence incorporated at least once into the genome of a stable mammalian cell, (i) Polypeptide of Interest (POI); (ii) one or more of the AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA gene products; and (iii) A second exogenous nucleic acid sequence comprising an inversion terminal repeat (ITR) adjacent to a coding sequence for a second selection marker. Claim 2 A stable mammalian host cell according to claim 1, wherein (a) the first exogenous nucleic acid sequence encodes the AAV REP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, and Ad VA gene products; and (b) the second exogenous nucleic acid encodes AAV CAP. Claim 3 A stable mammalian host cell in which the second exogenous nucleic acid encodes AAV CAP in claim 1. Claim 4 A stable mammalian host cell according to claim 1, comprising: A) one or more of the AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA gene products; and B) 1 to 10 additional exogenous nucleic acids comprising an inversion terminal repeat adjacent to a coding sequence for a selection marker; wherein each of the 1 to 10 additional exogenous nucleic acids is incorporated into the genome of the stable mammalian cell at least once. Claim 5 A stable mammalian host cell according to claim 4, wherein one or more of 1 to 10 additional exogenous nucleic acids further comprise a coding sequence for the polypeptide of interest (POI) within a lateral inversion terminal repeat. Claim 6 A stable mammalian host cell according to claim 1, further comprising a coding sequence for a second polypeptide of interest within a lateral inversion terminal repeat. Claim 7 A stable mammalian host cell according to claim 1, further comprising an expression cassette of a transcriptional activator (rTA) sequence, wherein the rTA sequence is fused to a first and / or second selection marker. Claim 8 In claim 7, a stable mammalian host cell in which rTA is fused to a first and / or second selection marker via a 2A peptide. Claim 9 In claim 8, a stable mammalian host cell in which the 2A peptide is P2A, T2A, E2A, or F2A. Claim 10 In any one of paragraphs 1 to 9, a stable mammalian host cell that is a stable human cell. Claim 11 In paragraph 10, the stable mammalian host human cell is a stable human cell that is an HEK293 cell. Claim 12 A stable mammalian host cell in which, in any one of claims 1 to 11, the integration of the first exogenous nucleic acid sequence is promoted by an exogenous nuclease. Claim 13 In paragraph 12, a stable mammalian host cell in which the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFN), ZFN dimers, transcription activator-like effector nucleases (TALEN), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly spaced short palindromic repeat (CRISPR) associated (Cas) endonucleases. Claim 14 A stable mammalian host cell in which one or more of the proteins encoded by the first exogenous nucleic acid are inductively expressed in claim 1. Claim 15 A stable mammalian host cell in which one or more of the AAV proteins encoded by the first exogenous nucleic acid sequence are inductively expressed in claim 1. Claim 16 A stable mammalian host cell in which one or more of the proteins encoded by the second exogenous nucleic acid sequence are inductively expressed in claim 1. Claim 17 A stable mammalian host cell in which one or more of the AAV proteins encoded by the second exogenous nucleic acid sequence are inductively expressed in claim 1. Claim 18 A stable mammalian host cell according to claim 1, wherein one or more of the proteins encoded by the first exogenous nucleic acid sequence are inductively expressed and one or more of the proteins encoded by the second exogenous nucleic acid sequence are constitutively expressed. Claim 19 A stable mammalian host cell according to claim 1, wherein one or more of the proteins encoded by the first exogenous nucleic acid sequence are inductively expressed, and one or more of the proteins encoded by the second exogenous nucleic acid sequence are inductively expressed. Claim 20 A stable mammalian host cell according to claim 1, wherein one or more of the proteins encoded by the first exogenous nucleic acid sequence are constitutively expressed and one or more of the proteins encoded by the second exogenous nucleic acid sequence are constitutively expressed. Claim 21 A stable mammalian host cell according to claim 1, wherein one or more of the proteins encoded by the first exogenous nucleic acid sequence are constitutively expressed and one or more of the proteins encoded by the second exogenous nucleic acid sequence are inductively expressed. Claim 22 A stable mammalian host cell in which a plurality of proteins encoded by the first and / or second exogenous nucleic acid sequences are inductively expressed in any one of claims 14 to 21. Claim 23 In paragraph 22, a stable mammalian host cell in which the inductively expressed protein is induced by the same inducer. Claim 24 A) a step of providing a stable mammalian host cell comprising a first exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian cell, wherein the first exogenous nucleotide sequence comprises two recombination recognition sequences (RRS) adjacent to a first selection marker; B) a step of introducing a second exogenous nucleic acid sequence into the stable mammalian host cell provided in (A), wherein the second exogenous nucleic acid sequence comprises two RRSs adjacent to a sequence encoding one or more of the AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, or Ad VA gene products and the second selection marker; C) a step of introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRS; D) simultaneously with B) and C), or sequentially after B) and C). A method for producing recombinant AAV, comprising the steps of: introducing one or more of the gene products of AAV REP, AAV CAP, Ad E2A, Ad E4, Ad E4orf6, Ad E4orf6 / 7, Ad E2DBP, and Ad VA, or a third exogenous nucleic acid sequence encoding a polypeptide of interest and a third selection marker into the genome of a stable mammalian cell through transposon-mediated genome integration; E) selecting a stable mammalian host cell that stably expresses the second and third selection markers; and F) culturing the selected stable mammalian host cell under conditions sufficient to produce recombinant AAV. Claim 25 A method according to claim 24, further comprising the step of recovering the AAV of interest from the cell culture medium. Claim 26 A method according to claim 24, wherein the second exogenous nucleic acid sequence encodes AAV REP, Ad E2A, Ad E4, and Ad VA. Claim 27 A method according to claim 24, wherein the nucleic acid sequence at step (D) further comprises a coding sequence encoding a polypeptide of interest. Claim 28 A method according to claim 24, wherein the first, second, and third exogenous nucleotide sequences further comprise an expression cassette of a transcriptional activator (rTA) sequence, wherein the rTA sequence is fused to one or more of the first, second, and third selection markers. Claim 29 A method according to claim 28 in which rTA is fused to one or more of the first, second, and third selection markers through a 2A peptide. Claim 30 In paragraph 29, the method in which the 2A peptide is P2A, T2A, E2A, or F2A. Claim 31 In paragraph 24, the method in which the third exogenous nucleic acid encodes AAV CAP. Claim 32 In paragraph 24, a method in which a stable mammalian host cell is a stable human cell. Claim 33 In paragraph 26, the method in which the stable human cell is an HEK293 cell. Claim 34 A method according to any one of claims 24 to 33, wherein the target integration of the first exogenous nucleotide sequence is promoted by an exogenous nuclease. Claim 35 A method according to claim 34, wherein the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFN), ZFN dimers, transcription activator-like effector nucleases (TALEN), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly spaced short palindromic repeat (CRISPR) associated (Cas) endonucleases. Claim 36 A method according to any one of claims 24 to 35, wherein the expression of one or more proteins is controlled by a regulated promoter. Claim 37 In paragraph 36, the method wherein the adjustable promoter is selected from the group consisting of SV40 and CMV promoters. Claim 38 A method according to claim 24 in which one or more of the proteins encoded by a first exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian cell are inductively expressed. Claim 39 A method according to claim 24 in which one or more of the proteins encoded by a second exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian cell are inductively expressed. Claim 40 A method according to claim 24 in which one or more of the proteins encoded by a transposon-mediated genome-integrated third exogenous nucleic acid are inductively expressed. Claim 41 A method according to claim 24 in which one or more of the proteins encoded by the transposon-mediated genome-integrated third exogenous nucleic acid are constitutively expressed. Claim 42 A method according to claim 24, wherein one or more of the proteins encoded by the first and / or second exogenous nucleotide sequences incorporated into the target locus of the genome of a stable mammalian cell are inductively expressed, and one or more of the proteins encoded by the third exogenous nucleic acid incorporated into the transposon-mediated genome are constitutively expressed. Claim 43 A method according to claim 24, wherein one or more proteins encoded by a first and / or second exogenous nucleotide sequence incorporated into a target locus of the genome of a stable mammalian cell are inductively expressed, and one or more proteins encoded by a third exogenous nucleic acid incorporated into a transposon-mediated genome are inductively expressed. Claim 44 A method according to claim 24, wherein one or more of the proteins encoded by the first and / or second exogenous nucleotide sequences incorporated into the target locus of the genome of a stable mammalian cell are constitutively expressed, and one or more of the proteins encoded by the third exogenous nucleic acid incorporated into the transposon-mediated genome are constitutively expressed. Claim 45 A method according to claim 24, wherein one or more of the proteins encoded by the first and / or second exogenous nucleotide sequences incorporated into the target locus of the genome of a stable mammalian cell are constitutively expressed, and one or more of the proteins encoded by the third exogenous nucleic acid incorporated into the transposon-mediated genome are inductively expressed. Claim 46 A method according to any one of claims 24 to 45, wherein a stable mammalian host cell selected to express a third selection marker comprises 1 to 10 copies of a transposon-mediated genome-integrated third exogenous nucleic acid sequence. Claim 47 A method according to any one of claims 24 to 46, wherein a plurality of proteins encoded by the first and / or second exogenous nucleic acid are inductively expressed. Claim 48 In paragraph 47, a method in which the inductively expressed protein is induced by the same inducer. Claim 49 A method in which, in any one of paragraphs 24 to 48, the cell culture is a suspension culture. Claim 50 A method in which, in any one of paragraphs 24 to 49, the cell culture is a serum-free culture. Claim 51 (a) obtaining a first plurality of TI pre-packaging clones by single-cell cloning by incorporating an exogenous nucleotide sequence encoding Rep and Helper genes into a TI host cell; (b) transfecting the first plurality of clones with rAAV encoding a gene of interest (GOI); (c) screening the transfected clones for viral genome replication to obtain a second plurality of TI pre-packaging clones that exhibit desirable viral genome replication efficiency upon induction of Rep and Helper genes; (d) obtaining a plurality of producer cell clones by performing transient transfection on the second plurality of clones using an exogenous nucleotide sequence encoding the Cap gene and GOI; and (e) screening the plurality of producer cell clones for rAAV production upon induction of Cap, Rep and Helper genes, comprising a method for screening rAAV producer cell clones. Claim 52 In paragraph 51, a method in which the TI host cell is a stable human cell. Claim 53 In paragraph 52, the method in which the stable human cell is an HEK293 cell. Claim 54 A method according to any one of claims 51 to 53, wherein the integration of exogenous nucleotide sequences encoding Rep and Helper genes is promoted by an exogenous nuclease. Claim 55 A method according to claim 54, wherein the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFN), ZFN dimers, transcription activator-like effector nucleases (TALEN), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly spaced short palindromic repeat (CRISPR) associated (Cas) endonucleases. Claim 56 A method according to claim 51 in which one or more of the proteins encoded by exogenous nucleotide sequences encoding Rep and Helper genes are inductively expressed. Claim 57 A method according to claim 51 in which one or more of the proteins encoded by the exogenous nucleotide sequence encoding the Cap gene and GOI are inductively expressed. Claim 58 A method according to claim 51, wherein one or more of the proteins encoded by the exogenous nucleotide sequence encoding the Rep and Helper genes are inductively expressed, and one or more of the proteins encoded by the exogenous nucleotide sequence encoding the Cap gene and GOI are constitutively expressed. Claim 59 A method according to claim 51, wherein one or more of the proteins encoded by the exogenous nucleotide sequence encoding the Rep and Helper genes are inductively expressed, and one or more of the proteins encoded by the exogenous nucleotide sequence encoding the Cap gene and GOI are inductively expressed. Claim 60 A method according to claim 51, wherein one or more of the proteins encoded by the exogenous nucleotide sequence encoding the Rep and Helper genes are constitutively expressed, and one or more of the proteins encoded by the exogenous nucleotide sequence encoding the Cap gene and GOI are constitutively expressed. Claim 61 A method according to claim 51, wherein one or more of the proteins encoded by exogenous nucleotide sequences encoding Rep and Helper genes are constitutively expressed, and one or more of the proteins encoded by exogenous nucleotide sequences encoding Cap gene and GOI are inductively expressed. Claim 62 A method according to any one of claims 51 to 59 and 61, wherein the inductively expressed protein is induced by the same inducer.

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