Methods for controlling adeno-associated virus production

Abstract

The subject matter of the present disclosure relates to compositions and methods for controlling recombinant adeno-associated virus (rAAV) production in cell culture. In particular, the subject matter of the present disclosure relates to a strategy to overcome AAV Rep protein-mediated cytotoxicity by reversible post-translational control of expression of AAV Rep and helper proteins resulting in controlled rAAV production.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 350,849, filed June 9, 2022, and U.S. Provisional Application No. 63 / 209,735, filed June 11, 2021, both of which are incorporated by reference in their entireties and to which priority is claimed.

[0002] 1. Field of the invention The subject matter of the present disclosure relates to compositions and methods for the control of recombinant adeno-associated virus (rAAV) production in cell culture. In particular, the subject matter of the present disclosure relates to a strategy to overcome AAV Rep protein-mediated cytotoxicity by reversible post-translational control of expression of the AAV Rep protein, resulting in the control of rAAV production. [Background technology]

[0003] 2.Background There are various AAV production systems used to produce rAAV in cell culture. These include plasmid transient transfection of human embryonic kidney (HEK) 293 cells, Hela producer cell lines, BHK21-based platforms, and baculovirus-based production systems. Each of these systems has advantages and disadvantages. For example, given the importance of the adenoviral E1a protein in initiating the production of rAAV, E1a-expressing cells, such as HEK293 cells, are attractive for producing rAAV because they eliminate the need to separately introduce the E1a gene into the host cell genome. E1a-expressing cells, such as HEK293 cells, can also offer ease of growth and the adaptability of growth in suspension. However, efforts to create stable and passivable rAAV-producing E1a-expressing cell lines have been hampered by the cytotoxicity caused by the E1a-induced accumulation of AAV Rep protein. In view of the above, there is a need in the art for new rAAV production strategies that can control the accumulation of Rep proteins, avoid Rep-mediated cytotoxicity, and result in controlled rAAV production. Summary of the Invention

[0004] 3. Overview of the Invention In certain embodiments, the disclosure is directed to a method of controlling the production of recombinant adeno-associated virus (rAAV) vector particles, the method comprising introducing into a cell a nucleic acid encoding a rAAV containing a gene of interest and a fusion protein, where the fusion protein comprises an AAV protein and a degradation ligand-dependent degradation domain, culturing the cells under conditions suitable for producing rAAV vector particles, and contacting the cells with a degradation ligand, where the degradation ligand binds to the degradation domain and controls expression of the AAV protein, thereby controlling production of the rAAV vector particles.

[0005] In certain embodiments, the nucleic acid encoding the fusion protein comprises a Rep protein, a linker, and a degradation ligand-dependent degradation domain. In certain embodiments, the ligand-dependent degradation domain is derived from FKBP. In certain embodiments, the degradation ligand-dependent degradation domain is DHFR. In certain embodiments, the degradation ligand-dependent degradation domain is an auxin-induced degradation domain. In certain embodiments, the degradation ligand is a small molecule ligand. In certain embodiments, the small molecule is Shield1. In certain embodiments, the small molecule is TMP. In certain embodiments, the small molecule is auxin. In certain embodiments, the small molecule is dTag13.

[0006] In certain embodiments, the nucleic acid encoding the fusion protein comprises a Cap protein, a linker, and a degradation ligand-dependent degradation domain. In certain embodiments, the ligand-dependent degradation domain is derived from FKBP. In certain embodiments, the degradation ligand-dependent degradation domain is DHFR. In certain embodiments, the degradation ligand-dependent degradation domain is an auxin-induced degradation domain. In certain embodiments, the degradation ligand is a small molecule ligand. In certain embodiments, the small molecule is Shield1. In certain embodiments, the small molecule is TMP. In certain embodiments, the small molecule is auxin. In certain embodiments, the small molecule is dTag13.

[0007] In certain embodiments, the nucleic acid encoding the fusion protein comprises a helper protein, a linker, and a degradation ligand-dependent degradation domain. In certain embodiments, the helper protein is E2. In certain embodiments, the ligand-dependent degradation domain is derived from FKBP. The degradation ligand-dependent degradation domain is DHFR. In certain embodiments, the degradation ligand-dependent degradation domain is an auxin-induced degradation domain. In certain embodiments, the degradation ligand is a small molecule ligand. In certain embodiments, the small molecule is Shield1. In certain embodiments, the small molecule is TMP. In certain embodiments, the small molecule is auxin. In certain embodiments, the small molecule is dTag13.

[0008] In certain embodiments, the cell is an E1a-expressing cell. In certain embodiments, the E1a-expressing cell is a HEK293 cell.

[0009] In certain embodiments, the Rep protein is a Rep78, Rep68, Rep52, or Rep40 protein.

[0010] In certain embodiments, the degradation ligand-dependent degradation domain is fused to the C-terminus of the AAV protein. In certain embodiments, the degradation ligand-dependent degradation domain is fused to the N-terminus of the AAV protein.

[0011] In certain embodiments, the linker is a flexible linker. In certain embodiments, the linker is a rigid linker.

[0012] In certain embodiments, the method of the present disclosure includes introducing a nucleic acid encoding a Cap protein into a cell. In certain embodiments, the nucleic acid encoding a fusion protein and the nucleic acid encoding a Cap protein are introduced into the cell using at least one plasmid. In certain embodiments, the nucleic acid encoding a fusion protein and the nucleic acid encoding a Cap protein are introduced into the cell using the same plasmid. In certain embodiments, the nucleic acid encoding a fusion protein and the nucleic acid encoding a Cap protein are introduced into the cell using separate plasmids. In certain embodiments, the AAV fusion protein-encoding gene and / or the cap gene are under the control of a control element. In certain embodiments, the control element is a promoter. In certain embodiments, the control element is a Tet response element.

[0013] In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is an animal cell. In certain embodiments, the animal cell is a mammalian cell. In certain embodiments, the mammalian cell is a HEK cell.

[0014] In certain embodiments, the present disclosure is directed to an rAAV production cell, the cell comprising a nucleic acid encoding a fusion protein comprising an AAV protein and a degradation ligand-dependent degradation domain. In certain embodiments, the nucleic acid encoding the fusion protein comprises an AAV protein, a linker, and a degradation ligand-dependent degradation domain. In certain embodiments, the degradation ligand-dependent degradation domain is derived from FKBP. In certain embodiments, the ligand-dependent degradation domain is derived from FKBP. In certain embodiments, the degradation ligand-dependent degradation domain is DHFR. In certain embodiments, the degradation ligand-dependent degradation domain is an auxin-induced degradation domain. In certain embodiments, the degradation ligand is a small molecule ligand. In certain embodiments, the small molecule is Shield1. In certain embodiments, the small molecule is TMP. In certain embodiments, the small molecule is auxin. In certain embodiments, the small molecule is dTag13.

[0015] In certain embodiments, the rAAV production cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is an animal cell. In certain embodiments, the animal cell is a mammalian cell. In certain embodiments, the mammalian cell is a HEK cell. In certain embodiments, the cell is an E1a expressing cell. In certain embodiments, the E1a expressing cell is a HEK293 cell.

[0016] In certain embodiments, the rAAV production cell comprises a ligand-dependent degradation domain fused to the C-terminus of AAV protein via a linker.In certain embodiments, the ligand-dependent degradation domain is fused to the N-terminus of AAV protein via a linker.In certain embodiments, the linker is a flexible linker.In certain embodiments, the linker is a rigid linker.

[0017] In certain embodiments, the cell comprises a nucleic acid encoding a Cap protein. In certain embodiments, the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using at least one plasmid. In certain embodiments, the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using the same plasmid. In certain embodiments, the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using separate plasmids. In certain embodiments, the AAV fusion protein-encoding gene and / or the cap gene are under the control of a control element. In certain embodiments, the control element is a promoter. In certain embodiments, the control element is a Tet response element. [Brief description of the drawings]

[0018] 4. Brief description of the drawings [Figure 1] FIG. 1 displays examples of plasmid constructs utilized in the methods and systems of the present disclosure. [Diagram 2] FIG. 2 displays a schematic diagram for the experimental flow to confirm rAAV production using degron constructs such as those shown in FIG. 1. [Diagram 3] FIG. 1 shows that rAAV can be produced by transfection of mammalian cells with the Rep and Cap genes on separate plasmids. [Figure 4] FIG. 1 shows that Rep-degron constructs can be used in AAV production and that accumulation of Rep proteins can be controlled by the addition of Shield-1 molecules. [Diagram 5] FIG. 1 shows Shield-1 mediated post-translational control of expression of Rep constructs, where the constructs encode Rep with a C-terminal degron fusion or Rep with an N-terminal degron fusion. [Figure 6]FIG. 1 shows Shield-1 mediated post-translational control of expression of Rep protein with or without a C-terminal degron fusion. Rep is present on the same construct as Cap or on a separate construct. [Figure 7] FIG. 1 shows that rAAV production can be regulated by fusing a C-terminal degron to Rep and adding increasing concentrations of Shield-1, and that increasing concentrations of the Rep-degron plasmid, where Rep expression is under the CMV promoter, results in reduced rAAV production. [Figure 8] FIG. 1 shows rAAV production using Rep protein with or without a C-terminal degron fusion and the incorporation of rigid or flexible linkers between Rep and the degron. [Figure 9] FIG. 1 shows rAAV production using Rep protein with or without N-terminal degron fusion and incorporation of rigid or flexible linkers between Rep and the degron. [Figure 10] FIG. 1 shows regulation of Rep constructs containing a C-terminal degron and Rep constructs containing an N-terminal degron using Shield-1. [Figure 11]Figure 1 displays possible size changes in Western blots after addition of a degron tag to the N- or C-terminus of Rep proteins. Addition of a degron to the N-terminus of Rep proteins (N-degron) altered the molecular weight of the large Rep protein but not the small Rep protein. The molecular weight of the small Rep protein is not affected by the N-terminal protein tag because the p19 promoter that drives expression of the small Rep is located within the Rep gene. The small Rep protein does not share the same N-terminal sequence as the large Rep protein. In contrast, addition of a degron to the C-terminus of a Rep protein (C-degron) alters the molecular weight of both the small and large Rep proteins since both C-termini are the same. Addition of small molecules such as Shield1 or TMP to cell cultures can inhibit protein degradation and band intensity will change accordingly. The example shown here is an FKBP-derived degron. [Figure 12A] Figure 1 shows the regulation of AAV production via Rep proteins containing an FKBP-derived degron.Figure 2 shows a Western blot of N-terminal FKBP degron-tagged Rep proteins. [Figure 12B] Figure 1 shows the regulation of AAV production via Rep proteins containing an FKBP-derived degron.Figure 2 shows a Western blot of C-terminal FKBP degron-tagged Rep proteins. [Figure 12C] 12A and 12B show the regulation of AAV production via Rep proteins containing the FKBP-derived degron.FIG. [Figure 12D] Figure 1 shows the control of AAV production via Rep protein containing an FKBP-derived degron.Figure 2 shows AAV titers with Rep and Cap plasmids transfected at different ratios. [Figure 13A]Figure 1 shows the control of AAV production via Rep proteins carrying an E. coli DHFR-derived degron (ecDHFR degron).Figure 2 shows a Western blot of N-terminal ecDHFR degron-tagged Rep proteins. [Figure 13B] Figure 1 shows the control of AAV production via Rep proteins carrying an E. coli DHFR-derived degron (ecDHFR degron).Figure 2 shows a Western blot of C-terminal ecDHFR degron-tagged Rep proteins. [Figure 13C] Figure 13A shows the control of AAV production via Rep proteins carrying the E. coli DHFR-derived degron (ecDHFR degron).Figure 13B shows the AAV titers of samples from Figures 13A and 13B. [Figure 14A] Figure 1 shows the control of AAV production via Rep proteins containing auxin-based and ecDHFR degrons.Figure 2 shows western blots of Rep tagged with auxin-inducible or ecDHFR degrons. [Figure 14B] Figure 14A shows the control of AAV production via Rep proteins, including the auxin-based and ecDHFR degrons.Figure 14B shows the AAV titers of the samples from Figure 14A. [Figure 15] Figure 1 shows the effect of different doses of Shield1 and dTag13 on AAV production. dTAG-13 is a small molecule that can target mutant FKBP sequences for ubiquitin-mediated degradation. It can function by linking the targeting protein sequence to the E3 ubiquitin ligase cereblon. dTAG-13 can result in the degradation of FKBP fusion proteins and proteins fused to it. [Figure 16A] FIG. 13 displays Western blots of Rep containing a C-terminal degron under exposure to control and Tet proteins and a single doxycycline concentration from a Tet response element-containing promoter (TRE3G). [Figure 16B]FIG. 16B displays the AAV titers of the samples from FIG. 16A. [Figure 17A] FIG. 13 shows Western blots of Rep containing a C-terminal degron under exposure to control and Tet proteins and a range of doxycycline concentrations under the TRE3G promoter. [Figure 17B] FIG. 17B displays the AAV titers of the samples from FIG. 17A. [Figure 18A] FIG. 1 shows the effect of the p5 promoter on Rep protein levels in the Tet system, particularly the TRE3G-Rep-degron system. [Figure 18B] FIG. 13 displays a Western blot of Rep constructs with a C-terminal degron and the TRE3G promoter. [Figure 18C] FIG. 18C displays the AAV titers of the samples from FIG. 18B. [Figure 19A] Figure 1 displays the DBP protein expression observed from E2A genes tagged with a degron motif from FKBP. A plasmid expressing the E2A-DBP-degron was transfected together with other plasmids expressing Rep / Cap, ITR-GOL, E4-E34K and VA2. At the end of 72 hours, cells were lysed and AAV titer levels were analyzed. Cells transfected only with the ITR-GOI plasmid (helper and Rep / Cap plasmids were omitted to prevent AAV production) were used as negative control samples. Without the addition of Shield1 molecules, AAV production is the same as background levels of qPCR results resulting from the negative control (dark red bars on the left vs. grey bars on the right). Addition of Shield1 molecules increases titer levels by approximately 3-fold, providing a control for AAV production using E2A-DBP with a degron tag (striped bars in the middle). [Figure 19B]1 displays the results of a Western blot showing a shift in protein size of E2A-DBP protein due to the addition of a degron tag. The tagged protein is approximately 12 kDa larger than the untagged DBP protein. The two samples on the left are untagged DBP, while the sample on the right is a sample of DBP containing a degron. [Figure 20A] Figure 1 shows that codon-modified Rep with a degron motif can be used for AAV production. The modified rep gene is under the control of regulatory elements, specifically the TRE3G-Tet system. The construct shown here has the large Rep protein produced only by the codon-modified Rep construct. The small Rep is expressed from another region on the same plasmid. The small Rep is also under the control of TRE3G and a degron on the same plasmid. [Figure 20B] Figure 1 shows that codon-modified Rep with degron motifs can be used for AAV production. Samples from cells with higher AAV titers were treated with Shield1 and Dox. This shows the inducible properties of the Tet promoter and multiple degron domains on multiple AAV genes, including the modified rep gene, in the context of the methods disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] 5. Detailed Description The subject matter of the present disclosure relates to compositions and methods for controlling recombinant rAAV production in cell culture. In particular, the subject matter of the present disclosure relates to compositions and methods for overcoming AAV Rep protein-mediated cytotoxicity by reversible post-translational control of expression of the AAV Rep protein.

[0020] In one aspect, the subject matter of the present disclosure is directed to a method of cell culture for post-translational control of the expression of rAAV.In certain embodiments, the reversible post-translational control of the expression of AAV Rep protein in cell culture is achieved by fusing a degradation domain to AAV Rep protein.In certain embodiments, fusing a degradation domain to AAV Rep protein allows the controlled degradation of AAV Rep protein based on the presence or absence of degradation ligand in cell culture.In certain embodiments, the controlled degradation of AAV Rep protein based on the presence or absence of degradation ligand results in the post-translational control of the expression of rAAV in cell culture.

[0021] In another aspect, the subject matter of the present disclosure is directed to an rAAV producing cell. For example, but not limited to, the present disclosure is directed to an rAAV producing cell in which expression of the AAV Rep protein is controlled by fusion of a degradation domain to the AAV Rep protein.

[0022] In another aspect, reversible post-translational control of AAV helper protein expression in cell culture is achieved by fusing a degradation domain to the AAV helper protein. In certain embodiments, fusing a degradation domain to the AAV helper protein allows for controlled degradation of the AAV helper protein based on the presence or absence of a degradation ligand in cell culture. In certain embodiments, controlled degradation of the AAV helper protein based on the presence or absence of a degradation ligand results in post-translational control of the expression of rAAV in cell culture.

[0023] For clarity, but not by way of limitation, the detailed description of the subject matter of this disclosure is divided into the following sections: 5.1.Definition 5.2. Methods for controlling AAV expression using degradation domains 5.3.rAAV-producing cells

[0024] 5.1.Definition The terms used herein generally have their usual meanings in the art, within the context of this disclosure, and in the specific context in which each term is used.Specific terms are described below or elsewhere in the specification to provide additional guidance to practitioners in describing the compositions and methods of the present disclosure and how to make and use them.

[0025] As used herein, the use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0026] "Comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0027] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by a person skilled in the art, but depends in part on how the value is measured or determined, for example, on the constraints of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, according to the practice in the art. Alternatively, "about" can mean within 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 with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0028] In certain embodiments, the cells of the present disclosure carry the rep gene integrated into the chromosome, but require helper virus functions to express the Rep protein. As used herein, "helper virus" or "helper virus functions" or "AAV helper" refers to at least one of adenovirus (Ad) E1 (e.g., Ad E1a or Ad E1b), Ad E2A, Ad E4 and VA RNA, or the corresponding functions of other viruses, such as herpesviruses and poxviruses, which can confer helper functions to assist in the replication and packaging of the AAV vector genome. In certain embodiments, hybrid viruses were made from adenoviruses that have E1 / E3 deletions but contain Ad E2A, Ad E4 and VA RNA that provide helper virus functions, and AAV ITRs flanking the heterologous nucleic acid. In other embodiments, the hybrid viruses include AAV ITRs flanking the heterologous nucleic acid along with helper virus functions from herpesviruses or poxviruses.

[0029] As used herein, the term "helper virus function(s)" refers to the function(s) encoded in the helper virus genome that (together with Rep and Cap) allows replication and packaging of the rAAV vector genome. As used herein, "helper virus function" may be provided in a number of different ways. For example, the helper virus function may be provided by the virus or provided in trans to the cell, for example, by a polynucleotide sequence encoding the essential helper function(s). In another example, a plasmid or other expression vector containing a polynucleotide sequence encoding one or more viral (e.g., adenovirus) proteins provides the helper function when transfected with the rAAV vector genome into a cell line of the invention, allowing replication and packaging of the rAAV vector genome into rAAV vector particles. In certain embodiments, the helper virus function is provided by a virus selected from an adenovirus, a herpesvirus, a poxvirus, or a hybrid virus thereof. In certain embodiments, the helper virus function includes one or more viruses, vectors, or plasmids that provide the helper virus function. In certain embodiments, the helper virus function comprises at least one of an Ad E1 protein (e.g., an Ad E1a protein or an Ad E1b protein), an Ad E2A protein, an Ad E4 protein, and an Ad VA RNA. In certain embodiments, the degradation ligand-dependent degradation domain is fused to a helper protein.

[0030] As used herein, the term "AAV protein" refers to any wild-type or modified protein derived from the AAV genome and required for AAV production. "AAV protein" includes any form of Rep, Cap, or helper protein, whether wild-type or modified. Modification of wild-type AAV genes does not necessarily result in a change in the amino acid sequence of the expressed protein. As used herein, "AAV fusion protein" refers to a fusion protein that includes an AAV protein in which the amino acid sequence of the AAV protein is fused to another amino acid sequence, such as a degron sequence, either directly or via a linker.

[0031] The term "vector" refers to a small carrier nucleic acid molecule, such as a plasmid, a virus (e.g., AAV vector), or other vehicle, that can be manipulated by inserting or incorporating a nucleic acid. Such vectors can be used in genetic engineering to introduce / transfer polynucleotides into cells, and to transcribe or translate the polynucleotides inserted into the cells (i.e., "cloning vectors"). An "expression vector" is a special vector that contains a gene or nucleic acid sequence together with the necessary control regions required for expression in a host cell.

[0032] The nucleic acid sequence of a vector generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as heterologous polynucleotide sequences, expression control elements (e.g., promoters, enhancers), introns, inverted terminal repeats (ITRs), selection markers (e.g., antibiotic resistance), polyadenylation signals.

[0033] Viral vectors are derived from or based on one or more nucleic acid elements that comprise a viral genome. A particular viral vector is the adeno-associated virus (AAV) vector.

[0034] The term "recombinant", as a modifier of vectors such as recombinant AAV vectors, and of sequences such as recombinant polynucleotides and polypeptides, generally means that the composition is manipulated (i.e., genetically engineered) in a manner that does not occur in nature. A specific example of a recombinant AAV vector would be when a click acid sequence (e.g., a heterologous nucleic acid sequence) that is not normally present in the wild-type AAV genome is inserted into the AAV genome. The term "recombinant" is not always used herein in conjunction with AAV vectors, as well as sequences such as polynucleotides, but recombinant forms that include polynucleotides are expressly included, despite such omission.

[0035] "Recombinant AAV vector" or "rAAV" is derived from the wild-type (wt or wild-type) genome of AAV using molecular methods to remove the wild-type genome from the AAV genome and replace it with a non-natural nucleic acid sequence, referred to as heterologous nucleic acid. Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the AAV vector. rAAV is distinguished from the AAV genome because all or part of the AAV genome is replaced with a non-natural sequence with respect to the AAV genome nucleic acid. Thus, the incorporation of the non-natural sequence defines the AAV vector as a "recombinant" vector, which can be referred to as a "rAAV vector".

[0036] The rAAV sequences can be packaged for subsequent infection (transduction) of cells ex vivo, in vitro or in vivo - referred to herein as "particles". When recombinant AAV vector sequences are encapsidated or packaged into an AAV particle, the particle can also be referred to as a "rAAV vector" or "rAAV particle". Such rAAV particles contain proteins that encapsidate or package the vector genome. In the case of AAV, these are referred to as capsid proteins.

[0037] Vector "genome" refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form a viral (e.g., AAV) particle. When a recombinant plasmid is used to construct or produce a recombinant vector, the vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is referred to as the "plasmid backbone", which is important for the processes required for plasmid cloning and amplification, propagation and recombinant virus production, but is not itself packaged or encapsidated in a viral (e.g., AAV) particle. Thus, vector "genome" refers to the nucleic acid that is packaged or encapsidated by a virus (e.g., AAV).

[0038] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA and antisense DNA, and spliced ​​or unspliced ​​mRNA, rRNA, tRNA, and inhibitory DNA or RNA (RNAi, e.g., short or small hairpin (sh)RNA, microRNA (miRNA), short or small interfering (si)RNA, trans-splicing RNA, or antisense RNA). Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides (e.g., variant nucleic acids). Nucleic acids, such as cDNA, genomic DNA, RNA, and fragments thereof, may be single-stranded or double-stranded.

[0039] Polynucleotides can be single-stranded, double-stranded, or triple-stranded, linear or circular, and can be of any length. In discussing polynucleotides, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0040] "Transgene" as used herein for convenience refers to a heterologous nucleic acid intended to be or that has been introduced into a cell or organism. Transgenes include any heterologous nucleic acid, such as a gene encoding a polypeptide or protein or encoding an inhibitory RNA.

[0041] Heterologous nucleic acid can be introduced / transferred into a cell through a vector such as AAV, "transduction" or "transfection". The term "transduce" and its grammatical variants refer to the introduction of a molecule, such as an rAAV vector, into a cell or host organism. The introduced heterologous nucleic acid can also be present extrachromosomally or only transiently in the recipient cell or host organism.

[0042] A "transduced cell" is a cell into which a transgene has been introduced. Thus, a "transduced" cell (e.g., of a mammal, such as a cell or tissue or tissue cell) refers to a genetic change in a cell following incorporation of an exogenous molecule, such as a nucleic acid (e.g., a transgene), into the cell. Thus, a "transduced" cell is a cell into which an exogenous nucleic acid has been introduced, or a progeny thereof. The cell(s) are propagated and the introduced protein is expressed or the nucleic acid is transcribed. For gene therapy applications and methods, the transduced cell may be in the subject's body.

[0043] "Expression control element" is a type of control element and refers to a nucleic acid sequence(s) that influences the expression of an operably linked nucleic acid. Regulatory elements include expression control elements as described herein, such as promoters and enhancers. A vector sequence, including an AAV vector, may include one or more "expression control elements". Typically, such elements are included to facilitate transcription and, if necessary, translation of the appropriate heterologous polynucleotide (e.g., promoters, enhancers, splicing signals of introns, maintaining the correct reading frame of the gene to allow in-frame translation of mRNA, and stop codons, etc.). Such elements typically act in cis and are referred to as "cis-acting" elements, but may also act in trans.

[0044] Expression regulation can occur at the level of transcription, translation, splicing, message stability, etc. Typically, expression regulatory elements that regulate transcription are juxtaposed near the 5' end of the transcribed nucleic acid (i.e., "upstream"). Expression regulatory elements can be located at the 3' end of the transcribed sequence (i.e., "downstream") or within the transcript (e.g., within an intron).

[0045] Functionally, the expression of the operably linked nucleic acid can be regulated, at least in part, by an element (e.g., a promoter) that regulates the transcription of the nucleic acid and, optionally, the translation of the transcript. A specific example of an expression control element is a promoter, which is usually located 5' of the transcribed nucleic acid sequence. A promoter typically increases the amount of expression from the operably linked nucleic acid compared to the amount expressed in the absence of the promoter.

[0046] Another type of control element includes Tet. In Tet-dependent induction, expression from the target transgene depends on an inducible promoter. The promoter can be controlled by the level of tetracycline or a tetracycline derivative, such as doxycycline (Dox). Activation of the Tet-On promoter depends on the presence of an additional activator protein that can bind to the promoter in the presence of Dox. In contrast, in the case of the Tet-Off system, transcription is inactive in the presence of Dox. Other examples of inducible systems include Cumate, abscisic acid (ABA), rapamycin, and tamoxifen inducible systems. The degradation ligand-dependent degradation domains disclosed herein can also be used with Cre-LoxP, CRISPR, riboswitch, and light-switchable transgene systems in a similar manner. As used herein, "enhancer" can refer to a sequence located adjacent to a heterologous nucleic acid. Enhancer elements are typically located upstream of a promoter element, but can also function and be located downstream or within the sequence. Enhancer elements typically increase expression of an operably linked nucleic acid above the expression conferred by a promoter element.

[0047] Expression control elements herein, such as promoters, are typically located far from the transcribed sequence. In certain embodiments, expression control elements, such as promoters, are located at least about 25 nucleotides 5' of the rep gene start codon, about 25-5,000 nucleotides 5' of the rep gene start codon, about 250-2,500 nucleotides 5' of the rep gene start codon, about 500-2,000 nucleotides 5' of the rep gene start codon, about 1,000-1,900 nucleotides 5' of the rep gene start codon, about 1,500-1,900 nucleotides 5' of the rep gene start codon, about 1,600-1,800 nucleotides 5' of the rep gene start codon, about 1,700-1,800 nucleotides 5' of the rep gene start codon, or about 1,750 nucleotides 5' of the rep gene start codon.

[0048] Expression control elements include ubiquitous or unregulated promoters / enhancers that can promote expression of polynucleotides in many different cell types. Such elements include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter / enhancer sequence, the Rous sarcoma virus (RSV) promoter / enhancer sequence, and other viral promoters / enhancers active in various mammalian cell types, or synthetic elements (see, for example, Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic β-actin promoter, and the phosphoglycerol kinase (PGK) promoter.

[0049] Expression regulatory elements also include the native element(s) for the heterologous polynucleotide.Natural regulatory elements (e.g., promoters) may be used when it is desired that the expression of the heterologous polynucleotide should mimic the native expression.Other native regulatory elements, such as introns, polyadenylation sites, or Kozak consensus sequences, may also be used.

[0050] The term "operably linked" means that regulatory sequences necessary for expression of a nucleic acid sequence are positioned in an appropriate location relative to the sequence to effect expression of the nucleic acid sequence. This same definition is sometimes applied to the arrangement of nucleic acid sequences and transcriptional regulatory elements (e.g., promoters, enhancers, and termination elements) in an expression vector, e.g., an rAAV vector.

[0051] In the example of an expression control element in operably linked to a nucleic acid, the relationship is such that the control element regulates expression of the nucleic acid. More specifically, for example, two DNA sequences that are operably linked means that the two DNA sequences are positioned (in cis or trans) in a relationship such that at least one of the DNA sequences can have a physiological effect on the other sequence.

[0052] As disclosed herein, a nucleic acid spacer sequence positioned between an expression control element and the AAV rep gene substantially reduces or eliminates expression of the rep gene, which in turn reduces or eliminates expression of the Rep protein, allowing the cell to survive while also expressing the adenovirus E1a protein. The addition of helper virus function to such a cell, for example, provided by a hybrid virus, adenovirus, poxvirus, or herpesvirus, can overcome the attenuating effect of the spacer nucleic acid on rep gene expression, which in turn promotes expression of the rep gene, thereby providing expression of the Rep protein.

[0053] Additional elements for rAAV vectors include, but are not limited to, 5' or 3' untranslated regions flanking sequences such as transcription termination signals or stop codons, one or more copies of AAV ITR sequences (e.g., polyadenylation (polyA) sequences), or introns.

[0054] Additional elements include, for example, filler or stuffer polynucleotide sequences, for example, to improve packaging and reduce the presence of contaminating nucleic acids. AAV vectors typically accept inserts of DNA having a size range that is generally about 4 kb to about 5.2 kb, or slightly more. Thus, for short sequences, a stuffer or filler is included to adjust the length to about the normal or normal size of the viral genomic sequence acceptable for AAV vector packaging into viral particles. In various embodiments, the filler / stuffer nucleic acid sequence is a non-translated (non-protein coding) segment of nucleic acid. For nucleic acid sequences less than 4.7 kb, the filler or stuffer polynucleotide sequence has a length that, when combined with the sequence (e.g., when inserted into a vector), has a total length of about 3.0 to 5.5 kb, or about 4.0 to 5.0 kb, or about 4.3 to 4.8 kb.

[0055] Where a wild-type heterologous nucleic acid or transgene is too large to be packaged within an AAV vector particle, the heterologous nucleic acid may be provided in a modified, fragmented, or truncated form for packaging within and delivery by an AAV vector, thereby ultimately providing a functional protein or nucleic acid product, such as a therapeutic protein or nucleic acid product.

[0056] In some embodiments, the heterologous nucleic acid encoding a protein (e.g., a therapeutic protein) is provided in a modified or truncated form, or the heterologous nucleic acid is provided in multiple constructs delivered by separate multiple AAV vectors.

[0057] In certain embodiments, the heterologous nucleic acid is provided as a truncated variant that maintains the functionality of the encoded protein (e.g., a therapeutic protein) by including removal of portions unnecessary for function, such that the encoding heterologous polynucleotide is reduced in size for packaging within an AAV vector.

[0058] In certain embodiments, the heterologous nucleic acid is provided in a split AAV vector, each of which provides nucleic acid encoding a different portion of a protein (e.g., a therapeutic protein), thereby delivering multiple portions of a protein (e.g., a therapeutic protein) that assemble and function in the cell.

[0059] In other aspects, the heterologous nucleic acid is provided by a dual AAV vector using overlapping vector, trans-splicing vector or hybrid trans-splicing dual vector technology. In certain embodiments, two overlapping AAV vectors are used that combine in cells to generate a complete expression cassette from which a full-length protein (e.g., a therapeutic protein) is expressed.

[0060] "Hemostasis-related disorder" refers to bleeding disorders such as hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, any of the following clotting factors: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor deficiency, combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, or gamma-carboxylase deficiency; bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulation, or disseminated intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparins, pentasaccharides, warfarin, or small molecule antithrombotic agents (i.e., FXa inhibitors); and platelet disorders, e.g., Bernard-Soulier syndrome, Glanzmann's thrombasthenia, and storage pool deficiency.

[0061] The term "isolated," when used as a modifier of a composition, means that the composition has been produced by the hand of man or has been completely or at least partially separated from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they are normally associated in nature, such as one or more proteins, nucleic acids, lipids, carbohydrates, cell membranes.

[0062] The term "isolated" does not exclude combinations produced by the hand of man, such as rAAV particles and pharmaceutical preparations that package or encapsidate rAAV sequences, or AAV vector genomes. The term "isolated" also does not exclude alternative physical forms of the composition, such as hybrids / chimers, multimers / oligomers, modified (e.g., phosphorylated, glycosylated, lipidated) or derivatized forms, or forms expressed in host cells produced by the hand of man.

[0063] The term "substantially pure" refers to a preparation that contains at least 50-60% by weight of the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). The preparation may contain at least 75%, or at least 85%, or about 90-99% by weight of the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g., chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0064] The phrase "consisting essentially of," when referring to a particular nucleotide or amino acid sequence, means a sequence having the characteristics of a given SEQ ID NO. For example, when referring to an amino acid sequence, the phrase includes the sequence itself and molecular modifications that do not affect the basic and novel characteristics of the sequence.

[0065] "Identity", "homology" and grammatical variations thereof mean that, in the case of "aligned" sequences, two or more referenced entities are the same. Thus, by way of example, if two protein sequences are identical, they have the same amino acid sequence, at least within the referenced region or portion. If two nucleic acid sequences are identical, they have the same nucleic acid sequence, at least within the referenced region or portion. Identity may exist over a defined area (region or domain) of the sequences.

[0066] An "area" or "region" of identity refers to a portion of two or more referenced entities that are the same. Thus, if two protein or nucleic acid sequences are identical over one or more sequence areas or regions, they share identity within that region. An "aligned" sequence often refers to a sequence of multiple proteins (amino acids) or nucleic acids that contains modifications of missing or additional bases or amino acids (gaps) compared to a reference sequence.

[0067] Identity can extend over the entire length of the sequence or over a portion of it. In certain embodiments, the length of the sequence that shares percent identity is 2, 3, 4, 5 or more contiguous amino acids or nucleic acids, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more contiguous nucleic acids or amino acids. In additional embodiments, the length of the sequence that shares identity is 21 or more contiguous amino acids or nucleic acids, such as 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more contiguous amino acids or nucleic acids. In further embodiments, the length of the sequence that shares identity is 41 or more contiguous amino acids or nucleic acids, such as 42, 43, 44, 45, 45, 47, 48, 49, 50 or more contiguous amino acids or nucleic acids. In yet another embodiment, the length of the sequences sharing identity is 50 or more contiguous amino acids or nucleic acids, for example, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-150, 150-200, 200-250, 250-300, 300-500, or 500-1,000 contiguous amino acids or nucleic acids.

[0068] The extent of identity (homology) or "percent identity" between two sequences can be ascertained using a computer program and / or a numerical algorithm. For the purposes of the present invention, comparison of nucleic acid sequences is carried out using the GCG Wisconsin Package version 9.1 available from the Genetics Computer Group in Madison, Wisconsin. For convenience, the default parameters (gap formation penalty=12, gap extension penalty=4) specified by the program are intended to be used herein to compare sequence identity. Alternatively, the Blastn2.0 program provided by the National Center for Biotechnology Information (found on the World Wide Web at ncbi.nlm.nih.gov / blast / ; Altschul et al., 1990, J Mol Biol 215:403-410), using gapped alignment with default parameters, can be used to determine the level of identity and similarity between nucleic acid and amino acid sequences. When comparing polypeptide sequences, typically, the BLASTP algorithm is used in combination with a scoring matrix such as PAM100, PAM250, BLOSUM62 or BLOSUM50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantify the degree of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol. Biol. 147:195 (1981)). Programs that use Delaunay-based topological mapping to quantify protein structural similarity have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

[0069] Nucleic acid molecules, expression vectors (e.g., AAV vector genomes), plasmids, including nucleic acids encoding the modified / variant AAV capsids of the invention and heterologous nucleic acids, can be prepared using recombinant DNA techniques. The availability of nucleic acid sequence information allows for the preparation of isolated nucleic acid molecules of the invention by various means. For example, nucleic acid sequences can be produced using a variety of standard cloning, recombinant DNA techniques, through cellular expression, or in vitro translation and chemical synthesis techniques. The purity of polynucleotides can be determined by sequencing, gel electrophoresis, and the like. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries using probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides with shared structural features, for example, using expression libraries; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the nucleic acid sequence of interest; (4) computer-assisted searches of sequence databases for related sequences; and (5) difference screening of subtracted nucleic acid libraries.

[0070] 5.2. Methods for controlling AAV production using degradation domains In one aspect, the subject matter of the present disclosure is directed to a method of cell culture for post-translational control of expression of rAAV. In certain embodiments, reversible post-translational control of AAV Rep protein expression in cell culture is achieved by fusing a degradation domain ("degron") to the AAV Rep protein. In certain embodiments, fusing a degradation domain to the AAV Rep protein allows for control of degradation of the AAV Rep protein based on the presence or absence of a degradation ligand or other "inhibitor", such as a small molecule ligand that binds to the degron and modifies its rate of degradation, or an inhibitor such as light or temperature that modifies the rate of degradation of the degron. In certain embodiments, the controlled degradation of the AAV Rep protein based on the presence or absence of a degradation ligand or other inhibitor results in post-translational control of expression of rAAV in cell culture. As used herein, the term "degradation ligand-dependent degradation domain" refers to a degron domain that binds to a degradation ligand. As used herein, the term "degradation ligand" refers to a ligand that binds to a degradation domain.

[0071] Any suitable degron can be used in conjunction with the methods of the present disclosure. For example, but not limited to, the degron is modified from a human gene encoding a protein called FK506 binding protein 12 ("FKBP"). In certain embodiments, the degron is derived from FKBP. In certain embodiments, the FKBP variant protein from which the FKBP degron is derived comprises an F36V amino acid substitution. In certain embodiments, the FKBP variant protein from which the FKBP degron is derived comprises an L106P amino acid substitution. In certain embodiments, the FKBP variant protein from which the FKBP degron is derived comprises both F36V and L106P amino acid substitutions. In certain embodiments, the modification of FKBP a) deepens the binding pocket beyond FK506 to improve its specificity for a degradation ligand, e.g., Shield1, and / or b) makes the protein more unstable in the absence of its degradation ligand. dTAG-13 is a small molecule that can target mutant FKBP12 (F36V) sequences for ubiquitin-mediated degradation. It can function by linking the targeting protein sequence to the E3 ubiquitin ligase cereblon. dTAG-13 can cause the degradation of FKBP12-F36V fusion proteins and proteins fused to it. As used herein, the FKBP12 domain is referred to as "FKBP".

[0072] In certain embodiments, the degron is a dihydrofolate reductase (DHFR)-based degron, an auxin-inducible degron (AID) domain, an ornithine decarboxylase (ODC)-based degron, a split-ubiquitin-based degron system, a protease-based degron system, a proteolysis-inducing chimeric molecule (PROTAC)-based degron system, an antibody-dependent protein degron system, a light-sensitive degron (psd), a phosphorylation-dependent degron, or a temperature-dependent degron.

[0073] In certain embodiments, the degron is regulated by the presence or absence of a degradation ligand or inhibitor.In certain embodiments, the degradation ligand is a small molecule ligand.In certain embodiments, for example, when the degron is an FKBP variant protein, the small molecule ligand is Shield1.

[0074] In certain embodiments, for example, when the degron is a dihydrofolate reductase (DHFR)-based degron, the ligand is trimethoprim (TMP). In certain embodiments, for example, when the degron is an auxin-inducible degron (AID), the ligand is auxin. In certain embodiments, for example, when the degron is an ornithine decarboxylase (ODC)-based degron, the ligand is antizyme. In certain embodiments, for example, when the degron is a split-ubiquitin-based degron system, the ligand is rapamycin. In certain embodiments, for example, when the degron is a protease-based degron system, the inhibitor can be an HCV protease inhibitor or TEV protease expression. In certain embodiments, for example, when the degron is a proteolysis-inducing chimeric molecule (PROTAC)-based degron system, the inhibitor is PROTAC expression. In certain embodiments, for example, when the degron is an antibody-dependent protein degron system, the ligand is a corresponding antibody. In certain embodiments, for example, when the degron is a light-sensitive degron (psd), the inhibitor is light. In certain embodiments, for example, when the degron is a phosphorylation-dependent degron, the inhibitor is a corresponding kinase activator. In certain embodiments, for example, when the degron is a temperature-dependent degron, the inhibitor is a change in temperature.

[0075] In certain embodiments, the degron sequence is linked to the C-terminus of the AAV Rep protein. In certain embodiments, the degron sequence is linked to the N-terminus of the AAV-Rep protein. In certain embodiments, the degron and the AAV Rep protein are linked through a flexible linker. In certain embodiments, the degron and the AAV Rep protein are linked through a rigid linker. In certain non-limiting embodiments, the flexible linker has the amino acid sequence: GGGSGGGGSGGGGGS. In certain non-limiting embodiments, the rigid linker has the amino acid sequence: AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEA AAKA.

[0076] In certain embodiments, a method of the disclosure is directed to controlling the production of recombinant adeno-associated (rAAV) virus, the method comprising introducing into a mammalian cell an rAAV comprising a nucleic acid encoding a gene of interest and a fusion protein, the fusion protein comprising a Rep protein, a linker, and a degradation ligand-dependent degradation domain; culturing the cells under conditions suitable for producing the rAAV virus; and contacting the cells with a degradation ligand, where the degradation ligand binds to the degradation domain and controls expression of the Rep protein, thereby controlling the production of the rAAV.

[0077] In certain embodiments, the cell is an E1a expressing cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a HEK293 cell. In certain embodiments, the cell is a HEK293F cell. In certain embodiments, the cell is a PERC6 cell.

[0078] In certain embodiments, the Rep protein is a Rep78, Rep68, Rep52, or Rep40 protein.

[0079] The disclosed method includes the use of suitable control elements, including promoters, to promote the expression of Rep and Cap proteins. In certain embodiments, the suitable promoters can be eukaryotic, prokaryotic, or viral promoters. Suitable promoters include non-inducible promoters and non-tissue specific promoters. In certain embodiments, the promoter is the AAV p5 promoter, which in its native state promotes the expression of Rep protein from the rep gene. In certain embodiments, the promoter is the cytomegalovirus (CMV) immediate early promoter / enhancer. Non-limiting examples of additional suitable promoters include ubiquitous or unregulated promoters that can promote the expression of polynucleotides in many different cell types. Such elements include, but are not limited to, the Rous sarcoma virus (RSV) promoter sequence and other viral promoters active in various mammalian cell types, or synthetic elements (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic β-actin promoter, and the phosphoglycerol kinase (PGK) promoter. In certain embodiments, the promoter is selected from the human elongation factor-1α EF1 alpha promoter, the CAG promoter, the CBA promoter, the SFFV promoter, the p19 promoter, and the herpes simplex virus thymidine kinase (HSV-TK) promoter. In certain embodiments, the promoter may be located proximal or distal to the Rep and Cap genes. In certain embodiments, the control elements, including the promoter, may be in cis or trans with respect to the Rep and Cap genes.In certain embodiments, the methods of the disclosure are directed to expression of a nucleic acid encoding an AAV Rep protein, where the AAV Rep protein is an AAV1 Rep protein, an AAV2 Rep protein, an AAV3 Rep protein, an AAV4 Rep protein, an AAV5 Rep protein, an AAV6 Rep protein, an AAV7 Rep protein, an AAV8 Rep protein, an AAV9 Rep protein, an AAV10 Rep protein, or an AAV11 Rep protein. The methods of the disclosure are applicable to any cell type that produces AAV, so that the AAV can be, for example, human, avian, bovine, canine, equine, primate, non-primate, ovine, or any derivative thereof.

[0080] In certain embodiments, the rAAV produced by the methods of the present disclosure comprises any virus strain or serotype. In certain non-limiting embodiments, the rAAV can be based on any AAV genome, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, Rh8, Rh10, Rh74, AAV3B, AAV-2i8, LK03, RHM4-1, DJ, DJ8, NP59, Anc-80 and variants thereof, including those described in Pulicherla et al. al., Mol. Ther., 19(6) 1070-1078 (2011) (specifically describing AAV9 variants, including AAV9.47), U.S. Pat. No. 7,906,111 (specifically describing AAV9(hu14)), U.S. Pat. No. 10,532,111 (specifically describing NP59), U.S. Pat. No. US10738087 (specifically describing Anc-80), WO2012 / 145601, WO2013 / 158879, WO2015 / 0 13313, WO2018 / 156654, US2013 / 0059732, U.S. Patent No. 9,169,299 (LK03 description), U.S. Patent No. 9,840,719 (RHM4-1 description), U.S. Patent No. 7,749,492, U.S. Patent No. 7,588,772 (DJ and DJ8 description), and U.S. Patent No. 9,587,282 AAV capsid variants, all of which are incorporated herein by reference in their entirety. Thus, rAAV vectors contain gene / protein sequences identical to those characteristic of specific serotypes, as well as mixed serotypes.

[0081] In certain embodiments, the nucleic acid encoding the AAV Rep protein and the Cap protein is arranged similarly to the natural AAV genome. In certain embodiments, the nucleic acid encoding the AAV Rep protein and the Cap protein is arranged similarly to the natural AAV genome, except that the nucleic acid encodes a linker and / or degron sequence at the N-terminus or C-terminus of the AAV Rep protein. In certain embodiments, the nucleic acid encoding the AAV Rep protein and the Cap protein is not arranged in a tandem arrangement, directly or indirectly. In certain embodiments, the nucleic acid encoding the AAV Rep protein and the Cap protein is not covalently linked. Additional suitable arrangements of the AAV Rep and Cap coding sequences can be used, for example, the order of the AAV Rep and Cap coding sequences can be reversed relative to their natural AAV genome order, and one or both of the AAV Rep and Cap coding sequences can be before or after a sequence that includes an IRES, a self-cleaving protein (e.g., 2A peptide) coding sequence, or a non-functional stuffer region. In certain embodiments, one or both of the AAV Rep and Cap coding sequences can be integrated into a cell genome. In certain embodiments, one or both of the AAV Rep and Cap coding sequences can be combined with factors that increase DNA sequences that increase maintenance of the episomal plasmid.

[0082] In certain embodiments, helper virus function is provided by a virus selected from adenovirus, herpesvirus, poxvirus, or their hybrid viruses.In certain embodiments, helper virus function comprises one or more viruses, vectors, or plasmids that provide helper virus function.In certain embodiments, helper virus function comprises at least one of adenovirus (Ad) E1 protein (e.g., Ad E1a or Ad E1b protein), Ad E2A protein, Ad E4 protein, and Ad VA RNA.

[0083] Helper virus functions can be provided in a number of different ways: In certain embodiments, helper virus functions can be provided by the virus or are provided in trans to the cell, for example, by a polynucleotide sequence encoding the essential helper function(s).

[0084] In certain embodiments, the degron sequence is linked to the C-terminus of the adenovirus (Ad) E1 protein (e.g., Ad E1a or Ad E1b protein), Ad E2A protein, or Ad E4 protein. In certain embodiments, the degron sequence is linked to the N-terminus of the Ad E1 protein (e.g., Ad E1a or Ad E1b protein), Ad E2A protein, or Ad E4 protein. In certain embodiments, the degron and the Ad E1 protein (e.g., Ad E1a or Ad E1b protein), Ad E2A protein, or Ad E4 protein are linked through a flexible linker. In certain embodiments, the degron and the Ad E1 protein (e.g., Ad E1a or Ad E1b protein), Ad E2A protein, or Ad E4 protein are linked through a rigid linker.

[0085] In certain embodiments, two or more AAV proteins, such as Rep, Cap, and / or helper proteins, are independently linked to a degron sequence to generate two or more AAV protein-degron fusion proteins. In certain embodiments, each of the two or more AAV protein-degron fusion proteins comprises a degron at the C-terminus or N-terminus of the AAV protein. In certain embodiments, each of the two or more AAV protein-degron fusion proteins comprises a different degron sequence. In certain embodiments, each of the two or more AAV protein-degron fusion proteins comprises the same degron sequence. In certain embodiments, the linkage between the AAV protein and its respective degron is a flexible linker. In certain embodiments, the linkage between the AAV protein and its respective degron is a rigid linker.

[0086] In certain embodiments, two or more AAV protein fusion proteins can be encoded by sequences on the same vector, e.g., a plasmid. In certain embodiments, two or more AAV protein-degron fusion proteins can be encoded by sequences on separate vectors, e.g., a first plasmid contains the coding sequence of a first AAV protein-degron fusion, and a second plasmid contains the coding sequence of a second AAV protein-degron fusion. In certain embodiments, the expression of at least one of the AAV protein-degron fusion proteins is under the control of a control element. In certain embodiments, the expression of at least two of the AAV protein-degron fusion proteins is under the control of a control element. In certain embodiments, the expression of each of the multiple AAV protein-degron fusion proteins is under the control of a different control element. In certain embodiments, the expression of all of the AAV protein-degron fusion proteins is under the control of a control element. In certain embodiments, the control element is a promoter. In certain embodiments, the control element is a Tet response element.

[0087] In certain embodiments, the cell of the present disclosure comprises a nucleic acid sequence that is not normally present in the wild-type AAV genome, such as a heterologous nucleic acid sequence, also referred to herein as gene of interest or (GOI).In certain non-limiting embodiments, the GOI comprises a nucleic acid sequence that encodes a therapeutic protein or an inhibitory nucleic acid sequence.In certain embodiments, the GOI can be introduced / transferred into the cell through transduction or transfection of a vector such as AAV.In certain embodiments, the introduced GOI can be present extrachromosomally or only transiently in the recipient cell or host organism.

[0088] In certain embodiments, the GOI encodes a protein (e.g., a therapeutic protein) that is provided in a modified or truncated form, or the GOI is provided in multiple constructs delivered by separate multiple AAV vectors.

[0089] In certain embodiments, the GOI is provided as a truncated variant that maintains the functionality of the encoded protein (e.g., a therapeutic protein) by including removal of portions unnecessary for function, whereby the GOI is reduced in size for packaging within an AAV vector.

[0090] In certain embodiments, the GOI is provided in a split AAV vector, each of which provides nucleic acid encoding a different portion of a protein (e.g., a therapeutic protein), thereby delivering multiple portions of a protein (e.g., a therapeutic protein) that assemble and function in the cell.

[0091] In certain embodiments, GOI is provided by dual AAV vectors using overlapping vector, trans-splicing dual vector or hybrid trans-splicing vector technology.In certain embodiments, two overlapping AAV vectors are used, which combine in cells to generate a complete expression cassette from which full-length protein (e.g., therapeutic protein) is expressed.

[0092] 5.3.AAV-producing cells In another aspect, the subject matter of the present disclosure is directed to an rAAV producing cell. For example, but not limited to, the present disclosure is directed to an rAAV producing cell in which expression of the AAV Rep protein is controlled by fusion of a degradation domain to the AAV Rep protein.

[0093] The embodiments of the rAAV producing cells of the present disclosure include any cell type or system that can produce rAAV or AAV. Some examples of various systems have been previously described, for example, in Conlon and Mavilio, Mol.Therapy,8:181-182 (2018), the contents of which are incorporated herein by reference in their entirety. The embodiments of the present disclosure include the creation of rAAV by transient transfection of plasmids in mammalian cells, the production of rAAV in stable cell lines, the production of rAAV by herpes simplex virus in mammalian cells, and the production of rAAV by baculovirus in Sf9 cells.

[0094] In certain embodiments, the rAAV producing cell of the present disclosure is a eukaryotic cell. In certain embodiments, the rAAV producing cell of the present disclosure is an animal cell. In certain embodiments, the rAAV producing cell of the present disclosure is an insect cell. In certain embodiments, the rAAV producing cell of the present disclosure is a mammalian cell. In certain embodiments, the rAAV producing cell of the present disclosure is a human cell. In certain embodiments, the rAAV producing cell of the present disclosure is a human embryonic kidney (HEK) cell. In certain embodiments, the rAAV producing cell of the present disclosure is a HEK293 cell, a HEK293F cell, or an Expi293 cell.

[0095] In certain embodiments, the rAAV production cells of the present disclosure are Chinese hamster ovary (CHO) cells.

[0096] In certain embodiments, the rAAV production cells of the present disclosure are insect (Sf9) cells.

[0097] In certain embodiments, the rAAV producing cells of the present disclosure do not express the SV40 large T antigen.

[0098] In certain embodiments, the rAAV producing cells of the present disclosure are suspension cells. In certain embodiments, the rAAV producing cells of the present disclosure are adherent cells.

[0099] In certain embodiments, the rAAV producing cells of the present disclosure are at least about 1 × 10 6 Cells / mL, at least approximately 5 x 10 6 Cells / mL, at least approximately 1 x 10 7 cells / mL, or at least about 2 x 10 7 In certain embodiments, the rAAV producing cells of the present disclosure can be cultured at a cell density of about 1×10 6 ~5×10 6 cells / mL, approximately 5×10 6 ~1×10 7 cells / mL, approximately 1×10 7 ~2×10 7 The cells can be cultured at a cell density of 1000 cells / mL.

[0100] In certain embodiments, the rAAV producing cells of the present disclosure are present in culture medium or growth medium.

[0101] In certain embodiments, the rAAV producing cells of the present disclosure are in a medium suitable for storage. In certain embodiments, the rAAV producing cells of the present disclosure are in a medium suitable for long-term storage at 0° C. or lower, -30° C. or lower, -80° C. or lower, or -160° C. or lower.

[0102] In certain embodiments, the present disclosure is directed to a mammalian rAAV production cell, the cell comprising a nucleic acid encoding a fusion protein comprising a Rep protein, a linker, and a degradation ligand-dependent degradation domain. In certain embodiments, the degradation ligand-dependent degradation domain is an FKBP variant protein. In certain embodiments, the degradation ligand is a small molecule ligand. In certain embodiments, the small molecule is Shield1.

[0103] In certain embodiments, the rAAV production cell of the present disclosure is an E1a-expressing cell. In certain embodiments, the E1a-expressing cell is a HEK293 cell.

[0104] In certain embodiments, the ligand-dependent degradation domain of the mammalian rAAV production cell of the present disclosure is fused to the C-terminus of the Rep protein via a linker. In certain embodiments, the ligand-dependent degradation domain of the mammalian rAAV production cell of the present disclosure is fused to the N-terminus of the Rep protein via a linker. In certain embodiments, the linker is a flexible linker. In certain non-limiting embodiments, the flexible linker has the amino acid sequence: GGGSGGGGSGGGGGS. In certain embodiments, the linker is a rigid linker. In certain non-limiting embodiments, the rigid linker has the amino acid sequence: AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAK EAAAKEAAAKA.

[0105] In certain embodiments, the mammalian rAAV production cell of the present disclosure comprises a nucleic acid encoding an AAV Rep protein and a Cap protein arranged similarly to the natural AAV genome. In certain embodiments, the nucleic acid encoding the AAV Rep protein and the Cap protein is arranged similarly to the natural AAV genome, except that the nucleic acid encodes a linker and / or a degron sequence at the N-terminus or C-terminus of the AAV Rep protein. In certain embodiments, the nucleic acid encoding the AAV Rep protein and the Cap protein is not arranged in a tandem arrangement, directly or indirectly. In certain embodiments, the nucleic acid encoding the AAV Rep protein and the Cap protein is not covalently linked. Additional suitable arrangements of the AAV Rep and Cap coding sequences can be used, for example, the order of the AAV Rep and Cap coding sequences can be reversed relative to their natural AAV genome order, and one or both of the AAV Rep and Cap coding sequences can be before or after a sequence that includes an IRES, a self-cleaving protein (e.g., 2A peptide) coding sequence, or a non-functional stuffer region. In certain embodiments, one or both of the AAV Rep and Cap coding sequences can be integrated into the cell genome. In certain embodiments, one or both of the AAV Rep and Cap coding sequences can be combined with factors that increase DNA sequences that increase maintenance of the episomal plasmid.

[0106] In certain embodiments, the mammalian rAAV production cell of the present disclosure comprises a virus capable of helper virus function.In certain embodiments, the virus is selected from adenovirus, herpesvirus, poxvirus, or hybrid virus thereof.In certain embodiments, the mammalian rAAV production cell of the present disclosure comprises one or more viruses, vectors, or plasmids that provide helper virus function.In certain embodiments, the helper virus function comprises at least one of adenovirus (Ad) E1 protein (e.g., Ad E1a or Ad E1b protein), Ad E2A protein, Ad E4 protein, and Ad VA RNA.

[0107] In certain embodiments, the mammalian rAAV production cell of the present disclosure comprises a GOI.In certain non-limiting embodiments, the GOI comprises a nucleic acid sequence encoding a therapeutic protein or an inhibitory nucleic acid sequence.In certain embodiments, the GOI can be introduced / transferred into the cell through transduction or transfection of a vector such as AAV.In certain embodiments, the introduced GOI can be present extrachromosomally or only transiently in the recipient cell or host organism.

[0108] In certain embodiments, the GOI encodes a protein (e.g., a therapeutic protein) that is provided in a modified or truncated form, or the GOI is provided in multiple constructs delivered by separate multiple AAV vectors.

[0109] In certain embodiments, the GOI is provided as a truncated variant that maintains the functionality of the encoded protein (e.g., a therapeutic protein) by including removal of portions unnecessary for function, whereby the GOI is reduced in size for packaging within an AAV vector.

[0110] In certain embodiments, the GOI is provided in a split AAV vector, each of which provides nucleic acid encoding a different portion of a protein (e.g., a therapeutic protein), thereby delivering multiple portions of a protein (e.g., a therapeutic protein) that assemble and function in a mammalian rAAV production cell of the present disclosure.

[0111] In certain embodiments, the GOI is provided by a dual AAV vector using overlapping vector, trans-splicing vector, or hybrid trans-splicing dual vector techniques. In certain embodiments, two overlapping AAV vectors are used that combine in a mammalian rAAV production cell of the present disclosure to generate a complete expression cassette from which a full-length protein (e.g., a therapeutic protein) is expressed.

[0112] Non-limiting examples of heterologous nucleic acids encoding gene products (e.g., therapeutic proteins) useful according to the present invention include those that are associated with "hemostasis" or blood clotting disorders, e.g., hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, deficiency of clotting factors VII, VIII, IX, and X, XI, V, XII, II, von Willebrand factor, combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, gamma-carboxylase deficiency; anemia, trauma, These include those that may be used in the treatment of diseases or disorders including, but not limited to, bleeding associated with injury, thrombosis, thrombocytopenia, stroke, coagulation, or disseminated intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparins, pentasaccharides, warfarin, small molecule antithrombotic agents (i.e., FXa inhibitors); and platelet disorders, such as Bernard-Soulier syndrome, Glanzmann's thrombasthenia, and storage pool deficiency.

[0113] In certain embodiments, the disease or disorder affects or originates in the central nervous system (CNS). In certain embodiments, the disease is a neurodegenerative disease. In certain embodiments, the CNS or neurodegenerative disease is Alzheimer's disease, Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, polyglutamine repeat disease, or Parkinson's disease. In certain embodiments, the CNS or neurodegenerative disease is a polyglutamine repeat disease. In certain embodiments, the polyglutamine repeat disease is spinocerebellar degeneration (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17).

[0114] In certain embodiments, the AAV particles are capable of binding to any of a wide variety of proteins, including insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGFa), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and IGF-II), TGFp, activin, inhibin, bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins, and the like. The heterologous nucleic acid encoding a gene product selected from the group consisting of NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0115] In certain embodiments, the AAV particle comprises a heterologous nucleic acid encoding a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1-IL-17), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factors alpha and beta, interferons alpha, beta, and gamma, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules.

[0116] In certain embodiments, the AAV particles are capable of detecting the following proteins: carbamoyl synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathione beta synthase, branched chain keto acid decarboxylase, albumin, The heterologous nucleic acid encoding a gene product selected from the group consisting of isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H protein, T protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin cDNA sequence.

[0117] In certain embodiments, the AAV particle comprises a heterologous nucleic acid encoding a polypeptide, a nucleic acid encoding a protein or transcribed into a transcript of interest, or a nucleic acid selected from the group consisting of an siRNA, an antisense molecule, an miRNA, a ribozyme, and an shRNA.

[0118] In certain embodiments, the AAV particles comprise a heterologous nucleus encoding a protein selected from the group consisting of GAA (acid alpha-glucosidase) for the treatment of Pompe disease; ATP7B (copper-transporting ATPase 2) for the treatment of Wilson disease; alpha-galactosidase for the treatment of Fabry disease; ASS1 (argininosuccinate synthase) for the treatment of citrullinemia type 1; beta-glucocerebrosidase for the treatment of Gaucher disease type 1; beta-hexosaminidase A for the treatment of Tay-Sachs disease; SERPING1 (C1 protease inhibitor or C1 esterase inhibitor) for the treatment of hereditary angioedema (HAE), also known as C1 inhibitor deficiency types I and II; and glucose-6-phosphatase for the treatment of glycogen storage disease type I (GSDI).

[0119] In certain embodiments, the heterologous nucleic acid is selected from the group consisting of CFTR (cystic fibrosis transmembrane conductance regulator protein), blood clotting (clotting) factors (such as factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C, etc.) gain-of-function blood clotting factors, antibodies, retinal pigment epithelium specific 65 kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporters, (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphoribosyltransferase, beta-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor-3 and -4, brain-derived neurotrophic factor, glial-derived growth factor, transforming growth factor α and β, cytokines, α-interferon, interferon, beta-interferon, interferon-gamma, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), peptides with immunomodulatory properties peptide, tolerogenic or immunogenic peptide or protein Tregitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (choroideremia), LCA5 (LCA-reversilin), ornithine ketoacid aminotransferase (gyriform atrophy), retinoschisin 1 (X-linked retinal detachment), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR type of RP: retinitis pigmentosa), DFNB1 (connexin 26 deafness), ACHM2,3,4 (color blindness), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid activating protein, one or more zinc finger nucleases for genome editing, or one or more donor sequences used as repair templates for genome editing.

[0120] Nucleic acid molecules, vectors such as cloning, expression vectors (e.g., vector genomes) and plasmids can be prepared using recombinant DNA technology methods. The availability of nucleic acid sequence information allows for the preparation of nucleic acid molecules by various means. For example, heterologous nucleic acids encoding factor IX (FIX), including vectors or plasmids, can be produced using a variety of standard cloning, recombinant DNA techniques, cellular expression, or through in vitro translation and chemical synthesis techniques. The purity of the polynucleotides can be determined by sequencing, gel electrophoresis, and the like. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries using probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides with shared structural features, for example, using expression libraries; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the nucleic acid sequence of interest; (4) computer-assisted searches of sequence databases for related sequences; and (5) difference screening of subtracted nucleic acid libraries. EXAMPLES

[0121] Working Example The following examples are merely illustrative of the subject matter of the present disclosure and should not be construed as limiting in any way.

[0122] Example 1: Rep-degron constructs and rAAV production Various constructs were designed to engineer mechanisms for controllable Rep accumulation and higher rAAV production. The Rep transgene was tagged at the N- or C-terminus with a destabilizing domain (degron) to confer instability and target the translated Rep protein product to the proteasome for degradation. Rep protein degradation can be reversed by addition of Shield1 ligand. The degron was engineered from the human gene encoding FK506 binding protein 12 ("FKBP12"). It was previously shown that F36V modification of the protein improves its specificity for Shield1 over FK506 (Clackson et al, PNAS 1998). Subsequent efforts to create a less stable degron motif resulted in the L107P mutation (Banaszynski et al, Cell 2006). The amino acid sequence of the degron used in this example, referred to herein as "FKBP", with the F36V and L107P mutations was as follows: TIFF2024520838000002.tif21170

[0123] Production of rAAV: Recombinant AAV was produced by transfection of human embryonic kidney (HEK293) cells grown in 6-well plates. Cells at 70-90% confluency were transfected with 3 μg of total DNA using jetOPTIMUS® DNA (PolyPlus) transfection reagent. Positive control rAAV produced with LK03Rep / Cap plasmid was produced via a triple transfection method. Plasmids were added in a 1:1:1 ratio (1 helper:1 ITR plasmid:1 rep / cap plasmid). rAAV plasmids produced with split Rep / Cap plasmids were produced using a quadruple plasmid transfection method, where Rep and Cap plasmids were used in equal amounts to each other, but half the amount of helper and ITR plasmids unless otherwise noted (1 helper:1 ITR plasmid:0.5 Rep:0.5 cap). For degron-containing constructs, the wtRep or Rep / Cap plasmid was replaced with the degron containing Rep or Rep / Cap plasmid. After transfection, the cell culture medium was changed the next day. After medium change, the small molecule Shield-1 (Takara) was added to the medium of the corresponding samples, and the cells were grown for an additional 24–36 h to allow AAV production. AAV in crude cell lysate extracts or in the medium of HEK293 cells was used to transduce target cells.

[0124] For the use of rAAV in crude extracts, cells were resuspended in the cell culture medium in which they were growing and the suspension was transferred to a 1.5 ml tube. Instead of detergent-based lysis, cells were lysed by four successive freeze-thaw cycles moving between dry ice and a 37°C water bath. Samples were vortexed after each lysis cycle to enhance cell lysis and release of promoter viral particles. Upon completion of the lysis step, samples were centrifuged at 13,500×g for 10 min in an Eppendorf centrifuge set at 4°C. The supernatant containing the rAAV particles was transferred to a new tube for use in the transduction assay.

[0125] Transduction of cells: Huh7 cells were transduced with equal volumes of cell lysate or cell media containing AAV vectors. Briefly, Huh7 cells were seeded in 24-well plates one day prior to transduction. Up to 50ul of unpurified virus preparation from HEK293 was added to the media of Huh7. Transduction efficiency was assessed in biological duplicates or triplicates. Media was changed the next day and samples were analyzed for transgene expression 48–72 hours after transduction. For analysis of luciferase expression, Huh7 cells were lysed in passive lysis buffer (Promega) and each biological sample was split and plated into four wells of a 96-well luminescence assay plate. Renilla luciferase levels were determined using a Renilla luciferase assay kit (Promega) and analyzed with a microplate luminometer equipped with an injector (Spectramax).

[0126] Cloning of plasmid constructs: Cloning of constructs carrying degron motifs was performed using the Gibson assembly method. Briefly, the designed degron-linker sequences with the desired homology regions were ordered as gBlocks fragments from Integrated DNA Technologies. The backbone containing Rep-only or Rep / Cap plasmids and the gblocks were ligated using the NEBuilder Hifi DNA Assembly Kit (New England Biolabs, #E5520). Upon plasmid purification and sequence verification, the plasmids with the correct sequences were used for rAAV production experiments.

[0127] As shown in Figures 1 and 2, the REP and Cap genes were cloned either into the same plasmid or into separate plasmids. The aim was to control Rep expression without altering Cap expression. Plasmids were constructed with degron sequences cloned either C-terminally or N-terminally to the Rep sequence (Figure 1). Two types of linkers were tested; a rigid linker and a flexible linker. The P40 promoter was used to drive expression of Cap. For Rep, in some cases the p5 promoter was used while in others the CMV promoter was used as indicated.

[0128] Transfection of cells with only the transgene plasmid, as shown in Figures 1 and 2, was used as a negative control. Shield1 was added to selected wells (Figure 2). Media or cell lysates from rAAV-producing HEK293 cells were used to transduce Huh7 cells (hepatocytes, hepatocellular carcinoma). Huh7 cells were then lysed and luciferase activity was measured for detection of transgene activity.

[0129] Example 2: rAAV produced by transfection of Rep and Cap on separate plasmids The method of Example 1 was used to determine whether rAAV could still be produced by transfection of Rep and Cap on separate plasmids, except as specifically noted. Rep / Cap plasmids without a degron, Rep-only plasmids without a degron, and Cap-only plasmids were tested. Rep was under the control of the CMV promoter. Cap contained the CMV promoter (the end of Rep is missing, CMV-Cap ( *) and the P40 promoter (P40-Cap with the Rep end present). As shown in Figure 3, P40-Cap can support rAAV production as determined by target cell transduction. However, CMV-Cap cannot support rAAV production. Figure 3 shows that rAAV can be produced by transfection of Rep and Cap on separate plasmids.

[0130] Example 3: Control of rAAV production by addition of Shield1 The method of Example 1 was used to determine whether the addition of Shield1 could exert post-translational control on the accumulation of Rep-degrons, except where specifically stated. Constructs were transfected into HEK293 cells with or without Cap plasmid (Figure 4). The addition of Shield1 aided in rAAV production and transduction of target cells. As shown in Figure 4, a Rep-only plasmid C-terminal degron was used. Transduction efficiency was approximately 15-20% of the wtRep / Cap plasmid.

[0131] Example 4: Regulation of various Rep constructs The method of Example 1 was used to determine whether the location of the degron (i.e., C-terminally located or N-terminally located) affects post-translational control of Rep expression, except where specifically stated. Huh7 cells were transduced with supernatant from HEK293 cells as described above. P40-Cap plasmid was used to drive Cap expression. As shown in Figures 5 and 10, rAAV production from Rep constructs with N-terminally attached degrons shows less tightly regulated expression compared to Rep constructs with C-terminally attached degrons.

[0132] The method of Example 1 was used to determine whether Shield1 affects post-translational control of Rep expression, except where specifically noted, in conjunction with constructs containing both Rep and Cap coding sequences versus constructs having only Rep coding sequences. The P40-Cap plasmid was used to drive Cap expression, provided with the "CapP plasmid" (denoted as "+"). As shown in FIG. 6, despite a slight increase in rAAV production when Rep containing a C-terminally linked degron is expressed in the presence of Cap (either expressed from a single Rep / Cap construct or Cap expressed in trans), rAAV production is significantly increased when Rep containing a C-terminally linked degron is expressed in the presence of both Cap and Shield1. Again, Cap can be expressed from a single Rep / Cap construct or Cap expressed in trans.

[0133] The method of Example 1 was used, except as specifically noted, to determine the effect on rAAV production of adding increasing amounts of a plasmid encoding a Rep containing a C-terminally linked degron. As shown in Figure 7, increasing amounts of a plasmid encoding a Rep containing a C-terminally linked degron reduced overall rAAV production and resulted in less differential regulation by Shield1.

[0134] Example 5: Control of Rep constructs with rigid or flexible linkers The method of Example 1 was used to determine the effect of the type of linker between the C-terminus of Rep and the degron, except where specifically stated. Two types of linkers were tested; rigid linker and flexible linker. Figure 8 shows the results for Rep lacking a degron (+ / -Shield1); Rep with a tightly bound C-terminal degron (+ / -Shield1); Rep with a flexibly bound C-terminal degron (+ / -Shield1); and a control where no Rep or Cap was provided and no dual Rep / Cap plasmid (expression is driven by the P5 distal promoter). Figure 9 shows the results for Rep lacking a degron (+ / -Shield1); Rep with a tightly bound N-terminal degron (+ / -Shield1); Rep with a flexibly bound N-terminal degron (+ / -Shield1); and a control where no Rep or Cap was provided and no dual Rep / Cap plasmid (expression is driven by the P5 distal promoter). In both Figures 8 and 9, the use of a flexible linker is associated with Shield1-mediated post-translational control of Rep expression, resulting in rAAV production.

[0135] Example 6: Analysis of Rep expression and AAV production A. Materials and Methods A. Transfection Expi293 cells were seeded at 1.4E6 viable cells / mL in Expi293 medium the day before transfection. Transfection was performed when viable cells were 2E6-3E6 cells / mL. PEIpro transfection reagent (Polyplus), OptiMEM (Gibco) was used. The PEI:DNA ratio was 2, and 0.6 μg of DNA per million cells was used. Triple transfections were performed with RHM4-1 Rep Cap, Helper and Gaussia luciferase plasmids. Split Rep and Cap plasmids were used in a ratio of 1:4 (Rep:Cap). In some cases, split Rep Cap plasmids and TIR1 or CMV-Tet were used in a ratio of 1:3:1 (Rep:Cap:TIR1 or CMV-Tet). 1.5 mM SAHA was added after the addition of DNA-PEI complexes. In some cases, Shield1 or TMP or auxin and / or doxycycline were added the next day. For doxycycline-treated samples, an additional treatment of Dox was performed 24 hours after the first treatment. Cells were harvested at 4, 8, and 24 hours after treatment and at 44, 68, or 72 hours after transfection, centrifuged, and washed with cold PBS. These cell pellets were stored in a -80° freezer. For qPCR, 1 mL of cells was harvested at 44, 68, or 72 hours after transfection.

[0136] B. Western blot Cell pellets were lysed in RIPA buffer (Pierce) containing protease and phosphatase inhibitors and EDTA for 30 min on ice. Lysates were centrifuged at maximum speed for 15 min and the supernatant was transferred to a new Eppendorf tube and used for Western blot. Protein concentration was measured with a BCA kit (Pierce). 30 μg of protein was loaded per lane of an SDS-PAGE gel. These gels were transferred to PVDF or nitrocellulose membranes. Membranes were blocked with blocking buffer (Li-Cor) for 30 min at room temperature. Anti-AAV Rep clone 303.9 (ARP, catalog no. 03-61069) purified mouse monoclonal antibody was diluted 500-fold in either blocking buffer (Li-Cor) or antibody diluent (Li-Cor). Membranes were incubated overnight in a cold room with or without shaking. The next day, the membrane was washed three times for 10 min each with 1x TBST buffer (Invitrogen). Goat anti-mouse Alexa Flour 680 secondary antibody was diluted 1000x in either blocking buffer (Li-Cor) or antibody diluent (Li-Cor). The membrane was incubated for 45 min at room temperature with rocking. The membrane was then washed six times for 10 min each with 1x TBST buffer. The membrane was scanned with an Odysset (Li-Cor). Anti-GAPDH rabbit antibody (Cell Signaling) was diluted 5000x in either blocking buffer (Li-Cor) or antibody diluent (Li-Cor) as a loading control. Primary incubation was 1 h at room temperature with rocking. Goat anti-rabbit Alexa Flour 800 secondary antibody was diluted 10000x in either blocking buffer (Li-Cor) or antibody diluent (Li-Cor).

[0137] c.qPCR Frozen harvested cells were thawed and sonicated. 300 μL of sonicated cells were treated with benzonase for 1 h in a 37°C incubator on a rotator / shaker. DNaseI treatment was then performed for 15 min. The reaction was stopped with stop reagent (0.2% SDS / 5 mM EDTA / 0.2 M NaCl), heated to 95°C for 10 min and centrifuged. Serial dilutions were performed to 10,000 or 100,000 fold. Taqman qPCR was performed using specific primers and probes using a Quant Studio real-time PCR machine.

[0138] B. FKBP degron-tagged Rep protein expression and AAV production In this study, we investigated the control of AAV production via Rep proteins containing FKBP-derived degrons. As a preliminary matter, Figure 11 illustrates the molecular weight change of Rep proteins upon tagging with degrons. Specifically, Figure 11 displays the possible size changes in Western blots after addition of a degron tag to the N- or C-terminus of Rep proteins. Addition of a degron to the N-terminus of Rep proteins (N-degron) altered the molecular weight of the large Rep protein but not the small Rep protein. The molecular weight of the small Rep protein is not affected by the N-terminal protein tag because the p19 promoter that drives expression of small Rep is located within the Rep gene. The small Rep protein does not share the same N-terminal sequence as the large Rep protein. In contrast, addition of a degron to the C-terminus of Rep protein (C-degron) alters the molecular weight of both the small and large Rep proteins because both C-termini are the same. Addition of small molecules such as Shield1 or TMP to cell cultures can inhibit protein degradation and band intensity will change accordingly. The example shown here is an FKBP-derived degron.

[0139] In this study, RHM4-1 was used as the AAV capsid according to the materials and methods presented in section 6A above. As displayed in Figure 12A-12D, "RHM4-1" and "RHM4-1v2" are plasmids expressing both the Rep protein and RHM4-1 Cap without the degron. In contrast, the "p40Cap plasmid" expresses only the RHM4-1 capsid, while the "pAAV2 Rep plasmid" expresses the Rep protein from AAV2. The ITR plasmids used in these studies carry Gaussia luciferase as a transgene.

[0140] Figure 12A displays a western blot of N-terminal FKBP degron-tagged Rep protein. In this study, a plasmid expressing an N-terminal degron-containing Rep (N-degron Rep) construct was used for AAV production. Samples were harvested 4, 8, or 24 hours after the addition of Shield1 to the medium. The molecular weight and expression of small Rep (Rep52) is not affected by the presence or absence of the degron, as described in Figure 11 (samples 1-6 vs. samples 7-11). Control samples produced using Rep without the degron are loaded in lanes 7-11. Due to the unique plasmid design, large Rep (Rep78) expression is much lower than small Rep expression in all samples. Large Rep expression from N-degron Rep samples is increased after the addition of Shield1 (samples 4-6) compared to untreated controls (samples 1-3). The molecular weight of the large Rep protein is larger for samples with N-degron Rep (samples 1-6 vs. 7-11), but no change in the molecular weight of the small Rep is observed in these samples, as illustrated in Figure 11.

[0141] Figure 12B displays a Western blot of C-terminal FKBP degron-tagged Rep proteins. In this study, a plasmid expressing a C-terminal degron-containing Rep construct (C-degron Rep) was used for AAV production, and samples were taken at different time points (4, 8, and 24 h) after induction with Shield1. Expression of small and large Rep is higher with Shield1 (samples 4–6) than with untreated controls (samples 1–3) at the same time points. The molecular weights of small and large Rep are larger with the C-terminal FKBP degron, and a shift was observed compared to samples with Rep proteins that do not contain a degron (samples 1–6 vs. 7–11).

[0142] Figure 12C displays the AAV titers of samples from Figures 12A and 12B. AAV titers increase upon addition of Shield1 molecules to the medium used for AAV production. In this study, cells were transfected with Rep-degron-containing plasmids along with other plasmids required for AA production. Addition of Shield1 molecules increases AAV titers for N- and C-terminal degron constructs compared to untreated controls.

[0143] FIG. 12D displays the AAV titers observed with Rep and Cap plasmids transfected at different ratios. In this study, degron-tagged Rep proteins are controlled in cells transfected with different ratios of Rep and Cap plasmids. As illustrated in the results, AAV production can be controlled through degron-tagged Rep proteins provided as separate plasmids transfected at different ratios into HEK293 cells, as listed at the bottom of the figure. Titers were normalized to titers from control transfections using Rep / Cap plasmids without degrons. 500 nM Shield1 was added to the samples to inhibit Rep degradation.

[0144] C. ecDHFR degron-tagged Rep protein expression and AAV production In this study, we investigated the regulation of AAV production mediated by the Rep protein, which contains an ecDHFR-derived degron with the following amino acid sequence: The TIFF2024520838000003.tif27170ecDHFR-derived degron has the following DNA sequence: TIFF2024520838000004.tif71170In this study, RHM4-1 was used as the AAV capsid according to the Materials and Methods presented in Section 6A above.

[0145] Figure 13A displays a Western blot of N-terminal ecDHFR degron tagged Rep protein. In this study, an N-terminal ec degron containing Rep (N-ec degron Rep) construct is used for AAV production, with the Cap gene provided as a separate plasmid. Small Rep expression is not affected by the N-terminal ec degron fusion (samples 1-6) as described in Figure 11. Large Rep expression is higher at the 24 hour time point for samples treated with 5 μM TMP (sample 3 vs. sample 6). The molecular weight of large Rep is larger when it is tagged (N-terminal) with an ecDHFR degron, and a shift is observed for those samples (samples 1-6). As expected, no shift is observed for the small Rep protein, as its molecular weight remains the same.

[0146] Figure 13B displays a Western blot of C-terminal ecDHFR degron-tagged Rep proteins. In this study, a C-terminal ec degron-containing Rep (C-ec degron Rep) construct is used for AAV production, with the Cap gene provided as a separate plasmid. Expression of small and large Rep is greater at 5 µM TMP (sample 3 vs. sample 6). The molecular weights of small and large Rep are greater with the C-terminal ecDHFR degron (samples 1-6), and a shift is observed for both proteins.

[0147] Figure 13C displays the AAV titers of samples from Figures 13a and 13b. Titer levels of AAV produced via plasmids containing ecDHFR degron-containing Rep were analyzed. Addition of TMP increases AAV titers of C-terminal degron constructs, correlating with the Western blot data for Rep expression.

[0148] D. Auxin or ecDHFR degron-tagged Rep protein expression and AAV production In this study, we investigated the control of AAV production via Rep proteins carrying either an auxin-derived degron or an ecDHFR-derived degron. The auxin-derived degron has the following amino acid sequence: TIFF2024520838000005.tif21170 The auxin-derived degron has the following DNA sequence: TIFF2024520838000006.tif71170In this study, RHM4-1 was used as the AAV capsid according to the Materials and Methods presented in Section 6A above.

[0149] Figure 14A displays a Western blot of Rep tagged with an auxin-inducible degron or an ecDHFR degron. The size of small and large Rep is larger in the C-terminal auxin-inducible degron-containing Rep plasmid (samples 3-6) and a shift is observed for both proteins. The change in Rep expression for the auxin-inducible degron is difficult to observe due to the presence of a non-specific band with the same size as the degron-tagged small Rep. This non-specific band is present even in the untransfected sample (sample 9). The expression of small and large Rep is more in TMP (samples 7 and 8) in the case of the C-terminally tagged ecDHFR degron. The size of small and large Rep is larger in the C-terminal ecDHFR degron (samples 7 and 8) and a shift is observed.

[0150] Figure 14B displays the AAV titers of the samples from Figure 14A. As illustrated in Figure 14B, the auxin-inducible degron and the ecDHFR degron can control AAV titers, where the auxin-inducible degron functions together with the auxin-dependent degron only if auxin and TIR1 ubiquitin ligase are present in the cell. The amino acid sequence of the TIR-1 protein is as follows: Unlike FKBP and ecDHFR-dependent degrons, which inhibited protein degradation upon addition of small molecules, the auxin-inducible degron leads to the degradation of the protein tagged to it. For the auxin-inducible degron to function, auxin, TIR1, and the degron must be present. AAV production is undetectable for the sample in which all components of the auxin-mediated degron (degron, auxin, and TIR1) are present (sample 4). This indicates the functionality of this degron for the control of AAV production at the C-terminus of the Rep protein. The ecDHFR degron-tagged Rep was able to produce AAV in the presence of TMP molecules (sample 8), which was undetectable without TMP molecules (sample 7).

[0151] E. Rep protein expression and AAV production in the presence of Shield1 or dTag13 Figure 15 displays the effect of different doses of Shield1 and dTag13 on AAV production. In this study, Rep constructs containing C-terminally tagged degrons were tested with different doses of Shield1 and dTag13. Different Shield1 levels were compared to see the effect on titer. Shield1 is more effective at a concentration of 500 nM to increase AAV production. dTag13 was also tested, which is a molecule that binds to several FKBP-derived domains and further causes their degradation. In this study, dTag13 did not decrease the basal level of AAV production.

[0152] F. Rep protein expression and AAV production under transcription level control system and / or degron level control Figure 16A displays a Western blot of Rep containing a C-terminal degron under the control of the TRE3G promoter. The DNA sequence of the TRE3G promoter is as follows: TIFF2024520838000008.tif56170In this study, as shown in Figure 18A, a Tet response element-containing promoter (TRE3G) is cloned in front of the Rep gene. This promoter can be activated in the presence of Tet protein and doxycycline induction. Rep containing a C-terminal degron under Tet response element promoter control has been shown to be functional for AAV production control. Large Rep is under the control of the Tet promoter, and expression of small Rep is provided by the p19 promoter (samples 1-5). Expression of large Rep is tightly controlled by this system and can be detected upon addition of doxycycline (samples 3 and 4). Shield1 can upregulate expression of small Rep (sample 1 vs. sample 2) and large Rep (sample 3 vs. sample 4). A separate plasmid was used to supply the Tet-On 3G transactivator protein, where expression of the transactivator protein is under the control of the ubiquitous CMV promoter (samples 1-4). The amino acid sequence of the transactivator protein is as follows: TIFF2024520838000009.tif34170The DNA sequence of the CMV promoter is as follows: TIFF2024520838000010.tif101170

[0153] Figure 16B displays the AAV titers of samples from Figure 16A. This study shows that using C-terminal degron-tagged Rep under control of the TRE3G promoter, AAV production can be controlled, and that doxycycline induction can increase AAV production approximately 29-fold from basal levels. The levels of AAV titers from these samples are comparable to control samples that do not contain a degron (samples 6-8).

[0154] Figure 17A displays a Western blot of Rep constructs containing a C-terminal degron. In this study, we tested the effect of different doxycycline levels on AAV production from Rep-degron constructs under TRE3G promoter control. Large Rep is under TRE3G promoter control (samples 1-10). Doxycycline effectively induces expression of large Rep (samples 3-8). Expression of large and small Rep is increased in the presence of Shield1 (samples 2, 4, 6, and 8 vs. samples 1, 3, 5, and 7). Omitting the plasmid expressing the Tet-On 3G transactivator protein from the transfection results in doxycycline-induced inactivation (samples 9 and 10).

[0155] Figure 17B displays the AAV titers of the samples from Figure 17A. This study illustrates that Rep under the control of TRE3G and degron can produce amounts of AAV comparable to the positive control uncontrolled Rep / Cap plasmid. Different doxycycline levels can increase AAV titers with or without Shield1 molecules, but basal levels are lower in the absence of Shield1 (sample 1), as evidence of tighter control by the dual system.

[0156] Figure 18A shows the effect of the p5 promoter on Rep protein levels in the TRE3G-Rep-degron system. In this study, plasmids containing degron-tagged Rep constructs under the control of the TRE3G promoter were generated with or without the p5 promoter 3' of the Rep gene.

[0157] Figure 18B displays a Western blot of Rep constructs containing a C-terminal degron and the TRE3G promoter. In this study, large Rep is under TRE3G promoter control (samples 1-8). Doxycycline effectively induces the expression of large Rep (samples 3-4 and 7-8), and the expression of large Rep is further increased by the addition of Shield1 (samples 4 and 8). As illustrated in the figure, both plasmids (with or without the p5 promoter in 3' of the Rep gene) are capable of expressing large and small Rep.

[0158] Figure 18C displays the AAV titers of the samples from Figure 18B. This study shows that in transient transfection using the TRE3G system, the P5 promoter may not be essential for AAV production. As illustrated in the figure, both plasmids can produce comparable levels of AAV titers. Again, Gaussia luciferase was used as the gene of interest (GOI).

[0159] Figures 20A-20B display codon-modified Rep with a degron motif and show that such constructs can be used for AAV production. The figures also show a second AAV rep gene, a small Rep under the control element and a degron domain. The results achieved with these constructs demonstrate that the disclosed method can be customized to work with multiple control elements (whether they are the same or different), multiple degron domains (whether they are the same or different), and modified or unmodified genes encoding AAV proteins.

[0160] Example 7: Degron-tagged helper genes for controlled AAV production As illustrated in this example, degron-tagged helper genes can be used to control AAV production. Classical helper plasmids used for AAV production contain E2A, E4 and VA genes. In this example, open reading frames from E2A, E4 and VA genes were cloned into separate plasmids. The open reading frames selected for E2A, E4 and VA are DBP, E4-E34K and VA2, respectively. The triple transfection method described in Examples 1-6 was modified to allow E2A-DBP, E4-E34K or VA2 genes to be supplied in separate plasmids instead of a single helper plasmid for use in triple production.

[0161] A. Materials and Methods For the present degron study utilizing helper genes, DNA binding protein (DBP), E4-E34K genes and VA2 were selected as open reading frames for E2A, E4 and VA genes, respectively. These selected open reading frames from E2A, E4 and VA genes were cloned into separate plasmids under the control of the CMV promoter. The degron from FKBP was cloned into the C-terminus of the E2A-DBP gene.

[0162] The triple transfection method was modified to allow the E2A-DBP, E4-E34K and VA2 genes to be supplied in three separate plasmids instead of a single helper plasmid. For transfection and cell lysis, the same reagents and methods were used as for the Rep-degron experiments described herein. The molar ratios for the plasmids used for quintuple transfection with separate helper plasmids (ITR-GOI:Rep / Cap:DBP:E34K:VA2) were 2:2:1:1:1. The molar ratios for the plasmids used for triple transfection with a single helper plasmid (ITR-GOI:Rep / Cap:Helper) were 2:2:1. Negative control samples in which AAV production was suppressed were achieved by transfecting cells with only the ITR-GOI plasmid (i.e., the helper and Rep / Cap plasmids were omitted). For DBP Western blot analysis of the experimental results, anti-DBP polyclonal antibody (CSB-PA365892ZA01HIL) from CusaBio was used.

[0163] B. Experimental Results As shown in FIG. 19A, the DBP protein expressed from the E2A gene was tagged with a FKBP-derived degron motif. The plasmid expressing the E2A-DBP-degron was transfected together with other plasmids expressing Rep / Cap, ITR-GOI, E4-E34K and VA2. At the end of 72 hours, the cells were lysed and the AAV titer levels were analyzed. Cells transfected only with the ITR-GOI plasmid (i.e., excluding the helper and Rep / Cap plasmids) were used as a negative control sample. Without the addition of Shield1 molecules, AAV production is observed at the same background level as the qPCR results resulting from the negative control (dark bars on the left vs. bars on the right). Upon addition of Shield1 molecules, the titer level increases approximately 3-fold, providing a control for AAV production using E2A-DBP with a degron tag (striped bars in the middle).

[0164] Figure 19B displays a Western blot highlighting the shift in protein size of the E2A-DBP protein due to the addition of the degron tag. The tagged protein is approximately 12 kDa larger than the untagged DBP protein. The two samples on the left are untagged DBP, while the sample on the right is a sample of DBP containing a degron.

[0165] C. Sequences of helper genes used E2A-DBP nucleic acid sequence TIFF2024520838000011.tif225170E2A-DBP amino acid sequence TIFF2024520838000012.tif71170E4-34K (ORF6) nucleic acid sequence TIFF2024520838000013.tif123170E4-34K (ORF6) amino acid sequence TIFF2024520838000014.tif58170VA2 Nucleic acid sequence: TIFF2024520838000015.tif64170VA2 Amino acid sequence: TIFF2024520838000016.tif21170

[0166] All publications, patents, and other references cited herein are incorporated by reference in their entirety into this disclosure.

Claims

1. A method for controlling the production of recombinant adeno-associated virus (rAAV) vector particles, the method comprising: a) introducing into a cell a. an rAAV containing a gene of interest, and b. a nucleic acid encoding a fusion protein, the fusion protein comprising an AAV protein and a degradation ligand-dependent degradation domain ; b) culturing the cell under conditions suitable for producing rAAV vector particles; c) contacting the cell with a degradation ligand, the degradation ligand binding to the degradation domain and controlling the expression of the AAV protein, thereby controlling the production of rAAV vector particles. A method comprising.

2. The method according to claim 1, wherein the nucleic acid encodes a fusion protein, the fusion protein comprising an AAV protein, a linker, and a degradation ligand-dependent degradation domain.

3. The method according to claim 1, wherein the AAV protein is Cap.

4. The method according to claim 1, wherein the AAV protein is a helper protein.

5. The method according to claim 4, wherein the helper protein is E2.

6. The method according to claim 1, wherein the AAV protein is Rep.

7. The degradation ligand-dependent degradation domain is (i) derived from FK506 binding protein (FKBP); (ii) dihydrofolate reductase (DHFR); or (iii) an auxin-inducible degradation domain. The method according to claim 1. **Claim 8**: The method according to claim 7, wherein the cleavage ligand is Shield1, trimethoprim (TMP), or auxin. **Claim 9** The method according to claim 1, wherein the cell is an E1a-expressing cell. **Claim 10** The method according to claim 9, wherein the E1a-expressing cell is a HEK293 cell. **Claim 11** The method according to claim 6, wherein the Rep protein is a Rep78, Rep68, Rep52, or Rep40 protein. **Claim 12** The method according to claim 6, wherein the cleavage ligand-dependent cleavage domain is fused to the C-terminus or N-terminus of the Rep protein. **Claim 13** The method according to claim 3, comprising introducing into the cell a nucleic acid encoding a Cap protein. **Claim 14** The method according to claim 13, wherein the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using at least one plasmid. **Claim 15** The method according to claim 13, wherein the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using the same plasmid. **Claim 16** The method according to claim 13, wherein the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using separate plasmids. **Claim 17** The method according to any one of claims 1 to 16, wherein the rep, cap, or helper gene is under the control of a control element. **Claim 18** The method according to claim 17, wherein the control element is a promoter. **Claim 19** The method according to claim 17, wherein the control element comprises a Tet-responsive binding element. **Claim 20** The method according to claim 1, wherein the cell is a eukaryotic cell. **Claim 21** The method according to claim 20, wherein the eukaryotic cell is an animal cell. **Claim 22** The method according to claim 21, wherein the animal cell is a mammalian cell. **Claim 23** The method according to claim 22, wherein the mammalian cell is a HEK cell. **Claim 24** The method according to claim 22, wherein the mammalian cell is a Chinese hamster ovary cell. **Claim 25** An rAAV-producing cell, wherein the cell comprises a nucleic acid encoding a fusion protein comprising an AAV protein and a degradation ligand-dependent degradation domain. **Claim 26** The rAAV-producing cell according to claim 25, wherein the nucleic acid encodes a fusion protein comprising an AAV protein, a linker, and a degradation ligand-dependent degradation domain. **Claim 27** The rAAV-producing cell according to claim 25, wherein the AAV protein is selected from the group consisting of Rep, Cap, and helper protein. **Claim 28** The rAAV-producing cell according to claim 27, wherein the AAV protein is Cap. **Claim 29** The rAAV-producing cell according to claim 27, wherein the AAV protein is a helper protein. **Claim 30** The rAAV-producing cell according to claim 27, wherein the AAV protein is Rep. **Claim 31** The rAAV-producing cell according to claim 25, wherein the degradation ligand is a small molecule ligand. **Claim 32** The cleavage ligand-dependent cleavage domain is (i) derived from FK506 binding protein (FKBP); (ii) dihydrofolate reductase (DHFR); or (iii) an auxin-inducible cleavage domain, The rAAV-producing cell according to claim 25.

33. The rAAV-producing cell according to claim 32, wherein the cleavage ligand is Shield1, trimethoprim (TMP), or auxin.

34. The rAAV-producing cell according to claim 25, wherein the cell is a eukaryotic cell.

35. The rAAV-producing cell according to claim 34, wherein the eukaryotic cell is an animal cell.

36. The rAAV-producing cell according to claim 35, wherein the animal cell is a mammalian cell.

37. The rAAV-producing cell according to claim 36, wherein the mammalian cell is a HEK cell.

38. The rAAV-producing cell according to claim 36, wherein the mammalian cell is a Chinese hamster ovary cell.

39. The rAAV-producing cell according to claim 25, wherein the cell is an E1a-expressing cell.

40. The rAAV-producing cell according to claim 39, wherein the E1a-expressing cell is a HEK293 cell.

41. The rAAV-producing cell according to any one of claims 25 to 33, wherein the ligand-dependent cleavage domain is fused to the C-terminus or N-terminus of the Rep protein via a linker.

42. The rAAV-producing cell according to claim 35, further comprising introducing a nucleic acid encoding a Cap protein into the cell.

43. The rAAV-producing cell according to claim 42, wherein the cell is a mammalian cell, and the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using at least one plasmid.

44. The rAAV-producing cell according to claim 42, wherein the cell is a mammalian cell, and the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using the same plasmid.

45. The rAAV-producing cell according to claim 42, wherein the cell is a mammalian cell, and the nucleic acid encoding the fusion protein and the nucleic acid encoding the Cap protein are introduced into the cell using separate plasmids.

46. The rAAV-producing cell according to claim 35, wherein the cell is a mammalian cell, and the rep, cap, or helper gene is under the control of a control element.

47. The rAAV-producing cell according to claim 46, wherein the control element is a promoter.

48. The rAAV-producing cell according to claim 46, wherein the control element comprises a Tet-responsive binding element.

49. (a) Two or more different AAV proteins are independently fused to a cleavage ligand-dependent cleavage domain; or (b) Each different AAV protein is independently fused to a different cleavage ligand-dependent cleavage domain, The rAAV-producing cell according to any one of claims 25 to 40 or 42 to 48.

50. (a) Two or more different AAV proteins are independently fused to a cleavage ligand-dependent cleavage domain; or (b) Each different AAV protein is independently fused to a different cleavage ligand-dependent cleavage domain, The method according to any one of claims 1 to 16.

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