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  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
  • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

• the present inventions provide eukaryotic cells that comprise adeno-associated virus (AAV) polynucleotides, including AAV capsid proteins (Cap).
• AAV adeno-associated virus
• Cap AAV capsid proteins
• the cells are capable of expressing the polypeptides encoded by the AAV polynucleotides, and thereby are capable of producing AAV, including recombinant AAV.
• the eukaryotic cells also may comprise adenovirus (Ad) polynucleotides.
• the present inventions also provide methods of expressing AAV polynucleotides, as well as Ad polynucleotides, in eukaryotic cells.
• the present inventions also provide methods for producing recombinant adeno-associated virus utilizing eukaryotic cells that express AAV and Ad polypeptides encoded by polynucleotides, as well as recombinant AAV produced by these inventive methods.
• the present inventions further provide other products and methods described herein.
• Adeno-associated virus is a non-enveloped, singlestranded DNA virus and is used as a gene delivery vector for both research and therapeutics.
• Gene transfer vectors based on AAV have demonstrated promise for human gene therapy based on their safety profile and potential to achieve long-term efficacy in animal models. Wang etal., Nature, 18: 358-78 (2019).
• a major challenge for advancing AAV-based therapies into clinical development is the difficulty and cost of producing sufficient quantities of AAV through transient methodologies.
• AAV has been produced in HEK 293, BHK, human amniotic (for example, epithelial cells such as HAEpiC) and SF9 lines.
• expression is transient due to the use of plasmid vectors containing the necessary AAV and helper virus genes.
• plasmid vectors containing the necessary AAV and helper virus genes For example, recombinant AAV production in HEK 293 cells using adenovirus helper gene products utilizes adenovirus E2A, E4, VA RNA and AAV Rep and Cap, along with AAV inverted terminal repeats (ITR) flanking the polynucleotide of interest.
• ITR AAV inverted terminal repeats
• the AAV genome includes a capsid gene referred to as “Cap” or “CAP”.
• Cap in nature is translated to produce, via alternative start codons and transcript splicing, three size-variant structural proteins referred to as VP1 (about 90kDa), VP2 (about 72kDa) and VP3 (about 60kDa).
• An AAV capsid contains 60 subunits total of the VP proteins.
• a ratio of 1 :1 :10 is considered the most typical ratio for VP1 :VP2:VP3, which is a stoichiometry of 5 VP1 subunits:5 VP2 subunits:50 VP3 subunits.
• Wbrner et al. Nature
• AAV polynucleotides and proteins, including CAP, can be selected from any serotype.
• the present inventions provide stable eukaryotic cells, such as mammalian cells (for example, primate, rodent and canine cells), comprising integrated AAV polynucleotides and Ad polynucleotides.
• AAV polynucleotides for example, primate, rodent and canine cells
• the protein and VA RNA products of the Ad polynucleotides act in a helper capacity. All AAV and Ad types are amenable for use according to the present inventions.
• the present inventions advantageously can employ site-specific integration into the cell genome, which refers to pre-selected genomic sites for exogenous DNA to be inserted into a cellular genome. Random insertion can be employed as well.
• polynucleotides where each can comprise (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, (iv) a polynucleotide encoding Adeno-associated virus (AAV) Cap protein, and (v) a polyadenylation site.
• the polynucleotide can be in a CHO cell and have the (i) promoter, (ii) intron, (iii) internal ribosome entry site and (iv) polynucleotide encoding AAV Cap protein can be operably linked.
• the polynucleotide can be integrated into a CHO cell genome, such as a CHO chromosome.
• the polynucleotide can be in a HEK 293 cell and have the (i) promoter, (ii) intron, (iii) internal ribosome entry site and (iv) polynucleotide encoding AAV Cap protein operably linked.
• the polynucleotide can be integrated into a HEK 293 cell genome, such as a HEK 293 cell chromosome.
• polynucleotide can be in a BHK cell and have the (i) promoter, (ii) intron, (iii) internal ribosome entry site and (iv) polynucleotide encoding AAV Cap protein operably linked.
• the polynucleotide can be integrated into a BHK cell genome, such as a BHK cell chromosome.
• the polynucleotide can be in a human amniotic cell and have the (i) promoter, (ii) intron, (iii) internal ribosome entry site and (iv) polynucleotide encoding AAV Cap protein operably linked.
• the polynucleotide can be integrated into a human amniotic cell genome, such as a human amniotic cell chromosome. Additionally, the polynucleotide can be integrated into non-chromosomal locations as known by the person skilled in the art, such as episomes.
• the polynucleotide can further comprise an operator.
• the promoter can be a CMV promoter and the operator can be a Tet operator.
• AAV proteins and polynucleotides, including CAP can be selected from any serotype.
• Cap is from serotype 5 (“Cap5”) and expressed in a CHO cell
• the polynucleotide allows for production of AAV Cap5 VP2 and VP3 protein, wherein the amount of VP3 produced is greater than the amount of VP2 produced.
• Production of VP1 can be less than 1% the level of VP2 production. Production ratios can vary based upon experimental conditions and analytical techniques.
• polynucleotides are described, wherein each can comprise (i) a promoter, (ii) an intron, (iii) a first internal ribosome entry site, (iv) a first polynucleotide encoding AAV Cap protein, (v) a second internal ribosome entry site, (vi) a second polynucleotide encoding AAV Cap protein, and (vii) a polyadenylation site.
• the polynucleotide can be in a CHO cell and the (i) promoter, (ii) intron, (iii) first internal ribosome entry site, (iv) first polynucleotide encoding AAV Cap protein, (v) a second internal ribosome entry site, and (vi) second polynucleotide encoding AAV Cap protein can be operably linked.
• the polynucleotide can be integrated into a CHO cell genome.
• the polynucleotide can be in a HEK 293 cell and the (i) promoter, (ii) intron, (iii) first internal ribosome entry site, (iv) first polynucleotide encoding AAV Cap protein, (v) a second internal ribosome entry site, and (vi) second polynucleotide encoding AAV Cap protein are operably linked.
• the polynucleotide can be integrated into a HEK 293 cell genome.
• the polynucleotide can be in a BHK cell and the (i) promoter, (ii) intron, (iii) first internal ribosome entry site, (iv) first polynucleotide encoding AAV Cap protein, (v) a second internal ribosome entry site, and (vi) second polynucleotide encoding AAV Cap protein are operably linked.
• the polynucleotide can be integrated into a BHK cell genome.
• the polynucleotide can be in a human amniotic cell and the (i) promoter, (ii) intron, (iii) first internal ribosome entry site, (iv) first polynucleotide encoding AAV Cap protein, (v) a second internal ribosome entry site, and (vi) second polynucleotide encoding AAV Cap protein are operably linked.
• the polynucleotide can be integrated into a human amniotic cell genome.
• the polynucleotide can further comprise an operator.
• the promoter can be a CMV promoter and the operator can be a Tet operator.
• AAV proteins and polynucleotides, including CAP can be selected from any serotype.
• Cap is from serotype 5 (“Cap5”) and expressed in a CHO cell
• the polynucleotide allows for production of AAV Cap5 proteins VP1 , VP2 and VP3.
• the amount of VP3 production can be greater than the amount of VP1 production and the amount of VP2 production.
• the amount of VP2 production can be greater than the amount of VP1 production. Production ratios can vary based upon experimental conditions and analytical techniques.
• each polynucleotide can comprise (i) a promoter, (ii) an intron, (iii) a polynucleotide encoding AAV Cap protein, and (iv) a polyadenylation site, wherein the polynucleotide allows for production of AAV Cap VP1 protein when expressed.
• the polynucleotide can be in a CHO cell and the (i) promoter, (ii) intron, and (iii) polynucleotide encoding AAV Cap protein can be operably linked.
• the polynucleotide can be integrated into a CHO cell genome.
• the polynucleotide can be in a HEK 293 cell and the (i) promoter, (ii) intron, and (iii) polynucleotide encoding AAV Cap protein can be operably linked.
• the polynucleotide can be integrated into a HEK 293 cell genome.
• the polynucleotide can be in a BHK cell and the (i) promoter, (ii) intron, and (iii) polynucleotide encoding AAV Cap protein can be operably linked.
• the polynucleotide can be integrated into a BHK cell genome.
• the polynucleotide can be in a human amniotic cell and the (i) promoter, (ii) intron, and (iii) polynucleotide encoding AAV Cap protein can be operably linked.
• the polynucleotide can be integrated into a human amniotic cell genome.
• the polynucleotide can further comprise an operator.
• the promoter can be a CMV promoter and the operator can be a Tet operator.
• polynucleotides wherein each can comprise (i) a promoter, (ii) an internal ribosome binding site, (iii) a polynucleotide encoding AAV Cap protein, and (iv) a polyadenylation site, wherein the polynucleotide allows for production of AAV VP1 Cap protein when expressed.
• the polynucleotide can be in a CHO cell and the (i) promoter, (ii) internal ribosome binding site, and (iii) polynucleotide encoding AAV Cap protein can be operably linked.
• the polynucleotide can be integrated into a CHO genome.
• the polynucleotide can be in a HEK 293 cell and the (i) promoter, (ii) internal ribosome binding site, and (iii) polynucleotide encoding AAV Cap protein can be operably linked.
• the polynucleotide can be integrated into a HEK 293 cell genome.
• the polynucleotide can be in a BHK cell and the (i) promoter, (ii) internal ribosome binding site, and (iii) polynucleotide encoding AAV Cap protein can be operably linked.
• the polynucleotide can be integrated into a BHK cell genome.
• the polynucleotide can be in a human amniotic cell and the (i) promoter, (ii) internal ribosome binding site, and (iii) polynucleotide encoding AAV Cap protein can be operably linked.
• the polynucleotide can be integrated into a human amniotic cell genome.
• the polynucleotide can comprise an operator.
• the promoter can be a CMV promoter and the operator can be a Tet operator.
• each cell can comprise a polynucleotide comprising (i) a promoter, (ii) an intron, (iii) a first internal ribosome entry site, (iv) a first polynucleotide encoding AAV Cap protein, (v) a second internal ribosome entry site, (vi) a second polynucleotide encoding AAV Cap protein, and (vii) a polyadenylation site.
• a polynucleotide comprising (i) a promoter, (ii) an intron, (iii) a first internal ribosome entry site, (iv) a first polynucleotide encoding AAV Cap protein, (v) a second internal ribosome entry site, (vi) a second polynucleotide encoding AAV Cap protein, and (vii) a polyadenylation site.
• the (i) promoter, (ii) intron, (iii) first internal ribosome entry site, (iv) first polynucleotide encoding AAV Cap protein, (v) a second internal ribosome entry site, and (vi) second polynucleotide encoding AAV Cap protein can be operably linked.
• the polynucleotide can be integrated into a cell genome.
• the cell can be a CHO cell, a HEK 293 cell, a BHK cell, a Human Amniotic Cell or other eukaryotic cell.
• the cell can further comprise an operator.
• the promoter can be a CMV promoter and the operator can be a Tet operator.
• the cell can further comprise: a polynucleotide encoding AAV Rep, a polynucleotide encoding Ad E1 A, a polynucleotide encoding Ad E1 B, a polynucleotide encoding Ad E2A or E2A orf, a polynucleotide encoding Ad E4 or E4 orf 6, a polynucleotide encoding VA RNA, and a polynucleotide encoding AAV ITRs and a protein of interest.
• each cell can comprise (A) a first polynucleotide comprising (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, (iv) a polynucleotide encoding AAV Cap protein, and (v) a polyadenylation site; and (B) a second polynucleotide comprising (i) a promoter, (ii) an intron, (iii) a polynucleotide encoding AAV Cap protein, and (iv) a polyadenylation site.
• the (i) promoter, (ii) intron, (iii) internal ribosome entry site and (iv) polynucleotide encoding AAV Cap protein of (A) first polynucleotide can be operably linked, and wherein the (i) promoter, (ii) intron, and (iii) polynucleotide encoding AAV Cap protein of (B) second polynucleotide can be operably linked.
• the cell can have at least one polynucleotide integrated into a cell genome.
• the cell can be a CHO cell, a HEK 293 cell, a BHK cell, a human amniotic cell or other eukaryotic cell.
• the cell can further comprise an operator.
• the promoter can be a CMV promoter and the operator can be a Tet operator.
• the cell can further comprise a polynucleotide encoding AAV Rep, a polynucleotide encoding Ad E1A, a polynucleotide encoding Ad E1 B, a polynucleotide encoding Ad E2A or E2A orf, a polynucleotide encoding E4 or E4 orf 6, a polynucleotide encoding VA RNA, and a polynucleotide encoding AAV ITRs and a protein of interest.
• each cell can comprise: (A) a first polynucleotide comprising (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, (iv) a polynucleotide encoding AAV Cap protein, and (v) a polyadenylation site; and (B) a second polynucleotide comprising (i) a promoter, (ii) an internal ribosome entry site, (iii) a polynucleotide encoding AAV Cap protein, and (iv) a polyadenylation site.
• the cells can have the (i) promoter, (ii) intron, (iii) internal ribosome entry site and (iv) polynucleotide encoding AAV Cap protein of (A) first polynucleotide operably linked, and the (i) promoter, (ii) internal ribosome entry site, and (iii) polynucleotide encoding AAV Cap protein of (B) second polynucleotide operably linked.
• At least one polynucleotide can be integrated into a cell genome.
• the cell can be a CHO cell, a HEK 293 cell, a BHK cell, a human amniotic cell or other eukaryotic cell.
• the cell can further comprise an operator.
• the promoter can be a CMV promoter and the operator can be a Tet operator.
• the cell can further comprise: a polynucleotide encoding AAV Rep, a polynucleotide encoding Ad E1 A, a polynucleotide encoding Ad E1 B, a polynucleotide encoding Ad E2A or E2A orf, a polynucleotide encoding Ad E4 or E4 orf 6, a polynucleotide encoding VA RNA, and a polynucleotide encoding AAV ITRs and a protein of interest.
• AAV adeno-associated virus
• a method comprises the steps of: providing eukaryotic cells, wherein a cell comprises a polynucleotide comprising (i) a promoter, (ii) an intron, (iii) a first internal ribosome entry site, (iv) a first polynucleotide encoding AAV Cap protein, (v) a second internal ribosome entry site, (vi) a second polynucleotide encoding AAV Cap protein, and (vii) a polyadenylation site; and culturing the cells in a culture medium to allow the cells to produce AAV Cap protein, wherein the polynucleotide allows for production of AAV Cap proteins VP1 , VP2 and VP3.
• the (i) promoter, (ii) intron, (iii) first internal ribosome entry site, (iv) first polynucleotide encoding AAV Cap protein, (v) a second internal ribosome entry site, and (vi) second polynucleotide encoding AAV Cap protein can be operably linked.
• the polynucleotide can be integrated into a cell genome.
• the cell can be a CHO cell, a HEK 293 cell, a BHK cell, a human amniotic cell or other eukaryotic cell.
• the cell can further comprise an operator.
• the cell can further comprise: a polynucleotide encoding AAV Rep, a polynucleotide encoding Ad E1 A, a polynucleotide encoding Ad E1 B, a polynucleotide encoding Ad E2A or E2A orf, a polynucleotide encoding Ad E4 or E4 orf 6, a polynucleotide encoding VA RNA, and a polynucleotide encoding AAV ITRs and a protein of interest, wherein the cell can produce recombinant AAV.
• a method comprises the steps of providing eukaryotic cells, where a cell comprises (a) a first polynucleotide comprising (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, (iv) a polynucleotide encoding AAV Cap protein, and (v) a polyadenylation site; and (b) a second polynucleotide comprising (i) a promoter, (ii) an intron, (iii) a polynucleotide encoding AAV Cap protein, and (iv) a polyadenylation site; and culturing the cells in a culture medium to allow the cells to produce AAV Cap protein, wherein the polynucleotide allows for production of AAV Cap proteins VP1 , VP2 and VP3.
• the (i) promoter, (ii) intron, (iii) internal ribosome entry site and (iv) polynucleotide encoding AAV Cap protein of (a) first polynucleotide can be operably linked, and the (i) promoter, (ii) intron, and (iii) polynucleotide encoding AAV Cap protein of (b) second polynucleotide can be operably linked.
• the polynucleotide can be integrated into a cell genome.
• the cell can be a CHO cell, a HEK 293 cell, a BHK cell, a human amniotic cell or other eukaryotic cell.
• the cell can further comprise an operator.
• the promoter can be a CMV promoter and the operator can be a Tet operator.
• the cell can further comprise: a polynucleotide encoding AAV Rep, a polynucleotide encoding Ad E1 A, a polynucleotide encoding Ad E1 B, a polynucleotide encoding Ad E2A or E2A orf, a polynucleotide encoding Ad E4 or E4 orf 6, a polynucleotide encoding VA RNA, and a polynucleotide encoding AAV ITRs and a protein of interest, wherein the cell can produce recombinant AAV.
• adeno-associated virus (AAV) Cap protein in cell culture, wherein a method comprises the steps of: providing eukaryotic cells, where a cell comprises (a) a first polynucleotide comprising (i) a promoter, (ii) an intron, (iii) an internal ribosome entry site, (iv) a polynucleotide encoding AAV Cap protein, and (v) a polyadenylation site; and (b) a second polynucleotide comprising (i) a promoter, (ii) an internal ribosome entry site, (iii) a polynucleotide encoding AAV Cap protein, and (iv) a polyadenylation site; and culturing the cells in a culture medium to allow the cells to produce AAV Cap protein, wherein the polynucleotide allows for production of AAV Cap proteins VP1 , VP2 and VP
• the (i) promoter, (ii) intron, (iii) internal ribosome entry site and (iv) polynucleotide encoding AAV Cap protein of (a) first polynucleotide can be operably linked, and the (i) promoter, (ii) internal ribosome entry site, and (iii) polynucleotide encoding AAV Cap protein of (b) second polynucleotide can be operably linked.
• the polynucleotide can be integrated into a cell genome.
• the cell can be a CHO cell, a HEK 293 cell, a BHK cell, a human amniotic cell or other eukaryotic cell.
• the cell can further comprise an operator.
• the promoter can be a CMV promoter and the operator can be a Tet operator.
• the cell can further comprise: a polynucleotide encoding AAV Rep, a polynucleotide encoding Ad E1A, a polynucleotide encoding Ad E1 B, a polynucleotide encoding Ad E2A or E2A orf, a polynucleotide encoding Ad E4 or E4 orf 6, a polynucleotide encoding VA RNA, and a polynucleotide encoding AAV ITRs and a protein of interest, wherein the cell can produce recombinant AAV.
• FIGURE 1 is a schematic diagram of a polynucleotide comprising a promoter, an intron, an internal ribosome entry site (IRES), a polynucleotide encoding AAV Cap protein and a polyadenylation site (poly A).
• IRS internal ribosome entry site
• poly A polyadenylation site
• FIGURE 2 is a schematic diagram of a polynucleotide comprising a promoter, an intron, two internal ribosome entry sites, two polynucleotides encoding AAV Cap protein and a polyadenylation site.
• FIGURE 3 is a schematic diagram of a polynucleotide comprising a promoter, an intron, a polynucleotide encoding AAV Cap protein and a polyadenylation site.
• FIGURE 4 is a schematic diagram of a polynucleotide comprising a promoter, an internal ribosome entry site, a polynucleotide encoding AAV Cap protein and a polyadenylation site.
• FIGURE 5 is a schematic diagram of a polynucleotide comprising a promoter with operator (Op), an intron, an internal ribosome entry site, a polynucleotide encoding AAV Cap protein and a polyadenylation site.
• Op promoter with operator
• FIGURE 6 is a schematic diagram of a polynucleotide comprising a promoter with operator (Op), an intron, two internal ribosome entry sites, two polynucleotides encoding AAV Cap protein and a polyadenylation site.
• Op promoter with operator
• FIGURE 7 is a schematic diagram of a polynucleotide comprising a promoter with operator (Op), an intron, a polynucleotide encoding AAV Cap protein and a polyadenylation site.
• Op promoter with operator
• FIGURE 8 is a schematic diagram of a polynucleotide comprising a promoter with operator (Op), an internal ribosome entry site, a polynucleotide encoding AAV Cap protein and a polyadenylation site.
• Op promoter with operator
• “Intron” is a section of DNA located between exons. An intron is removed to form a mature messenger RNA. Preferred introns are those that can affect the starting point of translation, and exemplars are the hCMV-IE intron (Human cytomegalovirus immediate early protein) and FMDV intron (Foot and Mouth Disease Virus). The globin gene intron also has been reportedly used for expression.
• a “nucleic acid moiety” includes any arrangement of single stranded or double stranded nucleotide sequences. Nucleic acid moieties can include, but are not limited to, polynucleotides, promoters, enhancers, operators, repressors, transcription termination signals, ribosomal entry sites and polyadenylation signals.
• a “DNA cassette” or “cassette” is a type of nucleic acid moiety that comprises at least a promoter, at least one open reading frame and optionally a polyadenylation signal, for example an SV40 polyadenylation signal. Other nucleic acid moieties, such as operators, also are optional.
• a DNA cassette thus is a polynucleotide that comprises two or more shorter polynucleotides.
• operably linked refers to one or more nucleotide sequences in functional relationships with one or more other nucleotide sequences. Such functional relationships can directly or indirectly control, cause, regulate, enhance, facilitate, permit, attenuate, repress or block an action or activity in accordance with the selected design. Exemplars include single-stranded or double-stranded nucleic acid moieties, and can comprise two or more nucleotide sequences arranged within a given moiety in such a way that sequence(s) can exert at least one functional effect on other(s). For example, a promoter operably linked to the coding region of a DNA polynucleotide sequence can facilitate transcription of the coding region.
• “Operator” indicates a DNA sequence that is introduced in or near a polynucleotide sequence in such a way that the polynucleotide sequence may be regulated by the interaction of a molecule capable of binding to the operator and, as a result, prevent or allow transcription of the polynucleotide sequence, as the case may be.
• the operator must be located sufficiently in proximity to the promoter such that it is capable of controlling or influencing transcription by the promoter, which can be considered a type of operable linkage.
• the operator may be placed either downstream or upstream of the promoter. These include, but are not limited to, the operator region of the Lex A gene of E.
• percent identity or “% identical,” in their various grammatical forms, when describing a sequence is meant to include homologous sequences that display the recited identity along regions of contiguous homology, but the presence of gaps, deletions, or insertions that have no homolog in the compared sequence are not taken into account in calculating percent identity.
• a “percent identity” or “% identical” determination between homologs would not include a comparison of sequences where the homolog has no homologous sequence to compare in an alignment.
• percent identity and % identical do not include penalties for gaps, deletions, and insertions.
• a “homologous sequence” in the context of nucleic acid sequences refers to a sequence that is substantially homologous to a reference nucleic acid sequence.
• two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding nucleotides are identical over a relevant stretch of residues.
• the relevant stretch is a complete (i.e., full) sequence.
• Polynucleotide includes a sequence of nucleotides covalently joined, and includes RNA and DNA. Oligonucleotides are considered shorter polynucleotides. Genes are DNA polynucleotides (polydeoxyribonucleic acid) that ultimately encode polypeptides, which are translated from RNA (polyribonucleic acid) that was typically transcribed from DNA. DNA polynucleotides also can encode RNA polynucleotides that is not translated, but rather function as RNA “products”. The type of polynucleotide (that is, DNA or RNA) is apparent from the context of the usage of the term.
• a polynucleotide referred to or identified by the polypeptide it encodes sets forth and covers all suitable sequences in accordance with codon degeneracy. Polynucleotides, including those disclosed herein, include percent identity sequences and homologous sequences when indicated.
• Polypeptide or “peptide” refers to sequence(s) of amino acids covalently joined. Polypeptides include natural, semi-synthetic and synthetic proteins and protein fragments. “Polypeptide” and “protein” can be used interchangeably. Oligopeptides are considered shorter polypeptides.
• Protein of interest or “polypeptide of interest” can have any amino acid sequence, and includes any protein, polypeptide, or peptide, and derivatives, components, domains, chains and fragments thereof. Included are, but not limited to, viral proteins, bacterial proteins, fungal proteins, plant proteins and animal (including human) proteins.
• Protein types can include, but are not limited to, antibodies, bi-specific antibodies, multi-specific antibodies, antibody chains (including heavy and light), antibody fragments, Fv fragments, Fc fragments, Fc- containing proteins, Fc-fusion proteins, receptor Fc-fusion proteins, receptors, receptor domains, trap and mini-trap proteins, enzymes, factors, repressors, activators, ligands, reporter proteins, selection proteins, protein hormones, protein toxins, structural proteins, storage proteins, transport proteins, neurotransmitters and contractile proteins. Derivatives, components, chains and fragments of the above also are included. The sequences can be natural, semi-synthetic or synthetic.
• Proteins of interest and polypeptides of interest are encoded by “genes of interest,” which also can be referred to as “polynucleotides of interest.” Where multiple genes (same or different) are integrated, they can be referred to as “first,” “second”, “third,” “fourth,” “fifth,” “sixth,” “seventh,” “eighth,” “ninth,” “tenth,” etc. as is apparent from the context of use.
• “Promoter” indicates a DNA sequence that cause transcription of a DNA sequence to which it is operably linked, i.e., linked in such a way as to permit transcription of the nucleotide sequence of interest when the appropriate signals are present and repressors are absent.
• the expression of a polynucleotide of interest may be placed under control of any promoter or enhancer element known in the art.
• a eukaryotic promoter can be operably linked to a TATA Box.
• the TATA Box is typically located upstream of the transcription start site.
• Useful promoters include, but are not limited to, the SV40 early promoter region, SV40 E/L (early late) promoter, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus, the regulatory sequences of the metallothionein gene, mouse or human cytomegalovirus major immediate early (CMV-MIE) promoter and other CMV promoters, including CMVmin promoters.
• SV40 E/L early late promoter
• CMV-MIE cytomegalovirus major immediate early
• Plant expression vectors comprising the nopaline synthetase promoter region, the cauliflower mosaic virus 35S RNA promoter, and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I; insulin; immunoglobulin; mouse mammary tumor virus; albumin; C.-feto protein; C.1 - antitrypsin; 3-globin, and myosin light chain-2.
• Various forms of the CMV promoter can be used according to the inventions.
• Minimal promoters such as CMVmin promoters
• CMVmin promoters can be truncated promoters or core promoters and are preferred for use in controlled expression systems.
• Minimal promoters and development approaches are widely known and disclosed in, for example, Saxena et al., Methods Molec. Biol. 1651 :263- 73 (2017); Ede etal., ACS Synth Biol. 5:395-404 (2016); Brown etal., Biotech Bioeng. 111 :1638-47 (2014); Morita et al., Biotechniques 0:1 -5 (2012); Lagrange et al., Genes Dev. 12:34-44 (1998).
• CMVmin promoters described in the field.
• reporter proteins refers to any protein capable of generating a detectable signal. Reporter proteins typically fluoresce, or catalyze a colorimetric or fluorescent reaction, and often are referred to as “fluorescent proteins” or “color proteins.” However, a reporter protein also can be non-enzymatic and non-fluorescent as long as it can be detected by another protein or moiety, such as a cell surface protein detected with a fluorescent ligand. A reporter protein also can be an inactive protein that is made functional through interaction with another protein that is fluorescent or catalyzes a reaction. Accordingly, any suitable reporter protein, as understood by one of skill in the art, could be used.
• the reporter protein may be selected from fluorescent protein, luciferase, alkaline phosphatase, [3-galactosidase, [3-lactamase, dihydrofolate reductase, ubiquitin, and variants thereof.
• Fluorescent proteins are useful for the recognition of gene cassettes that have or have not been successfully inserted and/or replaced, as the case may be. Fluid cytometry and fluorescence-activated cell sorting are suitable for detection.
• fluorescent proteins are well-known in the art, including, but not limited to Discosoma coral (DsRed), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyano fluorescent protein (CFP), enhanced cyano fluorescent protein (eCFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP) and far-red fluorescent protein (e.g. mKate, mKate2, mPlum, mRaspberry or E2-crimson. See, for example, U.S. Patent Nos. 9,816,110. Reporter proteins are encoded by polynucleotides, and are referred to herein as “reporter genes” or “reporter protein genes.” Reporters can be considered a type of marker. “Color” or “fluorescent,” in their various grammatical forms, also can be used the more specifically refer to a reporter protein or gene..
• a “repressor protein”, also referred to as a “repressor,” is a protein that can bind to DNA in order to repressor transcription.
• Repressors are of eukaryotic and prokaryotic origin. Prokaryotic repressors are preferred. Examples of repressor families include: TetR, LysR, Lacl, ArsR, IcIR, MerR, AsnC, MarR, DeoR, GntR and Crp families.
• Repressor proteins in the TetR family include: ArcR, Actll, AmeR, AmrR, ArpR, BpeR, EnvR, EthR, HemR, HydR, IfeR, LanK, LfrR, LmrA, MtrR, Pip, PqrA, QacR, RifQ, RmrR, SimReg2, SmeT, SrpR, TcmR, TetR, TtgR, TrgW, UrdK, VarR YdeS, ArpA., BarA, Aur1 B, CalR1 , CprB, FarA, JadR*, JadR2, MphB, NonG, PhlF, TylQ, VanT, TarA, TyIP, BM1 P1 , Bm3R1 , ButR, CampR, CamR, DhaR, KstR, LexA-like, AcnR, PaaRR, Psbl, Th1 R, UidR
• repressor genes include PurR, LacR, MetJ and PadR, Repressor proteins are encoded by genes referred to as “repressor genes” or “repressor protein genes.”
• “Selectable” or “selection” marker proteins include proteins conferring certain traits, including but not limited to drug resistance or other selective advantages. Selection markers can give the cell receiving the selectable marker gene resistance towards a certain toxin, drug, antibiotic or other compound and permit the cell to produce protein and propagate in the presence of the toxin, drug, antibiotic or other compound, and are often referred to as “positive selectable markers.” Suitable examples of antibiotic resistance markers include, but are not limited to, proteins that impart resistance to various antibiotics, such as kanamycin, spectinomycin, neomycin, gentamycin (G418), ampicillin, tetracycline, chloramphenicol, puromycin, hygromycin, zeocin, and/or blasticidin.
• antibiotic resistance markers include, but are not limited to, proteins that impart resistance to various antibiotics, such as kanamycin, spectinomycin, neomycin, gentamycin (G418), ampicillin, tetracycline, chlorampheni
• selectable markers There are other selectable markers, often referred to as “negative selectable markers,” which cause a cell to stop propagating, stop protein production and/or are lethal to the cell in the presence of the negative selectable marker proteins.
• Thymidine kinase and certain fusion proteins can serve as negative selectable markers, including but not limited to GyrB-PKR. See White et al., Biotechniques, 50: 303-309 (May 2011 ).
• Selectable marker proteins and corresponding genes can be referred to generically as first (1 ), second (2), third (3), fourth (4), fifth (5), sixth (6), seventh (7), eighth (8), ninth (9), tenth (10), etc., as is apparent from the context of usage..
• the inventions provide cells comprising AAV and optionally Ad polynucleotide sequences to allow production of recombinant AAV comprising a polynucleotide of interest, such as a gene or other sequence encoding a polypeptide of interest.
• AAV and Ad polynucleotides provide the requisite structural and helper products required for AAV production.
• AAV polynucleotides, and optionally Ad polynucleotides can be integrated using a recombinase-mediated cassette exchange (RMCE), for example.
• RCE recombinase-mediated cassette exchange
• “Stable” in the context of cell integration refers to a polynucleotide of interest, such as a gene, introduced into the genome of a cell and can be passed to subsequent generations of cells, and thereby can provide cell lines that are genetically homogeneous for a period of time.
• the cell line is a eukaryotic cell line such as a yeast cell line, insect cell line (for example, Sf9 and Sf21 cells) or a mammalian cell line.
• Preferred mammalian cells include primate cells (including human), canine cells and rodent cells.
• Cells can be primary cells or immortalized cells.
• Suitable cells can be selected from Vero cells, COS cells, HEK 293 cells, HeLa cells, CHO cells, BHK cells, MDCK cells, amniotic cells (human), embryonic cells, cell lines transfected with viral genes, for example, AD5 E1 , including but not limited to an immortalized human retinal cell transfected with an adenovirus gene, for example, a PER.C6 cell, or an NSO cell.
• the cell is a Chinese hamster ovary (CHO) cell line.
• CHO cells include, but are not limited to, CHO-ori, CHO-K1 , CHO-s, CHO-DHB11 , CHO-DXB1 1 , CHO-K1 SV, and mutants and variants thereof.
• the cell is a HEK293 cell.
• HEK293 cells include, but are not limited, to HEK293, HEK293A, HEK293E, HEK293F, HEK293FT, HEK293FTM, HEK293H, HEK293MSR, HEK293S, HEK293SG, HEK293SGGD, HEK293T and mutants and variants thereof.
• hamster cells such as CHO and BHK
• integration can be accomplished by inventions disclosed in U.S. Patent Nos. 7,771 ,997 (“Stable Site 1 ”) and 9,816,110 (“Stable Site 2”), which are hereby incorporated by reference, including sequence information.
• Regeneron provides a suite of goods and services referred to as EESYR®.
• CHO cells with integrated sequences in Stable Site 1 and Stable Site 2 are disclosed in US 2019/0233544 A1 , which is hereby incorporated by reference, including sequence information. Sequences set forth in these patents and Examples 14 and 15 can be used according to the inventions described and depicted herein.
• an AAVS1 -like region and the COSMC locus in hamster cells can be used according to the inventions.
• adeno-associated virus integration site 1 (AAVS1 ) can be undertaken. See Lou et al.,
• Modification of cellular genomes can be undertaken with known approaches, such as Cre/Lox, Flp/Frt, transcription activator-like effector nuclease (TALEN), a TAL effector domain fusion protein, zinc finger nuclease (ZFN), a ZFN dimer, or a RNA-guided DNA endonuclease system, such as CRISPR/Cas9.
• TALEN transcription activator-like effector nuclease
• ZFN zinc finger nuclease
• ZFN dimer a ZFN dimer
• RNA-guided DNA endonuclease system such as CRISPR/Cas9.
• Stable Integration Sites can be created using Genomic Safe Harbors and the like in a wide variety of cell types and lines according to the teachings of U.S. Serial No. 63/256,675.
• the descriptions (including examples) and figures providing methods and cells resulting from the methods of U.S. Serial No. 63/256,675 are hereby incorporated by reference.
• the inventions provide for integration of AAV Cap to produce size variants VP1 (about 90kDa), VP2 (about 72kDa) and VP3 (about 60kDa).
• the variants differ at their N-terminus.
• recombinant AAV will contain a gene-of interest (GOI) flanked by AAV ITRs (inverted terminal repeats),
• GOI gene-of interest
• AAV ITRs inverted terminal repeats
• seven additional polynucleotides namely adenovirus E1A, E1 B, E4, E2A, VA RNA and AAV Rep and Cap, are typically employed for production.
• Figures 1 -4 depict constructs that permit constitutive expression of Cap protein.
• Figures 5-8 depict constructs that permit controlled expression of Cap protein by inclusion of an operator downstream of the promoter.
• a preferred operator is the tetracycline operator (TetO), which binds the tetracycline repressor (TetR). Tetracycline, doxycycline and derivatives thereof can bind TetR so that TetR no longer binds TetO, and thus is permissive for transcription.
• TetO tetracycline operator
• TetR tetracycline repressor
• Cap-containing polynucleotides according to Figures 1 -8 are stably inserted into the CHO genome.
• a preferred promoter is the hCMV-IE promoter, and optionally a tet operator can be operably linked to the promoter for expression control.
• an intron can be located 3’ of the promoter.
• a preferred intron is an hCMV-IE intron.
• AAV Cap, Rep and ITRs can be obtained from any AAV serotype.
• Preferred AAV serotypes are AAV2 and AAV5.
• AAV polynucleotide sequences are set forth in Example 11 .
• Promoter, operator, IRES and intron sequences are set forth in Example 13.
• AAV ITRs flanking a gene of interest, AAV Rep and Ad E1 A, E1 B, E2A (or E2A partial sequence (E2A orf)), E4 (or E4 partial sequence (E4 orf 6)) and VA RNA can be randomly integrated, site-specif ically integrated or remain on a plasmid.
• Adenovirus polynucleotide sequences are available and are exemplified in Example 12.
• Adenovirus (Ad) proteins and polynucleotides can be selected from any serotype.
• Cap-containing polynucleotides according to Figures 1 -8 are stably inserted into the HEK 293 genome.
• a preferred promoter is the hCMV-IE promoter, and optionally a tet operator can be operably linked to the promoter for expression control.
• an intron can be located 3’ of the promoter.
• a preferred intron is an hCMV-IE intron.
• AAV Cap, Rep and ITRs can be obtained from any AAV serotype. Preferred AAV serotypes of AAV2 and AAV5.
• AAV polynucleotide sequences are set forth in Example 11 .
• AAV ITRs and Rep and Ad E1 A, E1 B, E2A (or E2A partial sequence (E2A orf)), E4 (or E4 partial sequence (E4 orf 6)) and VA RNA can be randomly integrated, site-specifically integrated or remain on a plasmid.
• Adenovirus polynucleotide sequences are available and are exemplified in Example 12.
• BHK cells are fibroblast cells from baby hamster kidneys.
• Cap-containing polynucleotides according to Figures 1 -8 are stably inserted into the BHK genome.
• a preferred promoter is the hCMV-IE promoter, and optionally a tet operator can be operably linked to the promoter for expression control.
• an intron can be located 3’ of the promoter.
• a preferred intron is an hCMV-IE intron.
• AAV Cap, Rep and ITRs can be obtained from any AAV serotype. Preferred AAV serotypes of AAV2 and AAV5.
• AAV polynucleotide sequences are set forth in Example 11 .
• AAV ITRs and Rep and Ad E1 A, E1 B, E2A (or E2A partial sequence (E2A orf)), E4 (or E4 partial sequence (E4 orf 6)) and VA RNA can be randomly integrated, site-specifically integrated or remain on a plasmid.
• Adenovirus polynucleotide sequences are available and are exemplified in Example 12.
• Embodiments of this construct are depicted in Figures 1 (constitutive) and 5 (controllable). In a cell, both embodiments can primarily produce VP2 and VP3, with more VP3 being produced than VP2. In an experiment in CHO cells using Cap5, an average ratio of about 1 :5.7 of VP2 to VP3 was observed by conducting densitometry analyses on western blots. Some VP1 also can be produced, but the level observed was typically less than 1 % the level of VP2 that is produced. Accordingly, this construct can be used with a construct that will produce primarily VP1 . See Examples 6 and 7. Production ratios can vary based upon experimental conditions and analytical techniques.
• a preferred IRES is the encephalomyocarditis virus (referred to as “EMCV” or “ECMV”) IRES.
• Embodiments of this construct are depicted in Figures 2 (constitutive) and 6 (controllable).
• This construct contains two IRES polynucleotides and two Cap polynucleotides.
• both embodiments can produce a VP1 , VP2 and VP3.
• the amount of VP3 production can be greater than the amount of VP1 production and the amount of VP2 production.
• the amount of VP2 production can be greater than the amount of VP1 production.
• an average ratio of about 1 :2:9.3 of VP1 to VP2 to VP3 was observed by conducting densitometry analyses on western blots. Production ratios can vary based upon experimental conditions and analytical techniques.
• a preferred IRES is the encephalomyocarditis virus (referred to as “EMCV” or “ECMV”) IRES.
• Embodiments of this construct are depicted in Figures 3 (constitutive) and 7 (controllable).
• This construct contains an Intron and Cap polynucleotide, and produces predominantly VP1 , and can be used with the constructs of Example 4 to produce VP1 , VP2 and VP3.
• Embodiments of this construct are depicted in Figures 4 (constitutive) and 8 (controllable).
• This construct contains an Intron and Cap polynucleotide, and produces predominantly VP1 , and can be used with the constructs of Example 4 to produce VP1 , VP2 and VP3.
• a preferred IRES is the encephalomyocarditis virus (referred to as “EMCV” or “ECMV”) IRES.
• CHO cells of Example 1 can comprise an Intron IRES CAP IRES CAP polynucleotide of Example 5 ( Figures 2 or 6).
• CHO cells of Example 1 can comprise CAP polynucleotides of Example 4 (Figs. 1 or 5) and Examples 6 (Figs. 3 or 7) or 6 (Figs.
• Example 9 HEK 293 Cells Comprising Cap constructs
• HEK 293 cells of Example 2 can comprise an Intron IRES CAP IRES CAP polynucleotide of Example 5 ( Figures 2 or 6).
• HEK 293 cells of Example 2 can comprise CAP polynucleotides of Example 4 (Figs. 1 or 5) and Examples 6 (Figs. 3 or 7) or 7 (Figs. 4 and 8). Such cells would express VP1 , VP2 and VP3, although possibly at different ratios than according to Example 5.
• BHK cells of Example 3 can comprise an Intron IRES CAP IRES CAP polynucleotide of Example 5 ( Figures 2 or 6).
• BHK cells of Example 3 can comprise CAP polynucleotides of Example 4 (Figs. 1 or 5) and Examples 6 (Figs. 3 or 7) or 7 (Figs. 4 and 8). Such cells would express VP1 , VP2 and VP3, although possibly at different ratios than according to Example 5.
• AAV Rep, Cap and ITR sequences are known in the art.
• the present inventions are amenable to all AAV serotypes.
• AAV sequences from various AAV serotypes are set forth below. Many of these sequences are available from the National Center for Biotechnology Information (NCBI).
• CapVP l ( SEQ ID NO : 1 )
• CapVP l ( SEQ ID NO : 5 )
• CapVP2 ( SEQ ID NO : 6 )
• CapVP 3 ( SEQ ID NO : 7 )
• CapAAP ( SEQ ID NO : 8 )
• CapVP l ( SEQ ID NO : 12 )
• CapVP l ( SEQ ID NO : 14 )
• CapVP l ( SEQ ID NO : 16 )
• CapVP l ( SEQ ID NO : 18 )
• CapVP l ( SEQ ID NO : 20 )
• CapVP l ( SEQ ID NO : 21 )
• CapVP l ( SEQ ID NO : 23 )
• CapVP l ( SEQ ID NO : 25 )
• CapVP l ( SEQ ID NO : 27 )
• CapVP l ( SEQ ID NO : 29 )
• Cap2 Sequence - contains sequentially VP1 , VP2 , AAP , VP3 (start codons underlined) ( SEQ ID NO : 32 )
• Sequence - contains sequentially VP1 , VP2 , AAP , VP3 (start codons underlined) ( SEQ ID NO : 33 )
• Adenovirus (Ad) polynucleotides can be selected from any serotype, and representative polynucleotides are exemplified below.
• E2A Full Sequence SEQ ID NO : 34 )
• VA Sequence (VA transcripts I and II are underlined) ( SEQ ID NO : 38 )
• Two proteins can be transcribed, a 32 kDa protein (first acces s ion number ) and a 27 kDa protein ( second acces s ion number ) . These are both spl ice variant s from the transcript :
• Ad5 E2A orf Ad5 E2A orf :
• the first i s a spl ice variant contained within the ORF .
• the second i s a non-spl iced transcript present in the ORF . Acces s ion 1 : QHX41659 . 1
• n is a, c, g, t or nucleotide is missing

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