Patent Application
20250109410
A Sequence Listing is provided herewith as Sequence Listing XML, “SHPE-006WO_SEQ_LIST,” created on Jan. 31, 2023, and having a size of 444,705 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.
Recombinant adeno-associated virus (rAAV) is the preferred vehicle for in vivo gene delivery. AAV has no known disease associations, infects dividing and non-dividing cells, rarely if ever integrates into the mammalian cell genome, and can persist essentially for the lifetime of infected cells as a transcriptionally active nuclear episome. The FDA has recently approved several rAAV gene therapy products and many other rAAV-based gene therapy and gene editing products are in development.
The most widely used method for producing rAAV virions is based on the helper-virus-free transient transfection of multiple plasmids, typically a triple transfection, into adherent cell lines. Although there is ongoing investment to increase production capacity, current AAV manufacturing processes are inefficient and expensive. In addition, they result in variable product quality, with low levels of encapsidation of a payload, such as a therapeutic payload.
There is, therefore, a need for improved methods for producing rAAV products. Any such solution must address the toxicity to the host production cell due to constitutive expression of AAV Rep protein and the toxicity to the host production cell due to constitutive expression of adenoviral helper protein.
Disclosed herein are stable mammalian cell lines, wherein the cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload. Disclosed herein are constructs that are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload, when introduced into a cell. The constructs may or may not be integrated into the genome of the cell.
Further provided herein is a stable mammalian cell line, wherein the cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload; and wherein a population of virions produced by the stable cell are more homogenous than a population of virions produced by an otherwise comparable cell producing rAAV virions upon transient transfection.
Further provided herein is a stable mammalian cell line and constructs, wherein the cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload; and production of virions is inducible upon addition of a triggering agent.
Further provided herein is a stable mammalian cell line and constructs, wherein the cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload; and production of virions is not conditioned on the presence of a plasmid within the cell.
In some aspects, a composition comprising one or more nucleic acids which together comprises: (i) a first recombinant nucleic acid sequence encoding an AAV Rep protein and an AAV Cap protein; and (ii) a second recombinant nucleic acid sequence encoding one or more adenoviral helper proteins, wherein when the one or more nucleic acids are integrated into the nuclear genome of a mammalian cell the AAV Rep protein, the AAV Cap protein, and/or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions. In some embodiments, the conditional expression of the AAV Rep protein, the AAV Cap protein, and/or the one or more adenoviral helper proteins is controlled by one or more excisable elements present in the one or more nucleic acids. In some embodiments, the one or more excisable elements comprise one or more introns and/or one or more exons. In some embodiments, the first recombinant nucleic acid sequence encodes: a) a first part of the AAV Rep protein coding sequence; b) the second part of the AAV Rep protein coding sequence; c) an excisable element between the first part of the AAV Rep protein coding sequence and the second part of the AAV Rep protein coding sequence; and d) the AAV Cap protein coding sequence. In some embodiments, the excisable element comprises: a) a first spacer segment comprising a first intron, b) a second spacer segment comprising a coding sequence of a detectable marker; and c) a third spacer segment comprising a second intron, and wherein the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery of a mammalian cell. In some embodiments, the excisable element comprises from 5′ to 3′: a) a 5′ splice site; b) a first spacer segment comprising a first intron; c) a second spacer segment comprising: i) a first lox sequence; ii) a 3′ splice site; iii) an exon; iv) a stop signaling sequence; and v) a second lox sequence; and d) a third spacer segment comprising a second intron. In some embodiments, the detectable marker is a luminescent marker, a radiolabel or a fluorescent marker, optionally a fluorescent marker which is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. In some embodiments, a) the first spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 1; and/or b) the second spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 2; and/or c) the third spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3. In some embodiments, the second spacer segment is capable of being excised by a Cre polypeptide. In some embodiments, the expression of the AAV Rep protein and/or the AAV Cap protein is driven by native promoters. In some embodiments, wherein: a) the native promoters P5 and/or P19 drive the expression of the AAV Rep protein; and/or b) the native promoter P40 drives the expression of the AAV Cap protein. In some embodiments, the second recombinant nucleic acid sequence encodes: a) one or more adenoviral helper proteins; b) a conditionally self-excising element; and c) an inducible promoter; wherein, once integrated into the nuclear genome of a mammalian cell, the expression of the one or more adenoviral helper protein coding sequences is under the control of the conditionally self-excising element and the inducible promoter. In some embodiments, the one or more adenoviral helper proteins comprise E2A and E4. In some embodiments, the self-excising element comprises a sequence which encodes a polypeptide, preferably a recombinase polypeptide, more preferably a Cre polypeptide. In some embodiments, the polypeptide encoded by the self-excising element is conditionally expressible and is expressed only in the presence of a triggering agent. In some embodiments, the triggering agent is a hormone, preferably tamoxifen. In some embodiments, the inducible promoter is a Tet inducible promoter. In some embodiments, the second recombinant nucleic acid sequence further comprises a sequence that encodes a Tet responsive activator protein, preferably Tet-on-3G. In some embodiments, the expression of Tet-On 3G activator protein is driven by an EF1alpha promoter. In some embodiments, the second recombinant nucleic acid sequence comprises a sequence with at least 80% homology, at least 90% homology, at least 95% homology, at least 99% homology, or a sequence identical to SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the one or more nucleic acids further comprises a nucleic acid sequence encoding a VA RNA sequence. In some embodiments, the expression of VA RNA is constitutive. In some embodiments, the expression of VA RNA is inducible. In some embodiments, the VA RNA sequence comprises one or more mutations in the VA RNA internal promoter, preferably G16A and G60A. In some embodiments, the expression of VA RNA is driven by a EF1alpha promoter, a U6 promoter, or a U7 promoter. In some embodiments, the expression of VA RNA is driven by a U6 promoter or a U7 promoter. In some embodiments, the U6 promoter or the U7 promoter comprises: a) a first part of a U6 or U7 promoter sequence, b) a stuffer sequence, and c) a second part of a U6 or U7 promoter sequence, and wherein the stuffer sequence is capable of being excised by a Cre polypeptide. In some embodiments, a serotype of the AAV Cap protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 and AAVhu68. In some embodiments, the serotype is an AAV5 and the Cap protein that comprises one or more mutations or insertions. In some embodiments, the one or more recombinant nucleic acids further encode a third recombinant nucleic acid sequence encoding a payload, optionally wherein the payload is: (a) a polynucleotide payload, such as a guide RNA for RNA editing, a guide RNA for Cas protein-directed DNA editing, a tRNA suppressor, or a gene for replacement gene therapy; or (b) a protein such as a therapeutic antibody or a vaccine immunogen. In some embodiments, the one or more recombinant nucleic acids comprise one or more mammalian cell selection elements. In some embodiments, one or more of the mammalian cell selection elements encodes an antibiotic resistance gene, optionally a blasticidin resistance gene. In some embodiments, one or more of the mammalian cell selection elements is an auxotrophic selection element which encodes an active protein. In some embodiments the auxotrophic selection element is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, one or more of the mammalian cell selection elements is a first auxotrophic selection element which encodes an inactive protein that requires expression of a second inactive protein from a second auxotrophic selection coding sequence for activity. In some embodiments, the first auxotrophic selection coding sequence encodes for DHFR Z-Cter (SEQ ID NO: 5) activity, and/or wherein the second auxotrophic selection coding sequence encodes for DHFR Z-Nter (SEQ ID NO: 4). In some embodiments, a) the first recombinant nucleic acid comprises a mammalian cell selection element which encodes an antibiotic resistance gene, preferably a blasticidin resistance gene; and b) the second recombinant nucleic acid comprises a first auxotrophic selection element which encodes an inactive protein that requires expression of a second inactive protein from a second auxotrophic selection coding sequence for activity; and c) the third recombinant nucleic acid comprises the second auxotrophic selection element which encodes the inactive protein that requires expression of the first inactive protein from the first auxotrophic selection coding sequence for activity; and wherein in (i) or (ii) the first auxotrophic selection coding sequence encodes for DHFR Z-Cter (SEQ ID NO: 5), and the second auxotrophic selection coding sequence encodes for DHFR Z-Nter (SEQ ID NO: 4) or wherein the first auxotrophic selection coding sequence encodes for DHFR Z-Nter (SEQ ID NO: 4), and the second auxotrophic selection coding sequence encodes for DHFR Z-Cter (SEQ ID NO: 5). In some embodiments, elements of the previous embodiments are capable of being in one or more separate constructs, in any combination, wherein the one or more constructs are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload, when introduced into a cell.
In some aspects, disclosed herein is a mammalian cell wherein the nuclear genome of the cell comprises a plurality of integrated recombinant nucleic acid constructs which together encode for a recombinant adeno-associated virus (rAAV) virions, wherein the rAAV virions can be conditionally expressed from the cell. In some embodiments, the plurality of integrated recombinant nucleic acid constructs comprise the one or more recombinant nucleic acids of any one of previous embodiment, wherein the AAV Rep protein, the AAV Cap protein and/or the adenoviral helper proteins can be conditionally expressed from the cell. In some embodiments, the cell line expresses adenoviral helper proteins E1A and E1B. In some embodiments, the plurality of integrated recombinant nucleic acid constructs comprise: (i) a first integrated polynucleotide construct comprising: a) a first part of an AAV Rep protein coding sequence; b) a second part of an AAV Rep protein coding sequence; c) an excisable element between the first part of the AAV Rep protein coding sequence and the second part of the AAV Rep protein coding sequence, wherein the excisable element comprises: i) a first spacer segment comprising a first intron; ii) a second spacer segment comprising a coding sequence of a detectable marker, wherein the second spacer segment is capable of being excised by a Cre polypeptide; and iii) a third spacer segment comprising a second intron; and d) an AAV Cap protein coding sequence; wherein the AAV Rep protein and the AAV Cap protein is driven by the native promoters P5, P19, and P40; (ii) a second integrated polynucleotide construct comprising a) a conditionally expressible VA RNA coding sequence which comprises a mutation in the VA RNA internal promoter, wherein the expression of VA RNA is driven by a U6 or a U7 promoter, optionally wherein the VA RNA sequence comprises G16A and G60A mutations; b) one or more adenoviral helper protein coding sequences, wherein the adenoviral helper proteins are E2A and E4; c) a conditionally self-excising element which encodes a Cre polypeptide which translocates to the nucleus and self-excises only in the presence of a triggering agent which is tamoxifen, and d) an inducible promoter which is a Tet inducible promoter, and wherein the expression of the one or more adenoviral helper protein coding sequences is under the control of the conditionally self-excising element and the inducible promoter; and (iii) a third integrated polynucleotide construct comprising encodes for the payload, wherein the payload is a polynucleotide payload.
In some aspects, a method of producing a population of rAAV virions comprises: (a) culturing the cell of any one of the embodiments disclosed herein in conditions which allow for the expression of the rAAV virions; and (b) isolating the rAAV virions from the cell culture.
In some embodiments, the prepurification rAAV viral genome (VG) to viral particle (VG:VP) ratio of greater than 0.5. In some embodiments, the population of rAAV virions produced by the cell has: (a) a ratio of viral genomes to transduction units of about 500 to 1 to 1 to 1; and/or (b) a ratio of vector genomes to infectious unit of 100:1.
In some aspects, a method of preparing the cell of any one of the previous embodiments comprises: i) providing a mammalian cell and the one or more nucleic acids of any one of the previous embodiments; and ii) integrating the one or more nucleic acids of any one of the previous embodiments into the nuclear genome of the mammalian cell.
In some aspects, a method of preparing the cell of any one of the previous embodiments comprises providing a mammalian cell and the one or more nucleic acids of any one of the previous embodiments. In some embodiments, the one or more nucleic acids of any one of the previous embodiments integrates into the nuclear genome of the mammalian cell. In some embodiments, the one or more nucleic acids of any one of the previous embodiments do not integrate into the nuclear genome of the mammalian cell.
In some aspects, a population of rAAV virions produced by the method of any one of the previous embodiments. In some embodiments, the infectivity of the virions is at least 50% at an MOI of 10000.
In some aspects, a pharmaceutical composition comprising a population of rAAV virions according to any one of the previous embodiments, for use as a medicament, optionally for use in treating a monogenic disorder. In some embodiments, the population of rAAV virions according to any one of the previous embodiments or the pharmaceutical composition according to any one of the previous embodiments, for use as a medicament, optionally for use in treating a monogenic disorder. In some embodiments, the population of rAAV virions or the pharmaceutical composition for use according to any one of the previous embodiments, wherein the rAAV virions are administered at a dosage of 4×1014 or lower.
Also provided herein are cells comprising: a) a first polynucleotide construct coding for an AAV Rep protein and an AAV Cap protein; b) a second polynucleotide construct coding for one or more adenoviral helper proteins; wherein when the one or more nucleic acids are integrated into the nuclear genome of a mammalian cell the AAV Rep protein, the AAV Cap protein, and/or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions.
In some embodiments, the second polynucleotide construct comprises a sequence coding for: a) one or more helper proteins; b) a self-excising element upstream of the one or more helper proteins; and c) an inducible promoter upstream of the self-excising element. In some embodiments, the self-excising element is operably linked to the inducible promoter. In some embodiments, expression of the self-excising element is driven by the inducible promoter.
In some embodiments, the inducible promoter is a tetracycline-responsive promoter element (TRE). In some embodiments, the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. In some embodiments, the minimal promoter is a human cytomegalovirus promoter. In some embodiments, the minimal promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 63-68. In some embodiments, the inducible promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 22, 46-48, or 50-62. In some embodiments, transcription is activated from the inducible promoter upon binding of an activator. In some embodiments, the activator binds to the inducible promoter in the presence of a first triggering agent. In some embodiments, the second polynucleotide construct further comprises a sequence coding for an activator. In some embodiments, the activator is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain. In some embodiments, the rTA comprises four mutations in the tetR DNA binding moiety. In some embodiments, the rTA comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 21, 40-45, or 69-85, or variants thereof.
In some embodiments, the inducible promoter is a cumate operator sequence. In some embodiments, the cumate operator sequence is downstream of a constitutive promoter. In some embodiments, the constitutive promoter is a human cytomegalovirus promoter. In some embodiments, the inducible promoter is bound by a cymR repressor in the absence of a first triggering agent. In some embodiments, the inducible promoter is activated in the presence of a first triggering agent. In some embodiments, the first triggering agent binds to the cymR repressor. In some embodiments, the second polynucleotide construct further comprises a cymR repressor. In some embodiments, the cymR repressor is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the first triggering agent is a cumate.
In some embodiments, the sequence coding for the self-excising element comprises a poly A sequence. In some embodiments, the self-excising element is a recombinase. In some embodiments, the recombinase is fused to a ligand binding domain. In some embodiments, the recombinase is Cre polypeptide or flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. In some embodiments, the self-excising element translocates to the nucleus in the presence of a second triggering agent. In some embodiments, the second triggering agent is an estrogen receptor ligand. In some embodiments, the second triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the second triggering agent is tamoxifen. In some embodiments, the recombinase is flanked by recombination sites. In some embodiments, the recombination sites are lox sites or flippase recognition target (FRT) sites. In some embodiments, the lox sites are loxP sites.
In some embodiments, the one or more adenoviral helper proteins comprise E2A and E4. In some embodiments, the E2A is FLAG-tagged E2A. In some embodiments, the sequence coding for E2A and the sequence coding for E4 are separated by an internal ribosome entry site (IRES) or by P2A.
In some embodiments, the second polynucleotide construct further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is an auxotrophic protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the auxotrophic protein or split intein linked to a C-terminus of the auxotrophic protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the auxotrophic protein or leucine zipper linked to a C-terminus of the auxotrophic protein. In some embodiments, the auxotrophic protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal fragment of the split intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal fragment of the split intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, the second polynucleotide construct further comprises a sequence coding for VA RNA. In some embodiments, the sequence coding for VA RNA is a transcriptionally dead sequence. In some embodiments, the sequence coding for VA RNA comprises at least two mutations in the internal promoter. In some embodiments, expression of VA RNA is driven by a U6 or U7 promoter. In some embodiments, the second polynucleotide construct further comprises upstream of the sequence coding for VA RNA gene sequence, from 5′ to 3′: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; and e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by the recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter.
In some embodiments, the first polynucleotide construct comprises: a) a sequence of a first part of a Rep gene; b) a sequence of a second part of the Rep gene; c) a sequence of a Cap gene; and d) an excisable element positioned between the first part of the sequence of Rep gene and the second part of the sequence of the Rep gene.
In some embodiments, the excisable element comprises a stop signaling sequence. In some embodiments, the excisable element comprises a rabbit beta globin intron. In some embodiments, the excisable element comprises an exon. In some embodiments, the excisable element comprises an intron and an exon. In some embodiments, the excisable element comprises an intron.
In some embodiments, two splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene. In some embodiments, the two splice sites are a 5′ splice site and a 3′ splice site. In some embodiments, the 5′ splice site is a rabbit beta globin 5′ splice site. In some embodiments, the 3′ splice site is a rabbit beta globin 3′ splice site. In some embodiments, three splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene. In some embodiments, the three splice sites are a 5′ splice site, a first 3′ splice site, and a second 3′ splice site. In some embodiments, a first 3′ splice site is a duplicate of the second 3′ splice site. In some embodiments, the first 3′ splice site is a rabbit beta globin 3′ splice site. In some embodiments, the second 3′ splice site is a rabbit beta globin 3′ splice site.
In some embodiments, the excisable element comprises a recombination site. In some embodiments, the recombination site is a lox site or FRT site. In some embodiments, the lox site is a loxP site.
In some embodiments, the excisable element comprises from 5′ to 3′: a) the 5′ splice site; b) a first recombination site; c) the first 3′ splice site; d) a stop signaling sequence; e) a second recombination site; and f) the second 3′ splice site.
In some embodiments, the excisable element comprises from 5′ to 3′: a) the 5′ splice site; b) a first spacer segment; c) a second spacer segment comprising: i) a first recombination site; ii) the first 3′ splice site; iv) a stop signaling sequence; and v) a second recombination site; and d) a third spacer segment comprising the second 3′ splice site. In some embodiments, the first spacer sequence comprises an intron. In some embodiments, the first spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the second spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the third spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the third spacer segment comprises an intron. In some embodiments, the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery. In some embodiments, the second spacer segment comprises an exon. In some embodiments, the second spacer segment further comprises a polyA sequence. In some embodiments, the poly A sequence is 3′ of the exon. In some embodiments, the polyA sequence comprises a rabbit beta globin (RBG) polyA sequence.
In some embodiments, the second spacer segment comprises from 5′ to 3′: a) a first recombination site; b) the first 3′ splice site; c) an exon; d) a stop signaling sequence; and e) a second recombination site. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP sequence and a second lox sequence is a second loxP sequence. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site. In some embodiments, the stop signaling sequence is a termination codon of the exon or a polyA sequence. In some embodiments, the poly A sequence comprises a rabbit beta globin (RBG) polyA sequence. In some embodiments, the exon encodes a detectable marker or a selectable marker. In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry.
In some embodiments, the second spacer segment is excisable by a recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the recombinase is a Cre-ERT2 polypeptide.
In some embodiments, the Rep gene codes for Rep polypeptides. In some embodiments, the Cap gene codes for Cap polypeptides. In some embodiments, transcription of the Rep gene and the Cap gene are driven by native promoters. In some embodiments, the native promoters comprise P5, P19, and P40.
In some embodiments, the Rep polypeptides are wildtype Rep polypeptides. In some embodiments, the Rep polypeptides comprise Rep78, Rep68, Rep52, and Rep40. In some embodiments, a truncated replication associated protein comprising a polypeptide expressed from the sequence of first part of a Rep gene and the exon is capable of being expressed in the absence of the recombinase.
In some embodiments, the Cap polypeptides are wildtype Cap polypeptides. In some embodiments, the Cap polypeptides are AAV capsid proteins. In some embodiments, the AAV capsid proteins comprise VP1, VP2, and VP3. In some embodiments, a serotype of the AAV capsid proteins is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2YF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, and AAVhu68.
In some embodiments, the first polynucleotide construct further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is DHFR, GS, TYMS, or PAH. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the auxotrophic selection element or split intein linked to a C-terminus of the auxotrophic selection element. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the active protein or split intein linked to a C-terminus of the active protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the auxotrophic selection element or leucine zipper linked to a C-terminus of the auxotrophic selection element. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the active protein or leucine zipper linked to a C-terminus of the active protein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
In some embodiments, the first polynucleotide construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1-SEQ ID NO: 3, SEQ ID 6-SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90-SEQ ID NO: 99, SEQ ID NO: 101-SEQ ID NO: 109, SEQ ID NO: 112-SEQ ID NO: 131, or SEQ ID NO: 136-SEQ ID NO: 138. In some embodiments, the second polynucleotide construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 9-SEQ ID NO: 19, SEQ ID 23-SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 90-SEQ ID NO: 99, SEQ ID NO: 101-SEQ ID NO: 109, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the Rep/Cap construct comprises SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins. In some embodiments, the Rep/Cap construct lacks SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins. In some embodiments, the Rep/Cap construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1-SEQ ID NO: 3, SEQ ID 6-SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90-SEQ ID NO: 99, SEQ ID NO: 101-SEQ ID NO: 109, SEQ ID NO: 112-SEQ ID NO: 131, or SEQ ID NO: 136-SEQ ID NO: 138, but wherein these sequences lack SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins. In some embodiments, the first polynucleotide construct and the second polynucleotide construct are stably integrated in the cell's genome.
In some embodiments, the cell further comprises a payload construct, wherein the payload construct is a polynucleotide coding for a payload. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33. In some embodiments, the payload construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, the payload construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 149. In some embodiments, the payload construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 151. In some embodiments, the payload construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 153. In some embodiments, a plasmid comprises the payload construct and comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33. In some embodiments, a plasmid comprises the payload construct and comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, a plasmid comprises the payload construct and comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 149. In some embodiments, a plasmid comprises the payload construct and comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 151. In some embodiments, a plasmid comprises the payload construct and comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 153. In some embodiments, the payload construct comprises a sequence of a payload flanked by ITR sequences. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 139. In some embodiments, expression of the sequence of the payload is driven by a constitutive promoter. In some embodiments, the constitutive promoter and sequence of the payload are flanked by ITR sequences. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 146. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 148. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 150. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 152.
In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a gene. In some embodiments, the gene codes for a selectable marker or detectable marker. In some embodiments, the gene codes for a therapeutic polypeptide or transgene.
In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a therapeutic polynucleotide. In some embodiments, the therapeutic polynucleotide is a tRNA suppressor or a guide RNA. In some embodiments, the guide RNA is a polyribonucleotide capable of binding to a protein. In some embodiments, the protein is nuclease. In some embodiments, the protein is a Cas protein, an ADAR protein, or an ADAT protein. In some embodiments, the Cas protein is catalytically inactive Cas protein. In some embodiments, the payload construct is stably integrated into the genome of the cell.
In some embodiments, a plurality of the payload construct are stably integrated into the genome of the cell. In some embodiments, the plurality of the payload constructs are separately stably integrated into the genome of the cell. In some embodiments, the payload construct further comprises a sequence coding for a selectable marker or detectable marker outside of the ITR sequences. In some embodiments, the payload construct is integrated into the genome of the cell.
In some embodiments, a method for increasing production of rAAV virions from a cell, comprises amplifying expression of AAV Rep and capsid proteins, helper proteins, and/or payload in the cell, wherein the amplifying comprises:
In some embodiments, the polynucleotide construct further comprises a selectable marker operably linked to an attenuated promoter. In some embodiments, the increasing copy number of the polynucleotide construct comprises culturing the cell under conditions that select for the presence of the selectable marker, thereby producing the cell comprising an increased copy number of the polynucleotide construct compared to the polynucleotide construct further comprising a selectable marker operably linked to a nonattenuated promoter. In some embodiments, the attenuated promoter is an attenuated EF1alpha promoter and the nonattenuated promoter is an EF1alpha promoter; optionally, wherein the attenuated EF1alpha promoter is SEQ ID NO: 132 and the EF1alpha promoter is SEQ ID NO: 133. In some embodiments, the polynucleotide construct further comprises a mutated selectable marker having decreased enzymatic activity compared to an unmutated selectable marker. In some embodiments, the increasing copy number of the polynucleotide construct comprises culturing the cell under conditions that select for the presence of the mutated selectable marker, thereby producing the cell comprising an increased copy number of the polynucleotide construct compared to the polynucleotide construct further comprising the unmutated selectable marker. In some embodiments, the mutated selectable marker is a mutated GS and the unmutated selectable marker is GS; optionally, wherein the mutated GS having a R324C, R324S, or R341C mutation as compared to SEQ ID NO: 112 and the GS is SEQ ID NO: 112; optionally, wherein the mutated GS is SEQ ID NO: 142, SEQ ID NO: 143, or SEQ ID NO: 144. In some embodiments, the polynucleotide construct further comprises a selectable marker. In some embodiments, the increasing copy number of the polynucleotide construct comprises culturing the cell under conditions that select for the presence of the selectable marker and in the presence of an inhibitor of the selectable marker, thereby producing the cell comprising an increased copy number of the polynucleotide construct compared to the polynucleotide construct further comprising the selectable marker cultured in the absence of the inhibitor of the selectable marker. In some embodiments, the polynucleotide construct is any polynucleotide construct as described herein.
Also provided herein are methods of producing a stable cell line comprising expanding a cell described above.
Also provided herein are methods of producing a plurality of rAAV virion comprising culturing a cell described above in the presence of a first triggering agent and a second triggering agent. In some embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. In some embodiments, the plurality of rAAV virion have an encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the plurality of rAAV virion have a F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the plurality of rAAV virion have a concentration of greater than 1×1011 or no less than 5×1011, 1×1012, 5×1012, 1×1013 or 1×1014 viral genomes per milliliter prior to purification. In some embodiments, the plurality of rAAV virion have an infectivity of no less than 50%, 60%, 70%, 80%, 90%, 95%, or 99% at an MOI of 1×105 vg/target cell or less. In some embodiments, the culturing is in a bioreactor.
Also provided herein are pharmaceutical compositions comprising the rAAV virion produced by the cell or the method described above and a pharmaceutically acceptable carrier. Also provided herein are methods of treating a condition or disorder, the method comprising administering a therapeutically effective amount of the pharmaceutical composition to a patient in need thereof.
To solve the problems presented by transient transfection approaches to rAAV production while addressing the toxicity of AAV Rep protein when constitutively expressed, disclosed herein are polynucleotide constructs and cell lines stably integrated with said polynucleotide constructs (referred to herein as “stable cell lines”) that enable conditional (also referred to herein as “inducible”) production of recombinant AAV (rAAV) virions. In some embodiments, the compositions and methods of use thereof as disclosed herein provide rAAV virions that encapsidate a desired expressible payload, such as an expressible therapeutic payload. Further provided herein is a stable mammalian cell line, wherein the cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload; and wherein a population of virions produced by the stable cell are more homogenous than a population of virions produced by an otherwise comparable cell producing rAAV virions upon transient transfection.
Further provided herein is a stable mammalian cell line, wherein the cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload; and production of virions is inducible upon addition of a triggering agent.
Further provided herein is a stable mammalian cell line, wherein the cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload; and production of virions is not conditioned on the presence of a plasmid within the cell.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the invention pertains.
“Recombinant”, as applied to an AAV virion, means that the rAAV virion (synonymously, rAAV virus particle) is the product of one or more procedures that result in an AAV particle construct that is distinct from an AAV virion in nature.
In some aspects, the disclosure provides transfected host cells. The term “transfection” is used to refer to the uptake of foreign DNA by a cell, and a cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.
A “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell is a mammalian cell. A host cell may be used as a recipient of an AAV helper construct, an AAV minigene plasmid, an accessory function vector, or other transfer DNA associated with the production of recombinant AAVs. The term includes the progeny of the original cell which has been transfected. Thus, a “host cell” may refer to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A “host cell” as used herein may refer to any mammalian cell which is capable of functioning as an adenovirus packaging cell, i.e., expresses any adenovirus proteins essential to the production of AAV, such as HEK 293 cells and their derivatives (HEK293T cells, HEK293F cells), HeLa, A549, Vero, CHO cells or CHO-derived cells, and other packaging cells.
As used herein, the term “cell line” refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.
As used herein, the terms “recombinant cell” refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.
The term “cell culture,” refers to cells grown adherent or in suspension, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks and the like, as well as the components of the supernatant or suspension itself, including but not limited to rAAV particles, cells, cell debris, cellular contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids, and lipids, and flocculants. Large scale approaches, such as bioreactors, including suspension cultures and adherent cells growing attached to microcarriers or macrocarriers in stirred bioreactors, are also encompassed by the term “cell culture.” Cell culture procedures for both large and small-scale production of proteins are encompassed by the present disclosure.
As used herein, the term “intermediate cell line” refers to a cell line that contains the AAV rep and cap components integrated into the host cell genome or a cell line that contains the adenoviral helper functions integrated into the host cell genome.
As used herein, the term “packaging cell line” refers to a cell line that contains the AAV rep and cap components and the adenoviral helper functions integrated into the host cell genome. A payload construct must be added to the packaging cell line to generate rAAV virions.
As used herein, the term “production cell line” refers to a cell line that contains the AAV rep and cap components, the adenoviral helper functions, and a payload construct. The rep and cap components and the adenoviral helper functions are integrated into the host cell genome. The payload construct can be stably integrated into the host cell genome or transiently transfected. rAAV virions can be generated from the production cell line upon the introduction of one or more triggering agents in the absence of any plasmid or transfection agent.
As used herein, the term “downstream purification” refers to the process of separating rAAV virions from cellular and other impurities. Downstream purification processes include chromatography-based purification processes, such as ion exchange (IEX) chromatography and affinity chromatography.
The term “prepurification yield” refers to the rAAV yield prior to the downstream purification processes. The term “postpurification yield” refers to the rAAV yield after the downstream purification processes. rAAV yield can be measured as viral genome (vg)/L.
The encapsidation ratio of a population of rAAV virions can be measured as the ratio of rAAV viral particle (VP) to viral genome (VG). The rAAV viral particle includes empty capsids, partially full capsids (e.g., comprising a partial viral genome), and full capsids (e.g., comprising a full viral genome).
The F:E ratio of a population of rAAV virions can be measured as the ratio of rAAV full capsids to empty capsids. The rAAV full capsid particle includes partially full capsids (e.g., comprising a partial viral genome) and full capsids (e.g., comprising a full viral genome). The empty capsids lack a viral genome.
The potency or infectivity of a population of rAAV virions can be measured as the percentage of target cells infected by the rAAV virions at a multiplicity of infection (MOI; viral genomes/target cell). Exemplary MOI values are 1×101, 1×102, 2×103, 5×104, or 1×105 vg/target cell. An MOI can be a value chosen from the range of 1×101 to 1×105 vg/target cell.
As used herein, the term “vector” includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. The use of the term “vector” throughout this specification refers to either plasmid or viral vectors, which permit the desired components to be transferred to the host cell via transfection or infection. For example, an adeno-associated viral (AAV) vector is a plasmid comprising a recombinant AAV genome. In some embodiments, useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter.
The phrases “operatively positioned,” “operatively linked,” “under control” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
The term “expression vector or construct” or “synthetic construct” means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In some embodiments, expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or functional RNA (e.g., guide RNA) from a transcribed gene.
The term “auxotrophic” or “auxotrophic selection marker” as used herein refers to the usage of a medium lacking a supplement, such as a medium lacking an essential nutrient such as the purine precursors hypoxanthine and thymidine (HT), or the like, for selection of a functional enzyme which allows for growth in the medium lacking the essential nutrient, e.g., a functional dihydrofolate reductase or the like.
The term cytostatic as used herein refers to a cellular component or agent/element or condition that inhibits cell growth. Cytostasis is the inhibition of cell growth and multiplication.
The term cytotoxic as used herein refers to quality of being toxic to cells. For instance, cells exposed to a cytotoxic agent or condition may undergo necrosis, in which they lose membrane integrity and die rapidly as a result of cell lysis. Cells exposed to a cytotoxic agent can also stop actively growing and dividing (a decrease in cell viability), or the cells can activate a genetic program of controlled cell death (apoptosis).
As used herein, a “monoclonal cell line” or “monoclonality” is used to describe cells produced from a single ancestral cell by repeated cellular replication. Thus, “monoclonal cells” can be said to form a single clone.
The terms “tetracycline” is used generically herein to refer to all antibiotics that are structurally and functionally related to tetracycline, including tetracycline, doxycycline, demeclocycline, minocycline, sarecycline, oxytetracycline, omadacycline, or eravacycline.
The terms “constitutive” or “constitutive expression” are used interchangeably herein. They refer to genes that are transcribed in an ongoing manner. In some embodiments, the terms refer to the expression of a therapeutic payload or a nucleic acid sequence that is not conditioned on addition of an expression triggering agent to the cell culture medium.
The term “expressible therapeutic polynucleotide or “expressible polynucleotide encoding a payload” or “payload polynucleotide” or “payload” refers to a polynucleotide that is encoded in an AAV genome vector (“AAV genome vector”) flanked by AAV inverted terminal repeats (ITRs). A payload disclosed herein may be a therapeutic payload. A payload may include any one or combination of the following: a transgene, a tRNA suppressor, a guide RNA, or any other target binding/modifying oligonucleotide or derivative thereof, or payloads may include immunogens for vaccines, and elements for any gene editing machinery (DNA or RNA editing). Payloads can also include those that deliver a transgene encoding antibody chains or fragments that are amenable to viral vector-mediated expression (also referred to as “vectored or vectorized antibody” for gene delivery). See, e.g., Curr Opin HIV AIDS. 2015 May; 10 (3): 190-197, describing vectored antibody gene delivery for the prevention or treatment of HIV infection. See also, U.S. Pat. No. 10,780,182, which describes AAV delivery of trastuzumab (Herceptin) for treatment of HER2+brain metastases. A payload disclosed herein may not be a therapeutic payload (e.g., a coding for a detectable marker such as GFP).
In particular, in some instances the payload polynucleotide refers to a polynucleotide that can be a homology element for homology-directed repair, or a guide RNA to be delivered for a variety of purposes. In some embodiments, the transgene refers to a nucleic acid sequence coded for expression of guide RNA for ADAR editing or ADAT editing. In some embodiments, the transgene refers to a transgene packaged for gene therapy. In some embodiments, the transgene refers to synthetic constructs packaged for vaccines.
The stable mammalian cell line relies on stable integration and maintenance of a plurality of synthetic nucleic acid constructs within the nuclear genome of the cell. One of these constructs permits inducible expression of a hormone-activated excising element. The excising element can be a recombinase. The recombinase can be a site-specific recombinase. The site-specific recombinase can be a Cre polypeptide or a flippase. Triggering of Cre expression leads to genomic rearrangements, which in turn lead to expression of adenovirus helper proteins, expression of AAV Rep and Cap proteins, and production of rAAV, optionally, encapsidating a therapeutic payload (e.g., transgene, a tRNA suppressor, a guide RNA, or other oligonucleotide). These elements can be in one or more constructs, in any combination that is capable of conditionally producing AAV virion.
An exemplary construct 2 is shown in greater detail in
In the pre-triggered state (top of
When the triggering agent (e.g., Dox) and tamoxifen are added to the culture medium, TetOn3G binds the Tet responsive basal promoter and estrogen response elements are activated, triggering Cre expression (bottom of
As shown at the top of
This approach provides numerous benefits over current AAV systems for delivery of payloads.
Maintaining constructs stably in the cellular genome requires selective pressure. To reduce the number of selective agents (and in particular, antibiotics) required to stably maintain three integrated constructs within the cell line genome, we have designed an approach that stably maintains all 3 constructs in the nuclear genome with a single antibiotic selection, plus a single auxotrophic selection. In some embodiments, the cell line stably maintains all 3 constructs in the nuclear genome with no antibiotic selection. In some embodiments, the cell line stably maintains all 3 constructs in the nuclear genome utilizing auxotrophic protein selection. In some embodiments, the auxotrophic protein selection is with two auxotrophic protein selections.
In some embodiments, the split auxotrophic selection system is a split intervening proteins (inteins) system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure. Inteins auto catalyze a protein splicing reaction that results in excision of the intein and joining of the flanking amino acids (extein sequences) via a peptide bond. Inteins exist in nature as a single domain within a host protein or, less frequently, in a split form. For split inteins, the two separate polypeptide fragments of the intein must associate in order for protein trans-splicing to occur to excise the intein. Split intein systems are described in: Cheriyan et al, J. Biol. Chem 288:6202-6211 (2013); Stevens et al, PNAS 114:8538-8543 (2017); Jillette et al., Nat Comm 10:4968 (2019); US 2020/0087388 A1; and US 2020/0263197 A1. In some embodiments, the split auxotrophic selection system described herein comprises a construct encoding an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of the split intein and a construct encoding the C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of the split intein. This N-terminal fragment is enzymatically nonfunctional and this C-terminal fragment is enzymatically nonfunctional. When both fragments are concurrently expressed in the cell, the split inteins can catalyze the joining of the N-terminal fragment of the auxotrophic protein and a C-terminal fragment of the auxotrophic protein to form a functional enzyme, such as any one of the enzymes disclosed herein (e.g., PAH, GS, TYMS, DHFR). In some embodiments, both constructs can be stably retained in the genome of a cell by growth in a medium lacking the product produced by the enzyme. In some embodiments, the split auxotrophic selection elements (e.g., a construct encoding an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of the split intein and a construct encoding the C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of the split intein) deployed on, for example, constructs 1 and 3, are part of a split intein system. A separate exemplary auxotrophic selection approach, e.g., a full length auxotrophic protein, can be deployed on construct 2. In some embodiments, a construct encoding an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of the split intein or a construct encoding the C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of the split intein further encodes a helper enzyme, wherein expression of the helper enzyme facilitates growth of the host cell in conjunction with the functional enzyme upon application of the single selective pressure.
Following triggering and Cre-mediated genomic rearrangement, the selection elements remain unchanged, allowing continued maintenance of the three post-triggering integrated constructs using a single antibiotic in medium lacking hypoxanthine and thymidine.
Viral proteins needed for AAV virion formation are inhibited by host cell mechanisms. Inhibition of these host cell mechanisms to maximize AAV viral titers in the stable cell lines described herein include, but are not limited to: knocking out PKR (PKR KO) (pathway is responsible for inhibition of viral proteins) in the starting cell line (P0), introducing a mutant EIF2alpha (in the PKR pathway) in the starting cell line (P0), and/or manipulating or modulating virus-associated (VA) RNAs (VA RNAs, an inhibitor of PKR). Virus-associated (VA) RNAs from adenovirus act as small-interference RNAs and are transcribed from the vector genome. These VA RNAs can trigger the innate immune response. Moreover, VA RNAs are processed to functional viral miRNAs and disturb the expression of numerous cellular genes. Therefore, VA-deleted adenoviral vector production constructs (AdVs) lacking VA RNA genes, or having modified VA RNA, would be advantageous. However, VA-deleted AdVs do not produce commercially sufficient quantities of AAV titers (e.g., resulting in fewer and poor-quality virions). Conversely, overexpressing VA RNA also results in a low titer of AAV production that would not be commercially feasible for scale-up. Thus, developing conditional VA RNA constructs, and combining any of those optimized constructs with the conditional helper constructs described herein, will provide commercially relevant, high-quality virions from the AAV production systems as described herein. All three of these strategies can be done in any combination.
VA RNA is also an inhibitor of PKR, which is involved in a pathway responsible for inhibiting AAV viral protein synthesis. In particular, PKR phosphorylates EIF2alpha, which results in inhibition of viral protein synthesis.
While the limited interactions between VA RNA, PKR, and EIF2alpha are understood, PKR is a major kinase that may self-phosphorylate and EIF2alpha may be phosphorylated by other kinases. As such, three strategies (PKR KO, EIF2alpha mutation, manipulation of VA RNA) are being developed for use in any combination in the AAV production systems described herein.
Thus, an option for overcoming the general antiviral effects of mammalian cell production of AAV virions is to modify expression of VA RNA. Therefore, VA-deleted adenoviral vector production constructs (AdVs) lacking VA RNA genes, or having modified VA RNA, have been designed and are described herein in
The constructs of this system can also be used in a vector system, wherein the constructs do not integrate into the genome of the cell.
In a first aspect, the stable cell lines are provided. In some embodiments, the stable cell lines are mammalian stable cell lines. The cells are capable of conditionally producing recombinant AAV (rAAV) virions. In some embodiments, the cells are capable of conditionally producing rAAV virions. In some embodiments, said rAAV virions package an expressible payload. In some embodiments, said rAAV virions package a sequence encoding a payload. In preferred embodiments, production of virions is not conditioned on the presence of an episome or independent plasmid within the cell.
In another aspect, the plasmids or episomes are provided comprising the constructs as disclosed herein. In some embodiments, the plasmids or episomes are transfected mammalian stable cell lines. In some embodiments, the plasmids or episomes further comprise Epstein-Barr virus (EBV) sequences to stably maintain the constructs extrachromosomally. The cells are capable of conditionally producing recombinant AAV (rAAV) virions. In some embodiments, the cells are capable of conditionally producing rAAV virions. In some embodiments, said rAAV virions package an expressible payload. In some embodiments, said rAAV virions package a sequence encoding a payload.
In some embodiments, expression of AAV Rep is conditional. In some embodiments, expression of AAV Rep and Cap proteins is conditional. In certain embodiments, expression of AAV Rep and Cap proteins is conditioned on addition of at least a first expression triggering agent to the cell culture medium. In certain embodiments, expression of AAV Rep and Cap proteins is conditioned on addition of a first expression triggering agent and a second expression triggering agent to the cell culture medium.
In a system with a triggering agent, doxycycline is a suitable agent. In certain embodiments, doxycycline is used to the control a Tet inducible promoter. Alternatively, other inducible promoters can be utilized instead of a Tet inducible promoter, such as, but not limited to, a cumate inducible promoter system, which is under the control of cumate as the triggering agent or an ecdysone-inducible promoter, which is under the control of ecdysone or ponasterone as the triggering agent.
Any suitable inducible excising agent (e.g., recombinase) can be utilized. An excising agent can be a recombinase. An excising agent can be a site-specific recombinase. Exemplary site-specific recombinase systems include, without limitation, Cre-loxP, Flp-FRT, PhiC31-att, Dre-rox, and Tre-loxLTR site-specific recombinase systems. The Cre-loxP system uses a Cre recombinase to catalyze site-specific recombination between two loxP sites. The Flp-FRT system uses a flippase (FLP) recombinase to catalyze site-specific recombination between two flippase recognition target (FRT) sites. The PhiC31-att system uses a phiC31 recombinase to catalyze site-specific recombination between two attachment (att) sites referred to as attB and attP. The Dre-rox system uses a DreO recombinase to catalyze site-specific recombination between two rox sites. The Tre-loxLTR system uses a Tre recombinase to catalyze site-specific recombination between two loxP sites that are modified with HIV long terminal repeats (loxLTR). For a description of various site-specific recombinase systems, see, e.g., Stark et al. (2011) Biochem. Soc. Trans. 39 (2): 617-22; Olorunniji et al. (2016) Biochem. J. 473 (6): 673-684; Birling et al. (2009) Methods Mol. Biol. 561:245-63; García-Otin et al. (2006) Front. Biosci. 11:1108-1136; Weasner et al. (2017) Methods Mol. Biol. 1642:195-209; herein incorporated by reference in their entireties.
An excising agent can target a recombination site. Examples of suitable inducible excising agents include Cre and a flippase. The Cre element can be hormone activated Cre, or light inducible Cre. A recombination site can be a lox site. A lox site can be a loxP site. A recombination site can be an FRT site.
The Flippase recombinase system is based on Flp-FRT recombination, a site-directed recombination technology used to manipulate DNA under controlled conditions in vivo. It is analogous to Cre-lox recombination but involves the recombination of sequences between short flippase recognition target (FRT) sites by the recombinase flippase (Flp) derived from the 2μ plasmid of baker's yeast Saccharomyces cerevisiae. The Flp protein, much like Cre, is a tyrosine family site-specific recombinase.
In typical embodiments, the cells do not express cytotoxic levels of Rep protein prior to addition of both the first expression and second triggering agents to the cell culture medium. In certain embodiments, the cells do not express cytostatic levels of Rep protein prior to addition of both the first and second expression triggering agents to the cell culture medium. In certain embodiments, the average concentration of Rep protein within the cells is less than the amount prior to addition of both of the first and second expression triggering agents to the cell culture medium. In some embodiments, expression of Rep and Cap proteins becomes constitutive after addition of all of the at least first expression triggering agents to the cell culture medium.
In some embodiments, expression of at least one adenoviral helper protein is conditional.
In certain embodiments, expression of the at least one adenoviral helper protein is conditioned on addition of at least a third expression triggering agent to the cell culture medium. In particular embodiments, the third expression triggering agent is the same as the first expression triggering agent. In certain embodiments, expression of adenoviral helper proteins is conditioned on addition of a third expression triggering agent and a fourth expression triggering agent to the cell culture medium. In particular embodiments, the fourth expression triggering agent is the same as the second expression triggering agent. In particular embodiments, the third expression triggering agent is the same as the first expression triggering agent and the fourth expression triggering agent is the same as the second expression triggering agent.
In some embodiments, continued expression of adenoviral helper proteins following triggering of expression by contact of the cell with the at least third expression triggering agent requires the presence of only the third expression triggering agent in the cell culture medium. In certain embodiments, the third triggering agent is the same as the first triggering agent.
In some embodiments, expression of at least one adenoviral helper RNA is conditional. In certain embodiments, the adenoviral helper proteins comprise Ad E2A. In certain embodiments, the adenoviral helper proteins comprise Ad E4. In some embodiments, the adenoviral helper protein is tagged. A tag can be a protein tag. A protein tag can be a FLAG tag. In some embodiments, E2A is FLAG-tagged. In some embodiments, E4 is FLAG-tagged.
In particular embodiments, the adenoviral helper RNA is a VA RNA. In particular embodiments, the adenoviral helper RNA is expressed from an inducible VA RNA construct. In some embodiments, the VA RNA is a mutant VA RNA. In some embodiments, the VA RNA is a transcriptionally dead VA RNA. In some embodiments, the VA RNA is under the control of an RNA polymerase III promoter. In some embodiments, the VA RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the VA RNA is under the control of a U6 or U7 promoter. In some embodiments, the VA RNA is under the control of an interrupted U6 or U7 promoter.
In some embodiments, the third expression triggering agent is a tetracycline. In certain embodiments, the tetracycline is doxycycline (“Dox”). In some embodiments, the fourth expression triggering agent is an estrogen receptor ligand. In certain embodiments, the estrogen receptor ligand is a selective estrogen receptor modulator (SERM). In particular embodiments, the estrogen receptor ligand is tamoxifen.
In some embodiments of the stable cell line, expression of the payload is conditioned on addition of at least a fifth expression triggering agent to the cell culture medium. In some embodiments, expression of the payload is not conditioned on addition of an expression triggering agent to the cell culture medium.
In some embodiments, expression of Rep and Cap proteins, adenoviral helper proteins, and the payload becomes constitutive after addition of only one expression triggering agent to the cell culture medium. In certain embodiments, expression of Rep and Cap proteins and the adenoviral helper proteins becomes constitutive after addition of only one expression triggering agent to the cell culture medium.
In certain embodiments, the one expression triggering agent is the first expression triggering agent. In certain embodiments, the first expression triggering agent is a tetracycline. In particular embodiments, the first expression triggering agent is doxycycline.
In typical embodiments, the nuclear genome of the cell of the stable cell line comprises a plurality of integrated synthetic nucleic acid constructs. Typically, each of the plurality of synthetic nucleic acid constructs is separately integrated into the nuclear genome of the cell. In some embodiments, only a single non-auxotrophic selection is required to maintain all of the plurality of synthetic nucleic acid constructs stably within the nuclear genome of the cells. In some embodiments, antibiotic resistance is required to maintain the plurality of synthetic constructs stably within the nuclear genomes of the cells. In some embodiments, both a non-auxotrophic selection and antibiotic resistance is required to maintain the plurality of synthetic constructs stably within the nuclear genomes of the cells. In some embodiments, auxotrophic selection and antibiotic resistance is required to maintain the plurality of synthetic constructs stably within the nuclear genomes of the cells. In some embodiments, auxotrophic selection is required to maintain the plurality of synthetic constructs stably within the nuclear genomes of the cells.
In some embodiments, the nuclear genome of the cell comprises two integrated synthetic constructs.
In some embodiments, the nuclear genome of the cell comprises three integrated synthetic constructs. In particular embodiments, the first integrated synthetic construct comprises conditionally expressible AAV Rep and Cap coding sequences; the second integrated synthetic construct comprises a conditionally expressible Cre coding sequence and conditionally expressible adenoviral helper protein coding sequences; and the third integrated synthetic construct comprises expressible coding sequences for the payload.
In some embodiments, the nuclear genome of the cell comprises four or more integrated synthetic constructs. In particular embodiments, the four or more integrated synthetic construct comprises, in various combinations, conditionally expressible AAV Rep coding sequences; conditionally expressible Cap coding sequences; a recombinase coding sequence (e.g., can be under a constitutive or inducible promoter; may or may not be self-excising); conditionally expressible adenoviral helper protein coding sequences; the expressible coding sequences for the payload; and expressible VA RNA coding sequence (e.g., can be under a constitutive or inducible promoter; can be wild-type VA RNA or a mutant thereof).
Disclosed herein are polynucleotide constructs encoding for a Rep and Cap polypeptide. Provided herein is a first polynucleotide construct, which encodes for Rep and Cap and comprises spacer or excisable elements. This first polynucleotide construct (Construct 1) is also referred to as a Rep/Cap construct, and/or “AAV Rep/Cap Construct.” In some embodiments, the elements of this first polynucleotide construct are in one or more separate constructs.
These polynucleotide constructs can be stably integrated into a cell line and are triggered to produce AAV Rep and Cap polypeptides in the presence of an excising element (e.g., a recombinase). In some embodiments, the first integrated synthetic construct comprises conditionally expressible AAV Rep and Cap coding sequences. In some embodiments, the polynucleotides do not integrate into the cell line and are triggered to produce AAV Rep and Cap polypeptides in the presence of an excising element (e.g., a recombinase. In some embodiments, the transfected plasmids or episomes comprise conditionally expressible AAV Rep and Cap coding sequences.
The Rep sequence can encode Rep from any desired AAV serotype. In some embodiments, the encoded Rep protein is drawn from the same serotype as the Cap protein. In some embodiments, the encoded Rep protein is drawn from a different serotype from the Cap protein. In particular embodiments, the encoded Rep protein includes, but is not limited to, a Rep protein from AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 and AAV-11, or chimeric combinations thereof.
The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol, 45:555-564 (1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Pat. Nos. 7,282,199 and 7,790,449 relating to AAV-8); the AAV-9 genome is provided in Gao et al. Virol, 78:6381-6388 (2004); the AAV-10 genome is provided in Mol Ther, 13 (1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330 (2): 375-383 (2004).
In the exemplary embodiments illustrated in
In certain embodiments, the intervening spacer segment comprises, from 5′ to 3′, a first spacer segment, a second spacer segment, and a third spacer segment. In some embodiments, the intervening spacer is inserted at CAG-G, CAG-A, AAG-G, AAG-A, wherein the dash (−) indicates the point of insertion of the intervening spacer, in the Rep coding sequence, and the intervening spacer is inserted downstream of the p19 promoter and upstream of the p40 promoter. In some embodiments, the intervening spacer is an excisable spacer.
In particular embodiments, the first spacer segment comprises a 5′ splice site (5′SS) 5′ to the first spacer element. In some embodiments, the first spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 1.
In some embodiments, the second spacer segment comprises a polynucleotide encoding a detectable protein marker flanked by lox sites. In certain embodiments, the detectable protein marker is a fluorescent protein. In particular embodiments, the fluorescent protein is a green fluorescent protein (GFP). In specific embodiments, the GFP is EGFP. In particular embodiments, the fluorescent protein is a blue fluorescent protein (BFP). Screening for the fluorescent marker can be used to confirm integration of the construct into the cell genome, and can subsequently be used to confirm excision of the intervening spacer segment. In some embodiments, the second spacer segment further comprises a polyA sequence. In certain embodiments, the poly A sequence comprises a rabbit beta globin (RBG) polyA. In some embodiments, the second spacer segment further comprises a first 3′ splice site (3′SS) between the first lox site and the polynucleotide encoding the protein marker.
In some embodiments, the second spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 2.
In some embodiments, the third spacer segment further comprises a second 3′ splice site (3′SS). In particular embodiments, the second 3′ splice site is positioned 3′ to the second lox site.
In some embodiments, the third spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3.
In various embodiments, the Rep coding sequences are operatively linked to an endogenous P5 promoter. In various embodiments, the Rep coding sequences are operatively linked to an endogenous P19 promoter. In some embodiments, the intervening spacer is inserted into the Rep coding sequence at a position downstream of the P19 promoter and upstream of the endogenous P40 promoter. In some embodiments, the intervening spacer is inserted at CAG-G, CAG-A, AAG-G, AAG-A downstream of the P19 promoter and upstream of the P40 promoter, wherein the dash (−) indicates the point of insertion of the intervening spacer, in the Rep coding sequence.
In some embodiments, the Rep coding sequences are operably linked to an inducible promoter. In some embodiments, the inducible promoter comprises a tetracycline-inducible promoter, a cumate-inducible promoter, or a cumate-inducible promoter. In some embodiments, the Rep coding sequences are operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the intervening spacer is inserted into the Rep coding sequence at a position downstream of the P19 promoter and upstream of the endogenous P40 promoter. In some embodiments, the intervening spacer is inserted at CAG-G, CAG-A, AAG-G, AAG-A downstream of the P19 promoter and upstream of the P40 promoter, wherein the dash (−) indicates the point of insertion of the intervening spacer, in the Rep coding sequence.
In some embodiments, the Rep coding sequence is 5′ to the Cap coding sequence. In certain embodiments, the Cap coding sequence is operatively linked to an endogenous P40 promoter.
In some embodiments, the Cap coding sequence is operably linked to a promoter. In some embodiments, the sequence coding for VP1, the sequence coding for VP2, and the sequence coding for VP3 are operably linked to a promoter. In some embodiments, a single construct or separate constructs comprise these sequences, in any combination. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter comprises a tetracycline-inducible promoter, a cumate-inducible promoter, or a cumate-inducible promoter. In some embodiments, the promoter is a constitutive promoter, wherein the sequences coding for the one or more cap proteins are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), the sequences coding for the one or more cap proteins are operably linked to the constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
In various embodiments, the Cap protein is selected from the capsid of an avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and modifications, derivatives, or pseudotypes thereof.
In some embodiments, the capsid is a capsid selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 or AAVhu68 (described in WO2020/033842, incorporated herein by reference in its entirety). The hu68 capsid is described in WO 2018/160582, incorporated herein by reference in its entirety.
In some embodiments, the capsid is a derivative, modification, or pseudotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV 13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 or AAVhu68.
In some embodiments, capsid protein is a chimera of capsid proteins from two or more serotype selected from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2YF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16 (described in WO2020/033842, incorporated herein by reference in its entirety). In certain embodiments, the capsid is an rh32.33 capsid, described in U.S. Pat. No. 8,999,678, incorporated herein by reference in its entirety.
In particular embodiments, the capsid is an AAV1 capsid. In particular embodiments, the capsid is an AAV5 capsid. In particular embodiments, the capsid is an AAV9 capsid.
In various embodiments, the first integrated construct further comprises a first mammalian cell selection element.
In some embodiments, the inducible Rep and Cap construct is as shown in
In some embodiments, the second spacer segment in the excisable element is excised by a recombinase. A recombinase can be Cre. Cre may be provided as any form of exogenous Cre, such as Cre gesicles. Cre may also be encoded for by a second polynucleotide construct or by any separate polynucleotide construct. In some embodiments, a construct encoding for adenoviral helper proteins also encodes for Cre. In some embodiments, the second polynucleotide construct is also inducible, for example, as described below. In some embodiments, a construct encoding for Rep/Cap proteins also encodes for Cre.
In some embodiments, expression of the Rep and Cap are driven by native promoters, including P5, P19, P40, or any combination thereof. In some embodiments, expression of the Rep and Cap are driven by inducible promoters. In some embodiments, expression of the Rep and Cap are driven by constitutive promoters. In some embodiments the exon of the excisable element may be any detectable marker. For example, detectable markers contemplated herein include luminescent markers, fluorescent markers, or radiolabels. Fluorescent markers include, but are not limited to, EGFP, GFP, BFP, RFP, or any combination thereof.
In some embodiments, the Rep/Cap construct is a polynucleotide construct comprising: a) a sequence of a first part of a Rep gene; b) sequence of a second part of the Rep gene; c) a sequence of a Cap gene; and d) an excisable element positioned between the first part of the sequence of Rep gene and the second part of the sequence of the Rep gene. In some embodiments, the excisable element comprises a stop signaling sequence. In some embodiments, the excisable element comprises a rabbit beta globin intron. In some embodiments, the excisable element comprises an exon. In some embodiments, the excisable element comprises an intron and an exon. In some embodiments, the excisable element comprises an intron. In some embodiments, two splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene. In some embodiments, the two splice sites are a 5′ splice site and a 3′ splice site. In some embodiments, the 5′ splice site is a rabbit beta globin 5′ splice site. In some embodiments, the 3′ splice site is a rabbit beta globin 3′ splice site. In some embodiments, three splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene. In some embodiments, the three splice sites are a 5′ splice site, a first 3′ splice site, and a second 3′ splice site. In some embodiments, a first 3′ splice site is a duplicate of the second 3′ splice site. In some embodiments, the first 3′ splice site is a rabbit beta globin 3′ splice site. In some embodiments, the second 3′ splice site is a rabbit beta globin 3′ splice site. In some embodiments, the excisable element comprises a recombination site. In some embodiments, the recombination site is a lox site or FRT site. In some embodiments, the lox site is a loxP site. In some embodiments, the excisable element comprises from 5′ to 3′: a) the 5′ splice site; b) a first recombination site; c) the first 3′ splice site; d) a stop signaling sequence; e) a second recombination site; and f) the second 3′ splice site. In some embodiments, the excisable element comprises from 5′ to 3′: a) the 5′ splice site; b) a first spacer segment; c) a second spacer segment comprising: i) a first recombination site; ii) the first 3′ splice site; iv) a stop signaling sequence; and v) a second recombination site; and d) a third spacer segment comprising the second 3′ splice site. In some embodiments, the first spacer sequence comprises an intron. In some embodiments, the first spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the second spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the third spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the third spacer segment comprises an intron. In some embodiments, the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery. In some embodiments, the second spacer segment comprises an exon. In some embodiments, the second spacer segment further comprises a poly A sequence. In some embodiments, the polyA sequence is 3′ of the exon. In some embodiments, the polyA sequence comprises a rabbit beta globin (RBG) polyA sequence. The polynucleotide construct of any one of claims, wherein the second spacer segment comprises from 5′ to 3′: a) a first recombination site; b) the first 3′ splice site; c) an exon; d) a stop signaling sequence; and e) a second recombination site. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP sequence and a second lox sequence is a second loxP sequence. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site. In some embodiments, the stop signaling sequence is a termination codon of the exon or a polyA sequence. In some embodiments, the poly A sequence comprises a rabbit beta globin (RBG) polyA sequence. In some embodiments, the exon encodes a detectable marker or a selectable marker. In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. In some embodiments, the second spacer segment is excisable by a recombinase. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. In some embodiments, the recombinase is encoded by a second polynucleotide construct or exogenously provided. In some embodiments, the Rep gene codes for Rep polypeptides. In some embodiments, the Cap gene codes for Cap polypeptides. In some embodiments, transcription of the Rep gene and the Cap gene are driven by native promoters. In some embodiments, the native promoters comprise P5, P19, and P40. In some embodiments, transcription of the Rep gene and the Cap gene are driven by inducible promoters. In some embodiments, the Rep polypeptides are wildtype Rep polypeptides. In some embodiments, the Rep polypeptides comprise Rep78, Rep68, Rep52, and Rep40. In some embodiments, a truncated replication associated protein comprising a polypeptide expressed from the sequence of first part of a Rep gene and the exon is capable of being expressed in the absence of the recombinase. In some embodiments, the Cap polypeptides are wildtype Cap polypeptides. In some embodiments, the Cap polypeptides are AAV capsid proteins. In some embodiments, the AAV capsid proteins comprise VP1, VP2, and VP3. In some embodiments, a serotype of the AAV capsid proteins is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, and AAVhu68.
In some embodiments, the Rep/Cap construct further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, the Rep/Cap construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the Rep/Cap construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the Rep/Cap construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, a selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109.
In some embodiments, the Rep/Cap construct is in a vector. In some embodiments, the Rep/Cap construct is in a plasmid. In some embodiments, the Rep/Cap construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, the Rep/Cap construct is a synthetic nucleic acid construct. In some embodiments, the Rep/Cap construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1-SEQ ID NO: 3, SEQ ID 6-SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90-SEQ ID NO: 99, SEQ ID NO: 101-SEQ ID NO: 109, SEQ ID NO: 112-SEQ ID NO: 131, or SEQ ID NO: 136-SEQ ID NO: 138, or any combination thereof. In some embodiments, the Rep/Cap construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1-SEQ ID NO: 3, SEQ ID 6-SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90-SEQ ID NO: 99, SEQ ID NO: 101-SEQ ID NO: 109, SEQ ID NO: 112-SEQ ID NO: 131, or SEQ ID NO: 136-SEQ ID NO: 138, or any combination thereof. In some embodiments, the Rep/Cap construct comprises SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins. In some embodiments, the Rep/Cap construct lacks SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins. In some embodiments, the Rep/Cap construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1-SEQ ID NO: 3, SEQ ID 6-SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90-SEQ ID NO: 99, SEQ ID NO: 101-SEQ ID NO: 109, SEQ ID NO: 112-SEQ ID NO: 131, or SEQ ID NO: 136-SEQ ID NO: 138, but wherein Rep/Cap construct lacks SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins.
In some embodiments, the Rep/Cap construct further comprises a sequence coding for VA RNA. In some embodiments, a sequence coding for VA RNA is in separate construct or in any separate construct coding for an element of the Rep/Cap construct. In some embodiments, a payload construct comprises a polynucleotide construct coding for a VA RNA. In some embodiments, the VA RNA is operably linked to a constitutive promoter or an inducible promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. In some embodiments, the sequence coding for VA RNA is a transcriptionally dead sequence. In some embodiments, the sequence coding for VA RNA comprises at least two mutations in the internal promoter. In some embodiments, the expression of the VA RNA is under the control of an RNA polymerase III promoter. In some embodiments, the expression of the VA RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the expression of the VA RNA is under the control of a U6 or U7 promoter. In some embodiments, the expression of the VA RNA is under the control of an interrupted U6 or U7 promoter. In some embodiments, the polynucleotide construct comprises upstream of the sequence coding for VA RNA gene sequence, from 5′ to 3′: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by the recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter.
In some embodiments the gene codes for a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109.
A major advantage of the inducible polynucleotide constructs disclosed herein encoding for Rep and Cap include that upon stable integration into a mammalian cell line, expression of Rep and Cap is inducible even in the absence of a transfection agent or a plasmid. In some embodiments, the stable cell line populations disclosed herein are homogeneous. For example, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the stable cell population comprises the stably integrated polynucleotide construct encoding for Rep and Cap proteins.
Provided herein is a second polynucleotide construct (also referred to as Construct 2), which encodes for one or more adenoviral helper proteins. This second polynucleotide construct is also referred to as an inducible helper construct (e.g., Adenoviral Helper Construct provides one or more helper proteins selected from E1A, E1B, E2A and E4 absent from a host cell) to be used in production of rAAV virions. In some embodiments, the sequence encoding E4 is a sequence encoding E4orf6. In some embodiments, the elements of an inducible helper construct (e.g., one or more helper proteins selected from E1A, E1B, E2A and E4 absent from a host cell) are in one or more separate constructs to be used in production of rAAV virions. In some embodiments, the host cell provides, one, two, or three of the four helper proteins. For example, for a host cell expressing E1A and E1B, the adenoviral helper construct provides E2A and E4. For a host cell expressing E2A and E4, the adenoviral helper construct provides E1A and E1B. For a host cell expressing E1B, the adenoviral helper construct provides E1A, E2A and E4. For a host cell expressing E2A, the adenoviral helper construct provides E1B, E1A and E4. For a host cell expressing E4, the adenoviral helper construct provides E1B, E2A and E1A. For a host cell expressing E1A, E2A and E4, the adenoviral helper construct provides E1B. For a host cell expressing E1B, E1A and E4, the adenoviral helper construct provides E2A. For a host cell expressing E1B, E2A and E1A, the adenoviral helper construct provides E4. In some embodiments, E4 is E4orf6.
In some embodiments, the sequences coding for E1A, E1B, E2A, and E4 are operably linked to separate promoters. In some embodiments, the sequences coding for E1A, E1B, E2A, and E4 are operably linked to one promoter. In some embodiments, the sequences coding for E1A, E1B, E2A, and E4 are operably linked, in any combination, to one promoter or separate promoters. The separate promoters can be the same promoters or different promoters. A combination of the separate promoters and the one promoter can be the same promoters or different promoters. The one promoter can be a native promoter, a constitutive promoter, or an inducible promoter. The separate promoters can be native promoters, constitutive promoters, inducible promoters, or any combination thereof. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, a cumate-inducible promoter, or an ecdysone-inducible promoter. For example, some the sequence coding for E1A and E1B are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E1A and E2A are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E1A and E4 are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E1B and E2A are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E1B and E4 are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E2A and E4 are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequences coding for the helper proteins are in different orientations. In some embodiments, the sequences coding for the helper proteins are bidirectional. In some embodiments, the E1A is operably linked to a natural or constitutive promoter, E1B is operably linked to a natural or constitutive promoter, and E2A and E4 are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E2A and E4 are operably linked to the constitutive promoter. In some embodiments, the E1A is operably linked to a natural or constitutive promoter, E1B is operably linked to a natural or constitutive promoter, and E2A and E4 are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and inducible promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E2A and E4 are operably linked to the inducible promoter. In some embodiments, the E2A is operably linked to a natural or constitutive promoter, E4 is operably linked to a natural or constitutive promoter, and E1A and E1B are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E1A and E1B are operably linked to the constitutive promoter. In some embodiments, the E2A is operably linked to a natural or constitutive promoter, E4 is operably linked to a natural or constitutive promoter, and E1A and E1B are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and inducible promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E1A and E1B are operably linked to the inducible promoter.
In certain embodiments, the adenoviral helper construct provides inducible production of one or more of the helper proteins. In some embodiments, an adenoviral helper protein further comprises a protein tag. A protein tag can be a FLAG tag. In some embodiments, E2A is a FLAG tagged E2A. In some embodiments, E4 is a FLAG tagged E4. A protein tag, such as a FLAG tag, can be used to screen for or to confirm integration of the second polynucleotide construct and expression of the adenoviral helper protein from the second polynucleotide construct in a cell after induction.
In some embodiments, the second integrated synthetic construct comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins. In some embodiments, the second synthetic construct comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins. In some embodiments, the one or more separate integrated constructs comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins. In some embodiments, the one or more separate constructs comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins. In some embodiments, the second integrated synthetic construct comprises conditionally expressible Cre recombinase and conditionally expressible adenovirus helper proteins. In the exemplary embodiments illustrated in
In typical embodiments, the Cre coding sequence is operatively linked to the inducible promoter. In various embodiments, the inducible promoter comprises an element responsive to the third expression triggering agent. In some embodiments, the inducible promoter contains a regulatory sequence that allows for control of the promoter. The regulatory sequence can be operably linked to the promoter and positioned upstream of the promoter. Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. The regulatory sequence used to control expression may be endogenous or exogenous to the host cell. In some embodiments, bacterial gene control elements in combination with viral transactivator proteins are used to provide mammalian inducible expression. Examples of mammalian-compatible regulatory sequences include those capable of controlling an engineered promoter to adjust transcription in response to antibiotics including, without limitation, tetracyclines, streptogramins, and macrolides. For a description of various inducible expression systems, see, e.g., Weber et al. (2004) Methods Mol. Biol. 267:451-66, Das et al. (2016) Curr. Gene Ther. 16 (3): 156-67, Chruscicka et al. (2015) J. Biomol. Screen. 20 (3): 350-8, Yarranton (1992) Curr. Opin. Biotechnol. 3 (5): 506-11, Gossen & Bujard (1992) Proc. Natl. Acad. Sci. U.S.A. 89 (12): 5547-51, Gossen et al. (1995) Science 268 (5218): 1766-9; herein incorporated by reference.
In some embodiments, a bacterial tetracycline response element (TRE) is included in a construct to allow mammalian expression to be induced by tetracycline or a derivative thereof (e.g., doxycycline). In certain embodiments, the inducible promoter comprises a plurality of tetracycline (Tet) operator elements capable of binding to a Tet responsive activator protein in the presence of a tetracycline. In some embodiments, the plurality of tetracycline (Tet) operator elements form a Tetracycline Responsive element (TRE). In some embodiments, the TRE comprises seven repeats of a 19 base pair operator sequence. In further embodiments, the TRE comprises seven repeats of a 19 base pair operator sequence upstream of a minimal CMV promoter sequence.
In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid
In some embodiments, a Tet responsive activator protein comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid
In some embodiments, a Tet responsive activator protein comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid
In some embodiments, a Tet responsive activator protein comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid
In some embodiments, a Tet responsive activator protein comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence:
In some embodiments, a Tet responsive activator protein is a variant of a Tet responsive activator protein and comprises the sequence set forth in SEQ ID NO:40 but with the following amino acid substitutions: 86Y A209T; V9I F86Y A209T; F67S F86Y A209T; G138D F86Y A209T; E157K F86Y A209T; R171K F86Y A209T; V9I G138D F86Y A209T; V9I E157K F86Y A209T; V9I R171K F86Y A209T; F67S R171K F86Y A209T; V9I F67S F86Y A209T; F67S G138D F86Y A209T; F67S E157K F86Y A209T; V9I F67S G138D F86Y A209T; V9I F67S E157K F86Y A209T; V9I F67S R171K F86Y A209T; V9I G138D E157K F86Y A209T; V9I G138D R171K F86Y A209T; F86Y; F86Y A209T; F67S F86Y A209T; G138d F86Y A209T; E157K F86Y A209T; R171K F86Y A209T; V9I G138D F86Y A209T; V9I E157K F86Y A209T; V9I R171K F86Y A209T; F177L F86Y A209T; F67S F177L F86Y A209T; C195S F86Y A209T; G138S F86Y A209T; C68R F86Y A209T; V9I F67S F86Y A209T; F67S G138D F86Y A209T; F67S E157K F86Y A209T; F67S R171K F86Y A209T; V9I F67S G138D F86Y A209T; V9I F67S E157K F86Y A209T; V9I F67S R171K F86Y A209T; V9I G138D E157K F86Y A209T; V9I G138D R171K F86Y A209T; S12G F67S F86Y A209T; G19M F67S F86Y A209T; E37Q F67S F86Y A209T; C68R G138D F86Y A209T; G19M G138D F86Y A209T; E37Q G138D F86Y A209T; V9I C68R G138D F86Y A209T; V9I G19M G138D F86Y A209T; V9I E37Q G138D F86Y A209T; F67S; G138D; E157K; R171K; V9I G138D; V9I E157K; V9I R171K; F177L; F67S F177L; C195S; G138S; C68R; V9I F67S; F67S G138D; F67S E157K; F67S R171K; V9I F67S G138D; V9I F67S E157K; V9I F67S R171K; V9I G138D E157K; V9I G138D R171K; S12G F67S; G19M F67S; E37Q F67S; V9I C68R G138D; V9I G19M G138D; V9I E37Q G138D; V9I G19M F67S G138D; V9I S12G F67S G138D; V9I F67S C68R G138D; F67S F86Y; G138D F86Y; E157K F86Y; R171K F86Y; V9I G138D F86Y; V9I E157K F86Y; V9I R171K F86Y; F177L F86Y; F67S F177L F86Y; C195S F86Y; G138S F86Y; C68R F86Y; V9I F67S F86Y; F67S G138D F86Y; F67S E157K F86Y; F67S R171K F86Y; V9I F67S G138D F86Y; V9I F67S E157K F86Y; V9I F67S R171K F86Y; V9I G138D E157K F86Y; V9I G138D R171K F86Y; S12G F67S F86Y; G19M F67S F86Y; E37Q F67S F86Y; V9I C68R G138D F86Y; V9I G19M G138D F86Y; V9I E37Q G138D F86Y; V9I G19M F67S G138D F86Y; V9I S12G F67S G138D F86Y; V9I F67S C68R G138D F86Y; F67S A209T; G138D A209T; E157K A209T; R171K A209T; V9I G138D A209T; V9I E157K A209T; V9I R171K A209T; F177L A209T; F67S F177L A209T; C195S A209T; G138S A209T; C68R A209T; V9I F67S A209T; F67S G138D A209T; F67S E157K A209T; F67S R171K A209T; V9I F67S G138D A209T; V9I F67S E157K A209T; V9I F67S R171K A209T; V9I G138D E157K A209T; V9I G138D R171K A209T; S12G F67S A209T; G19M F67S A209T; E37Q F67S A209T; V9I C68R G138D A209T; V9I G19M G138D A209T; V9I E37Q G138D A209T; V9I G19M F67S G138D A209T; V9I S12G F67S G138D A209T; V9I F67S C68R G138D A209T; G19M F67S V9I G138D F86Y A209T; S12G F67S V9I G138D F86Y A209T; or C68R F67S V9I G138D F86Y A209T; where the numbering of the substituted amino acids is based on the numbering of amino acids in SEQ ID NO:40.
In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence:
In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence:
In some embodiments, a Tet Repressor binding protein may comprise a sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence:
In some embodiments, a Tet Repressor binding protein may comprise a sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence:
In some embodiments, the minimal promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 63-68. In some embodiments, the inducible promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 22, 46-48, or 50-62. In some embodiments, the rTA comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 21, 40-45, or 69-86, or variants thereof.
The Tet responsive activator protein or variant thereof, the Tet Repressor binding protein or variant thereof, TRE sequence, or any tetracycline-inducible promoter sequence or variant thereof can be any of those disclosed in U.S. Pat. Nos. 7,541,446; 8,383,364; 6,136,954; 5,814,618; 6,271,348; 5,789,156; 7,666,668; 6,914,124; 5,650,298; 5,922,927; 5,464,758; 5,866,755; 5,589,362; 5,654,168; 6,242,667; 5,912,411; 6,783,756; 5,888,981; 6,004,941; 6,252,136; 5,859,310; 6,271,341; 6,087,166; US2003022315; US20050037335; U.S. Pat. No. 9,181,556; and WO03056021, which are each herein incorporated by reference in their entirety.
Any of the proteins described herein may be expressed from a nucleotide sequence that has been codon-optimized to increase expression in a host cell, e.g., a mammalian cell or a human cell line.
In other embodiments, an insect gene control element is used to provide mammalian inducible expression. For example, an ecdysone-responsive element and a gene encoding the ecdysone receptor can be included in a construct to allow mammalian expression to be induced by the insect hormone ecdysone or analogs or derivatives thereof, such as ponasterone. In mammalian cells, the ecdysone receptor heterodimerizes with the retinoid X receptor (RXR). The ecdysone-responsive element comprises a binding site for the RXR-ecdysone receptor heterodimer, which is typically a synthetic recognition site for the heterodimer that preferably does not bind any endogenous transcription factors or natural nuclear hormone receptors. In the presence of ecdysone or an analog or derivative thereof, the RXR-ecdysone receptor heterodimer binds to the ecdysone-responsive element to activate transcription from the promoter. For a description of ecdysone-responsive promoters, see, e.g., No et al. (1996) Proc. Natl. Acad. Sci. USA 93 (8): 3346-51, Oehme et al. (2006) Cell Death and Differentiation (2006) 13:189-201; herein incorporated by reference.
In some embodiments, the second construct or an additional separate construct comprises an element responsive to a fourth expression triggering agent. In certain embodiments, the fourth expression triggering agent-responsive element comprises a plurality of hormone-response elements. In particular embodiments, the hormone-response elements are estrogen responsive elements (EREs). In various embodiments, the third expression triggering element is the same as the first expression triggering element, and the fourth expression triggering element is the same as the second expression triggering element.
In some embodiments, the inducible promoter comprises a plurality of Tet operator elements capable of binding to a Tet responsive activator protein in the presence of a third expression triggering agent. In particular embodiments, the third expression triggering agent is the same as the first expression triggering agent.
In some embodiments, the recombinase coding sequence is flanked by a first recombinase site and a second recombinase site. In some embodiments, the recombinase is Cre. In some embodiments, the Cre coding sequence is flanked by a first lox site and a second lox site. In some embodiments, the first polyA sequence is positioned between the Cre coding sequence and adenoviral helper protein coding sequences that encode one or both of adenovirus E2A and E4. The strong 3′ polyadenylation signal positioned upstream (5′ to) the coding sequences for the adenovirus helper proteins prevents basal expression of the downstream adenoviral helper genes, E2A and E4.
In some embodiments, helper construct does not comprise a recombinase that is self-excising. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is fused to a ligand binding domain. In some embodiments, the recombinase is Cre polypeptide or flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. In some embodiments, the recombinase translocates to the nucleus in the presence of a triggering agent. In some embodiments, the triggering agent is an estrogen receptor ligand. In some embodiments, the triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the triggering agent is tamoxifen. In some embodiments, the recombinase is self-excising to reduce toxicity of the expressed recombinase to cell after induction of expression of the recombinase. In some embodiments, the Cre is self-excising to reduce toxicity of the expressed Cre to cell after induction of expression of the Cre. In some embodiments, the Cre-ERT2 is self-excising to reduce toxicity of the expressed Cre-ERT2 to cell after induction of expression of the Cre-ERT2. When the recombinase is not self-excisable, the sequence or sequences encoding one or more helper proteins can be operably linked to an inducible promoter. When the recombinase is not self-excisable, the sequence or sequences encoding one or more helper proteins can be downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), the sequence or sequences encoding one or more helper proteins are operably linked to the constitutive promoter.
In some embodiments, the further segment shown in
In this embodiment, the further segment includes a Cre-inducible U6 or U7 promoter. The U6 or U7 promoter is split into 2 parts separated by a Lox flanked stuffer sequence. The U6 or U7 promoter is inactive because of the presence of the stuffer sequence. Cre mediated excision of the stuffer activates the U6 or U7 promoter. The U6 or U7 promoter drives the expression of transcriptionally dead mutants of VA RNA1 (a preferred embodiment is a double point mutant G16A-G60A). Other embodiments provide for alternative sources of VA-RNA.
In various embodiments, the coding sequence for the first expression triggering agent-responsive protein is operatively linked to a CMV promoter. In some embodiments, the coding sequence for the first expression triggering agent-responsive protein comprises a coding sequence for the Tet responsive activator protein. In particular embodiments, the Tet responsive activator protein is Tet-on-3G activator protein.
In various embodiments, the second mammalian cell selection element confers antibiotic resistance. In particular embodiments, the antibiotic resistance conferring element is a blasticidin resistance gene.
In some embodiments, the inducible helper polynucleotide construct is as shown at left or at right in
In other embodiments, instead of a mutated VA RNA gene sequence, the inducible helper constructs may comprise a constitutively expressed VA RNA that is not mutated, for example, as shown in
In some embodiments, the inducible helper polynucleotide construct also encodes for one or more helper proteins, a self-excising element upstream of the one or more helper proteins, and an inducible promoter upstream of the self-excising element. Expression of the self-excising element may be driven by a Tet-On-3G system. For example, the construct may comprise a Tet-On 3G gene sequence, wherein expression is driven by an EF1alpha promoter. The EF1alpha promoter may be a mutated EF1alpha promoter. The mutated EF1alpha promoter can have a sequence of:
In the presence of a first triggering agent, such as doxycycline, Tet-On-3G is able to bind the Tet inducible promoter. Upon this binding event, the Tet inducible promoter drives expression of the self-excising element. In some embodiments, the self-excising element is a hormone activated Cre. In the presence of a second triggering agent, such as tamoxifen, and upon expression of Cre, Cre self-excises itself leading to expression of downstream adenoviral helper proteins. Thus, mammalian cell lines stably integrated with the inducible helper constructs disclosed herein only express adenoviral helper proteins in the presence of at least two triggering agents (e.g., doxycycline and tamoxifen).
In some embodiments, an inducible helper construct is a polynucleotide construct coding for: a) one or more helper proteins; and b) a self-excising element upstream of the one or more helper proteins. In some embodiments, the self-excising element is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1 alpha promoter or human cytomegalovirus promoter. In some embodiments, a sequence coding for the self-excising element comprises a poly A sequence. In some embodiments, the self-excising element is a recombinase. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is fused to a ligand binding domain. In some embodiments, the recombinase is Cre polypeptide or flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. In some embodiments, the self-excising element translocates to the nucleus in the presence of a triggering agent. In some embodiments, the triggering agent is an estrogen receptor ligand. In some embodiments, the triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the triggering agent is tamoxifen. In some embodiments, the recombinase is flanked by recombination sites. In some embodiments, the recombination sites are lox sites or flippase recognition target (FRT) sites. In some embodiments, the lox sites are loxP sites. In some embodiments, the self-excising element is excised upon administration of the triggering agent, thereby operably linking the constitutive promoter to the one or more helper proteins. In some embodiments, the inducible helper construct lacks sequences coding for a tetracycline inducible system (e.g., a tetracycline-responsive promoter element (TRE) and/or a reverse tetracycline-controlled transactivator (rTA)). In some embodiments, the inducible helper construct lacks sequences coding for a tetracycline-inducible system, an ecdysone-inducible system, or a cumate-inducible system.
In some embodiments, an inducible helper construct is a polynucleotide construct coding for: a) one or more helper proteins; b) a self-excising element upstream of the one or more helper proteins; and c) an inducible promoter upstream of the self-excising element. In some embodiments, the self-excising element is operably linked to the inducible promoter. In some embodiments, expression of the self-excising element is driven by the inducible promoter.
In some embodiments, an inducible helper construct is a polynucleotide construct coding for: a) one or more helper proteins; b) a recombinase; and c) an inducible promoter upstream of the one or more helper proteins; and d) an inducible promoter upstream the recombinase. In some embodiments, the recombinase is operably linked to the inducible promoter. In some embodiments, the one or more helper proteins are operably linked to the inducible promoter. In some embodiments, expression of the recombinase is driven by the inducible promoter. In some embodiments, expression of the one or more helper proteins is driven by the inducible promoter.
In some embodiments, the inducible promoter is a tetracycline-responsive promoter element (TRE). In some embodiments, the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. In some embodiments, the minimal promoter is a human cytomegalovirus promoter. In some embodiments, the minimal promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 63-68. In some embodiments, the inducible promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 22, 46-48, or 50-62. In some embodiments, transcription is activated from the inducible promoter upon binding of an activator. In some embodiments, the activator binds to the inducible promoter in the presence of a first triggering agent. In some embodiments, further comprising an activator. In some embodiments, the activator is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1 alpha promoter or human cytomegalovirus promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain. In some embodiments, the rTA comprises four mutations in the tetR DNA binding moiety. In some embodiments, the rTA comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 21, 40-45, or 69-86, or variants thereof.
In some embodiments, the inducible promoter is bound by a repressor in the absence of a first triggering agent. In some embodiments, the inducible promoter is activated in the presence of a first triggering agent. In some embodiments, the first triggering agent binds to the repressor. In some embodiments, the repressor is a tetracycline-controlled transactivator. In some embodiments, further comprising the repressor. In some embodiments, the repressor is operably linked to a constitutive promoter. In some embodiments, further comprising a tetracycline-controlled transactivator. In some embodiments, the tetracycline-controlled transactivator is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1 alpha promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the tetracycline-controlled transactivator is unbound in the presence of a first triggering agent. In some embodiments, the tetracycline-controlled transactivator does not bind to the inducible promoter in the presence of a first triggering agent. In some embodiments, the constitutive promoter is EF1alpha promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, transcription is activated from the inducible promoter upon binding of the first triggering agent to the repressor. In some embodiments, the repressor binds to the first triggering agent. In some embodiments, the first triggering agent is a tetracycline. In some embodiments, the tetracycline is doxycycline.
In some embodiments, wherein the inducible promoter is a cumate operator sequence. In some embodiments, the cumate operator sequence is downstream of a constitutive promoter. In some embodiments, the constitutive promoter is a human cytomegalovirus promoter. In some embodiments, wherein the inducible promoter is bound by a cymR repressor in the absence of a first triggering agent. In some embodiments, the inducible promoter is activated in the presence of a first triggering agent. In some embodiments, the first triggering agent binds to the cymR repressor. In some embodiments, the cumate inducible system further comprises a cymR repressor. In some embodiments, the cymR repressor is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the first triggering agent is a cumate.
In some embodiments, a sequence coding for the self-excising element comprises a poly A sequence. In some embodiments, the self-excising element is a recombinase. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is fused to a ligand binding domain. In some embodiments, the recombinase is Cre polypeptide or flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. In some embodiments, the self-excising element translocates to the nucleus in the presence of a second triggering agent. In some embodiments, the second triggering agent is an estrogen receptor ligand. In some embodiments, the second triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the second triggering agent is tamoxifen. In some embodiments, the recombinase is flanked by recombination sites. In some embodiments, the recombination sites are lox sites or flippase recognition target (FRT) sites. In some embodiments, the lox sites are loxP sites.
In some embodiments, the one or more adenoviral helper proteins comprise E2A and E4. In some embodiments, the one or more adenoviral helper proteins further comprises a protein tag. In some embodiments, the protein tag is a FLAG-tag. In some embodiments, the E2A is FLAG-tagged E2A. In some embodiments, the sequence coding for E2 and the sequence coding for E4 are separated by an internal ribosome entry site (IRES) or by P2A.
In some embodiments, the inducible helper construct further comprises a sequence coding for a selectable marker.
In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, the helper construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the helper construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109.
In some embodiments, an inducible helper construct further comprises a sequence coding for VA RNA. In some embodiments, the VA RNA is on a separate construct from the sequences encoding one or more helper proteins. In some embodiments, the VA RNA is operably linked to a constitutive promoter or an inducible promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. In some embodiments, the sequence coding for VA RNA is a transcriptionally dead sequence. In some embodiments, the sequence coding for VA RNA comprises at least two mutations in the internal promoter. In some embodiments, the expression of the VA RNA is under the control of an RNA polymerase III promoter. In some embodiments, the expression of the VA RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the expression of the VA RNA is under the control of a U6 or U7 promoter. In some embodiments, the expression of the VA RNA is under the control of an interrupted U6 or U7 promoter. In some embodiments, the polynucleotide construct comprises upstream of the sequence coding for VA RNA gene sequence, from 5′ to 3′: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by the recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter.
In some embodiments, the gene encodes a detectable marker or a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109.
In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP site and the second lox sequence is a second loxP site. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site.
In some embodiments, an inducible helper construct is in a vector. In some embodiments, an inducible helper construct is in a plasmid. In some embodiments, an inducible helper construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, an inducible helper construct is a synthetic nucleic acid construct. In some embodiments, an inducible helper construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 9-SEQ ID NO: 19, SEQ ID 23-SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 90-SEQ ID NO: 99, SEQ ID NO: 101-SEQ ID NO: 109, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138, or any combination thereof. In some embodiments, an inducible helper construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 9-SEQ ID NO: 19, SEQ ID 23-SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 90-SEQ ID NO: 99, SEQ ID NO: 101-SEQ ID NO: 109, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138, or any combination thereof.
In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA RNA. In some embodiments, the VA RNA is on a separate construct from the sequences encoding one or more helper proteins. In some embodiments, the VA RNA is operably linked to a constitutive promoter or an inducible promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA RNA, wherein a sequence coding for the VA RNA comprises at least two mutations in an internal promoter. In some embodiments, a separate polynucleotide construct codes for a VA RNA, wherein a sequence coding for the VA RNA comprises at least two mutations in an internal promoter. In some embodiments, the sequence coding for the VA RNA comprises a sequence coding for a transcriptionally dead VA RNA. In some embodiments, the sequence coding for the VA RNA comprises a deletion of from about 5-10 nucleotides in the promoter region. In some embodiments, the sequence coding for the VA RNA comprises at least one mutation. In some embodiments, the at least one mutation is in the A Box promoter region. In some embodiments, the at least one mutation is in the B Box promoter region. In some embodiments, the at least one mutation is G16A and G60A. In some embodiments, the expression of the VA RNA is under the control of an RNA polymerase III promoter. In some embodiments, the expression of the VA RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the expression of the VA RNA is under the control of a U6 or U7 promoter. In some embodiments, the expression of the VA RNA is under the control of an interrupted U6 or U7 promoter. In some embodiments, the polynucleotide construct comprises upstream of the VA RNA gene sequence, from 5′ to 3′: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by a recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the gene encodes a detectable marker or a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109.
In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA RNA or the VA RNA construct further comprising a sequence coding for a recombinase. In some embodiments, the recombinase is exogenously provided. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP site and the second lox sequence is a second loxP site. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site. In some embodiments, the construct comprising the VA RNA as described herein further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, the construct comprising the VA RNA further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the construct comprising the VA RNA further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109.
In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA RNA or the VA RNA construct is in a vector. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA RNA or the VA RNA construct is in a plasmid. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA RNA or the VA RNA construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA RNA or the VA RNA construct is a synthetic nucleic acid construct. In some embodiments, an inducible helper construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13-SEQ ID NO: 19 or SEQ ID 23-SEQ ID NO: 26. In some embodiments, an inducible helper construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13-SEQ ID NO: 19 or SEQ ID 23-SEQ ID NO: 26. In some embodiments, a VA RNA construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13-SEQ ID NO: 19 or SEQ ID 23-SEQ ID NO: 26. In some embodiments, a VA RNA construct has a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13-SEQ ID NO: 19 or SEQ ID 23-SEQ ID NO: 26.
In some embodiments, the third integrated synthetic construct (also referred to as Construct 3) comprises the coding sequence for an expressible payload and a third mammalian cell selection element. In the exemplary embodiments shown in
In some embodiments, the expressible payload encodes a guide RNA. In certain embodiments, the guide RNA directs RNA editing. In some embodiments, the guide RNA directs Cas-mediated DNA editing. In some embodiments, the guide RNA directs ADAR-mediated RNA editing. In some embodiments, the third integrated synthetic construct comprises a sequence encoding for any of the expressible payloads disclosed herein. For example, said sequence can encode for any therapeutic. For example, the therapeutic may be a transgene, a guide RNA, an antisense RNA, an oligonucleotide, an mRNA, a miRNA, a shRNA, a tRNA suppressor, a CRISPR-Cas protein, any gene editing enzyme, or any combination thereof. In some embodiments, the transgene encodes for progranulin. In some embodiments, the tRNA suppressor is capable of suppressing an opal stop codon. In some embodiments, the tRNA suppressor is capable of suppressing an ochre stop codon. In some embodiments, the tRNA suppressor is capable of suppressing an amber stop codon. In some embodiments, the third integrated synthetic construct comprises sequences encoding for more than one of the expressible payloads disclosed herein. For example, the third integrated synthetic construct comprise 2 gRNA, 3 gRNA, 4 gRNA, 5 gRNA, 6 gRNA, 7 gRNA, 8 gRNA, 9 gRNA, or 10 gRNA. These gRNAs can all be the same, all be different, or any combination of the same and different. For example, the third integrated synthetic construct comprise 2 suppressor tRNAs, 3 suppressor tRNAs, 4 suppressor tRNAs, 5 suppressor tRNAs, 6 suppressor tRNAs, 7 suppressor tRNAs, 8 suppressor tRNAs, 9 suppressor tRNAs, or 10 suppressor tRNAs. These suppressor tRNAs can all be the same, all be different, or any combination of the same and different.
In some embodiments, the expressible payload encodes a protein. In certain embodiments, the expressible payload is an enzyme, useful for replacement gene therapy. In some embodiments, the protein is a therapeutic antibody. In some embodiments, the protein is a vaccine immunogen. In particular embodiments, the vaccine immunogen is a viral protein.
In some embodiments, the expressible payload is a homology construct for homologous recombination.
In various embodiments, the third mammalian cell selection element is an auxotrophic selection element.
In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 139. In some embodiments, the payload construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, the payload construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 149. In some embodiments, the payload construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 151. In some embodiments, the payload construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 153. In some embodiments, a plasmid comprises the payload construct and comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33. In some embodiments, a plasmid comprises the payload construct and comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, a plasmid comprises the payload construct and comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 149. In some embodiments, a plasmid comprises the payload construct and comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 151. In some embodiments, a plasmid comprises the payload construct and comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 153. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 139, wherein SEQ ID NO: 34 in SEQ ID NO: 33 or SEQ ID NO: 139 is replaced with a sequence of the payload of interest. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 146. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 148. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 150. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 152.
In some embodiments, the payload construct comprises a sequence of a payload flanked by ITR sequences. In some embodiments, expression of the sequence of the payload is driven by a constitutive promoter or an inducible promoter. In some embodiments, the promoter and sequence of the payload are flanked by ITR sequences. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 146. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 148. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 150. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 152.
In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a gene. In some embodiments, the gene codes for a selectable marker or detectable marker. In some embodiments, the gene codes for a therapeutic polypeptide or transgene. In some embodiments, the therapeutic polypeptide or transgene is progranulin. In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a therapeutic polynucleotide. In some embodiments, the therapeutic polynucleotide is a tRNA suppressor or a guide RNA. In some embodiments, the tRNA suppressor is capable of suppressing an opal stop codon. In some embodiments, the tRNA suppressor is capable of suppressing an ochre stop codon. In some embodiments, the tRNA suppressor is capable of suppressing an amber stop codon. In some embodiments, the guide RNA is a polyribonucleotide capable of binding to a protein. In some embodiments, the protein is nuclease. In some embodiments, the protein is a Cas protein, an ADAR protein, or an ADAT protein. In some embodiments, the guide RNA, when bound to a target RNA, recruits an ADAR protein for editing of the target RNA. In some embodiments, the Cas protein is catalytically inactive Cas protein. In some embodiments, the payload construct is stably integrated into the genome of the cell. In some embodiments, a plurality of the payload construct are stably integrated into the genome of the cell. In some embodiments, the plurality of the payload constructs are separately stably integrated into the genome of the cell.
In some embodiments, the payload construct further comprises a sequence coding for a selectable marker or detectable marker outside of the ITR sequences. In some embodiments, expression of the selectable marker or detectable marker outside of the ITR sequences is driven by a promoter. The promoter can be a constitutive promoter or an inducible promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. In some embodiments, the selectable marker is a mammalian cell selection element (e.g., a third mammalian cell selection element). In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, the payload construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the payload construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109.
In some embodiments, the selectable marker is outside of the ITR sequences on the payload construct. In some embodiments, the selectable marker outside of the ITR sequences is a split intein linked to an N-terminus of the auxotrophic protein or split intein linked to a C-terminus of the auxotrophic protein. In some embodiments, the selectable marker outside of the ITR sequences is a leucine zipper linked to an N-terminus of the auxotrophic or leucine zipper linked to a C-terminus of the auxotrophic. In some embodiments, the selectable marker outside of the ITR sequences is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker outside of the ITR sequences is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the payload construct further comprises a spacer between the 5′ ITR and the promoter/selectable marker or promoter/detectable marker outside of the ITR sequences. In some embodiments, the payload construct further comprises a spacer between the 3′ ITR and the promoter/selectable marker or promoter/detectable marker outside of the ITR sequences. In some embodiments, the spacer ranges in length from 500 base pairs to 5000 base pairs, including any length within this range such as 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1250, 1500, 1750, 2000, 2225, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 base pairs. In some embodiments, the spacer length is a sufficient length for decreasing reverse packaging of the selectable marker or detectable marker that is outside the ITR sequences.
In some embodiments, the third integrated synthetic construct comprising the coding sequence for a payload and a selectable marker or detectable marker is further engineered to remove locations having the potential for Rep-mediated nicking. For example, a location having the potential for Rep-mediated nicking is a location having the sequence CAGTGAGCGAGCGAGCGCGCAG (SEQ ID NO: 87); a sequence comprising GAGC repeats; or the sequence GATGGAGTTGGCCACTCCCTC (SEQ ID NO: 89). These sequences can be engineered to prevent binding of Rep proteins for Rep-mediated nicking. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 100 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 200 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 300 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 400 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 500 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1250, 1500, 1750, 2000, 2225, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 nucleotides of an ITR sequence.
In some embodiments, a payload construct comprises a polynucleotide construct coding for a VA RNA. In some embodiments, the VA RNA is operably linked to a constitutive promoter or an inducible promoter. In some embodiments, the constitutive promoter is EF1 alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. In some embodiments, a payload construct comprises a polynucleotide construct coding for a VA RNA, wherein a sequence coding for the VA RNA comprises at least two mutations in an internal promoter. In some embodiments, a separate polynucleotide construct codes for a VA RNA, wherein a sequence coding for the VA RNA comprises at least two mutations in an internal promoter. In some embodiments, the sequence coding for the VA RNA comprises a sequence coding for a transcriptionally dead VA RNA. In some embodiments, the sequence coding for the VA RNA comprises a deletion of from about 5-10 nucleotides in the promoter region. In some embodiments, the sequence coding for the VA RNA comprises at least one mutation. In some embodiments, the at least one mutation is in the A Box promoter region. In some embodiments, the at least one mutation is in the B Box promoter region. In some embodiments, the at least one mutation is G16A and G60A. In some embodiments, the expression of the VA RNA is under the control of an RNA polymerase III promoter. In some embodiments, the expression of the VA RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the expression of the VA RNA is under the control of a U6 or U7 promoter. In some embodiments, the expression of the VA RNA is under the control of an interrupted U6 or U7 promoter. In some embodiments, the polynucleotide construct comprises upstream of the VA RNA gene sequence, from 5′ to 3′: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by a recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the gene encodes a detectable marker or a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109.
In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA RNA or the VA RNA construct further comprising a sequence coding for a recombinase. In some embodiments, the recombinase is exogenously provided. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP site and the second lox sequence is a second loxP site. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site. In some embodiments, the construct comprising the VA RNA as described herein further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, the construct comprising VA RNA further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the construct comprising the VA RNA further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109.
In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA RNA or the VA RNA construct is in a vector. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA RNA or the VA RNA construct is in a plasmid. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA RNA or the VA RNA construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA RNA or the VA RNA construct is a synthetic nucleic acid construct. In some embodiments, an inducible helper construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13-SEQ ID NO: 19 or SEQ ID 23-SEQ ID NO: 26. In some embodiments, an inducible helper construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13-SEQ ID NO: 19 or SEQ ID 23-SEQ ID NO: 26. In some embodiments, a VA RNA construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13-SEQ ID NO: 19 or SEQ ID 23-SEQ ID NO: 26. In some embodiments, a VA RNA construct has a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13-SEQ ID NO: 19 or SEQ ID 23-SEQ ID NO: 26.
The present disclosure further provides host cells comprising the vector system described herein. A subject host cell can be an isolated cell, e.g., a cell in in vitro culture. A subject host cell is useful for producing rAAV virions, as described below. A subject host cell useful for producing rAAV virions can be any cell that is capable of expressing proteins from a p5 promoter. Where a subject host cell is used to produce rAAV virions, it is referred to as a “packaging cell.” In some cases, a subject host cell is stably genetically modified with the vector system. In other cases, a subject host cell is transiently genetically modified with the vector system.
The vector system described herein can be used in a variety of host cells for rAAV virion production. For example, suitable host cells that have been transfected with the vector system are rendered capable of producing rAAV virions. The first and second, and third polynucleotide constructs of the vector system can be introduced into a host cell, either simultaneously or serially, using established transfection techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like. In some embodiments, the first and second polynucleotide constructs of the vector system are introduced into a host cell, and the third polynucleotide construct comprising the expressible payload is introduced later when production of the payload is desired.
A subject host cell is generated by introducing the vector system into any of a variety of cells, e.g., mammalian cells, including, without limitation, murine cells, and primate cells (e.g., human cells). Suitable mammalian cells include, but are not limited to, primary cells and cell lines, where suitable cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1/2 fibroblasts, CHO cells, and the like. Non-limiting examples of suitable host cells include, e.g., HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. A subject host cell can also be made using a baculovirus to infect insect cells such as Sf9 cells, which produce AAV (see, e.g., U.S. Pat. Nos. 7,271,002 and 8,945,918). In some embodiments, a host cell is any cell capable of activating a p5 of sequence encoding a Rep protein.
In typical embodiments, the production host cell is a mammalian cell line that expresses adenovirus E1A and E1B. In particular embodiments, the cell is a human embryonic kidney (HEK) 293 cell line or derivatives thereof (HEK293T cells, HEK293F cells), a human HeLa cell line that expresses E1A and E1B, a Chinese hamster ovary (CHO) cell line that expresses E1A and E1B, or a Vero cell that expresses adenovirus E1A and E1B. In particular embodiments, the host cell is a HEK293 cell line.
In certain embodiments, the host cell is or is genetically altered to be deficient in an enzyme required for production of a molecule required for cell growth, for example, an enzyme required for catalyzing production of a cofactor or nutrient. In certain embodiments, the host cell is DHFR null. In specific embodiments, the host cell is a DHFR null HEK293 cell. In some embodiments, the host cell is GS null. In some embodiments, the host cell is a GS null HEK293.
In some embodiments, the host cell expresses or is genetically modified to express GTP-CH1.
In some embodiments, the HEK293 cell expresses AAV E1A and E1B. In the presence of doxycycline and tamoxifen, the ER2 Cre is excised from the first integrated synthetic construct, thereby permitting expression of AAV E2A and E4. The self-excised ER2 Cre recombines by virtue of the lox sites flanking the EGFP cassette in the second integrated synthetic construct, thereby removing the EGFP segment from the second spacer element in the integrated second synthetic construct. As such, any cells comprising only the second integrated synthetic construct will be EGFP signal positive whereas cells comprising both the first and second integrated synthetic constructs will be EGFP signal negative, following the addition of the triggering agents. Absence of EGFP signal indicates successful transfection of both the first and second integrated synthetic constructs in a cell. This is further ensured by antibiotic resistance selection, e.g., blasticidin resistance.
Additionally, removal of the EGFP cassette provides for the functional expression of Rep and Cap proteins, which can be linked to a first selectable marker, e.g., a first DHFR selection element, e.g., Z-Cter DHFR. The first selectable marker is capable of associating with a second selectable marker, e.g., a second DHFR selection element, e.g., a Z-Cter DHFR is capable of associating with a second DHFR selection element, e.g., Z-Nter DHFR, present in the third integrated synthetic construct to form an active molecule that allows the cell to survive in a selection medium, e.g., HT lacking media selection. The first selectable marker and second selectable marker can be any selectable marker as described herein, wherein expression of the first selectable marker and expression of the second selectable marker form an active molecule (e.g., a functional enzyme) that allows the cell to survive in a selection medium (e.g., a selection media deficient in the product produced by the functional enzyme).
In some embodiments, the third integrated synthetic construct comprises a payload. The payload can be a guide RNA (
In some embodiments, one or more of the synthetic nucleic acid constructs are integrated into the genome of a production host cell. In some embodiments, the integration of a construct into a chromosome is site-specific. Any method known in the art for directing integration into the genome may be used. For example, a polynucleotide construct can be cloned into a lentivirus vector that integrates into the nuclear genome of the cell. Alternatively, a transposon system, a clustered regularly interspersed short palindromic repeats (CRISPR) system, or a site-specific recombinase can be used to integrate a polynucleotide construct into the host cell genome, as described further below.
In some embodiments, a polynucleotide construct is integrated into the genome using a transposon system comprising a transposase and transposon donor DNA. The transposase can be provided to a host cell with an expression vector or mRNA comprising a coding sequence encoding the transposase. The transposon donor DNA can be provided with a vector comprising transposon terminal inverted repeats (TIRs). The polynucleotide construct is cloned into the transposon donor vector between the TIRs. The host cell is cotransfected with an expression vector or mRNA encoding the transposase and the transposon donor vector containing the polynucleotide construct insert, wherein the polynucleotide construct is excised from the transposon donor vector and integrated into the genome of the host cell at a target transposon insertion site. Transposition efficiency may be improved in a host cell by codon optimization of the transposase, using engineered hyperactive transposases, and/or introduction of mutations in the transposon terminal repeats. Any suitable transposon system can be used including, without limitation, the piggyBac, Tol2, or Sleeping Beauty transposon systems. For a description of various transposon systems, see, e.g., Kawakami et al. (2007) Genome Biol. 8 Suppl 1 (Suppl 1): S7, Tipanee et al. (2017) Biosci Rep. 37 (6): BSR20160614, Yoshida et al. (2017) Sci Rep. 7:43613, Yusa et al. (2011) Proc. Natl. Acad. Sci. USA 108 (4): 1531-1536, Doherty et al. (2012) Hum. Gene Ther. 23 (3): 311-320; herein incorporated by reference in their entireties.
In some embodiments, a construct is integrated at a target chromosomal locus by homologous recombination using site-specific nucleases or site-specific recombinases. For example, a construct can be integrated into a double-strand DNA break at the target chromosomal site by homology-directed repair. A DNA break may be created by a site-specific nuclease, such as, but not limited to, a Cas nuclease (e.g., Cas9, Cpf1, or C2c1), an engineered RNA-guided FokI nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), a restriction endonuclease, a meganuclease, a homing endonuclease, and the like. Any site-specific nuclease that selectively cleaves a sequence at the target site for integration of the construct may be used. See, e.g., Targeted Genome Editing Using Site-Specific Nucleases: ZFNs, TALENs, and the CRISPR/Cas9 System (T. Yamamoto ed., Springer, 2015); Genome Editing: The Next Step in Gene Therapy (Advances in Experimental Medicine and Biology, T. Cathomen, M. Hirsch, and M. Porteus eds., Springer, 2016); Aachen Press Genome Editing (CreateSpace Independent Publishing Platform, 2015); herein incorporated by reference in their entireties.
The construct sequence to be integrated is flanked by a pair of homology arms responsible for targeting the construct to the target chromosomal locus. A 5′ homology arm that hybridizes to a 5′ genomic target sequence and a 3′ homology arm that hybridizes to a 3′ genomic target sequence can be introduced into a polynucleotide construct. The homology arms are referred to herein as 5′ and 3′ (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms in the polynucleotide construct. The 5′ and 3′ homology arms hybridize to regions within the target locus where the construct is integrated, which are referred to herein as the “5′ target sequence” and “3′ target sequence,” respectively.
The homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the construct and genomic DNA at the target locus. For example, a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein construct is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5′ and 3′ homology arms.
In certain embodiments, the corresponding homologous nucleotide sequences in the genomic target sequence (i.e., the “5′ target sequence” and “3′ target sequence”) flank a specific site for cleavage and/or a specific site for integrating the construct. The distance between the specific cleavage site and the homologous nucleotide sequences (e.g., each homology arm) can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher gene targeting rate.
A homology arm can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc. In some instances, the 5′ and 3′ homology arms are substantially equal in length to one another, e.g., one may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology arm, or only a few nucleotides less than the other homology arm. In other instances, the 5′ and 3′ homology arms are substantially different in length from one another, e.g., one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.
An RNA-guided nuclease can be targeted to a particular genomic sequence (i.e., genomic target sequence for insertion of a polynucleotide construct) by altering its guide RNA sequence. A target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site. For example, the gRNA can be designed selectively bind to the chromosomal target site where integration of the construct is desired. In certain embodiments, the RNA-guided nuclease used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease. Any RNA-guided Cas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of polynucleotide constructs by the HDR mechanism can be used for selective integration at a target chromosomal site, including CRISPR system type I, type II, or type III Cas nucleases. Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, and homologs or modified versions thereof.
In certain embodiments, a type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to selectively integrate a construct at a chromosomal target site as described herein. The Cas9 need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP_002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP_002344900), Campylobacter coli (WP_060786116); Campylobacter fetus (WP_059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP_033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisI (NC_018721); Streptococcus thermophilus (YP_820832), Streptococcus mutans (WP_061046374, WP_024786433); Listeria innocua (NP_472073); Listeria monocytogenes (WP_061665472); Legionella pneumophila (WP_062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP_033919308); Lactobacillus rhamnosus (WP_048482595, WP_032965177); and Neisseria meningitidis (WP_061704949, YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res. 42 (4): 2577-90; Kapitonov et al. (2015) J. Bacteriol. 198 (5): 797-807, Shmakov et al. (2015) Mol. Cell. 60 (3): 385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42 (10): 6091-6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.
The CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA. The bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzes cleavage to produce a double-stranded break. Targeting of Cas9 typically further relies on the presence of a 5′ protospacer-adjacent motif (PAM) in the DNA at or near the gRNA-binding site.
The genomic target site may comprise a nucleotide sequence that is complementary to the gRNA, and may further comprise a protospacer adjacent motif (PAM). In certain embodiments, the target site comprises 20-30 base pairs in addition to a 3 base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide. In certain embodiments, the allele targeted by a gRNA comprises a mutation that creates a PAM within the allele, wherein the PAM promotes binding of the Cas9-gRNA complex to the allele.
In certain embodiments, the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. The guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.
In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpf1) may be used. Cpf1 is another class II CRISPR/Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cpf1 does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cpf1 for targeting than Cas9. Cpf1 is capable of cleaving either DNA or RNA. The PAM sites recognized by Cpf1 have the sequences 5′-YTN-3′ (where “Y” is a pyrimidine and “N” is any nucleobase) or 5′-TTN-3′, in contrast to the G-rich PAM site recognized by Cas9. Cpf1 cleavage of DNA produces double-stranded breaks with a sticky-ends having a 4 or 5 nucleotide overhang. For a discussion of Cpf1, see, e.g., Ledford et al. (2015) Nature. 526 (7571): 17-17, Zetsche et al. (2015) Cell. 163 (3): 759-771, Murovec et al. (2017) Plant Biotechnol. J. 15 (8): 917-926, Zhang et al. (2017) Front. Plant Sci. 8:177, Fernandes et al. (2016) Postepy Biochem. 62 (3): 315-326; herein incorporated by reference.
C2c1 is another class II CRISPR/Cas system RNA-guided nuclease that may be used. C2c1, similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites. For a description of C2c1, see, e.g., Shmakov et al. (2015) Mol Cell. 60 (3): 385-397, Zhang et al. (2017) Front Plant Sci. 8:177; herein incorporated by reference.
In yet another embodiment, an engineered RNA-guided FokI nuclease may be used. RNA-guided FokI nucleases comprise fusions of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on FokI. For a description of engineered RNA-guided FokI nucleases, see, e.g., Havlicek et al. (2017) Mol. Ther. 25 (2): 342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32 (6): 569-576; herein incorporated by reference.
The RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a gRNA, or provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector) that is introduced into the host cell. Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell or organism. For example, a nucleic acid encoding an RNA-guided nuclease can be modified to substitute codons having a higher frequency of usage in a yeast cell, a bacterial cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the RNA-guided nuclease is introduced into cells, the protein can be transiently, conditionally, or constitutively expressed in the cell.
In some embodiments, a polynucleotide construct is site-specifically integrated into the genome of a host cell using a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, wherein the construct is integrated into a Cas9-induced double-strand break at the target chromosomal site. A vector encoding Cas9 and a gRNA targeting the desired chromosomal site for integration is introduced into the host cell. Sequences with homology to the target locus are introduced into the polynucleotide construct to allow for integration by homology-directed repair. For a description of the use of a CRISPR-Cas9 systems for targeted genomic integration of AAV constructs, see, e.g., Nat. Commun. (2019) 10 (1): 4439; herein incorporated by reference.
Alternatively, site-specific recombinases can be used to selectively integrate a polynucleotide construct at a target chromosomal site. A target chromosomal site for integration of one or more polynucleotide constructs disclosed herein may include a transcriptionally active chromosomal sites. Examples of transcriptionally active chromosomal sites include DNaseI hypersensitive sites (DHSs). A polynucleotide construct can be site-specifically integrated into the genome of a host cell by introducing a first recombination site into the construct and expressing a site-specific recombinase in the host cell. The target chromosomal site of the host cell comprises a second recombination site, wherein recombination between the first and second recombination sites mediated by the site-specific recombinase results in integration of the vector at the target chromosomal locus. The target chromosomal site may comprise either a recombination site native to the genome of the host cell or an engineered recombination site recognized by the site-specific recombinase. Various recombinases may be used for site-specific integration of vector constructs, including, but not limited to phi C31 phage recombinase, TP901-1 phage recombinase, and R4 phage recombinase. In some cases, a recombinase engineered to improve the efficiency of genomic integration at the target chromosomal site may be used. For a description of various site-specific recombinase systems and their use in site-specific recombination and genomic integration of constructs, see, e.g., U.S. Pat. No. 6,632,672; Olivares et al. (2001) Gene 278:167-176; Stoll et al. (2002) J. Bacteriol. 184 (13): 3657-3663; Thyagarajan et al. (2001) Mol. Cell Biol. 21 (12): 3926-3934; Sclimenti et al. (2001) Nucleic Acids Res. 29 (24): 5044-5051; Stark et al. (2011) Biochem. Soc. Trans. 39 (2): 617-22; Olorunniji et al. (2016) Biochem. J. 473 (6): 673-684; Birling et al. (2009) Methods Mol. Biol. 561:245-63; García-Otin et al. (2006) Front. Biosci. 11:1108-1136; Weasner et al. (2017) Methods Mol. Biol. 1642:195-209; herein incorporated by reference in their entireties).
In some embodiments, one or more of the polynucleotide constructs are not integrated into the genome of the production host cell, and instead are maintained in the cell extrachromosomally. Examples of extrachromosomal polynucleotide constructs include those that persist as stable/persistent plasmids or episomal plasmids. In some embodiments, a construct comprises Epstein-Barr virus (EBV) sequences, including the EBV origin of replication. oriP, and the EBV gene, EBNA1, to provide stable extrachromosomal maintenance and replication of the construct. For a description of methods of using EBV sequences to stably maintain vectors extrachromosomally, see, e.g., Stoll et al. (2010) Mol. Ther. 4 (2): 122-129 and Deutsch et al. (2010) J. Virol. 84 (5): 2533-2546; herein incorporated by reference in their entireties. In some embodiments, the polynucleotide constructs of the present disclosure may be introduced into a cell in manner similar to the currently used triple-transfection method for production of rAAV virions.
In a preferred embodiment, this system requires only one antibiotic resistance marker, and two split auxotrophic constructs for selection of all three plasmids, each being transformed just once into the DHFR knockout strain-producing a master cell line for virion production which can be stored and then utilized for scaled-up production without further transformations. This approach provides inducible control over expression of the Rep/Cap products avoiding the toxicity typically associated with Rep/Cap production and also avoids selection with multiple antibiotics, which is not preferred for therapeutic products. Both overexpression of Rep/Cap and selection with multiple antibiotics can be toxic and result in diminished virion yield. The transformed cells can be frozen for storage and thawed for subsequent applications.
Disclosed herein are payloads that may be encoded for by polynucleotide construct 3, which encodes for a payload. This third polynucleotide is referred to herein as a “payload construct” or “therapeutic payload.” Thus, disclosed herein are stable mammalian cell lines that encapsidate a payload. The payload may be an expressible payload. The polynucleotide may encode for any therapeutic. For example, the therapeutic may be a transgene, a guide RNA, an antisense RNA, an oligonucleotide, an mRNA, a miRNA, a shRNA, a tRNA suppressor, a CRISPR-Cas protein, any gene editing enzyme, or any combination thereof. In some embodiments, the payload is guide RNA, wherein the guide RNA, when bound to a target RNA, recruits an ADAR enzyme for editing of the target RNA. In some embodiments, the payload is progranulin. In some embodiments, any one of SEQ ID NO: 146-SEQ ID NO: 153 comprise the progranulin payload. In some embodiments, the payload is progranulin and is flanked by ITRs, e.g., is an ITR flanked progranulin payload of SEQ ID NO: 146 or SEQ ID NO: 147. In some embodiments, the ITR flanked progranulin payload has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the ITR flanked progranulin payload of any one of SEQ ID NO: 146-SEQ ID NO: 153. In some embodiments, the stable mammalian cell lines disclosed herein can conditionally produce rAAV virions that encapsidate more than one payload. Any combination of payloads disclosed herein is contemplated.
Production of Single-Stranded or Self-Complementary rAAV Virion DNA
The region of the third polynucleotide construct between the two inverted terminal repeats (3′ ITR and 5′ ITR) is packaged into rAAV virions. In some embodiments, the rAAV virions comprise wild-type inverted terminal repeats, wherein the rAAV virion DNA that is generated is single-stranded (i.e., ssAAV virion). In other embodiments, a terminal resolution site in the 3′ ITR is deleted, resulting in formation of an rAAV virion comprising DNA that is self-complementary (i.e., scAAV virion). The scAAV forms a single-stranded DNA molecule during replication in which two single-stranded genomes comprising a plus DNA strand and a minus DNA strand are concatenated to form a self-complementary intramolecular dsDNA genome. Unlike ssAAV virions, the scAAV virions do not need to perform second-strand DNA synthesis, which increases the efficiency of scAAV transgene expression relative to ssAAV. However, the maximum cargo capacity of scAAV (i.e., maximum length of region between the 5′ ITR and 3′ ITR of the third polynucleotide construct) that can be packaged into the rAAV virion is about half that of ssAAV because the scAAV DNA packaged into a viral particle is a concatemer of two single-stranded genomes of opposite strands. For a description of methods of producing scAAV virions, see, e.g., Raj et al. (2011) Expert Rev. Hematol. 4 (5): 539-549, McCarty (2008) Mol. Ther. 16 (10): 1648-1656, McCarty et al. (2003) Gene Ther. 10 (26): 2112-2118; herein incorporated by reference in their entireties.
Maintaining constructs stably in the cellular genome requires selective pressure.
Typically, each integrated nucleic acid construct comprises a mammalian cell selection element. In some embodiments, the stable cell line comprises three integrated nucleic acid constructs, wherein the first nucleic acid construct comprises a first mammalian cell selection element, the second nucleic acid construct comprises a second mammalian cell selection element, and the third nucleic acid construct comprises a third mammalian cell selection element.
In some embodiments, the mammalian selection elements are components of a split auxotrophic selection system. In some embodiments, a first mammalian selection element, a second mammalian selection element, or a third mammalian selection element can be a component of a split auxotrophic selection system. For example, a first mammalian selection element can be a first component of the split auxotrophic selection system and second mammalian selection element can be a second component of the split auxotrophic selection system. As another example, a second mammalian selection element can be a first component of the split auxotrophic selection system and third mammalian selection element can be a second component of the split auxotrophic selection system.
A split auxotrophic system can be a leucine zipper based system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure. In some embodiments, components of the split auxotrophic selection system described herein comprise a C-terminal fragment of the auxotrophic protein Z-Cter and an N-terminal fragment of an auxotrophic protein Z-Nter. In some embodiments, a split auxotrophic system is a split intervening proteins (inteins) system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure. Inteins auto catalyze a protein splicing reaction that results in excision of the intein and joining of the flanking amino acids (extein sequences) via a peptide bond. Inteins exist in nature as a single domain within a host protein or, less frequently, in a split form. For split inteins, the two separate polypeptide fragments of the intein must associate in order for protein trans-splicing to occur to excise the intein. Split intein systems are described in: Cheriyan et al, J. Biol. Chem 288:6202-6211 (2013); Stevens et al, PNAS 114:8538-8543 (2017); Jillette et al., Nat Comm 10:4968 (2019); US 2020/0087388 A1; and US 2020/0263197 A1. In some embodiments, components of the split auxotrophic selection system described herein comprises a construct encoding an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of the split intein and a construct encoding the C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of the split intein. This N-terminal fragment is enzymatically nonfunctional and this C-terminal fragment is enzymatically nonfunctional. When both fragments are concurrently expressed in the cell, the split inteins can catalyze the joining of the N-terminal fragment of the auxotrophic protein and a C-terminal fragment of the auxotrophic protein to form a functional enzyme, such as any one of the enzymes disclosed herein (e.g., PAH, GS, TYMS, DHFR). In some embodiments, both constructs can be stably retained in the genome of a cell by growth in a medium lacking the product produced by the enzyme.
In some embodiments, a construct encoding for a component of a split auxotrophic system further encodes a helper enzyme, wherein expression of the helper enzyme facilitates growth of the host cell in conjunction with the functional enzyme upon application of the single selective pressure.
In some embodiments, the first nucleic acid construct comprises a first mammalian cell selection element, and the first mammalian cell selection element is a first auxotrophic selection element. In certain embodiments, the first auxotrophic selection element encodes an active protein. In some embodiments, the first auxotrophic selection element is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the first auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the first auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the first auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the first auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the first auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the first auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the first auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, first nucleic acid construct further comprises a sequence coding for a first auxotrophic selection element and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the first nucleic acid construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a first auxotrophic selection element comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the first auxotrophic selection element and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109.
In various embodiments, the second nucleic acid construct comprises a second mammalian cell selection element, and the second mammalian cell selection element encodes antibiotic resistance. In particular embodiments, the antibiotic resistance gene is a blasticidin resistance gene. In certain embodiments, the second mammalian cell selection element encodes an active protein. In some embodiments, t the second mammalian cell selection element is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
In various embodiments, the third nucleic acid construct comprises a third mammalian cell selection element. In some embodiments, the third mammalian cell selection element is a second auxotrophic selection element. In certain embodiments, the second auxotrophic selection element encodes an active protein. In some embodiments, the second auxotrophic selection element is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the second auxotrophic selection element codes for an inactive protein that requires expression of a first auxotrophic selection element for activity. In some embodiments, the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the first auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the second auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the first auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the first auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the second auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the second auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, third nucleic acid construct further comprises a sequence coding for second auxotrophic element and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the third nucleic acid construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a second auxotrophic selection element comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the second auxotrophic selection element and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109.
In various embodiments, the selectable marker of the first nucleic acid construct, the selectable marker of the second nucleic acid construct, and the selectable marker of the third nucleic acid construct are interchangeable between each other. For example, the first nucleic acid construct can comprise the second auxotrophic selection element, the second nucleic acid construct can comprise the first auxotrophic selection element, and the third nucleic acid construct can comprise the mammalian selection element.
In various embodiments, the stable mammalian cell line can be propagated in growth media lacking hypoxanthine and thymidine.
Described herein are many different combinations of elements in constructs, in combination, are capable of conditionally producing AAV virion in a cell. An exemplary complete system is described as follows, which is a non-limiting example. It is understood that variations of the elements described herein can be used in any of constructs described below, and the elements of the constructs described below may be split into one or more separate constructs. The first integrated synthetic construct comprises an intervening spacer sequence inserted into the coding sequence of AAV2 Rep protein. The intervening spacer sequence comprises an enhanced green fluorescent protein (EGFP) and a rabbit beta globin (RBG) polyadenylation (polyA) signal, flanked by two lox sites, are inserted into an RBG intron. The RBG intron includes the 5′ splice site (5′SS) and the 3′ splice site (3′SS) (as shown in
As described herein, expression of functional Rep protein is induced only in the presence of a first expression triggering agent, e.g., the addition of doxycycline which results in the production of Cre. In the presence of Cre, the intervening spacer is excised thereby resuming intact coding sequencing of the Rep protein. This approach provides controlled and inducible Rep expression.
This is driven by the second integrated synthetic construct, which comprises an estrogen inducible Cre (ER2 Cre) gene and adenoviral helper genes, E2A and E4orf6 (E4) (See,
In certain embodiments, the third integrated synthetic construct (“construct three”) comprises a polynucleotide flanked by AAV inverted terminal repeats (ITRs, shown by brackets in
In one aspect, provided herein is a stable mammalian cell line, wherein the cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload; and production of virions is not conditioned on the presence of an episome within the cell.
In various embodiments, expression of AAV rep and cap proteins is conditional. In some embodiments, expression of AAV rep and cap proteins is conditioned on addition of at least a first expression triggering agent to the cell culture medium. In some embodiments, expression of AAV Rep and Cap proteins is conditioned on addition of a first expression triggering agent and a second expression triggering agent to the cell culture medium.
In some embodiments, the cells do not express cytotoxic levels of Rep protein prior to addition of the at least a first expression triggering agent to the cell culture medium. In some embodiments, the cells do not express cytostatic levels of Rep protein prior to addition of the at least first expression triggering agent to the cell culture medium.
In some embodiments, the average concentration of Rep protein within the cells is less than between 1-99%, 10-90%, 20-80%, 30-70%, 40-60% prior to addition of the at least first expression triggering agent to the cell culture medium. In some embodiments, the average concentration of Rep protein within the cells is less than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% prior to addition of the at least first expression triggering agent to the cell culture medium.
In various embodiments, expression of Rep and Cap proteins becomes constitutive after addition of the at least first expression triggering agent to the cell culture medium. The stable cell lines include those wherein expression of adenoviral helper proteins is conditional. In some embodiments, expression of adenoviral helper proteins is conditioned on addition of at least a first expression triggering agent to the cell culture medium. In some embodiments, expression of adenoviral helper proteins is conditioned on addition of a first expression triggering agent and a second expression triggering agent to the cell culture medium. In some embodiments, continued expression of adenoviral helper proteins following triggering of expression requires presence of only the first expression triggering agent in the cell culture medium.
In some embodiments, the adenoviral helper proteins include E2A and E4.
In some embodiments, the first expression triggering agent is a tetracycline. In some embodiments, the tetracycline is doxycycline.
In some embodiments, the second expression triggering agent is an estrogen receptor ligand. In some embodiments, the estrogen receptor ligand is a selective estrogen receptor modulator (SERM). In some embodiments, the estrogen receptor ligand is tamoxifen.
In some embodiments, expression of the payload is not conditioned on addition of an expression triggering agent to the cell culture medium.
In various embodiments, the nuclear genome of the cell comprises a plurality of integrated synthetic nucleic acid constructs. In some embodiments, the nuclear genome of the cell comprises two integrated synthetic constructs. In some embodiments, the nuclear genome of the cell comprises three integrated synthetic constructs. In some embodiments, each of the plurality of synthetic nucleic acid constructs is separately integrated into the nuclear genome of the cell.
In some embodiments, only a single non-auxotrophic selection agent is required to be present in the cell culture medium to maintain all of the plurality of synthetic nucleic acid constructs stably within the nuclear genome of the cells.
In some embodiments, the first integrated synthetic construct comprises conditionally expressible AAV Rep and Cap coding sequences; the second integrated synthetic construct comprises a conditionally expressible Cre coding sequence and conditionally expressible adenoviral helper protein coding sequences; and the third integrated synthetic construct comprises expressible coding sequences for the payload.
In some embodiments, the first integrated construct comprises a Rep coding sequence interrupted by an intervening spacer. In some embodiments, the intervening spacer comprises, from 5′ to 3′, a first spacer, a second spacer and a third spacer. In some embodiments, the intervening spacer comprises nucleic acid sequences of a rabbit beta globin (RBG) intron and a rabbit beta globin (RBG) poly A. In some embodiments, the first spacer comprises a nucleic acid sequence of at least 80% identity to SEQ ID NO: 1. In some embodiments, the first spacer comprises a 5′ splice site (5′SS) 5′ to the first spacer. In some embodiments, the second spacer comprises a nucleic acid sequence of at least 80% identity to SEQ ID NO: 2. In some embodiments, the second spacer comprises, from 5′ to 3′ a first lox site, an enhanced green fluorescent protein (EGFP), the RBG polyA sequence, and a second lox site. In some embodiments, the second spacer further comprises a first 3′ splice site (3′SS) flanked by the first lox site and the EGFP. In some embodiments, the third spacer comprises a nucleic acid sequence of at least 80% identity to SEQ ID NO: 3. In some embodiments, the third spacer further comprises a second 3′ splice site (3′SS) 3′ to the third spacer.
In some embodiments, the Rep coding sequence comprises a polynucleotide sequence operatively linked to an endogenous P5 promoter. In some embodiments, the Rep coding sequence comprises a polynucleotide sequence operatively linked to an endogenous P19 promoter. In some embodiments, the intervening spacer is inserted into the Rep coding sequence at a position downstream of the P19 promoter. In some embodiments, the intervening spacer is inserted into the Rep coding sequence at a position in frame with the protein produced from activation of the P5 promoter and the P19 promoter. In some embodiments, wherein the Rep coding sequence is 5′ to the Cap coding sequence. In some embodiments, the Cap coding sequence is operatively linked to an endogenous P40 promoter.
In some embodiments, the second integrated construct comprises, from 5′ to 3′, a Cre coding sequence and a first polyA sequence, adenoviral helper protein coding sequences and a second polyA sequence, a first expression triggering agent responsive element, and an antibiotic selection element. In some embodiments, the Cre coding sequence is flanked by a first lox site and a second lox site. In some embodiments, the Cre coding sequence is operatively linked to an inducible promoter. In some embodiments, the inducible promoter comprises a plurality of tetracycline (Tet) operator elements capable of binding to a Tet responsive activator protein in the presence of a first expression triggering agent. In some embodiments, the inducible promoter comprises a plurality of Tet operator elements capable of binding to a Tet responsive activator protein in the presence of a first expression triggering agent and a second expression triggering agent responsive element. In some embodiments, the adenoviral helper protein coding sequences comprise E2A and E4 sequences. In some embodiments, the first expression triggering agent responsive element is operatively linked to a CMV promoter. In some embodiments, the first expression triggering agent responsive element comprises the Tet responsive activator protein (Tet-on-3G). In some embodiments, the antibiotic selection element is blasticidin resistance.
In some embodiments, the third integrated synthetic construct comprises a coding sequence for the expressible payload and a first element of an auxotrophic selection agent and the first integrated synthetic construct comprises coding sequences for a second element of the auxotrophic selection agent. In some embodiments, the first element of a auxotrophic selection agent comprises a first dihydrofolate reductase (DHFR) selectable marker (SEQ ID NO: 4). In some embodiments, the first DHFR comprises a leucine zipper (Nter). In some embodiments, the second element of the auxotrophic selection agent comprises a second DHFR (SEQ ID NO: 5). In some embodiments, the second DHFR comprises a leucine zipper (Cter). In some embodiments, the DHFR selection comprises the ability to grow in media lacking hypoxantine-thymidine.
In some embodiments, the mammalian cell line is selected from the group consisting of a human embryonic kidney (HEK) 293 cell line, a human HeLa cell line, and a Chinese hamster ovary (CHO) cell line. In some embodiments, the mammalian cell line is a HEK293 cell line. In some embodiments, the mammalian cell line expresses adenovirus helper functions E1A and E1B.
Alternative constructs as described herein can be used in a complete system. The complete system can be integrated into the host cell genome to produce a stable cell line. The complete system can be transfected into the host cell and then conditional production of AAV virion from the plasmids can be induced. In some embodiments, the complete system comprises episomes in the host cell and conditional production of AAV virion from the episomes is induced. In some embodiments, a complete system that lacks the elements of a tetracycline-inducible system is induced by adding tamoxifen to cause translocation of the Cre to the nucleus and subsequent production of AAV virion.
As described herein, the stable mammalian cell or cell line can be a human derived cell or cell line such as a human embryonic kidney (HEK) 293 cell line or a human HeLa cell line, or a mammalian cell or cell line such as Chinese hamster ovary (CHO) cell line. In some embodiments, the mammalian cell line is a HEK293 cell line. In some embodiments, the mammalian cell line expresses adenovirus helper functions E1A and E1B. In some embodiments, the mammalian cell line expresses adenovirus helper functions E2A and E4 (e.g., E4orf6).
Shown are exemplary designs of the first integrated synthetic construct (
As described in
Alternative Rep/Cap constructs as described herein can be integrated into a host cell genome to produce the stable cell line. Alternative Rep/Cap constructs that are non-integrating as described herein can be introduced into a host cell genome.
Shown are exemplary designs of the second integrated synthetic construct (
As shown in
In some embodiments, the Cre coding sequencing is an estrogen inducible Cre that has a strong polyadenylation signal (stop signal) at its 3′ end. Following this is a bicistronic E2A, E4orf6 cassette. The plasmid also has a constitutive promoter (CMV) which drives the expression of the Tet responsive activator protein (Tet-on 3G).
In the off state when doxycycline (Dox) is absent, the Tet-on 3G cannot bind to the Tet operator elements in the Tet-regulatable promoter so the promoter is not active. Estrogen responsive Cre is used instead of simple Cre to counteract the basal or leaky expression of the Tet-regulatable promoter. In the off state if there is leaky expression of Cre gene, the expressed Cre protein will be held inactive in the cytoplasm. The strong polyadenylation signal, 3′ of the cre gene will prevent basal expression of adenoviral helper genes, E2A and E4. To induce expression, doxycycline and tamoxifen are added to the cell culture (
Alternative helper constructs as described herein can be integrated into a host cell genome to produce the stable cell line. Alternative helper constructs that are non-integrating as described herein can be introduced into a host cell genome.
Shown are exemplary designs of the third integrated synthetic construct (
Alternative payload constructs as described herein can be integrated into a host cell genome to produce the stable cell line. Alternative payload constructs that are non-integrating as described herein can be introduced into a host cell genome.
In some embodiments, a cell comprises two constructs (any combination of Rep/Cap construct, inducible helper construct, and the payload construct). In some embodiments, a cell comprises the Rep/Cap construct and the inducible helper construct. In some embodiments, the inducible helper construct comprises a VA RNA construct as described herein. In some embodiments, cell further comprises the VA RNA construct.
In some embodiments, a cell comprises any combination of Rep/Cap construct(s) and inducible helper construct(s) as described herein. In some embodiments, a cell comprises any combination of Rep/Cap construct(s) and payload construct(s) as described herein. In some embodiments, a cell comprises any combination of inducible helper construct(s) and payload construct(s) as described herein. In some embodiments, a cell comprises any combination of Rep/Cap construct(s), inducible helper construct(s), and the payload construct(s) as described herein that are capable of producing AAV virion upon induction. In some embodiments, a cell comprises the Rep/Cap construct(s) and the inducible helper construct(s) of the complete system capable of producing AAV virion upon induction. In some embodiments, a cell comprises the Rep/Cap construct(s) and the payload construct(s). In some embodiments, a cell comprises the payload construct(s) and the inducible helper construct(s) of the complete system capable of producing AAV virion upon induction. In some embodiments, a Rep/Cap construct, an inducible helper construct, or a payload construct further comprises a VA RNA construct as described herein. In some embodiments, the cell further comprises a separate VA RNA construct as described herein.
In some embodiments, a cell comprises all three constructs (Rep/Cap construct, inducible helper construct, and the payload construct). In some embodiments, a cell comprises any combination of Rep/Cap construct(s), inducible helper construct(s), and the payload construct(s) as described herein that are capable of producing AAV virion upon induction. In some embodiments, the inducible helper construct comprises a VA RNA construct as described herein. In some embodiments, a Rep/Cap construct, an inducible helper construct, or a payload construct further comprises a VA RNA construct as described herein. In some embodiments, cell further comprises the VA RNA construct. In some embodiments, this cell is capable of producing an rAAV virion upon addition of at least one triggering agent. In some embodiments, the rAAV virion comprising the capsid protein and the payload nucleic acid sequence have an infectivity of no less than 50%, 60%, 70%, 80%, 90%, 95%, or 99% at an MOI of 1×105 vg/target cell or less. In some embodiments, the rAAV virions have an increased infectivity compared rAAV virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the rAAV virions have at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% greater infectivity compared rAAV virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the rAAV virions have at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared to AAV virions produced by a cell having wildtype AAV at the same MOI. In some embodiments, the rAAV virions have at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared to AAV virions at the same MOI. In some embodiments, the AAV virions are wildtype AAV virions produced by a cell having wildtype AAV. In some embodiments, the MOI is 1×101, 1×102, 2×103, 5×104, or 1×105 vg/target cell. In some embodiments, the MOI is selected from a range of 1×101 to 1×105 vg/target cell. In some embodiments, the cell is conditionally capable of producing rAAV virions having a payload encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the rAAV virions have a payload encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the rAAV virions have a concentration of greater than 1×1011 or no less than 5×1011, 1×1012, 5×1012, 1×1013 or 1×1014 viral genomes per milliliter prior to purification. In some embodiments, the cell is capable of producing rAAV virions comprising the payload nucleic acid sequence at a titer of greater than 1× 1011 or no less than 5×1011, 1×1012, 5×1012, 1×1013 or 1×1014 viral genomes per milliliter. In some embodiments, the cell is capable of producing rAAV virions comprising the payload nucleic acid sequence at a concentration of greater than 1×1011 or no less than 5×1011, 1×1012. 5×1012, 1×1013 or 1×1014 viral genomes per milliliter prior to purification. In some embodiments, this cell is expanded to produce a population of cells. In some embodiments, the population of cells produces a stable cell line as described herein. In some embodiments, this cell is passaged at least three times. In some embodiments, this cell can be passaged up to 60 times. In some embodiments, this cell can be passage more than 60 times. In some embodiments, the cell maintains the ability to be conditionally induced after each passage.
In some embodiments, a cell comprises one construct (Rep/Cap construct, inducible helper construct, and the payload construct). In some embodiments, the one construct is stably integrated into the genome of the cell. In some embodiments, the one construct is not stably integrated into the genome of the cell. In some embodiments, a plurality of the one construct is stably integrated into the genome of the cell. In some embodiments, a plurality of the one construct is not stably integrated into the genome of the cell. In some embodiments, a cell comprises two constructs (any combination of Rep/Cap construct, inducible helper construct, and the payload construct). In some embodiments, the two constructs are stably integrated into the genome of the cell. In some embodiments, the two constructs are not stably integrated into the genome of the cell. In some embodiments, the two constructs are separately stably integrated into the genome of the cell. In some embodiments, the two constructs are not separately stably integrated into the genome of the cell. In some embodiments, a plurality of the two constructs are stably integrated into the genome of the cell. In some embodiments, a plurality of the two constructs are not stably integrated into the genome of the cell. In some embodiments, a plurality of the two constructs are separately stably integrated into the genome of the cell. In some embodiments, a plurality of the two constructs are not separately stably integrated into the genome of the cell. In some embodiments, a cell comprises the Rep/Cap construct and the inducible helper construct. In some embodiments, a cell comprises the Rep/Cap construct(s) as disclosed herein, the inducible helper construct(s) as disclosed herein, payload construct(s) as disclosed herein, or any combination thereof. In some embodiments, the cell, the inducible helper construct comprises a VA RNA construct as described herein. In some embodiments, cell further comprises the VA RNA construct as described herein. In some embodiments, the VA RNA construct is stably integrated into the genome of the cell. In some embodiments, the VA RNA construct is not stably integrated into the genome of the cell.
In some embodiments, a cell comprises all three constructs (Rep/Cap construct, inducible helper construct, and the payload construct). In some embodiments, the three constructs are stably integrated into the genome of the cell. In some embodiments, the three constructs are not stably integrated into the genome of the cell. In some embodiments, the three constructs are separately stably integrated into the genome of the cell. In some embodiments, the three constructs are not separately stably integrated into the genome of the cell. In some embodiments, a plurality of the three constructs are stably integrated into the genome of the cell. In some embodiments, a plurality of the three constructs are not stably integrated into the genome of the cell. In some embodiments, a plurality of the three constructs are separately stably integrated into the genome of the cell. In some embodiments, a plurality of the three constructs are not separately stably integrated into the genome of the cell. In some embodiments, the cell, the inducible helper construct comprises a VA RNA construct as described herein. In some embodiments, cell further comprises the VA RNA construct.
In some embodiments, a VA RNA construct is a polynucleotide construct coding for a VA RNA, wherein a sequence coding for the VA RNA comprises at least two mutations in an internal promoter. In some embodiments, the sequence coding for the VA RNA comprises a sequence coding for a transcriptionally dead VA RNA. In some embodiments, the sequence coding for the VA RNA comprises a deletion of from about 5-10 nucleotides in the promoter region. In some embodiments, the sequence coding for the VA RNA comprises at least one mutation. In some embodiments, the at least one mutation is in the A Box promoter region. In some embodiments, the at least one mutation is in the B Box promoter region. In some embodiments, the at least one mutation is G16A and G60A. In some embodiments, the expression of the VA RNA is under the control of an RNA polymerase III promoter. In some embodiments, the expression of the VA RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the expression of the VA RNA is under the control of a U6 or U7 promoter. In some embodiments, the expression of the VA RNA is under the control of an interrupted U6 or U7 promoter. In some embodiments, the polynucleotide construct comprises upstream of the VA RNA gene sequence, from 5′ to 3′: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by a recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the gene encodes a detectable marker or a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109. In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. In some embodiments, the VA RNA construct further comprises a sequence coding for a recombinase. In some embodiments, the recombinase is exogenously provided. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP site and the second lox sequence is a second loxP site. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site. The polynucleotide construct of any one of any embodiment disclosed herein, further comprising a sequence coding for a selectable marker.
In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, the polynucleotide construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the polynucleotide construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109. In some embodiments, the VA RNA construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13-SEQ ID NO: 19 or SEQ ID 23-SEQ ID NO: 26. In some embodiments, VA RNA construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13-SEQ ID NO: 19 or SEQ ID 23-SEQ ID NO: 26.
In some embodiments, the cell is a mammalian cell or insect cell. In some embodiments, the cell is a HEK293 cell, HeLa cell, CHO cell, or SF9 cell. In some embodiments, the cell expresses E1A protein and E1B protein. In some embodiments, the cell further comprises a payload construct. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33. In some embodiments, the payload construct comprises a sequence of a payload flanked by ITR sequences. In some embodiments, expression of the sequence of the payload is driven by a constitutive promoter. In some embodiments, the constitutive promoter and sequence of the payload are flanked by ITR sequences. In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a gene. In some embodiments, the gene codes for a selectable marker or detectable marker. In some embodiments, the gene codes for a therapeutic polypeptide or transgene. In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a therapeutic polynucleotide. In some embodiments, the therapeutic polynucleotide is a tRNA suppressor or a guide RNA. In some embodiments, the guide RNA is a polyribonucleotide capable of binding to a protein. In some embodiments, the protein is nuclease. In some embodiments, the protein is a Cas protein, an ADAR protein, or an ADAT protein. In some embodiments, the Cas protein is catalytically inactive Cas protein. In some embodiments, the payload construct is stably integrated into the genome of the cell. In some embodiments, a plurality of the payload construct are stably integrated into the genome of the cell. In some embodiments, the plurality of the payload constructs are separately stably integrated into the genome of the cell. In some embodiments, the payload construct further comprises a sequence coding for a selectable marker or detectable marker outside of the ITR sequences. In some embodiments, the selectable marker is a mammalian cell selection element.
In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, the payload construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the payload construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109.
In some embodiments, the selectable marker is outside of the ITR sequences on the payload construct. In some embodiments, the selectable marker outside of the ITR sequences is a split intein linked to an N-terminus of the auxotrophic protein or split intein linked to a C-terminus of the auxotrophic protein. In some embodiments, the selectable marker outside of the ITR sequences is a leucine zipper linked to an N-terminus of the auxotrophic or leucine zipper linked to a C-terminus of the auxotrophic. In some embodiments, the selectable marker outside of the ITR sequences is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker outside of the ITR sequences is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the payload construct is in a plasmid. In some embodiments, the payload construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, the payload construct is stably integrated into the genome of the cell. In some embodiments, the payload construct is a synthetic nucleic acid construct. In some embodiments, the cell is capable of producing an rAAV virion that encapsidates the sequence of the payload. In some embodiments, the cell is capable of producing an rAAV virion upon addition of at least one triggering agent.
In some embodiments, the cell is selected to comprise a high copy number of a construct integrated into the cell. The construct can be the helper construct, the Rep/Cap construct, the payload construct, or any combination thereof. In some embodiments, the construct comprises an attenuated promoter that drives expression the selectable marker results in selection of a cell having integrated a high copy number of the construct into the cell genome. An attenuated promoter can be a mutated EF1alpha promoter, such as an attenuated EF1alpha promoter comprising SEQ ID NO: 132. In some embodiments, the construct comprises a selectable marker having weak activity, such as a selectable marker mutated to have decreased enzymatic activity, results in selection of a cell having integrated a high copy number of the construct into the cell genome. For example, the selectable marker can be a mutated GS, having a mutation at R324C, R324S, or R341C mutation as compared to SEQ ID NO: 112. In some embodiments, culturing the cell comprising a construct having a selectable marker with an inhibitor of the selectable marker results in selection of the cell having integrated a high copy number of the construct into the cell genome. For example, the selectable marker can be GS and the cell can be cultured with methionine sulfoximine (MSX). In some embodiments, the selectable maker is DHFR and the cell can be cultured with methotrexate, ochratoxin A, alpha-methyl-tyrosine, alpha-methyl-phenylalanine, beta-2-thienyl-DL-alanine, or fenclonine. The selectable marker can be any auxotrophic protein or any antibiotic resistance protein. The selectable marker can any auxtrophic selection element as described herein. The selectable marker can be any selectable marker as described herein. In some embodiments, the selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the construct further comprises a helper enzyme, such as GTP-CH1. In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the selectable marker and helper enzyme of the construct comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109. In some embodiments, the selection occurs in media comprising, for example, an antibiotic, or lacking nutrient required for cell growth accordingly for the selectable marker being used. In some embodiments, the media is supplemented with a cofactor or a cofactor precursor accordingly for the selectable marker being used and/or the helper enzyme being used. In some embodiments, the cofactor or cofactor precursor is tetrahydrobiopterin (BH4) or 7,8-dihydrobiopterin (7,8-BH2).
In some embodiments, this cell is capable of producing an rAAV virion upon addition of at least one triggering agent. In some embodiments, the rAAV virion comprising the capsid protein and the payload nucleic acid sequence have an infectivity of no less than 50%, 60%, 70%, 80%, 90%, 95%, or 99% at an MOI of 1×105 vg/target cell or less. In some embodiments, the rAAV virions have an increased infectivity compared rAAV virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the rAAV virions have at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% greater infectivity compared rAAV virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the rAAV virions have at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared to AAV virions produced by a cell having wildtype AAV at the same MOI. In some embodiments, the rAAV virions have at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared to AAV virions at the same MOI. In some embodiments, the AAV virions are wildtype AAV virions produced by a cell having wildtype AAV. In some embodiments, the MOI is 1×101, 1×102, 2×103, 5×104, or 1×105 vg/target cell. In some embodiments, the MOI is selected from a range of 1×101 to 1×105 vg/target cell. In some embodiments, the cell is conditionally capable of producing rAAV virions having a F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the rAAV virions have a F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the cell is conditionally capable of producing rAAV virions having a encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the rAAV virions have a payload encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the rAAV virions have a concentration of greater than 1×1011 or no less than 5×1011, 1×1012, 5×1012, 1×1013 or 1×1014 viral genomes per milliliter prior to purification. In some embodiments, the cell is capable of producing rAAV virions comprising the payload nucleic acid sequence at a titer of greater than 1× 1011 or no less than 5×1011, 1×1012, 5×1012, 1×1013 or 1×1014 viral genomes per milliliter. In some embodiments, the cell is capable of producing rAAV virions comprising the payload nucleic acid sequence at a concentration of greater than 1×1011 or no less than 5×1011, 1×1012, 5×1012, 1×1013 or 1×1014 viral genomes per milliliter prior to purification. In some embodiments, this cell is expanded to produce a population of cells. In some embodiments, the population of cells produces a stable cell line as described herein. In some embodiments, this cell is passaged at least three times. In some embodiments, this cell can be passaged up to 60 times. In some embodiments, this cell can be passage more than 60 times. In some embodiments, the cell maintains the ability to be conditionally induced after each passage.
In some embodiments, a population of cells comprise one construct (Rep/Cap construct, inducible helper construct, and the payload construct). In some embodiments, the one construct is stably integrated into the genomes of the cells. In some embodiments, the one construct is not stably integrated into the genomes of the cells. In some embodiments, a plurality of the one construct is stably integrated into the genomes of the cells. In some embodiments, a plurality of the one construct is not stably integrated into the genomes of the cells. In some embodiments, a population of cells comprises two constructs (any combination of Rep/Cap construct, inducible helper construct, and the payload construct). In some embodiments, the two constructs are stably integrated into the genomes of the cells. In some embodiments, the two constructs are not stably integrated into the genomes of the cells. In some embodiments, the two constructs are separately stably integrated into the genomes of the cells. In some embodiments, the two constructs are not separately stably integrated into the genomes of the cells. In some embodiments, a plurality of the two constructs are stably integrated into the genome of the cell. In some embodiments, a plurality of the two constructs are not stably integrated into the genome of the cell. In some embodiments, a plurality of the two constructs are separately stably integrated into the genomes of the cells. In some embodiments, a plurality of the two constructs are not separately stably integrated into the genomes of the cells. In some embodiments, a cell comprises the Rep/Cap construct and the inducible helper construct. In some embodiments, the cell, the inducible helper construct comprises a VA RNA construct as described herein. In some embodiments, cell further comprises the VA RNA construct as described herein. In some embodiments, the VA RNA construct is stably integrated into the genomes of the cells. In some embodiments, the VA RNA construct is not stably integrated into the genomes of the cells.
In some embodiments, a population of cells comprises all three constructs (Rep/Cap construct, inducible helper construct, and the payload construct). In some embodiments, the three constructs are stably integrated into the genomes of the cells. In some embodiments, the three constructs are not stably integrated into the genomes of the cells. In some embodiments, the three constructs are separately stably integrated into the genomes of the cells. In some embodiments, the three constructs are not separately stably integrated into the genomes of the cells. In some embodiments, a plurality of the three constructs are stably integrated into the genomes of the cells. In some embodiments, a plurality of the three constructs are not stably integrated into the genomes of the cells. In some embodiments, a plurality of the three constructs are separately stably integrated into the genomes of the cells. In some embodiments, a plurality of the three constructs are not separately stably integrated into the genomes of the cells. In some embodiments, the inducible helper construct comprises a VA RNA construct as described herein. In some embodiments, a population of cells further comprises the VA RNA construct separately integrated into the genomes of the cells. In some embodiments, a population of cells further comprises the VA RNA construct that are not separately integrated into the genomes of the cells.
In some embodiments, one or more constructs comprising the elements of the Rep/Cap construct, the helper construct, and/or the payload construct are stably integrated into the genomes of the cells. In some embodiments, one or more constructs comprising the elements of the Rep/Cap construct, the helper construct, and/or the payload construct are not stably integrated into the genomes of the cells. In some embodiments, one or more constructs comprising the elements of the Rep/Cap construct, the helper construct, and/or the payload construct are separately stably integrated into the genomes of the cells. In some embodiments, one or more constructs comprising the elements of the Rep/Cap construct, the helper construct, and/or the payload construct are not separately stably integrated into the genomes of the cells. In some embodiments, a plurality of the one or more constructs comprising the elements of the Rep/Cap construct, the helper construct, and/or the payload construct are separately stably integrated into the genomes of the cells. In some embodiments, the plurality of the one or more constructs comprising the elements of the Rep/Cap construct, the helper construct, and/or the payload construct are not separately stably integrated into the genomes of the cells. In some embodiments, cell further comprises the VA RNA construct as described herein. In some embodiments, the VA RNA construct is stably integrated into the genomes of the cells. In some embodiments, the VA RNA construct is not stably integrated into the genomes of the cells.
In some embodiments, the population of cells is selected to comprise a high copy number of a construct integrated into the population of cells. The construct can be the helper construct, the Rep/Cap construct, the payload construct, or any combination thereof. In some embodiments, the construct comprises an attenuated promoter that drives expression the selectable marker results in selection of the population of cells having integrated a high copy number of the construct into the cell genome. An attenuated promoter can be a mutated EF1alpha promoter, such as an attenuated EF1alpha promoter comprising SEQ ID NO: 132. In some embodiments, the construct comprises a selectable marker having weak activity, such as a selectable marker mutated to have decreased enzymatic activity, results in selection of the population of cells having integrated a high copy number of the construct into the cell genome. For example, the selectable marker can be a mutated GS, having a mutation at R324C, R324S, or R341C mutation as compared to SEQ ID NO: 112. In some embodiments, culturing the population of cells comprising the construct having a selectable marker, with an inhibitor of the selectable marker results in selection of the population of cells having integrated a high copy number of the construct into the cell genome of the population of cells. For example, the selectable marker can be GS and the cell can be cultured with methionine sulfoximine (MSX). In some embodiments, the selectable maker is DHFR and the cell can be cultured with methotrexate, ochratoxin A, alpha-methyl-tyrosine, alpha-methyl-phenylalanine, beta-2-thienyl-DL-alanine, or fenclonine. The selectable marker can be any auxotrophic protein or any antibiotic resistance protein. The selectable marker can any auxtrophic selection element as described herein. The selectable marker can be any selectable marker as described herein. In some embodiments, the selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the construct further comprises a helper enzyme. In some embodiments, the selectable marker and helper enzyme of the construct comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109. In some embodiments, the selection occurs in media comprising, for example, an antibiotic, or lacking nutrient (e.g., a cofactor) required for cell growth accordingly for the selectable marker being used.
A population of cells as disclosed herein can be capable of producing rAAV virions having a encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the population of cells are a population of mammalian cells or a population of insect cells. In some embodiments, the population of cells are a population of HEK293 cells, HeLa cells, CHO cells, or SF9 cells. In some embodiments, the cell expresses E1A protein and E1B protein. In some embodiments, the population of cells further comprises a payload construct. In some embodiments, the payload construct comprises a sequence of a payload flanked by ITR sequences. In some embodiments, expression of the payload is driven by a constitutive promoter. In some embodiments, the constitutive promoter and sequence of the payload are flanked by ITR sequences. In some embodiments, the payload comprises a polynucleotide sequence encoding a gene. In some embodiments, the gene codes for a selectable marker or detectable marker. In some embodiments, the gene codes for a therapeutic polypeptide or transgene. In some embodiments, the payload comprises a polynucleotide sequence coding for a therapeutic polynucleotide. In some embodiments, the therapeutic polynucleotide is a tRNA suppressor or a guide RNA. In some embodiments, the guide RNA is a polyribonucleotide capable of binding to a protein. In some embodiments, the protein is nuclease. In some embodiments, the protein is a Cas protein, an ADAR protein, or an ADAT protein. In some embodiments, the Cas protein is catalytically inactive Cas protein. In some embodiments, the payload construct is stably integrated into the genome of the cell. In some embodiments, the payload construct further comprises a sequence coding for a selectable marker or detectable marker outside of the ITR sequences.
In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
In some embodiments, the payload construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the payload construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90-SEQ ID NO: 98, SEQ ID NO: 112-SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101-SEQ ID NO: 109.
In some embodiments, the selectable marker is outside of the ITR sequences on the payload construct. In some embodiments, the selectable marker outside of the ITR sequences is a split intein linked to an N-terminus of the auxotrophic protein or split intein linked to a C-terminus of the auxotrophic protein. In some embodiments, the selectable marker outside of the ITR sequences is a leucine zipper linked to an N-terminus of the auxotrophic or leucine zipper linked to a C-terminus of the auxotrophic. In some embodiments, the selectable marker outside of the ITR sequences is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker outside of the ITR sequences is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the payload construct is in a plasmid. In some embodiments, the payload construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, the payload construct is stably integrated into the genomes of the population of cells. A population of cells produced by expanding a cell of any one of any embodiment disclosed herein. In some embodiments, expanding comprises passaging the cell at least three times. In some embodiments, a cell of the population of cells is capable of conditionally producing recombinant AAV (rAAV) virions upon addition of at least two triggering agents. In some embodiments, the cell is capable of conditionally producing rAAV virions upon addition of at least two triggering agents. In some embodiments, the at least two triggering agents comprise doxycycline and tamoxifen. In some embodiments, the at least two triggering agents induce the expression and translocation of an excising element to the nucleus. In some embodiments, a cell of the population of cells is capable of conditionally producing rAAV virions upon addition of an excising element. In some embodiments, the excising element is a recombinase. In some embodiments, the excising element is a site-specific recombinase. In some embodiments, the excising element is a Cre polypeptide or a flippase polypeptide. In some embodiments, the excising element is hormone regulated. In some embodiments, the population of cells are conditionally capable of producing rAAV virions within which are packaged an expressible polynucleotide encoding a payload; and wherein a population of virions produced by the population of cells are more homogenous than a population of virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection. In some embodiments, the population of virions produced by the population of cells has a ratio of viral genomes to transduction units of about 500:1 to 1:1. In some embodiments, the population of virions produced by the population of cells has a ratio of vector genomes to infectious unit of 100:1. In some embodiments, production of virions is inducible upon addition of a triggering agent. In some embodiments, production of virions is inducible upon addition of at least two triggering agents. In some embodiments, the population of cells is conditionally capable of producing rAAV virions having a payload encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the rAAV virions have a payload encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the population of cells are capable of reaching a viable cell density of no less than 1×106, 2×106, 5×106, or 1×107 cells per milliliter. In some embodiments, the rAAV virions have a concentration of greater than 1×1011 or no less than 5×1011, 1×1012, 5×1012, 1×1013 or 1×1014 viral genomes per milliliter prior to purification. In some embodiments, the population of cells is capable of producing rAAV virions comprising the payload nucleic acid sequence at a titer of greater than 1×1011 or no less than 5×1011, 1×1012, 5×1012, 1×1013 or 1× 1014 viral genomes per milliliter. In some embodiments, the population of cells is capable of producing rAAV virions comprising the payload nucleic acid sequence at a concentration of greater than 1×1011 or no less than 5×1011, 1×1012, 5×1012, 1×1013 or 1×1014 viral genomes per milliliter prior to purification. In some embodiments, the rAAV virions comprising the capsid protein and the payload nucleic acid sequence have an infectivity of no less than 50%, 60%, 70%, 80%, 90%, 95%, or 99% at an MOI of 1×105 vg/target cell or less. In some embodiments, the rAAV virions have an increased infectivity compared rAAV virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the rAAV virions have at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% greater infectivity compared rAAV virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the rAAV virions have at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared AAV virions AAV at the same MOI. In some embodiments, the AAV virions are wildtype AAV virions produced by a cell having wildtype AAV. In some embodiments, the MOI is 1×101, 1×102, 2×103, 5×104, or 1×105 vg/target cell. In some embodiments, the MOI is selected from a range of 1×101 to 1×105 vg/target cell. In some embodiments, the cell is conditionally capable of producing rAAV virions having a F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the rAAV virions have a F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the cells are cryopreserved. In some embodiments, the cells are comprised within a vial, flask, syringe, or other suitable cell-storage container. In some embodiments, production of rAAV virions is inducible in the absence of a plasmid. In some embodiments, expression of AAV Rep and Cap proteins is inducible in the absence of a plasmid. In some embodiments, expression of the at least one or more helper proteins is inducible in the absence of a plasmid. In some embodiments, production of rAAV virions is inducible in the absence of a transfection agent. In some embodiments, expression of AAV Rep and Cap proteins is inducible in the absence of a transfection agent. In some embodiments, expression of the at least one or more helper proteins is inducible in the absence of a transfection agent. A second population of cell produced by expanding the population of cells of any one of the preceding embodiments. The second population of cells, wherein expanding the population of cells comprises passaging the population of cells at least three times. In some embodiments expanding the population of cells comprises passaging the population of cells from 3 to 60 times. In some embodiments, expanding the population of cells comprises passaging the population of cells at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 times.
In some embodiments, an rAAV virion produced by the methods described herein have increased infectivity compared to a comparable rAAV virion produced by transient transfection methods.
In some embodiments, a stable cell line is produced from the cell as described herein. In some embodiments, a stable cell line is produced from the population of cells as described herein. In some embodiments, the stable cell line is derived from a single cell and is monoclonal. The stable cell line can be a mammalian stable cell line. The stable cell line can be produced by expanding or passaging a cell as described herein. The stable cell line produced by expanding or passaging a single cell and is a monoclonal stable cell line can have high reproducibility of titer (see, e.g.,
The stable cell line produced by expanding or passaging a single cell and is a monoclonal stable cell line can have high stability of titer (see, e.g.,
In some embodiments, a stable cell line comprises the population of cells as disclosed herein. In some embodiments, the population of cells are derived from a single cell. In some embodiments, at least 70%, 80%, 90%, 95%, 99%, or 100% of the cells of the stable cell line are the population of cells as disclosed herein. A stable cell line derived from a cell as disclosed herein. A stable cell line expanded from a cell as disclosed herein. In some embodiments, the stable cell line is a mammalian stable cell line. In some embodiments, expression of one or more helper proteins is inducible in the absence of a plasmid. In some embodiments, expression of one or more helper proteins is inducible in the absence of a transfection agent. In some embodiments, expression of AAV Rep and Cap proteins is inducible in the absence of a plasmid. In some embodiments, expression of AAV Rep and Cap proteins is inducible in the absence of a transfection agent. In some embodiments, production of rAAV virions is inducible in the absence of a plasmid. In some embodiments, production of rAAV virions is inducible in the absence of a transfection agent. In some embodiments, the stable cell line is capable of producing rAAV virions comprising the payload nucleic acid sequence at a concentration of greater than 1×1011 or no less than 5×1011, 1×1012, 5×1012, 1×1013 or 1×1014 viral genomes per milliliter. In some embodiments, the stable cell line is capable of producing rAAV virions comprising the payload nucleic acid sequence at a concentration of greater than 1×1011 or no less than 5×1011, 1×1012, 5×1012, 1×1013 or 1×1014 viral genomes per milliliter prior to purification. In some embodiments, the stable cell line is conditionally capable of producing rAAV virions having a encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the rAAV virions have a encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the rAAV virions comprising the capsid protein and the payload nucleic acid sequence have an infectivity of no less than 50%, 60%, 70%, 80%, 90%, 95%, or 99%, at an MOI of 1×105 vg/target cell or less. In some embodiments, the rAAV virions have an increased infectivity compared rAAV virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the rAAV virions have at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% greater infectivity compared rAAV virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the rAAV virions have at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared to AAV virions produced by a cell having wildtype AAV at the same MOI. In some embodiments, the rAAV virions have at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared to AAV virions at the same MOI. In some embodiments, the AAV virions are wildtype AAV virions produced by a cell having wildtype AAV. In some embodiments, the MOI is 1×101, 1×102, 2×103, 5×104, or 1×105 vg/target cell. In some embodiments, the MOI is selected from a range of 1×101 to 1×105 vg/target cell. In some embodiments, the stable cell line is conditionally capable of producing rAAV virions having a F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the rAAV virions have a F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, at least one cell of the stable cell line is cryopreserved. In some embodiments, at least one cell of the stable cell line is in a vial, flask, syringe, or other suitable cell-storage container.
In some embodiments, a method of producing a stable cell line comprises contacting a cell to the Rep/Cap construct as described herein, and expanding the cell to produce the stable cell line. In some embodiments, a method of producing a stable cell line comprises contacting a cell to the inducible helper construct as described herein, and expanding the cell to produce the stable cell line. In some embodiments, a method of producing a stable cell line comprises contacting a cell to the Rep/Cap construct, contacting the cell to the inducible helper construct as described herein, and expanding the cell to produce the stable cell line. In some embodiments, a method of producing a stable cell line comprises contacting a cell to the Rep/Cap construct, contacting the cell to inducible helper construct as described herein, contacting the cell to the payload construct, and expanding the cell to produce the stable cell line.
In some embodiments, a cell, population of cells, or stable cell line as disclosed herein is in a cell culture. In some embodiments, a cell culture composition comprising: a) suspension-adapted cells, b) serum-free cell culture media, and c) recombinant AAV (rAAV) virions, wherein the cell culture composition is free of herpes simplex virus, baculovirus, and adenovirus, and wherein the cell culture composition is free of plasmid and transfection agent. In some embodiments, the cell culture composition is free of polyethylenimine (PEI). In some embodiments, the suspension-adapted cells are suspension-adapted mammalian cells. In some embodiments, the suspension-adapted cells are suspension-adapted HEK293 cells or derivatives thereof. In some embodiments, the suspension-adapted mammalian cells are cells from the stable cell line of as disclosed herein, the population of cells as disclosed herein, or comprise a cell as disclosed herein. In some embodiments, the cell culture composition has a prepurification rAAV concentration of no less than 1×1014, 2×1014, 3×1014, 4×1014, 5×1014, 6×1014, 7×1014, 8×1014, 9×1014, 1×1015, or 5×1015 viral genome (vg)/L. In some embodiments, the cell culture composition has a prepurification rAAV encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99.
In some embodiments, rAAV virion from the stable cells as disclosed herein is produced in a bioreactor. In some embodiments, a bioreactor comprises the stable cell line as disclosed herein. In some embodiments, a bioreactor comprising the population of cells of as disclosed herein. In some embodiments, a bioreactor comprising the cell as disclosed herein. In some embodiments, a bioreactor contains the cell culture as disclosed herein. In some embodiments, the bioreactor is a 1L bioreactor. In some embodiments, the IL bioreactor has a total rAAV yield of greater than 1×1014 viral genome (vg). In some embodiments, the bioreactor is a 5L bioreactor. In some embodiments, the 5L bioreactor has a total rAAV yield of greater than 5×1014 viral genome (vg). In some embodiments, the bioreactor is a 50L bioreactor. In some embodiments, the 50L bioreactor has a total rAAV yield of greater than 5×1015 viral genome (vg). In some embodiments, the bioreactor is a 100L bioreactor. In some embodiments, the 100L bioreactor has a total rAAV yield of greater than 1×1016 viral genome (vg). In some embodiments, the bioreactor is a 500L bioreactor. In some embodiments, the 500L bioreactor has a total rAAV yield of greater than 5×1016 viral genome (vg). In some embodiments, the bioreactor is a 2000L bioreactor. In some embodiments, the 2000L bioreactor has a total rAAV yield of greater than 2×1017 viral genome (vg). In some embodiments, a bioreactor comprises a plurality of rAAV virions having a concentration of greater than 1×1014, 2×1014, 3×1014, 4×1014, 5×1014, 6×1014, 7×1014, 8×1014, 9×1014, 1×1015, or 5×1015 viral genome (vg)/L. In some embodiments, a bioreactor comprises a plurality of rAAV virions having a prepurification concentration of greater than 1×1014, 2×1014, 3×1014, 4×1014, 5×1014, 6×1014, 7×1014, 8×1014, 9×1014, 1×1015, or 5×1015 viral genome (vg)/L. In some embodiments, the bioreactor is a 1L, 5L, 50L, 100L, 500L, or 2000L bioreactor. In some embodiments, the bioreactor is a single use bioreactor.
Compositions of rAAV
In some embodiments, the cell, population of cells, or stable cell line as disclosed herein is induced (as disclosed herein, e.g., after administration of a first and a second triggering agent in a bioreactor) to produce a plurality of rAAV virions. In some embodiments, a composition comprises a plurality of rAAV virions encapsidating a viral genome, wherein the composition has a prepurification concentration of greater than 1×1011 or no less than 5×1011, 1× 1012, 5×1012, 1×1013 or 1×1014 viral genomes per milliliter. In some embodiments, a composition comprises a plurality of rAAV virions encapsidating a viral genome, wherein the composition has a prepurification encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, a composition comprises a plurality of rAAV virions encapsidating a viral genome, wherein the composition has a prepurification F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, a composition comprises an rAAV virion encapsidating a viral genome, wherein the composition has an infectivity of no less than 50%, 60%, 70%, 80%, 90%, 95%, or 99% at an MOI of 1×105 vg/target cell or less. In some embodiments, the rAAV virion has an increased infectivity compared an rAAV virion produced by an otherwise comparable cell capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the rAAV virion has at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% greater infectivity compared an rAAV virion produced by an otherwise comparable cell capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the rAAV virion has at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared an AAV virion produced by a cell having wildtype AAV at the same MOI. In some embodiments, the rAAV virion has at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared an AAV virion produced by a cell having wildtype AAV at the same MOI. In some embodiments, the compositions further comprises a plurality of the rAAV virion. In some embodiments, the plurality of rAAV virions have a prepurification concentration of greater than 1×1011 or no less than 5×1011, 1×1012, 5×1012, 1×1013 or 1×1014 viral genomes per milliliter. In some embodiments, the plurality of rAAV virions have a prepurification encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the plurality of rAAV virions have a prepurification F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the plurality of rAAV virions have an infectivity of no less than 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the plurality of rAAV virions have an increased infectivity compared a plurality of rAAV virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the plurality of rAAV virions have at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% greater infectivity compared a plurality of rAAV virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the plurality of rAAV virions have at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared a plurality of AAV virions produced by a cell having wildtype AAV at the same MOI. In some embodiments, the MOI is 1×101, 1×102, 2×103, 5×104, or 1×105 vg/target cell. In some embodiments, the MOI is selected from a range of 1×101 to 1×105 vg/target cell. In some embodiments, the viral genome comprises a sequence coding for a payload. In some embodiments, expression of the sequence of the payload is driven by a constitutive promoter. In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a gene. In some embodiments, the gene codes for a selectable marker or detectable marker. In some embodiments, the gene codes for a therapeutic polypeptide or transgene. In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a therapeutic polynucleotide. In some embodiments, the therapeutic polynucleotide is a tRNA suppressor or a guide RNA. In some embodiments, the guide RNA is a polyribonucleotide capable of binding to a protein. In some embodiments, the protein is nuclease. In some embodiments, the protein is a Cas protein, an ADAR protein, or an ADAT protein. In some embodiments, the Cas protein is catalytically inactive Cas protein. In some embodiments, the rAAV virion comprises a Cap polypeptide. In some embodiments, the Cap polypeptide is an AAV capsid protein. In some embodiments, the AAV capsid protein is VP1, VP2, or VP3. In some embodiments, a serotype of the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2YF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, and AAVhu68.
In some embodiments, rAAV virions as disclosed herein are in a first composition and a second composition. In some embodiments, the first composition and the second composition have an encapsidation ratio that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the first composition and the second composition have an F:E ratio that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the first composition and the second composition have a concentration of viral genomes per milliliter that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the first composition and the second composition have an infectivity that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the first composition is a first dose and the second composition is a second dose. In some embodiments, the first composition is produced at least 1, 2, 3, 4, 5, 6, or 7 days before the second composition is produced. In some embodiments, a plurality of rAAV virions of the first composition is produced at least 1, 2, 3, 4, 5, 6, or 7 days before a plurality of rAAV virions of the second composition is produced. In some embodiments, the first composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months before the second composition is produced. In some embodiments, a plurality of rAAV virions of the first composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months before the second composition is produced. In some embodiments, the first composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years before the second composition is produced. In some embodiments, a plurality of rAAV virions of the first composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years before the second composition is produced. In some embodiments, the first composition is produced from a plurality of virions from a first bioreactor and the second composition is produced from a plurality of virions from a second bioreactor. In some embodiments, a third composition or more compositions are produced from the rAAV as disclosed herein. In some embodiments, the first composition, the second composition, and the third composition have an encapsidation ratio that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the first composition, the second composition, and the third composition have an F:E ratio that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the first composition, the second composition, and the third composition have a concentration of viral genomes per milliliter that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the first composition, the second composition, and the third composition have an infectivity that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the third composition is a third dose. In some embodiments, the third composition is produced at least 1, 2, 3, 4, 5, 6, or 7 days after the second composition is produced. In some embodiments, a plurality of rAAV virions of the third composition is produced at least 1, 2, 3, 4, 5, 6, or 7 days after a plurality of rAAV virions of the second composition is produced. In some embodiments, the third composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the second composition is produced. In some embodiments, a plurality of rAAV virions of the third composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the second composition is produced. In some embodiments, the third composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after the second composition is produced. In some embodiments, a plurality of rAAV virions of the third composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after the second composition is produced. In some embodiments, the third composition is produced from a plurality of virions from a third bioreactor.
In some embodiments, a pharmaceutical composition comprises the plurality of rAAV virions of any one of the embodiments as disclosed herein and a pharmaceutically acceptable carrier. In some embodiment, a plurality of pharmaceutical doses each independently comprise the plurality of rAAV virions of any one of the embodiments as disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the encapsidation ratio has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses. In some embodiments, the F:E ratio has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses. In some embodiments, the concentration of viral genomes has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses. In some embodiments, the concentration of vector genomes has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses. In some embodiments, the rAAV virion infectivity has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses.
Method of Producing rAAV
In another aspect, methods of producing rAAV from stable cell lines is provided. The method comprises adding the at least first and at least second expression triggering agents to the medium within which the stable mammalian cell lines described above are being cultured.
In another aspect, methods of producing rAAV from a cell or population of cells as described herein is provided. The method comprises adding the at least first and at least second expression triggering agents to the medium within which the cell or population of cells described above are being cultured.
In particular embodiments, the first expression triggering agent is a tetracycline. In specific embodiments, the first expression triggering agent is Dox. In particular embodiments, the second expression triggering agent is an estrogen agonist or selective estrogen receptor modulator. In specific embodiments, the second expression triggering agent is tamoxifen.
In some embodiments, the method further comprises a later step of culturing the stable mammalian cell line only in the presence of the first expression triggering agent. In some embodiments, the method further comprises a later step of culturing the cell or population of cells only in the presence of the first expression triggering agent.
In some embodiments, the method further comprises purifying rAAV from culture medium. In some embodiments, the purifying comprises performing chromatographic purification. In some embodiments, the chromatographic purification comprises using a positively charged anion exchange resin, using a negatively charged anion exchange resin, using cation exchange chromatography, using affinity chromatography, using size exclusion chromatography, or a combination thereof. In some embodiments, the chromatographic purification comprises using column chromatographic fractionation.
In some embodiments, rAAV is produced in a bioreactor as described herein.
In some embodiments, a method of inducing the cell as described herein, the population of cells as described herein, or the stable cell line as described herein comprises administering a first triggering agent to the cell, population of cells, or the stable cell line, thereby inducing expression of the Rep polypeptides, Cap polypeptides, and one or more adenoviral helper proteins, in the cell, population of cells, or stable cell line. In some embodiments, the first triggering agent binds to an activator or a repressor. In some embodiments, activation of an inducible promoter is induced. In some embodiments, the activated inducible promoter transcribes a recombinase. In some embodiments, the first triggering agent is tetracycline or cumate. In some embodiments, the tetracycline is doxycycline. The methods described herein further comprise culturing the cell, population of cells, or the stable cell line with a second triggering agent. In some embodiments, the second triggering agent is an estrogen receptor ligand. In some embodiments, the second triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the second triggering agent is tamoxifen. In some embodiments, the second triggering agent binds to the recombinase. In some embodiments, the second triggering agent induces the recombinase to translocate to a nucleus of the cell, of a cell of the population of cells, of a cell of the stable cell lines.
In some embodiments, a method of producing rAAV virion comprises administering a first triggering agent to the cell, population of cells, or the stable cell line, administering a second triggering agent to the cell, population of cells, or stable cell line, thereby producing the rAAV virion in the cell, population of cells, or stable cell line. In some embodiments, the first triggering agent binds to an activator or a repressor. In some embodiments, activation of an inducible promoter is induced. In some embodiments, the activated inducible promoter transcribes a recombinase. In some embodiments, the first triggering agent is tetracycline or cumate. In some embodiments, the tetracycline is doxycycline. In some embodiments, the method further comprises culturing the cell, population of cells, or the stable cell line with a second triggering agent. In some embodiments, the second triggering agent is an estrogen receptor ligand. In some embodiments, the second triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the second triggering agent is tamoxifen. In some embodiments, the second triggering agent binds to the recombinase. In some embodiments, the second triggering agent induces the recombinase to translocate to a nucleus of the cell, of a cell of the population of cells, of a cell of the stable cell lines. In some embodiments, the recombinase cuts at recombinase sites. In some embodiments, the at least one adenoviral help proteins, the Rep polypeptides, and the Cap polypeptides are expressed. In some embodiments, the Rep polypeptides and the Cap polypeptides assemble into an rAAV virion. In some embodiments, the rAAV virion encapsidates a sequence of a payload. In some embodiments, the cell, population of cells, or stable cell line do not express cytotoxic levels of Rep polypeptides prior to administration of both the first triggering agent and the second triggering agent. In some embodiments, the cell, population of cells, or stable cell line do not express cytotoxic levels of Cap polypeptides prior to administration of both the first triggering agent and the second triggering agent. In some embodiments, the cell, population of cells, or stable cell line do not express cytostatic levels of Rep polypeptides prior to administration of both the first triggering agent and the second triggering agent. In some embodiments, the average concentration of Rep polypeptides within the cell, population of cells, or stable cell line is less than the amount prior to administration of both of the first triggering agent and second triggering agent. In some embodiments, expression of Rep polypeptides and Cap polypeptides becomes constitutive after administration of both the first triggering agent and the second triggering agent. In some embodiments, the method further comprises performing at least a portion of the method in a bioreactor. In some embodiments, the bioreactor is not less than 20 L, 30, L, 40 L, 50 L, 100 L, 250 L, 300 L, or 500 L.
In some embodiments, the method further comprises producing the rAAV virions in a plurality of batches. In some embodiments, the method further comprises producing the rAAV virions having a difference in the encapsidation ratio of not more than 20%, 15%, 10%, 5%, 3%, 2%, or 1% between a first batch and a second batch. In some embodiments, the method further comprises producing the rAAV virions having a difference in the F:E ratio of not more than 20%, 15%, 10%, 5%, 3%, 2%, or 1% between a first batch and a second batch. In some embodiments, the method further comprises producing the rAAV virions having a difference in the concentration of viral genomes of not more than 20%, 15%, 10%, 5%, 3%, 2%, or 1% between a first batch and a second batch. In some embodiments, the method further comprises producing the rAAV virions having a difference in the concentration of vector genomes of not more than 20%, 15%, 10%, 5%, 3%, 2%, or 1% between a first batch and a second batch. In some embodiments, the method further comprises producing the rAAV virions having a difference in infectivity of not more than 20%, 15%, 10%, 5%, 3%, 2%, or 1% between a first batch and a second batch. In some embodiments, the method further comprises performing the method according to good manufacturing practice (GMP) standards. In some embodiments, the method further comprises performing the method in a GMP facility. In some embodiments, the method further comprises comprising culturing the cells in a culture medium and collecting a portion of the plurality of rAAV virions from the culture medium. In some embodiments, the method further comprises purifying at least some of the plurality of rAAV virions collected from the culture medium to obtain a purified rAAV population. In some embodiments, the purifying comprises performing chromatographic purification. In some embodiments, the chromatographic purification comprises using a positively charged anion exchange resin, using a negatively charged anion exchange resin, using cation exchange chromatography, using affinity chromatography, using size exclusion chromatography, or a combination thereof. In some embodiments, the chromatographic purification comprises using column chromatographic fractionation.
In some embodiments, an rAAV virion is made by the methods as disclosed herein. In some embodiments, a composition comprising a plurality of rAAV virions is made by the methods as disclosed herein. In some embodiments, the rAAV virion produced as disclosed herein has increased infectivity compared to an rAAV virion produced by comparable transient transfection methods.
In another aspect, methods of treatment are provided. In various embodiments, the method comprises administering rAAV produced by the process described above to a patient in need thereof. In some embodiments, the administering is by intravenous administration, intramuscular administration, intrathecal administration, intracisternal administration, or administration via brain surgery.
In some embodiments, a method of treating a condition or disorder comprises administering a therapeutically effective amount of the pharmaceutical composition of as disclosed herein to a patient in need thereof. In some embodiments, the disorder is a monogenic disorder. In some embodiments, the treatment results in at least one undesirable side effect and wherein the undesirable side effect is reduced relative to administering a daily dose that deviates more than 50%, 40%, 30%, 30%, 15%, 10%, 5%, or 2% from an expected dose. In some embodiments, the administering is by injection. In some embodiments, the injection is an infusion. In some embodiments, the daily dose is administered to the patient once. In some embodiments, the daily dose is administered to the patient two or more times. In some embodiments, the treatment results in at least one undesirable side effect and wherein the undesirable side effect is reduced relative to administering a plurality of rAAV virions produced from a triple transfection method.
In some embodiments, the methods reduce the immunogenicity of a dose of rAAV having a predetermined number of viral genomes (VG) as compared to the same rAAV VG dose prepared by transient triple transfection. In some embodiments, the immunogenicity is measured by the titer or concentration of neutralizing antibodies in a subject. In some embodiments, a concentration of rAAV virion neutralizing antibody in the blood serum of the patient is reduced relative to a concentration of rAAV virion neutralizing antibody in the blood serum of a patient after administering a plurality of rAAV virions produced from a triple transfection method. In some embodiments, the concentration of rAAV virion neutralizing antibodies is measured by an ELISA assay.
In some embodiments, the methods reduce the number or intensity of adverse effects caused by administering a dose of rAAV having a predetermined number of viral genomes (VG) as compared to the same rAAV VG dose prepared by transient triple transfection. In some embodiments, the methods reduce the number of adverse effects. In some embodiments, the predetermined number of VG in a dose is no greater than 3×1014 vg/kg. In some embodiments, the predetermined number of VG in a dose is no greater than 1×1014 vg/kg. In some embodiments, the predetermined number of VG in a dose is no greater than 5×1013 vg/kg. In some embodiments, the methods reduce the intensity of adverse effects. In some embodiments, the methods reduce both the number and the intensity of adverse events.
In some embodiments, a method of administering a dose of rAAV virions having a predetermined number of viral genomes (VG) to a subject with reduced number or intensity of adverse effects as compared to administration of the same rAAV VG dose prepared by transient triple transfection comprises: administering a dose of rAAV produced in the cell as disclosed herein, the population of cells disclosed herein, or the stable cells as disclosed herein. In some embodiments, the adverse effect is selected from the group consisting of: liver dysfunction, liver inflammation, gastrointestinal infection, vomiting, bacterial infection, sepsis, increases in troponin levels, decreases in red blood cell counts, decreases in platelet counts, activation of the complement immune system response, acute kidney injury, cardio-pulmonary insufficiency, and death. In some embodiments, the adverse effect is an increase in serum levels of one or more proinflammatory cytokines. In some embodiments, the adverse effect is an increase in serum levels of one or more of interferon gamma (IFNγ), interleukin 1β (IL-1β), and interleukin 6 (IL-6).
In another aspects, a method of repeatedly administering a dose of rAAV to a subject in need thereof are provided. In some embodiments, the method comprises administering a first dose of rAAV produced by the cell lines and the processes described above, and then administering at least a second dose of rAAV produced by the cell lines and the processes described above. In some embodiments, the method comprises administering a first dose and a second dose of rAAV produced by the cell lines and the processes described above. In some embodiments, the method comprises administering a first dose, a second dose, and a third dose of rAAV produced by the cell lines and the processes described above. In some embodiments, the method comprises administering more than three doses of rAAV produced by the cell lines and the processes described above. In some embodiments, the first dose of rAAV and the at lease second dose of rAAV are administered through the same route of administration. In some embodiments, the first dose of rAAV and the at least second dose of rAAV are administered through different routes of administration. In some embodiments, the route of administration is intravenous administration, intramuscular administration, intrathecal administration, intracisternal administration, or administration via brain surgery.
In some embodiments, a method of treating a condition or disorder comprises administering a first therapeutically effective amount of the pharmaceutical composition of as disclosed herein having a predetermined number of viral genomes to a patient in need thereof and a second therapeutically effective amount of the pharmaceutical composition as disclosed herein having the predetermined number of viral genomes to the patient in need thereof. In some embodiments, the first therapeutically effective amount and the second therapeutically effective amount vary by no more than 1%, 5%, 10%, or 15%.
In another aspect, components or embodiments described herein for the system are provided in a kit. For example, any of the plasmids, as well as the mammalian cells, related buffers, media, triggering agents, or other components related to cell culture and virion production can be provided, with optional components frozen and packaged as a kit, alone or along with separate containers of any of the other agents and optional instructions for use. In some embodiments, the kit may comprise culture vessels, vials, tubes, or the like.
The methods for producing and packaging recombinant vectors in desired AAV capsids to produce the rAAVs are not meant to be limiting and other suitable methods will be apparent to the skilled artisan.
The below items disclose various aspects of the invention. Each of the aspects described below can be combined with other aspects and embodiments disclosed elsewhere herein, including the claims, where the combinations are clearly compatible. For example, described herein are three exemplary, non-limiting, constructs, referred to as “construct 1”, “construct 2” and “construct 3”. The disclosure provided herein describes these constructs in specific and general detail.
In the following aspects, the first recombinant nucleic acid sequence encoding an AAV Rep protein and an AAV Cap protein corresponds to the specific and general disclosures of “construct 1” provided herein. It is intended that any aspects described below relating to the first recombinant nucleic acid may be combined with any of the specific and general disclosures of “construct 1” provided herein where the combinations are clearly compatible.
In the following aspects, the second recombinant nucleic acid sequence encoding one or more adenoviral helper proteins corresponds to the specific and general disclosures of “construct 2” provided herein. It is intended that any aspects described below relating to the second recombinant nucleic acid may be combined with any of the specific and general disclosures of “construct 2” provided herein where the combinations are clearly compatible.
In the following aspects, the third recombinant nucleic acid sequence encoding a payload corresponds to the specific and general disclosures of “construct 3” provided herein. It is intended that any aspects described below relating to the third recombinant nucleic acid may be combined with any of the specific and general disclosures of “construct 3” provided herein where the combinations are clearly compatible.
It is intended that any aspects and disclosures provided herein relating to the first, second and third recombinant nucleic acids, and relating to the specific and general disclosures of constructs 1, 2 and 3 may be combined together where the combinations are clearly compatible.
The cell of any one of embodiments 1-56, wherein the first polynucleotide construct further comprises:
NpuDnaE intein
GNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVD
GQMLPIDEIFERELDLMRVDNLPN
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCTGFRLRP
NpuDnaE
intein/C-terminal
terminal NpuDnaE
intein
LSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPV
AQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPI
DEIFERELDLMRVDNLPN
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCTQYIRHG
NpuDnaE intein/C-
terminal NpuDnaE
intein
NGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTV
DGQMLPIDEIFERELDLMRVDNLPN
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCHELLGH
NpuDnaE intein/C-
terminal NpuDnaE
intein
VDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKF
MTVDGQMLPIDEIFERELDLMRVDNLPN
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCKQGDSI
NpuDnaE intein/C-
VEENPGP
MEKGPVRAPAEKPRGARCSNGFPERDPPRPGPSRPAE
KPPRPEAKSAQPADGWKGERPRSEEDNELNLPNLAAAYSSILSS
LGENPQRQGLLKTPWRAASAMQFFTKGYQETISDVLNDAIFDE
DHDEMVIVKDIDMFSMCEHHLVPFVGKVHIGYLPNKQVLGLS
KLARIVEIYSRRLQVQERLTKQIAVAITEALRPAGVGVVVEATH
MCMVMRGVQKMNSKTVTSTMLGVFREDPKTREEFLTLIRS
terminal NpuDnaE
intein-P2A-
QPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQML
PIDEIFERELDLMRVDNLPN
GSGATNFSLLKQAGDVEENPG
PMEK
GPVRAPAEKPRGARCSNGFPERDPPRPGPSRPAEKPPRPEAKSAQP
ADGWKGERPRSEEDNELNLPNLAAAYSSILSSLGENPQRQGLLKTP
WRAASAMQFFTKGYQETISDVLNDAIFDEDHDEMVIVKDIDMFSM
CEHHLVPFVGKVHIGYLPNKQVLGLSKLARIVEIYSRRLQVQERLT
KQIAVAITEALRPAGVGVVVEATHMCMVMRGVQKMNSKTVTSTM
LGVFREDPKTREEFLTLIRS
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCTGFRLRP
NpuDnaE intein/C-
GPVRAPAEKPRGARCSNGFPERDPPRPGPSRPAEKPPRPEAKSAQP
ADGWKGERPRSEEDNELNLPNLAAAYSSILSSLGENPQRQGLLKTP
WRAASAMQFFTKGYQETISDVLNDAIFDEDHDEMVIVKDIDMFSM
CEHHLVPFVGKVHIGYLPNKQVLGLSKLARIVEIYSRRLQVQERLT
KQIAVAITEALRPAGVGVVVEATHMCMVMRGVQKMNSKTVTSTM
LGVFREDPKTREEFLTLIRS
terminal NpuDnaE
intein-P2A-
LLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFE
YC
L
EDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNL
PN
GSGATNFSLLKQAGDVEENPGP
MEKGPVRAPAEKPRGARCSNG
FPERDPPRPGPSRPAEKPPRPEAKSAQPADGWKGERPRSEEDNEL
NLPNLAAAYSSILSSLGENPQRQGLLKTPWRAASAMQFFTKGYQET
ISDVLNDAIFDEDHDEMVIVKDIDMFSMCEHHLVPFVGKVHIGYL
PNKQVLGLSKLARIVEIYSRRLQVQERLTKQIAVAITEALRPAGVGV
VVEATHMCMVMRGVQKMNSKTVTSTMLGVFREDPKTREEFLTLI
RS
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCTQYIRHG
NpuDnaE intein/C-
LKQAGDVEENPGP
MEKGPVRAPAEKPRGARCSNGFPERDPPRPGP
SRPAEKPPRPEAKSAQPADGWKGERPRSEEDNELNLPNLAAAYSSI
LSSLGENPQRQGLLKTPWRAASAMQFFTKGYQETISDVLNDAIFDE
DHDEMVIVKDIDMFSMCEHHLVPFVGKVHIGYLPNKQVLGLSKLA
RIVEIYSRRLQVQERLTKQIAVAITEALRPAGVGVVVEATHMCMVM
RGVQKMNSKTVTSTMLGVFREDPKTREEFLTLIRS
terminal NpuDnaE
intein-P2A-
QPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQML
PIDEIFERELDLMRVDNLPN
GSGATNFSLLKQAGDVEENPGP
MEK
GPVRAPAEKPRGARCSNGFPERDPPRPGPSRPAEKPPRPEAKSAQP
ADGWKGERPRSEEDNELNLPNLAAAYSSILSSLGENPQRQGLLKTP
WRAASAMQFFTKGYQETISDVLNDAIFDEDHDEMVIVKDIDMFSM
CEHHLVPFVGKVHIGYLPNKQVLGLSKLARIVEIYSRRLQVQERLT
KQIAVAITEALRPAGVGVVVEATHMCMVMRGVQKMNSKTVTSTM
LGVFREDPKTREEFLTLIRS
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCHELLGHV
NpuDnaE intein/C-
PVRAPAEKPRGARCSNGFPERDPPRPGPSRPAEKPPRPEAKSAQPA
DGWKGERPRSEEDNELNLPNLAAAYSSILSSLGENPQRQGLLKTP
WRAASAMQFFTKGYQETISDVLNDAIFDEDHDEMVIVKDIDMFSM
CEHHLVPFVGKVHIGYLPNKQVLGLSKLARIVEIYSRRLQVQERLT
KQIAVAITEALRPAGVGVVVEATHMCMVMRGVQKMNSKTVTSTM
LGVFREDPKTREEFLTLIRS
terminal NpuDnaE
intein-P2A-
YTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQ
MLPIDEIFERELDLMRVDNLPN
GSGATNFSLLKQAGDVEENPGP
MEKGPVRAPAEKPRGARCSNGFPERDPPRPGPSRPAEKPPRPEAK
SAQPADGWKGERPRSEEDNELNLPNLAAAYSSILSSLGENPQRQGL
LKTPWRAASAMQFFTKGYQETISDVLNDAIFDEDHDEMVIVKDID
MFSMCEHHLVPFVGKVHIGYLPNKQVLGLSKLARIVEIYSRRLQVQ
ERLTKQIAVAITEALRPAGVGVVVEATHMCMVMRGVQKMNSKTV
TSTMLGVFREDPKTREEFLTLIRS
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCKQGDSIK
NpuDnaE intein/C-
RAPAEKPRGARCSNGFPERDPPRPGPSRPAEKPPRPEAKSAQPADG
WKGERPRSEEDNELNLPNLAAAYSSILSSLGENPQRQGLLKTPWRA
ASAMQFFTKGYQETISDVLNDAIFDEDHDEMVIVKDIDMFSMCEH
HLVPFVGKVHIGYLPNKQVLGLSKLARIVEIYSRRLQVQERLTKQIA
VAITEALRPAGVGVVVEATHMCMVMRGVQKMNSKTVTSTMLGVF
REDPKTREEFLTLIRS
terminal NpuDnaE
YTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQ
intein
MLPIDEIFERELDLMRVDNLPN
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCVEELPEW
NpuDnaE intein/C-
terminal NpuDnaE
intein
KIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLED
GSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCKRIMDM
terminal NpuDnaE
intein
QPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQML
PIDEIFERELDLMRVDNLPN
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCLYAGVKI
NpuDnaE intein/C-
terminal NpuDnaE
intein
TQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQM
LPIDEIFERELDLMRVDNLPN
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCEDFGVIA
NpuDnaE intein/C-
terminal NpuDnaE
intein
GKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLE
DGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCHTNFSTK
NpuDnaE intein/C-
ETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWH
terminal NpuDnaE
DRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERE
intein
LDLMRVDNLPN
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCGVRKDD
NpuDnaE intein/C-
terminal NpuDnaE
intein
EKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSL
IRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCHALCQFY
NpuDnaE intein/C-
GKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLE
DGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCQFYVVNS
NpuDnaE intein/C-
terminal NpuDnaE
intein
EILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDR
GEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELD
LMRVDNLPN
C-terminal
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCQLYQRSG
NpuDnaE intein/C-
All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference in its entirety, for all purposes. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. § 1.57 (b) (1), to relate to each and every individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. § 1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.
While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.
This application claims priority to U.S. Provisional Application No. 63/437,553, filed on Jan. 6, 2023; U.S. Provisional Application No. 63/427,037, filed on Nov. 21, 2022; U.S. Provisional Application No. 63/404,434, filed on Sep. 7, 2022; U.S. Provisional Application No. 63/316,308, filed on Mar. 3, 2022; and U.S. Provisional Application No. 63/305,662, filed on Feb. 1, 2022, the disclosures of which applications are herein incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/061667 | 1/31/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63437553 | Jan 2023 | US | |
| 63427037 | Nov 2022 | US | |
| 63404434 | Sep 2022 | US | |
| 63316308 | Mar 2022 | US | |
| 63305662 | Feb 2022 | US |
