Producer cells for safer AAV production

Abstract

The present disclosure provides compositions and methods for the production of recombinant adeno-associated virus (rAAV) particles that are safer for clinical administration. Among other things, the present disclosure describes producer cells comprising immortalization gene(s) that are too large to be packaged into rAAV (e.g., unpackable).

Description

PRODUCER CELLS FOR SAFER AAV PRODUCTIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 666,352 filed on July 1, 2024, the entire contents of which are hereby incorporated by reference in its entirety.BACKGROUND

[0002] Adeno-associated virus (AAV) technology has quickly become a dominant form of gene therapy for genetic diseases.

[0003] Despite much advancement over the last two decades, concerns regarding the cost and safety of AAV production continue to limit the therapeutic potential of AAV technology. These concerns are due in part to potential carcinogenic risk associated with remnant material from the AAV production process.SUMMARY

[0004] In some embodiments, the present disclosure provides, among other things, a producer cell (e.g., an rAAV producer cell). In some embodiments, the present disclosure provides a producer cell wherein AAV particles produced therefrom are safer for clinical administration relative to those known in the art. In some embodiments, the present disclosure provides a producer cell wherein AAV particles produced therefrom have decreased carcinogenic risk relative to those known in the art.

[0005] Among other things, the present disclosure provides a producer cell comprising a potentially carcinogenic immortalization gene that is too large to be packaged into an AAV particle (e.g., an oversized gene). In some embodiments, the present disclosure provides a rAAV producer cell with a genome comprising a region that includes two or more exons encoding SV40 large T-antigen and one or more intron(s) located between the two or more exons, wherein the combined size of the sequence encoding SV40 large T-antigen and the one or more intron(s) is larger than the packaging capacity of an rAAV.

[0006] In some embodiments, the combined size of the sequence encoding SV40 large T-antigen is about 1000 to about 3000 base pairs, about 1500 to about 3000 base pairs, about 2000 to about 3000 base pairs, about 2000 to about 2200 base pairs. In someembodiments, the combined size of the sequence encoding SV40 large T-antigen is about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2050, about 2100, about 2120, about 2130, about 2140, about 2150, about 2200, about 2250, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900, or is about 3000 base pairs.. In some embodiments, the combined size of the sequence encoding SV40 large T-antigen is about 2127 base pairs.

[0007] In some embodiments, the size of the one or more intron(s) is greater than about 1000, greater than about 2000, greater than about 3000, greater than about 4000, greater than about 5000, or greater than about 6000 base pairs in length. In some embodiments, the size of the one or more intron(s) is about 1000 to about 6000, about 2000 to about 6000, about 3000 to about 6000, about 4000 to about 6000, or about 5000 to about 6000 base pairs in length. In some embodiments, the size of the one or more intron(s) is greater than about 5000 base pairs in length. In some embodiments, the size of the one or more intron(s) is about 5410 base pairs in length.

[0008] In some embodiments, the region encoding a SV40 large T-antigen comprises an intron that is or is the complement of intron 1 of human eukaryotic translation elongation factor 1 alpha 1 (EFl Al).

[0009] In some embodiments, the region encoding a SV40 large T-antigen comprises an intron that is or is the complement of introns 2, 3, and 4 of human ribosomal protein S27a (RPS27A).

[0010] In some embodiments, the region encoding a SV40 large T-antigen comprises an intron that is or is the complement of intron 1 of human Foxf2.

[0011] In some embodiments, the region encoding a SV40 large T-antigen comprises an intron that is or is the complement of intron 1 of human EFl Al, an intron that is or is the complement of introns 2, 3, and 4 of human ribosomal protein S27a (RPS27A) and an intron that is or is the complement of intron 1 of human Foxf2.

[0012] In some embodiments, the chimeric intron has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 5 as disclosed herein.

[0013] In some embodiments, the chimeric intron has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 5 as disclosed herein.

[0014] In some embodiments, the chimeric intron has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 as disclosed herein.

[0015] In some embodiments, the combined size of the two or more exons encoding SV40 large T-antigen and the one or more intron(s) is at least about 7000, at least about 7100, at least about 7200, at least about 7300, at least about 7400, at least about 7500, at least about 7600, at least about 7700, at least about 7800, at least about 7900, or is at least about 8000 base pairs in length.

[0016] In some embodiments, the combined size of the two or more exons encoding SV40 large T-antigen and the one or more intron(s) is about 7000 to about 8000, about 7100 to about 8000, about 7200 to about 8000, about 7200 to about 8000, about 7300 to about 8000, about 7400 to about 8000, about 7500 to about 8000, about 7600 to about 8000, about 7000 to about 7600, about 7100 to about 7600, about 7200 to about 7600, about 7300 to about 7600, about 7400 to about 7600, or is about 7500 to about 7600 base pairs in length.

[0017] In some embodiments, the combined size of the two or more exons encoding SV40 large T-antigen and the one or more intron(s) located between the two or more exons is about 7000, about 7100, about 7200, about 7300, about 7400, about 7500, about 7600, about 7700, about 7800, about 7900, or is about 8000 base pairs in length. In some embodiments, the combined size of the two or more exons encoding SV40 large T-antigen and the one or more intron(s) is about 7537 base pairs in length.

[0018] In some embodiments, the two or more exons encoding SV40 large T-antigen and the one or more intron(s) located between the two or more exons has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6 as disclosed herein.

[0019] In some embodiments, the two or more exons encoding SV40 large T-antigen and the one or more intron(s) located between the two or more exons has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 6 as disclosed herein.

[0020] In some embodiments, the two or more exons encoding SV40 large T-antigen and the one or more intron(s) has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6 as disclosed herein.

[0021] In some embodiments, the region of the producer cell further comprises a nucleotide sequence encoding human telomerase reverse transcriptase hTERT. In some embodiments, the nucleotide sequence encoding hTERT is 5’ of the two or more exons encoding the SV40 large T-antigen. In some embodiments, the nucleotide sequence encoding hTERT is located upstream of the two or more exons encoding the SV40 large T- antigen.

[0022] In some embodiments, the region of the producer cell further comprises a nucleotide sequence comprising an EFl a promoter. In some embodiments, the nucleotide sequence comprising an EFl a promoter is 5’ of the nucleotide sequence encoding hTERT. In some embodiments, the nucleotide sequence comprising an EFl a promoter is located upstream of the nucleotide sequence encoding hTERT. In some embodiments, the nucleotide sequence comprising an EFl a promoter is operably linked to the nucleotide sequence encoding hTERT.

[0023] In some embodiments, the present disclosure provides a producer cell with a genome comprising a region having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 12 as disclosed herein.

[0024] In some embodiments, the present disclosure provides a producer cell with a genome comprising a region having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at leastabout 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to SEQ ID NO: 12 as disclosed herein.

[0025] In some embodiments, the present disclosure provides a producer cell with a genome comprising a region having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12 as disclosed herein.

[0026] In some embodiments, the present disclosure provides a producer cell that is mammalian. In some embodiments, the present disclosure provides a producer cell that is human. In some embodiments, the present disclosure provides a producer cell that is a HEK293 cell. In some embodiments, the present disclosure provides a producer cell that is a HEK293 cell which has been transfected with an immortalization plasmid (e.g., one as described herein).

[0027] In some embodiments, the present disclosure provides a population of rAAV particles that have reduced risk of carcinogenicity relative to those known in the art. In some embodiments, the present disclosure provides a population of rAAV particles that have substantially no risk of carcinogenicity relative to those known in the art. In some embodiments, the present disclosure provides a population of rAAV particles that have no risk of carcinogenicity relative to those known in the art.

[0028] In some embodiments, the present disclosure provides a method for producing a population of rAAV particles that are safer for clinical administration relative to those known in the art. In some embodiments, a method for producing a population of rAAV particles comprises culturing a producer cell (e.g., one as described herein). In some embodiments, a method for producing a population of rAAV particles comprises culturing a producer cell adherently or in suspension.

[0029] In some embodiments, the present disclosure provides a method for producing a population of rAAV particles at a higher titer of rAAV relative to those known in the art. In some embodiments, a method for producing a population of rAAV particles yields an rAAV titer of greater than about IxlO10, greater than about 5xl010, or greater than about IxlO11VG / mL.

[0030] In another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identical to SEQ ID NO: 5 as disclosed herein.

[0031] In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or is 100% identical to SEQ ID NO: 5 as disclosed herein.

[0032] In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or is 100% identical to SEQ ID NO: 5 as disclosed herein. In some embodiments, the present disclosure provides a nucleic acid vector comprising this nucleic acid. In some embodiments, the present disclosure provides a producer cell comprising this nucleic acid vector.

[0033] In another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identical to SEQ ID NO: 6 as disclosed herein.

[0034] In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or is 100% identical to SEQ ID NO: 6 as disclosed herein.

[0035] In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or is 100% identical to SEQ ID NO: 6 as disclosed herein. In some embodiments, the present disclosure provides anucleic acid vector comprising this nucleic acid. In some embodiments, the present disclosure provides a producer cell comprising this nucleic acid vector.

[0036] In another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identical to SEQ ID NO: 12 as disclosed herein.

[0037] In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or is 100% identical to SEQ ID NO: 12 as disclosed herein.

[0038] In some embodiments, the present disclosure provides a nucleic acid comprising a nucleotide sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or is 100% identical to SEQ ID NO: 12 as disclosed herein. In some embodiments, the present disclosure provides a nucleic acid vector comprising this nucleic acid. In some embodiments, the present disclosure provides a producer cell comprising this nucleic acid vector.

[0039] These, and other aspects encompassed by the present disclosure, are described in more detail below and in the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 shows an exemplary map of a nucleic acid sequence comprising an hTERT gene and an oversized SV40 large T-antigen gene which comprises a chimeric intron. Components of the chimeric intron are identified in the lower portion of the figure.

[0041] Figure 2 shows exemplary Western Blot probing of protein expression in total cell lysates from early passage HEK293 cells (EPSI) comprising an hTERT gene and an oversized SV40 large T-antigen gene. SV40 large T-antigen, hTERT, and P-actin protein expression were probed.

[0042] Figure 3 shows exemplary EGFP-AAV titer production in clones of EPS 1 cells comprising an hTERT gene and an oversized SV40 large T-antigen gene compared to original EPSI cells and HEK293T cells.

[0043] Figure 4 shows a schematic of an exemplary oversized SV40 large T-antigen, referred to herein as “SafeT”.

[0044] Figure 5 shows an exemplary timeline for generation of exemplary cell line Ig2-SafeT.

[0045] Figures 6A-6B shows plasmid maps of pUC57-PB-EFla-SV40 T-PGK- Hygro. Figure 6A shows a circularized plasmid map of pUC57-PB-EFla-SV40 T-PGK- Hygro. Figure 6B shows a linearized plasmid map of pUC57-PB-EFla-SV40 T-PGK- Hygro.

[0046] Figures 7A-7B shows plasmid maps of pUC-CMV-CO hpBase 8-18 (003).Figure 7A shows a circularized plasmid map of pUC-CMV-CO hpBase 8-18 (003). Figure 7B shows a linearized plasmid map of pUC-CMV-CO hpBase 8-18 (003).

[0047] Figure 8 shows an exemplary agarose gel of pUC57-PB-EFla-SV40 T-PGK- Hygro linearized with a restriction digest.

[0048] Figure 9 shows an exemplary microscope image of a single clone verification of “Clone Sil” for exemplary cell line Ig2-SafeT.

[0049] Figure 10 shows an exemplary microscope image of clonal growth verification of “Clone Sil” for exemplary cell line Ig2-SafeT.

[0050] Figure 11 shows an exemplary Jess Simple western blot of SV40 Large T- Antigen protein for screening of adherent exemplary Ig2-SafeT clonal cell lines.

[0051] Figure 12 shows an exemplary graph of cell viability and FBS weaning during suspension adaption of exemplary cell line Ig2-SafeT Clone Sil.

[0052] Figures 13A-13B shows T-antigen protein expression and quantification of Ig2-SafeT clonal cell lines in suspension. Figure 13A shows an exemplary Jess Simple western blot of SV40 Large T-Antigen protein expression. Figure 13B shows an exemplary graph of SV40 Large T-Antigen protein expression quantification.

[0053] Figure 14 shows an exemplary graph of lentivirus production in Ig2-SafeT clonal cell lines in suspension infectious titer.

[0054] Figure 15 shows exemplary titer data from clonal Ig2-SafeT cell lines in suspension at 75 mL scale (qPCR titer).

[0055] Figure 16 shows exemplary titer data from clonal Ig2-SafeT cell lines in suspension at 75 mL scale (qPCR titer).

[0056] Figures 17A-17B show exemplary titer and yield data from Ig2-SafeT clonal cell lines clarified lysate. Figure 17A shows exemplary qPCR titer data. Figure 17B shows exemplary yield data.

[0057] Figures 18A-18B show exemplary titer and yield data from Ig2-SafeT clonal cell lines clarified lysate. Figure 18A shows exemplary qPCR titer data. Figure 18B shows exemplary yield data.

[0058] Figure 19 shows an exemplary table of elution peak stunner data from Ig2- SafeT clonal cell lines.

[0059] Figure 20 shows exemplary CsCl ultracentrifugation band data from Ig2- SafeT clonal cell lines.

[0060] Figures 21A-21B shows exemplary qPCR titration and yield data from final formulation Ig2-SafeT clonal cell lines. Figure 21A shows exemplary qPCR titration data from final formulation Ig2-SafeT clonal cell lines. Figure 21B shows exemplary qPCR yield data from final formulation Ig2-SafeT clonal cell lines.

[0061] Figure 22 shows an exemplary table of stunner data from final formulation Ig2-SafeT clonal cell lines.

[0062] Figure 23 shows exemplary analytical ultracentrifugation results from final formulation Ig2-SafeT clonal cell lines in suspension.

[0063] Figure 24 shows an exemplary western blot confirming of SV40 T-Antigen Removal in final formulation Ig2-SafeT clonal cell lines.DEFINITIONS

[0064] Agent: In general, the term “agent”, as used herein, is used to refer to an entity (e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc, or complex, combination, mixture or system [e.g., cell, tissue, organism] thereof), or phenomenon (e.g., heat, electric current or field, magnetic force or field, etc). In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and / or is obtained fromnature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and / or produced through action of the hand of man and / or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. In some cases, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and / or is substantially free of any polymer and / or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.

[0065] Approximately / about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0066] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets ofcircumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0067] Corresponding to. As used herein, the term “corresponding to” may be used to designate the position / identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, Hhpred / Hhsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, S SEARCH, SWAPHI, SWAPHLLS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and / or nucleic acids in accordance with the present disclosure.

[0068] Downstream. As used herein, the term “downstream” refers to the location or position of a nucleic acid sequence relative to a reference nucleic acid sequence, particularly a position that, during RNA transcription, is closer to the 3’ end of the transcribed RNA molecule encoded by the reference sequence. For example, for two sequences, A and B, such that sequence A is downstream of sequence B, transcription of sequence B proceeds toward sequence A.

[0069] Nucleic acid. As used herein, in its broadest sense, the term “nucleic acid” refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments,"nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5 -iodouridine, C5-propynyl-uridine, C5 -propynyl- cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, anucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.

[0070] Operably linked. As used herein, the term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control element "operably linked" to a functional element is associated in such a way that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, "operably linked" control elements are contiguous (e.g., covalently linked) with the coding elements of interest; in some embodiments, control elements act in trans to or otherwise at a distance from the functional element of interest.

[0071] Producer cell. As used herein, the term “producer cell” refers to any cell used to produce recombinant AAV (rAAV). In some embodiments, a producer cell is a mammalian cell. In some embodiments, a producer cell is a transformed mammalian cell. In some embodiments, a producer cell is a Vero, HeLa, HEK293, HEK293T cell or derivative thereof.

[0072] Transformation. As used herein, the term “transformation” refers to any process by which exogenous DNA is introduced into a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. In some embodiments, a particular transformation methodology is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, mating, lipofection. In some embodiments, a "transformed" cell is stably transformed in that the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. In some embodiments, a transformed cell transiently expresses introduced nucleic acid for limited periods of time.

[0073] Upstream. As used herein, the term “upstream” refers to the location or position of a nucleic acid sequence relative to a reference nucleic acid sequence, particularly a position that, during RNA transcription, is closer to the 5’ end of the transcribed RNA molecule encoded by the reference sequence. For example, for two sequences, A and B, suchthat sequence A is upstream of sequence B, transcription of sequence B proceeds away from sequence A.

[0074] Vector. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0075] Prior studies have suggested that immortalized cells are not only capable of extended proliferation, but also possess identical genotype and phenotype compared to the parental cells. Commonly used methods for cell immortalization include expressing SV40 large T-antigen and / or human telomerase reverse transcriptase (hTERT) (see, e.g., Exp Cell Res. 1998;245(l): l-7, Mol Cell Endocrinol. 2004;216(l-2):83-94, Oncogene. 2008 Aug 18, etc.). Using an SV40 large T-antigen encoding sequence for cell immortalization raises safety concerns due to its carcinogenicity. It has been suggested that SV40-transformed human cells produce tumors when administered into nude mice and deregulate host cellularpathways see, e.g., Lab Invest. 1988;58(5):518-23, Cancer Res. 1993;53(5):985-91, J Virol. 1997; 71(11): 8157-8166, J Cell Sci. 2009; 122:3190-8, J Virol. 2010;84(16):8007-20, etc.). The presence of residual nucleic acid sequence encoding the SV40 large T-antigen in vector production poses significant safety concern.

[0076] The present disclosure, among other things, appreciates that a producer cell (e.g., a HEK293 cell) can be engineered to reduce risk (e.g., oncogenic risk) associated with viral vectors produced by the producer cell. In some embodiments, a rAAV producer cell with a genome comprising a region that includes two or more exons encoding SV40 large T- antigen and one or more intron(s) located between the two or more exons, wherein the combined size of the sequence encoding SV40 large T-antigen and the one or more introns is larger than the packaging capacity of an rAAV. In some embodiments, an intron is a chimeric intron.

[0077] In some embodiments, a producer cell comprises a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 12.

[0078] In some embodiments, a producer cell comprises a nucleotide sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to SEQ ID NO: 12.

[0079] In some embodiments, a producer cell comprises a nucleotide sequence having about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% sequence identity to SEQ ID NO: 12.

[0080] In some embodiments, a producer cell comprises a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12.

[0081] In some embodiments, rAAV particles produced by a producer cell as described herein are used for clinical administration (e.g., gene therapy).Producer Cells

[0082] The present disclosure describes, among other things, various embodiments of producer cells that are useful in the production of recombinant AAV (rAAV) particles. In some embodiments, a producer cell produces rAAV particles. In some embodiments, a producer cell is a eukaryotic cell. In some embodiments, a producer cell is a mammalian cell. In some embodiments, a mammalian producer cell is a human cell, a mouse cell, a rat cell, a monkey cell, or a hamster cell. In some embodiments, a mammalian producer cell is a human embryonic kidney (HEK) cell, a Chinese hamster ovarian (CHO) cell, a HeLa cell, or an A549 cell. In some embodiments, a producer cell is a HEK293 cell.

[0083] In some embodiments, a producer cell is an immortalized cell. In some embodiments, a produced cell is immortalized by transfection with a nucleotide sequence. In some embodiments, a produced cell is immortalized by transfection with a nucleotide sequence as described herein. In some embodiments, a producer cell comprises SV40 large T-antigen and / or hTERT encoding sequence(s). In some embodiments, a nucleotide sequence encoding hTERT is upstream, preceding, or 5’ of a nucleotide sequence encoding SV40 T-antigen. In some embodiments, a producer cell is a HEK293T cell. In some embodiments, a producer cell is a variant of a parent cell (e.g., one as described above).

[0084] In some embodiments, a producer cell is engineered. One of skill in the art will appreciate the methods available engineering a cell (e.g., a producer cell). In some embodiments, a producer cell described herein is engineered to comprise an SV40 T-antigen and / or hTERT encoding sequence(s) with a PiggyBac transposon system.

[0085] In some embodiments, a producer cell is cultured. In some embodiments, a producer cell culture is adherent culture or suspension culture. In some embodiments, a producer cell is adapted from adherent culture to suspension culture. One of skill in the art will appreciate the methods available for adapting an adherent producer cell to suspension culture.

[0086] In some embodiments, a producer cell comprises nucleic acid molecules encoding various components that contribute to the production of rAAV. In some embodiments, a producer cell comprises nucleic acid molecules encoding various components that contribute to the replication and / or packaging of rAAV. In some embodiments, a producer cell comprises rep and cap genes. In some embodiments, a producer cell comprises a heterologous gene of interest flanked by inverted terminal repeats (ITRs).

[0087] In some embodiments, a producer cell comprises one or more adenoviral (Ad) helper genes. In some embodiments, helper genes are permanently expressed in a producer cell. In some embodiments, helper genes are transiently expressed in a producer cell. In some embodiments, certain helper genes are permanently expressed, and other helper genes are transiently expressed in a producer cell. In some embodiments, a producer cell comprises one or more helper genes encoded by a plasmid or vector. In some embodiments, helper genes are encoded by the same nucleic acid vector (e.g., a plasmid). In some embodiments, helper genes are encoded on different nucleic acid vectors in trans.

[0088] In some embodiments, helper genes are provided by an adenoviral helper plasmid as described in WO2022 / 241215 the entire contents of which are hereby incorporated by reference. In some embodiments, an adenoviral helper plasmid comprises a modified L433K and / or L22K region. In some embodiments, an adenoviral helper plasmid lacks an L433K and / or L22K region. In some embodiments, a producer cell comprises an adenoviral helper plasmid with or without an L433K and / or L22K region and an L433K and / or L22K region provided in trans (e.g., encoded on a separate plasmid).

[0089] In some embodiments, a producer cell comprises a resistance gene. In some embodiments, an adenoviral helper plasmid of the present invention comprises an ampicillin resistance gene (e.g., a nucleotide sequence encoding a protein conferring resistance to ampicillin). In some embodiments, an adenoviral helper plasmid of the present invention does not comprise an ampicillin resistance gene. In some embodiments, an adenoviral helper plasmid of the present invention comprises a kanamycin resistance gene (e.g., a nucleotide sequence encoding a protein conferring resistance to kanamycin). In some embodiments, an adenoviral helper plasmid of the present invention does not comprise a kanamycin resistance gene. In some embodiments, an adenoviral helper plasmid of the present invention comprises a puromycin resistance gene (e.g., a nucleotide sequence encoding a protein conferring resistance to puromycin). In some embodiments, an adenoviral helper plasmid of the present invention does not comprise a puromycin resistance gene.

[0090] One of skill in the art will appreciate that producer cells as described herein may be used to produce AAV of any serotype. In some embodiments, an AAV serotype useful in the present disclosure is selected from any of the following serotypes, and variants thereof, including, but not limited to: AAV1, AAV10, AAV106.1 / hu.37, AAV11, AAV114.3 / hu.4O, AAV 12, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.1 / hu.43,AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV16.12 / hu. ll, AAV16.3, AAV16.8 / hu.lO, AAV161.1O / hu.6O, AAV161.6 / hu.61, AAV1- 7 / rh.48, AAVl-8 / rh.49, AAV2, AAV2.5T, AAV2- 15 / rh.62, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV2-3 / rh.61, AAV24.1, AAV2-4 / rh.5O, AAV2-5 / rh.51, AAV27.3, AAV29.3 / bb. 1, AAV29.5 / bb.2, AAV2G9, AAV-2-pre-miRNA-101, AAV3A, AAV3B, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-1 l / rh.53, AAV3-3, AAV33.12 / hu.l7, AAV33.4 / hu.l5, AAV33.8 / hu.l6, AAV3-9 / rh.52, AAV3a, AAV3b, AAV4, AAV4-19 / rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42- 15, AAV42-lb, AAV42- 2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42- aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4- 4, AAV44.1, AAV44.2, AAV44.5, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV4-8 / r 11.64, AAV4- 8 / rh.64, AAV4-9 / rh.54, AAV5, AAV52.1 / hu.2O, AAV52 / hu.l9, AAV5- 22 / rh.58, AAV5- 3 / rh.57, AAV54.1 / hu.21, AAV54.2 / hu.22, AAV54.4R / hu.27, AAV54.5 / hu.23, AAV54.7 / hu.24, AAV58.2 / hu.25, AAV6, AAV6.1, AAV6.1.2, AAV6.2, AAV7, AAV7.2, AAV7.3 / hu.7, AAV8, AAV-8b, AAV-8h, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAVA3.3, AAVA3.4, AAVA3.5, AAV A3.7, AAV-b, AAVC1, AAVC2, AAVC5, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAV-h, AAVH-l / hu.l, AAVH2, AAVH- 5 / hu.3, AAVH6, AAVhEl. l, AAVhER1.14, AAVhErl.16, AAVhErl.18, AAVhER1.23, AAVhErl.35, AAVhErl.36, AAVhErl.5, AAVhErl.7, AAVhErl.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhEr2.4, AAVhEr3.1, AAVhu.l, AAVhu.10, AAVhu. ll, AAVhu.12, AAVhu.13, AAVhu.14 / 9, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.l 9, AAVhu.2, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.3, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.4, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.5, AAVhu.51, AAVhu.52, AAVhu.53, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.6, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.68, AAVhu.7, AAVhu.8, AAVhu.9, AAVhu.t 19, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVLG- 9 / hu.39, AAV-LK01, AAV- LK02, AAVLKO3, AAV-LKO3, AAV-LK04, AAV-LKO5, AAV- LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV- LK13, AAV-LK14, AAV- LK15, AAV-LK17, AAV-LK18, AAV-LK19, AAVN721-8 / rh.43, AAV-PAEC, AAV-PAEC11, AAV- PAEC12, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAVpi.1, AAVpi.2, AAVpi.3, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.2, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.2R, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.44, AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.5O, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533 A mutant, BAAV, B P61 AAV, B P62 AAV, B P63AAV, bovine AAV, caprine AAV, Japanese AAV10, true type AAV (ttAAV), UPENN AAV 10, AAV-LK 16, AAAV, AAV Shuffle 100-1, AAV Shuffle 100-2, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10- 2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV SM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, AAV SM 10-8, AAV-PHP.B, AAV-PHP.N, AAV-PHP.S, AAVrh.74, AAV-HSC 1-17, AAV-CBr, AAV-CLv, AAV-CLg, and / or AAV. CAP-B1 to AAV.CAP-B25.Reducing Risk Associated with rAAV Production

[0091] One of skill in the art will recognize there can be risks associated with clinical use of rAAV due to remnants of material from cells that produced the rAAV. One of skill in the art will recognize that producer cells can contain material other than rAAV components that can be packaged in rAAV. For example, producer cells can contain nucleotide sequence encoding an SV40 large T-antigen. Accordingly, a nucleotide sequence encoding an SV40 large T-antigen could be packaged in an rAAV. In some embodiments SV40 large T-antigen and / or the sequence encoding the SV40 large T-antigen in an rAAV formulation may be carcinogenic if administered to human subjects. The present disclosure, among other things, describes rAAV producer cells that are engineered to reduce risk associated with clinical use of rAAV produced therefrom.

[0092] In some embodiments, risk associated with clinical use of rAAV produced from engineered producer cells described herein is reduced, substantially eliminated, or entirely eliminated. In some embodiments, risk associated with clinical use of rAAVproduced from engineered producer cells described herein is carcinogenicity, tumorigenesis, transformation, gene transfer, and / or toxicity. One of skill in the art, upon reading the present disclosure, will appreciate that the insights contained herein are applicable to AAV vectors of different serotypes and rAAV comprising various different gene products.Oversized Nucleic Acid

[0093] In some embodiments, a producer cell of the present disclosure comprises a heterologous nucleic acid (e.g., a gene that encodes for an SV40 large T-antigen). In some embodiments, an oversized heterologous nucleic acid has a particular length such that it cannot be packaged into an rAAV particle. One of skill in the art will appreciate that AAV particles have a packaging limit. In some embodiments, an AAV packaging limit is dependent on AAV serotype and / or physical size of formed AAV capsid. In some embodiments, an AAV packaging limit is about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, or about 5.2 kilobases (kb). In some embodiments, a nucleic acid is unpackable in an AAV particle if the nucleic acid is of a greater length than the packaging limit associated with the AAV particle.

[0094] In some embodiments, a nucleotide sequence is too large and is unpackable in a particular AAV serotype or multiple AAV serotypes. In some embodiments, a nucleotide sequence is too large and is unpackable in all AAV serotypes.

[0095] In some embodiments, a nucleotide sequence of the present disclosure encodes for a protein product that is identical to a protein encoded by a control and / or wildtype version of the gene. In some embodiments, a nucleotide sequence of the present disclosure encodes a SV40 large T-antigen.

[0096] In some embodiments, an rAAV producer cell with a genome comprising a region that includes two or more exons encoding SV40 large T-antigen and one or more intron(s) located between the two or more exons, wherein the combined size of sequence encoding SV40 large T-antigen and the one or more introns is larger than the packaging capacity of an rAAV. In some embodiments, the combined size of the sequence encoding SV40 large T-antigen is about 1000 to about 3000 base pairs, e.g., about 1000, about 2000, or about 3000 base pairs. In some embodiments, the combined size of the sequence encoding SV40 large T-antigen have a combined length of about 2000 to 2200 base pairs, e.g., about 2000, about 2100, or about 2200 base pairs. In some embodiments, the combinedsize of the sequence encoding SV40 large T-antigen have a combined length of about 2124 base pairs.

[0097] In some embodiments, a nucleotide sequence of the present disclosure is increased in length relative to a control and / or wild-type version of the gene by addition of nucleotides. In some embodiments, a nucleotide sequence of the present disclosure is increased in length relative to a control and / or wild-type version of the gene by addition of noncoding nucleotides. In some embodiments, a nucleotide sequence of the present disclosure comprises one or more noncoding stuffer sequences. In some embodiments, a nucleotide sequence of the present disclosure comprises one or more intron(s).

[0098] In some embodiments, a producer cell with a genome comprising a nucleotide sequence encoding a SV40 large T-antigen wherein a nucleotide sequence further comprises one or more intron(s). In some embodiments, the size of the one or more intron(s) is greater than about 1000, greater than about 2000, greater than about 3000, greater than about 4000, greater than about 5000, or greater than about 6000 base pairs in length. In some embodiments, the size of the one or more intron(s) is about 1000 to about 6000, about 2000 to about 6000, about 3000 to about 6000, about 4000 to about 6000, or about 5000 to about 6000 base pairs in length. In some embodiments, the size of the one or more intron(s) is greater than about 5000 base pairs in length. In some embodiments, the size of the one or more intron(s) is about 5410 base pairs in length.

[0099] In some embodiments, a nucleotide sequence of the present disclosure comprises one or more introns. In some embodiments, a nucleotide sequence of the present disclosure comprises a natural intron. In some embodiments, a nucleotide sequence of the present disclosure comprises an intron comprising natural intronic sequences. One of skill in the art will appreciate that many different intronic sequences may be used in the context of the present disclosure to reduce risk associated with rAAV production. In some embodiments, an intron of any sequence is used to make an oversized gene unpackable.

[0100] In some embodiments, an intron, e.g., one as described herein, is increased in length relative to a control and / or wild-type version of an intronic sequence. In some embodiments, an intron is engineered to comprise one or more stuffer sequences. In some embodiments, an intron, e.g., one as described herein, comprises two, three, four, or more stuffer sequences in tandem. In some embodiments, an intron comprising one or more stuffer sequences of different origin is a chimeric intron.

[0101] In some embodiments, a stuffer sequence is not derived from any natural sequence. In some embodiments, a stuffer sequence is derived from a natural intronic sequence. In some embodiments, a stuffer sequence is derived from human eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), human ribosomal protein S27A (RPS27A), or human forkhead box protein F2 (FOXF2). In some embodiments, a stuffer sequence is or comprises intron 1 of EEF1A1, internal region of intron 3, 2, 4 of RPS27A, or internal region of intron 1 of FOXF2.

[0102] In some embodiments, a stuffer sequence is not G-C enriched relative a control and / or wild-type intronic sequence. In some embodiments, a stuffer sequence is derived from a natural intron sequence that is A-T enriched.

[0103] In some embodiments, an intron, e.g., one as described herein, comprises a single splicing donor site and a single splicing acceptor site. In some embodiments, a stuffer sequence does not comprise a splicing donor site nor a splicing acceptor site. In some embodiments, a chimeric intron has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 5.

[0104] In some embodiments, a chimeric intron has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 5.

[0105] In some embodiments, a chimeric intron has about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO: 5.

[0106] In some embodiments, a chimeric intron has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 as disclosed herein.

[0107] In some embodiments, the combined size of the two or more exons encoding SV40 large T-antigen and the one or more intron(s) is about 7000 to about 8000, about 7100 to about 8000, about 7200 to about 8000, about 7200 to about 8000, about 7300 to about8000, about 7400 to about 8000, about 7500 to about 8000, about 7600 to about 8000, about 7000 to about 7600, about 7100 to about 7600, about 7200 to about 7600, about 7300 to about 7600, about 7400 to about 7600, or is about 7500 to about 7600 base pairs in length.

[0108] In some embodiments, the combined size of the two or more exons encoding SV40 large T-antigen and the one or more intron(s) is about 7000, about 7100, about 7200, about 7300, about 7400, about 7500, about 7600, about 7700, about 7800, about 7900, or is about 8000 base pairs in length. In some embodiments, the combined size of the two or more exons encoding SV40 large T-antigen and the one or more intron(s) is about 7537 base pairs in length.

[0109] In some embodiments, the two or more exons encoding SV40 large T-antigen and the one or more intron(s) located between the two or more exons has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6 as disclosed herein.

[0110] In some embodiments, the two or more exons encoding SV40 large T-antigen and the one or more intron(s) located between the two or more exons has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 6 as disclosed herein.[OHl] In some embodiments, the two or more exons encoding SV40 large T-antigen and the one or more intron(s) has about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% sequence identity to SEQ ID NO: 6 as disclosed herein.

[0112] In some embodiments, the two or more exons encoding SV40 large T-antigen and the one or more intron(s) has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6 as disclosed herein.Methods of rAAV Production

[0113] One of skill in the art will appreciate the methods available for rAAV production. In some embodiments, a method of producing rAAV comprises transfection of a producer cell with an AAV vector plasmid, an AAV Rep-Cap expressing plasmid, and an adenoviral helper plasmid. In some embodiments, an AAV vector plasmid comprises AAV inverted terminal repeats (ITRs) and a transgene of interest. In some embodiments, a method of producing rAAV comprises transfection of a producer cell stably expressing Rep- Cap. In some embodiments, a method of producing a rAAV comprises transfection of a producer cell stably expressing Rep-Cap with an AAV vector plasmid and an adenoviral helper plasmid.

[0114] In some embodiments, a method of producing rAAV comprises culturing a producer cell (e.g., one as described herein). In some embodiments, a method of producing rAAV comprises culturing a producer cell under appropriate conditions for rAAV production. In some embodiments, a method of producing rAAV comprises culturing a producer cell adherently or in suspension.EXEMPLIFICATION

[0115] The present disclosure provides producer cells for rAAV production, in which the produced rAAV are associated with fewer safety concerns (e.g., reduced oncogenic risk) relative to rAAV produced by the most commonly used producer cells.

[0116] The plasmids provided in this disclosure were synthesized de novo, were sequence-verified, and were scaled up for use in large-scale rAAV manufacturing.Production of rAAV studies were performed to compare vector yields when using the provided producer cells versus other commercially available producer cells. Taken together, these following examples demonstrate that provided producer cells generate rAAV of high yield and quality, in a potentially safer design.Example 1 : Exemplary rAAV producer cells comprising an oversized SV40 large T-antigen

[0117] The present Example describes engineering safer immortalized producer cells for rAAV production by insertion of an oversized SV40 large T-antigen. An oversized SV40 large T-antigen is, for example, two or more exons encoding SV40 large T-antigen and one or more intron(s) located between the two or more exons, wherein the combined size of thetwo or more exons encoding SV40 large T-antigen and the one or more introns is larger than the packaging capacity of an rAAV (Figure 4).

[0118] An immortalization plasmid provided by the present disclosure was designed for insertion into a producer cell. The immortalization plasmid was designed to comprise an oversized SV40 large T-antigen so as to reduce oncogenic risk associated with adeno- associated virus (AAV) produced by the producer cell. The immortalization plasmid was designed to comprise (in a 5’ to 3’ direction) a 5’ ITR, EFla promoter, hTERT gene, oversized SV40 large T-antigen gene, puromycin resistance gene, and 3’ ITR. The 5’ and 3’ ITRs are derived from a PiggyBac DNA integration system. The gene-encoding sequences were separated by sequences encoding cleavage peptides (e.g., P2A or T2A). The SV40 large T-antigen gene was designed to comprise a chimeric intron, as characterized by SEQ ID NO: 5. An exemplary plasmid map of the immortalization plasmid is shown in Figure 1.

[0119] The immortalization plasmid was inserted into genome of early passage HEK293 cells (EPSI). EPSI cells are early passaged from the original HEK293 cell line. Single clones of EPS 1 cells comprising the inserted immortalization passage (T-safe EPSI cells) were isolated and cultured under appropriate conditions to allow for SV40 T-antigen and hTERT expression. Protein expression of SV40 T-antigen and hTERT from single T- safe EPSI clones is shown in Figure 2.

[0120] Single T-safe EPSI clones were transfected with a gene of interest plasmid, a packaging plasmid comprising rep and cap genes, and an adenoviral helper plasmid and cultured under appropriate conditions to allow for EGFP-AAV production, relative to control cells of original EPSI cells and HEK293T cells. EGFP-AAV titer was measured in VG / mL as shown in Figure 3. Four T-safe EPSI clones (B4, C2, C5, and D6) showed a statistically significant increase in AAV production yield compared to original EPSI cells and HEK293 T cells. Select clones were about 3-4 fold more efficient in AAV production relative to HEK293T cells and 27-41 fold more efficient compared to original EPSI cells.Table 1: Exemplary Components of Immortalization Plasmids

[0121] The below Table of Sequences lists and describes the sequences of various components of immortalization plasmids discussed herein. Unless stated otherwise, all sequences are recited with 5’ to 3’ directionality of the positive strand of a plasmid. This directionality is preserved irrespective of the orientation of a gene or element described to be associated with a sequence. Asterisks as used herein indicate a stop codon.Table 2: Exemplary Immortalization Plasmids

[0122] The below Table of Sequences lists and describes the sequences of various immortalization plasmids discussed herein. Unless stated otherwise, all sequences are recited with 5’ to 3’ directionality of the positive strand of a plasmid. This directionality is preserved irrespective of the orientation of a gene or element described to be associated with a sequence. Asterisks as used herein indicate a stop codon.Example 2: Process development of exemplary rAAV producer cell line comprising an oversized SV40 large T-antigen

[0123] This Example describes generation and validation of an exemplary rAAV producer cell described herein. The present Example describes generation and validation of Ignition 2 Efla-SV40 Safe T Clone Sil (Ig2-SafeT) Cell Line, a safer immortalized producer cell line for rAAV production by insertion of an oversized SV40 large T-antigen, as well as generation of a pre-Master Cell Bank (pre-MCB).1. Ignition 2.0 Parental Cell Line

[0124] Ignition 2.0 cell line is an exemplary HEK293 based clonal cell line selected based on superior growth in suspension and high yield AAV production. An exemplary timeline for generation of a clonal Ignition “Safe-T” cell line is provided in Figure 5.2. Ig2-SafeT Passage History

[0125] A vial of Ignition 2.0 Research Working Cell Bank (RCB) cells was thawed into suspension media and passaged until recovery. RCB IG2 Cells were converted to an adherent state and passaged until recovery prior to transfection. A person of skill in the art will appreciate, upon reading the present disclosure, that adherent adaption of suspension cells offers certain advantages, including but not limited to, efficient antibiotic selection and / or transfection with small amounts of DNA (e.g., linearized DNA). A plasmid, as described by the present disclosure, comprising a PiggyBac terminal repeat bound Efl a promoter driven SV40 large T-Antigen with an oversized “Safe” intron pUC57-PB-EFla- S V40 T-PGK-Hygro was synthesized by GenScript. A linear DNA fragment was prepared by restriction digest and agarose gel purification prior to co-transfection with a PiggyBac transposase. A polyclonal population with stable genomic integration was established using hygromycin selection. Clonal lines were isolated by limiting dilution and expanded before screening for SV40 T-antigen protein expression. Positive clones were banked, adapted to suspension as clones, and weaned off anti-clumping agent and FBS in suspension.Intermediate banks were prepared throughout this process.2.1 Process Parameters UsedTable 3: Media TypesTable 4: Vial Thaw ParametersTable 5: Adherent Incubator and Passaging ParametersTable 6: Suspension Incubator and Passaging ParametersTable 7: Banking ParametersTable 8: Adherent PiggyBac Transfection Parameters2.2 Preparation of Ignition 2 Cells for Adherent Transfection

[0126] To improve transfection efficiency and allow for efficient antibiotic selection, suspension Ignition 2.0 cells were converted back to an adherent state prior to transfection. A vial of Ignition 2.0 Clone 7, RCB for development use, was thawed according to vial thaw parameters into prewarmed Suspension F17 Media on Day 1. Cells were passaged 4 times and expanded in suspension until viability was > 98%.

[0127] To convert suspension cells back to an adherent state, 1.6E6 passage 4 Ignition 2.0 cells were transferred into a T75 flask containing Adherent DMEM Media with 10% FBS. Adherent Ignition cells were allowed to adhere and adapt for 3 days prior to seeding for transfection. Adherent cells were dissociated using TrypLE Select and seeded into a 6 well plate 24 hours prior to transfection on Day 4.2.3 Preparation of a Linearized DNA Fragment for Transfection and PiggyBac Integration

[0128] A plasmid, described by the present disclosure, comprising a PiggyBac terminal repeat bound Efl a promoter driven SV40 large T- Antigen with an oversized “Safe” intron pUC57-PB-EFla-SV40 T-PGK-Hygro, (Figures 6A-6B) was synthesized by GenScript. Atransposase plasmid pUC57-CMV-CO_hPBase, described by the present disclosure, was designed to express PiggyBac transposase codon optimized for human expression under regulation of the CMV promoter and enhancer (Figures 7A-7B). pUC57- CMV-CO_hPBase was cloned and synthesized by GenScript.

[0129] On Day 5, 50pg of transposon containing plasmid, pUC57-PB-EFla-SV40 T- PGK-Hygro, was linearized with a 24-hour Sall-HF restriction digest. Linearized transposon (Figure 8, Upper Band) was isolated and plasmid backbone DNA was removed using agarose gel electrophoresis. Linearized transposon DNA was extracted from the gel purified using a Zymoclean Gel DNA Recovery Kit (#D4001). DNA was quantified by nanodrop and stored at 4°C prior to transfection.2.4 Transfection and Selection to Generate a Polyclonal Ig2-SafeT Cell Line

[0130] A transfection of transposase plasmid and linearized transposon DNA was performed for one well of adherent Ignition 2 cells at a 6 well plate scale according to adherent PiggyBac transfection parameters on Day 19. Cells were dissociated using TrypLE Select and transferred to a T25 flask in preparation for selection. After allowing 24 hours for cells to adhere, Hygromycin B (Invitrogen 10687010) was added to cells in adherent DMEM media at an initial concentration of 200 pg / mL.

[0131] On Day 25, a bank of adherent polyclonal Ig2-SafeT cells was generated by freezing cells in a controlled rate freezing device at -80°C. Cells were passaged to a new T25 flask, maintaining a Hygromycin B selection concentration of 200 pg / mL.

[0132] Selection appeared to be nearly complete due to detachment of unmodified cells, so Hygromycin B concentration was reduced to 25 pg / mL on Day 29. Hygromycin B selection was maintained at 25 pg / mL until Day 36.

[0133] On Day 36, a bank of adherent polyclonal Ig2-SafeT cells was generated by freezing cells in a controlled rate freezing device at -80°C. Remaining adherent Ig2-SafeTpolyclonal cells were expanded into two T75 flasks in Adherent DMEM media in preparation for single cell cloning.2.5 Single Cell Cloning by Limiting Dilution

[0134] Polyclonal Ig2-Safe cells were clonally isolated into 96 well plates by limiting dilution on Day 38. Cells were rinsed with PBS and dissociated with 0.25% Trypsin EDTA according to adherent incubator and passaging parameters. Serial dilutions were prepared in adherent DMEM media to a target seeding dilution of 2.0 cells per well.

[0135] Clonality was verified by imaging of individual wells using a Leica DMi8 automated microscope on Day 40 (Figure 9). Clonal population growth was verified by a second round of Leica DMi8 imaging prior to expansion on Day 47 (Figure 10).2.6 Expansion and Protein Screening of Adherent Clonal Ig2-SafeT Cell Lines

[0136] Clonal Ig2-SafeT cells were dissociated from 96 well plates using TrypLE and expanded into 24 well plates on Day 54. On Day 60 Ig2-SafeT clonal lines were expanded into T25 flasks.

[0137] On Day 65, SafeT Clone 11 cells were frozen to -80°C at a controlled rate to generate Bank 11.0. Remaining cells were expanded to a T75 flask for protein isolation.

[0138] On Day 67 samples of clonal cell populations were detached using TrypLE select dissociation reagent, centrifuged and washed with PBS before lysing cells using Pierce RIPA buffer (ThermoFisher 89900), and removing cellular debris by centrifugation. SV40 Large T-Antigen protein was quantified using Jess Simple western blotting on Day 73 (Figure 11). T-Antigen was detected using anti-SV40 T rabbit MAB (Cell Signaling Technologies D1E9E). Additional cell lines were included as relative reference controls. Positive clones were selected for expansion and adaptation.2. 7 Adaptation of Ig2-SafeT Clone Sil to Suspension

[0139] On Day 81, a vial of Ig2-SafeT clone Sil cells was thawed into a T25 containing adherent DMEM media. Media was replaced with fresh adherent DMEM after 24 hours to prevent toxicity from residual DMSO.

[0140] On Day 85, cells were dissociated with TrypLE Select and transferred into two T75 flasks into adherent F17 media.

[0141] Suspension adaptation began on Day 99. Cells were grown slightly past 100% confluency in two T75 flasks to achieve sufficient density. Cells were washed withPBS before dissociation with .25% Trypsin EDTAto according to adherent process parameters. Use of a stronger dissociation reagent results in a single cell suspension, optimal for suspension seeding. Cells were resuspended in 30mL suspension seeding F17 media and grown according to process parameters. Cells were periodically counted using a Vi -CELL BLU cell analyzer (Beckman C19196) and passaged to a density between 0.5- 1.0 X 106VC / mL into F17 suspension media (Figure 12). No additional FBS or anti-clumping agent was added after the initial seeding.

[0142] On Day 152, suspension adaptation and weaning from FBS and antidumping was considered complete for Ig2-SafeT clone SIL A suspension cell bank was generated by freezing cells in a controlled rate device at -80°C.3. Clonal Selection

[0143] Following completion of suspension adaptation, Ig2-SafeT clonal suspension cell lines were evaluated for productivity in AAV production, SV40 T- Antigen protein expression and for productivity in lentivirus production. Atop clone Ig2-SafeT Sil was selected due to superior yields in AAV production and the highest level of SV40 T-Antigen protein expression.3.1 Process Parameters UsedTable 9: Suspension Lentivirus Transfection ParametersTable 10: Suspension AAV Transfection Parameters3.2 SV40-T Antigen Protein Expression in Suspension Clonal Ig2-SafeT Cell Lines

[0144] On Day 122, samples of clonal cell populations were collected, centrifuged and washed with PBS before lysing cells using Pierce RIPA buffer (ThermoFisher 89900), and removing cellular debris by centrifugation. Total protein was quantified by DC Protein Assay (Biorad 5000111). SV40 Large T-Antigen protein was quantified using Jess Simple western blotting (Figures 13A-13B). T-Antigen was detected using anti-SV40 T rabbit MAB (Cell Signaling Technologies D1E9E). Additional suspension cell lines were included as expression reference controls. Ig2-SafeT clone Sil expressed the highest level of SV40 T-Antigen protein.3.3 Evaluation of Lentivirus Production in Clonal Ig2-SafeT Suspension Cell Lines

[0145] Based on SV40-T protein quantitation, positive clones Ig2-SafeT S7, S9, SI 1, and S12 were selected for a lentivirus production study. Clone S6 was included as a negative control and additional polyclonal cell lines were included as reference controls.

[0146] Transfections were performed according to suspension lentivirus transfection process parameters on Day 138. Productions were performed at 30mL suspension scale with 3 replicates of each cell line. Viral supernatant was harvested at 72 hours post transfection and used to transduce HT1080 cells for infectious titer assessment. Infectious titer was assessed by GFP flow cytometry 48 hours after transduction (Figure 14). Ig2-SafeT clonallines positive for SV40 T-Antigen expression generated 2- to 4- fold more infectious lentivirus particles than unmodified Ignition 2.0 cells.3.4 Evaluation of AAV Production in Clonal Ig2-SafeT Suspension Cells at 75mL Scale

[0147] Positive clones Ig2-SafeT S7, S9, Si l, and S12 were selected for an AAV production study. Clone S6 was included as a negative control and additional polyclonal cell lines were included as reference controls.

[0148] Transfections were performed according to suspension AAV transfection process parameters on Day 144. Productions were performed at 75mL suspension scale with 3 replicates of each cell line. Cells were lysed and clarified 96 hours after transfection on Day 148. Clarified lysates were titered by qPCR.

[0149] In the initial AAV production study, Ig2-SafeT clones positive for SV40-T antigen expression were more productive than unmodified Ignition 2.0 cells (Figure 15). Clones S7 and Sil were the most productive and were selected for further evaluation.

[0150] A repeat AAV production study was performed to evaluate the performance of Ig2-SafeT clones S7 and Sil. Unmodified Ignition 2.0 and Dharmacon 293T cells were included as controls. Transfections were performed according to suspension AAV transfection process parameters on Day 165. Productions were performed at 75mL suspension scale with 3 replicates of each cell line. Cells were lysed and clarified 96 hours after transfection on Day 169. Clarified lysates were titrated by qPCR. Clarified lysate EGFP qPCR titers were significantly higher for Ig2-SafeT clones S7 and Sil than for unmodified ignition 2 cells (Figure 16). In a repeat production study, Ig2-SafeT clone Sil cells were most productive (e.g., had the highest titer), whereas in a pilot study, Ig2-SafeT clone S7 were most productive (e.g., had the highest titer).3.5 Evaluation of Large Scale AAV Production in Top Performing Clones of Ig2- SafeT

[0151] To confirm results of two 75mL scale AAV production studies, a larger volume AAV productivity study was performed to compare Ig2-SafeT Clone S7 and Ig2- SafeT Clone Sil with unmodified ignition 2.0 cells. Transfections were performed according to suspension AAV transfection process parameters shown in Table 10.

[0152] Cells were lysed and lysates were clarified after 96 hours. Aliquots of clarified lysate were collected and titrated by EGFP qPCR (Figures 17A-17B). Clarified lysates were pooled into a single 1500mL volume prior to purification and analysis. Both Ig2-SafeT clone S7 and Ig2 SafeT yielded significantly higher clarified lysate titers than unmodified Ignition 2.0 cells. As shown in Figures 17A-17B, Ig2-SafeT clone Sil was most productive (e.g., had the highest titer).

[0153] Clarified lysates were purified using FPLC using an Acta Pure 150 and 2mL AAVX column. Aliquots were collected and titrated by qPCR (Figures 18A-18B) and analyzed by stunner to assess empty to full particle ratios (Figure 19). Both Ig2-SafeT clone S7 and Ig2 SafeT clone Sil yielded significantly higher elution peak titers than unmodified Ignition 2.0 cells. As shown in Figures 18A-18B, Ig2-SafeT clone Sil was most productive (e.g., had the highest yield). The percentage of full AAV particles was much higher for Ig2- SafeT clones than for unmodified Ignition 2.0 cells.

[0154] Following FPLC purification, full particles were isolated by Cesium Chloride ultracentrifugation. Banding after centrifugation was imaged (Figure 20). Full (lower) bands were larger for both Ig2-SafeT clones, indicative of a higher total yield. Partially packaged capsids (Intermediate Region) appeared to be reduced in Ig2-SafeT cells. Residual cesium chloride was removed, and final formulation was produced through dialysis. Aliquots were collected for product analysis. Final formulation was titrated by qPCR (Figures 21A-21B). Empty to full capsid ratio was assessed by Stunner (Figure 22).

[0155] Analytical ultracentrifugation (“AUC”) was performed to further evaluate the empty full ratio in the final product and to detect the packaging frequency of truncated AAV genomes. Titer and total AAV yield was highest for Ig2-SafeT clone SI 1. The percentage of full AAV capsids present in final formulation was improved for productions using Ig2-SafeT clones. Analytical ultracentrifugation of final formulations detected a higher proportion of full AAV capsids and a significant reduction in the packaging of truncated intermediately full capsids for productions using Ig2-SafeT clone S7 and Ig2-SafeT clone Sil (Figure 23). Ig2-SafeT clone Sil was selected as the top candidate due to superior AAV production in the scale up study.3.6 Analysis of AAV Produced in Ig2 -SafeT Cells for Residual SV40 T- AntigenProtein

[0156] A study was performed to assess the ability of the AAV purification to remove all residual SV40 T- Antigen Protein from final formulation. Total protein was quantified by DC assay from clarified lysate and final formulation samples from the scale up study by DC assay. SV40 Large T- Antigen protein was quantified using Jess Simple western blotting probing with anti-SV40 T rabbit MAB (Cell Signaling Technologies D1E9E).SV40-T antigen was present in clarified lysates from IG2-SafeT clones. No residual SV40 T- Antigen protein was detected in any formulation (Figure 24).

[0157] Together, these results show that Ignition Safe-T cell line is an improvement over unmodified Ignition 2.0 cells and commercially available 293T cells by improving quality of rAAV production (e.g., yield, purity) and offers a safer immortalized producer cell line for rAAV production.EQUIVALENTS

[0158] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMSWe claim:

1. An rAAV producer cell with a genome comprising a region that includes two or more exons encoding SV40 large T-antigen and one or more intron(s) located between the two or more exons, wherein the combined size of the sequence encoding SV40 large T-antigen and the one or more intron(s) is larger than the packaging capacity of an rAAV.

2. The rAAV producer cell of claim 1, wherein the combined size of the sequence encoding the SV40 large T-antigen is about 1000 to 3000 base pairs, e.g., about 1000, 2000, or 3000 base pairs.

3. The rAAV producer cell of claim 2, wherein the combined size of the sequence encoding the SV40 large T-antigen is about 2000 to 2200 base pairs, e.g., about 2000, 2100, or 2200 base pairs.

4. The rAAV producer cell of claim 3, wherein the combined size of the sequence encoding SV40 large T-antigen is about 2124 base pairs.

5. The rAAV producer cell of claim 1, wherein the size of the one or more intron(s) is greater than about 1000, 2000, 3000, 4000, 5000, or 6000 base pairs in length, e.g., about 1000 to about 6000 base pairs in length.

6. The rAAV producer cell of claim 5, wherein the size of the one or more intron(s) is greater than about 5000 base pairs in length.

7. The rAAV producer cell of claim 6, wherein the size of the one or more intron(s) is about 5410 base pairs in length.

8. The rAAV producer cell of any one of the preceding claims, wherein the region comprises an intron that is or is the complement of intron 1 of human eukaryotic translation elongation factor 1 alpha 1 (EF1A1).

9. The rAAV producer cell of any one of the preceding claims, wherein the region comprises an intron that is or is the complement of introns 2, 3, and 4 of human ribosomal protein S27a (RPS27A).

10. The rAAV producer cell of any one of the preceding claims, wherein the region comprises an intron that is or is the complement of intron 1 of human Foxf2.

11. The rAAV producer cell of any one of the preceding claims, wherein the region comprises each of an intron that is or is the complement of intron 1 of human EFl Al, an intron that is or is the complement of introns 2, 3, and 4 of human ribosomal protein S27a (RPS27A) and an intron that is or is the complement of intron 1 of human Foxf2.

12. The rAAV producer cell of any one of the preceding claims, wherein the region comprises a chimeric intron having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 5.

13. The rAAV producer cell of any one of the preceding claims, wherein the combined size of the two or more exons encoding SV40 large T-antigen and the one or more intron(s) is about 7000 to about 8000 base pairs in length, e.g., about 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, or 8000 base pairs in length.

14. The rAAV producer cell of claim 12, wherein the combined size of the two or more exons encoding SV40 large T-antigen and the one or more intron(s) is 7537 base pairs in length.

15. The rAAV producer cell of any one of the preceding claims, wherein the two or more exons encoding the SV40 large T-antigen and one or more intron(s) located between the two or more exons has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6.

16. The rAAV producer cell of any one of the preceding claims, wherein the region further comprises a nucleotide sequence encoding hTERT.

17. The rAAV producer cell of claim 16, wherein the nucleotide sequence encoding hTERT is located upstream of the two or more exons encoding the SV40 large T-antigen.

18. The rAAV producer cell of claim 17, wherein the region further comprises a nucleotide sequence comprising an EFl a promoter.

19. The rAAV producer cell of claim 18, wherein the nucleotide sequence comprising the EFl a promoter is located upstream of the nucleotide sequence encoding hTERT.

20. The rAAV producer cell of claim 19, wherein the nucleotide sequence comprising the EFl a promoter is operably linked to the nucleotide sequence encoding hTERT.

21. The rAAV producer cell of any one of the preceding claims, wherein the genome comprises a region having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 12.

22. The rAAV producer cell of any one of the preceding claims, wherein the rAAV producer cell is mammalian.

23. The rAAV producer cell of claim 22, wherein the rAAV producer cell is human.

24. The rAAV producer cell of claim 23, wherein the rAAV producer cell is a HEK293 cell.

25. A population of rAAV particles produced by the rAAV producer cell of any one of claims 1-24.

26. A method of producing rAAV comprising: culturing the rAAV producer cell of any one of claims 1-24.

27. The method of claim 26, wherein the rAAV producer cell is cultured adherently or in suspension.

28. The method of claim 27, wherein the rAAV producer cell yields a higher titer of rAAV relative to a control producer cell that lacks the nucleotide sequence encoding a SV40 large T-antigen.

29. The method of claim 28, wherein the rAAV producer cell yields an rAAV titer of greater than about IxlO10, 5xlO10, or IxlO11VG / mL.

30. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5.

31. A nucleic acid vector comprising the nucleic acid of claim 30.

32. An rAAV producer cell comprising the nucleic acid vector of claim 31.

33. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6.

34. A nucleic acid vector comprising the nucleic acid of claim 33.

35. An rAAV producer cell comprising the nucleic acid vector of claim 34.

36. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12.

37. A nucleic acid vector comprising the nucleic acid of claim 36.

38. An rAAV producer cell comprising the nucleic acid vector of claim 37.

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