Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14151—Methods of production or purification of viral material

Definitions

• the vector encoding adenovirus helper proteins lacks Adenoviral structural and replication genes.
• the AAV rep and capsid (cap) genes are from different serotypes.
• the AAV rep and capsid genes from the same serotype. Examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 13 and the like.
• the AAV rep gene is from a serotype selected from the group consisting of AAV2, 3, 8, 9 and 10.
• the AAV cap gene is from a serotype selected from the group consisting of AAV2, 3, 8, 9 and 10.
• the AAV rep gene is an AAV2 rep gene and the AAV capsid gene is an AAV8 capsid gene.
• the AAV inverted terminal repeat (ITR) sequences are adeno-associated virus 2 inverted terminal repeat (ITR) sequences. Virtually any other combination of serotypes can be used.
• the transfected host cell is lysed. In some other embodiments, the transfected cell is not lysed. In certain embodiments, the titer of the rAAV vector particles is at least 1 x 10 10 to at least 1 x10 16 vector genomes/1.0 x 10 8 to lx 10 11 viable cells transfected. In certain embodiments, the titer of rAAV vector particles is at least 1 x 10 11 vector genomes/1.0 x 10 9 to 2x 10 11 viable cells transfected. In certain embodiments, the titer of rAAV vector particles is at least 1 x 10 12 vector genomes/2.0 x 10 9 to 3x 10 11 viable cells transfected.
• the titer of rAAV vector particles is at least 1 x 10 13 vector genomes/2.0 x 10 9 to 2x 10 11 viable cells transfected. In certain embodiments, the titer of rAAV vector particles is at least 1 x 10 14 vector genomes/2.0 x 10 9 to 4x 10 11 viable cells transfected. In certain embodiments, the titer of rAAV vector particles is at least 2 x 10 14 vector genomes/3.0 x 10 9 to 5x 10 11 viable cells transfected. In certain embodiments, the titer of rAAV vector particles is at least 3 x 10 14 vector genomes/4.0 x 10 9 to 5x 10 11 viable cells transfected.
• Certain embodiments of the methods described herein include use of a) a nucleic acid sequence encoding helper proteins, b) a nucleic acid sequence encoding rep and cap genes, and c) a close ended linear duplexed rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements.
• the polycationic polymer contains one or more amine residues, such as polyethylene imine (PEI) or a poly amino acid such as polyomithme, polyarginine, and poiylysine.
• PES polyethylene imine
• the polycationic polymer is PEL
• the molecular weight of the polycatiomc polymer can be varied in view of the identity of the one or more nucleic acids. Accordingly, in some embodiments, the poly cationic polymer has a molecular weight of between about 5,000 Daltons and about 100,000 Daltons, more preferably between about 5,000 and about 50,000 Daltons, most preferably between about 10,000 and about 35,000 Daltons
• the purified recombinant AAV has a particle to infectivity ratio of less than 9X10 2 vg/TCID50. In some embodiments, the purified recombinant AAV (rAAV) has a particle to infectivity ratio of less than 8X10 2 vg/TCID50. In some embodiments, the purified recombinant AAV (rAAV) has a particle to infectivity ratio of less than 7X10 2 vg/TCID50. In some embodiments, the purified recombinant AAV (rAAV) has a particle to infectivity ratio of less than 6X10 2 vg/TCID50. In some embodiments, the purified recombinant AAV (rAAV) has a particle to infectivity ratio of less than 5X10 2 vg/TCID50.
• Embodiments of the methods described herein include incubating the inoculated cell culture medium, e.g., the transfected host cells for a period of time to produce rAAV.
• the inoculated cell culture medium e.g., the transfected host cells can be incubated for a period of at least 24 hours.
• the transfected host cells can be incubated for a period of at least 30 hours.
• the inoculated cell culture medium e.g., the transfected cells are cultured for no more than 1000 hours, or no more than 950 hours, or no more than 900 hours, or no more than 850 hours, or no more than 800 hours, or no more than 750 hours, or no more than 700 hours, or no more than 650 hours, or no more than 600 hours, or no more than 550 hours, or no more than 500 hours, or no more than 450 hours, or no more than 400 hours, or no more than 350 hours, or no more than 300 hours, or no more than 250 hours, or no more than 200 hours, or no more than 150 hours, or no more than 100 hours.
• the volume of the cell culture comprising the host cells can be increased prior to transfection.
• the cell culture volume can be increased from about 10 to 20, 30, 40 or 50 ml volumes to about 4000 liter volumes.
• the cell culture volume can be increased from about 10 to 20, 30, 40 or 50 ml volumes to about 2000 liters.
• the cell culture volume can be increased from about 10 to 20, 30, 40 or 50 ml volumes to about 250 liters.
• the cell culture volume can be increased from about 10 to 20, 30, 40 or 50 ml volumes to about 50 liters or about 100 liters.
• the cell culture volume can be increased from about 100 liter volumes.
• the cell culture volume can be increased from about 50 ml volumes to about 50 liter volumes.
• the cell culture volume can be increased from about 50 ml volumes to about 10 liter volumes.
• the culturing volumes are progressively increased from about 50 ml volumes to about 4000 liter volumes. In certain embodiments, the culturing volumes are progressively increased from about 50 ml volumes to about 2000 liter volumes. In certain embodiments, the culturing volumes are progressively increased from about 10 to 20, 30, 40 or 50 ml volumes to about 250 liter volumes. In certain embodiments, the culturing volumes are progressively increased from about 10 to 20, 30, 40 or 50 ml volumes to about 100 liter volumes. In certain embodiments, the culturing volumes are progressively increased from about 10 to 20, 30, 40 or 50 ml volumes to about 50 liter volumes.
• the infectious particle titer is at least 1 x 10 6
• the infectious particle titer is at least 2.5 x 10 8
• the infectious particle titer is at least 7.5 x 10 8
• the infectious particle titer is at least 9 x 10 8
• the infectious particle titer is at least 3.5 10 9
• the infectious particle titer is at least 8 x 10 9
• the infectious particle titer is at least 8.5 x 10 9
• the AAV inverted terminal repeat (ITR) sequences are adeno-associated virus 2 inverted terminal repeat (ITR) sequences.
• the nucleic acid sequences added to the transfection composition comprise: about 0.1 ⁇ g to about 1 ⁇ g of Ad helper DNA, Rep/Cap DNA, or transgene per 0.5 x10 6 to about 5 x10 6 cells.
• the transfection is performed over a time course of about 10 minutes to about 60 minutes. In certain embodiments, the transfection is performed over a time course of about 10 minutes to about 120 minutes.
• a recombinant adeno-associated virus comprises a protelomerase target sequence.
• the protelomerase target sequence comprises a double stranded palindromic sequence of at least 10 base parrs in length.
• the rAAV comprises a transgene.
• AAV sequences may be obtained from a variety of sources.
• a suitable AAV sequence may be obtained as described in WO 2005/033321 or from known sources, e.g., the American Type Culture Collection, or a variety of academic vector core facilities.
• suitable sequences are synthetically generated using known techniques with reference to published sequences.
• the disclosure also provides for a method of producing a population of purified recombinant adeno-associated virus (rAAV) that lacks prokaryotic sequences, comprises: transfecting the mammalian cell line in suspended in culture medium with a transfection composition; wherein, the transfection composition comprises a) a nucleic acid sequence encoding helper proteins sufficient for rAAV replication ; b) a nucleic acid sequence encoding rep and cap genes, and c) a close ended linear duplexed rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements, and d) a stable cationic polymer; and wherein, the ratio of the stable cationic polymer to the total amount of nucleic acid contents from a), b) and c) is at least 1.5:1; culturing the transfected cell line for at least 40 hours; harvesting the transfected cell line of step ii); purifying the
• the cell lines are derived from cell biopsies and include, for example, lymph node cells, bone marrow cells, cord blood cells. In certain embodiments, the cell lines are derived from circulating tumor cells. In certain embodiments, the cell lines are derived from blood cell lines, for example, Jurkat and Molt4 T cell lines, U937 and THP pro- monocytes cell lines, B cell hybridomas. In certain embodiments, the cell lines are derived from stem cells.
• Another type of host cell is one that is stably transformed with the sequences encoding rep and cap, and which is transfected with the adenovirus El, E2A, and E40RF6 DNA and a construct carrying the expression cassette as described above.
• Stable rep and/or cap expressing cell lines such as B-50 (International Patent Application Publication No. WO 99/15685), or those described in U.S. Pat. No. 5,658,785, may also be similarly employed.
• Another desirable host cell contains the minimum adenoviral DNA which is sufficient to express E4 ORF6.
• Yet other cell lines can be constructed using the novel modified cap sequences of the invention.
• Regulatable promoters allow control of gene expression by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only.
• Regulatable promoters and systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art.
• the transgene operably linked to a tissue-specific promoter.
• a promoter active in muscle should be used.
• These include non-limiting examples of promoters from genes encoding skeletal b-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, as well as synthetic muscle promoters with activities higher than naturally-occurring promoters (see Li et al, Nat. Biotech., 17:241-245 (1999)).
• Examples of promoters that are tissue-specific are known for liver (albumin, Miyatake et al, J.
• This DNA arises by a cleaving-joining reaction, which is exerted by a single enzyme, a protelomerase, for example, TelN (prokaryotic telomerase) [Deneke J, Ziegelin G, Lurz R, Lanka E (2000) The protelomerase of temperate Escherichia coli phage N15 has cleaving-joining activity. Proc Natl Acad Sci U S A 97:7721-7726], A protelomerase such as TelN recognizes a target sequence in double-stranded DNA.
• TelN prokaryotic telomerase
• an in vivo cell system is used to produce_close ended linear duplex nucleic acids.
• the method comprises using a cell that expresses a protelomerase, such as TelN, or other protelomerase, wherein the protelomerase gene is under the control of a regulatable promoter.
• a regulatable promoter such as a small molecule regulated promoter or a temperature sensitive promoter, e.g. a heat shock promoter.
• variants of palindrome sequences described herein include truncated palindrome sequences that preserve the perfect repeat structure, and remain capable of allowing for formation of closed linear DNA.
• variant protelomerase target sequences may be modified such that they no longer preserve a perfect palindrome, provided that they are able to act as substrates for protelomerase activity.
• the closed linear expression cassette DNA product may comprise, consist or consist essentially of a eukaryotic promoter operably linked to a coding sequence of interest, and optionally a eukaryotic transcription termination sequence.
• the closed linear expression cassette DNA product may additionally lack one or more bacterial or vector sequences, typically selected from the group consisting of: (i) bacterial origins of replication; (ii) bacterial selection markers (typically antibiotic resistance genes) and (iii) unmethylated CpG motifs.
• Closed circular dsDNA templates are particularly preferred for use with RCA (rolling circle amplification) DNA polymerases.
• a circular dsDNA template may be in the form of a plasmid or other vector typically used to house a gene for bacterial propagation.
• the process of the invention may be used to amplify any commercially available plasmid or other vector, such as a commercially available DNA medicine, and then convert the amplified vector DNA into closed linear DNA.
• any desired non-ionic surfactant can be used for lysing the transfected host ceils.
• Exemplary non-ionic surfactants and classes of non-ionic surfactants for lysing the transfected host cells can include polyarylphenol polyethoxy ethers; polyalkylphenoi polyethoxy ethers; polyglycol ether derivatives of saturated fatty acids; polyglycol ether derivatives of unsaturated fatty acids; polyglycol ether derivatives of aliphatic alcohols, polyglycol ether derivatives of cycloaliphatic alcohols; fatty acid esters of polyoxyethylene sorbitan; alkoxylated vegetable oils; alkoxy!aied acetylenic dials; polyalkoxy!aied alkyiphenois; fatty acid alkoxyiates; sorbitan alkoxylates; sorbitol esters; C x to C 22 alkyl or alkenyl poly glycosides; polyalkoxy
• the non-ionic surfactant for lysing the host cells is selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethy!ene-po!yoxypropylene block copolymers, alkyiglucosides, alkylphenol ethoxylates, preferably polysorbates, polyoxyethylene alkyl phenyl ethers, and any combinations thereof.
• a zwitterionic surfactant can be added to the cell culture for lysing the transfected host cell.
• exemplary zwitterionic surfactants include, but are not limited to, sulfonates, such as CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1- propanesulfonate), CHAPSO (3- ⁇ (3-cholamidopropyl)dimethylammonio ⁇ -2-hydroxy-1- propane-sulfonate), 3-(decyldimethylammonio)propanesulfonate, 3-
• a nuclease e.g., an endonuclease is added to the lysate for reducing or removing impurities such as hcDNA.
• exemplary endonucleases include endonucleases derived from both Prokaryotes and Eukaryotes.
• the nuclease is BENZONASE ® or a salt active nuclease (SAN).
• Embodiment 12 The method of any one of claims 1-11, wherein the total amount of nucleic acid transfected from a), b), and c) per 1 x 10 6 cells is less than 2 ⁇ g, optionally, the total amount of nucleic acid transfected from a), b), and c) per 1 x 10 6 cells is less than 1 Eg ⁇
• Embodiment 39 The method of any one of claims 34-38, wherein a), b) and c) are transfected using a transfection composition comprising a), b) and c), and a stable cationic polymer, wherein the ratio of stable cationic polymer to total amount of nucleic acid from a), b) and c), is from about 1:1 to about 3:1 (weight/ weight), e.g., the ratio of stable cationic polymer to total amount of nucleic acid from a), b) and c), is about 1.5:1.
• Embodiment 43 The method of any one of claims 34-42, wherein, the amount of total of DNA from a), b) and c) are optionally 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.2, 1.4, 1.6 or 1.8 Eg ⁇
• Embodiment 48 The method of any one of claims 34-47, wherein the amino acid is L-glutamine.
• Embodiment 59 The method of any one of claims 57-58, wherein the mammalian cell line is derived from a human embryonic kidney cell line.
• Embodiment 68 The method of any one of claims 57-67, wherein the culture media is added after step i) and before step ii).
• Embodiment 81 The method of any one of claims 57-80, wherein the culturing volume comprises a concentration of an amino acid from about 1 mM to about 20 mM.
• Embodiment 87 The method of any one of claims 57-86, wherein the transfection composition comprises at least about 5% volume/volume (v/v) to about 20% v/v of the culture media.
• Embodiment 91 The method of any one of claims 57-90, wherein the AAV Rep and the AAV Cap genes are from same AAV serotype.
• Embodiment 97 The method of any one of claims 1-96, wherein the packaged nucleic acid of the rAAV further lacks eukaryotic DNA sequences.
• Embodiment 98 The method of any one of claims 1-97, wherein the closed ended linear duplexed nucleic acid comprises 1 ⁇ 2 of a protelomerase binding site.
• Embodiment 10 The method of any one of Embodiments 1-9, wherein the total amount of the nucleic acids from a), b), and c) per 1 x 10 6 cells is less than about 1.5 ⁇ g.
• Embodiment 19 The method of any one of Embodiments 1-18, wherein the polycationic polymer is linear polyethylenimine.
• Embodiment 22 The method of any one of Embodiments 1-21, wherein the ratio of the stable cationic polymer to total amount of nucleic acid from a), b) and c), is from about 1.9:1 to about 2.6:1.
• Embodiment 31 The method of any one of Embodiments 1-30, wherein the culture medium comprises an amino acid at a concentration of from about 1 mM to about 20 mM.
• Embodiment 40 The method any one of Embodiments 1-39, wherein the culture medium is subjected to an air sparge at a flow rate between about 0.25 LPM to about 0.75 LPM.
• Embodiment 45 The method of any one of Embodiments 1-44, wherein the host cell line is a serum free cell line.
• Embodiment 46 The method of any one of Embodiments 1-45, wherein the host cell line is suspension-adapted.
• Embodiment 47 The method of any one of Embodiments 1-46, wherein the host cell line is suspended in the culture medium.
• Embodiment 49 The method of any one of Embodiments 1-48, wherein the host cell line comprises a cell density of about 3.0 x10 6 to about lx10 8 viable cells/ml.
• Embodiment 55 The method of any one of Embodiments 1-54, wherein the closed ended linear duplexed nucleic acid comprises 1 ⁇ 2 of a protelomerase binding site, and wherein the 1 ⁇ 2 of the protelomerase binding site is formed by protelomerase digestion of a target binding site. Comprising a double-stranded palindromic sequence of at least 10 base pairs in length.
• Embodiment 59 The method of any one of Embodiments 1-58, wherein the AAV ITR sequences and AAV cap gene are from different serotypes.
• helper virus or “contaminating helper virus” refers to a virus used when producing copies of a helper virus-dependent viral vector, such as adeno- associated virus, which does not have the ability to replicate on its own.
• the helper virus is used to co-infect cells alongside the viral vector and provides the necessary proteins for replication of the genome of the viral vector.
• the term encompasses intact viral particles, empty capsids, viral DNA and the like.
• Helper viruses commonly used to produce rAAV particles include adenovirus, herpes simplex virus, cytomegalovirus, Epstein-Barr virus, and vaccinia virus.
• telomeres refers to preferential entry of the virus into certain cells or tissues, optionally followed by expression (e.g., transcription and, optionally, translation) of a sequence(s) carried by the viral genome in the cell, e.g., for a recombinant virus, expression of a heterologous nucleic acid(s) of interest.
• the primary aim of the small-scale screening experiments was to identify near- optimal transfection conditions for the 50L scaled portion of the experimental plan.
• cells were thawed, cultured and progressively expanded until inoculation into the 50L production bioreactor.
• the cell culture expansion process continued in the production bioreactor prior to transient transfection being performed.
• the transfected cell culture was incubated in the production bioreactor for approximately 72-hpt. At harvest, the transfected cell culture was lysed and clarified via depth and membrane filtration followed by purification.
• ⁇ g DNA per 1 x 10 6 viable cells and PETDNA ratio were studied in a range between 0.5 -2 ⁇ gand 1 -3, respectively.
• the design was a custom response surface model (RSM) with three levels for each factor allowing for the interpretation of both linear and quadratic effects. Duplicate center points in the design space were used to estimate the significance of each effect.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biomedical Technology (AREA)
• Organic Chemistry (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Wood Science & Technology (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• Virology (AREA)
• Molecular Biology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Biophysics (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Production Of Liquid Hydrocarbon Mixture For Refining Petroleum (AREA)

Priority Applications (8)

Applications Claiming Priority (2)

Publications (2)

Family

ID=76864730

Family Applications (1)

Country Status (8)

Cited By (3)

Families Citing this family (2)

Family Cites Families (8)

• 2021
  • 2021-01-15 WO PCT/US2021/013689 patent/WO2021146591A2/en not_active Ceased
  • 2021-01-15 AU AU2021207683A patent/AU2021207683A1/en active Pending
  • 2021-01-15 CA CA3162520A patent/CA3162520A1/en active Pending
  • 2021-01-15 CN CN202180023263.2A patent/CN115315518A/zh active Pending
  • 2021-01-15 EP EP21741128.9A patent/EP4090750A4/en active Pending
  • 2021-01-15 US US17/793,196 patent/US20230048994A1/en active Pending
  • 2021-01-15 JP JP2022543381A patent/JP7846625B2/ja active Active
  • 2021-01-15 IL IL294775A patent/IL294775A/en unknown
• 2025
  • 2025-11-14 JP JP2025195186A patent/JP2026032042A/ja active Pending

Also Published As

Similar Documents

Legal Events