WO2017189901A1 - Silençage de l'expression des transgènes lors de la production de vecteurs - Google Patents

Silençage de l'expression des transgènes lors de la production de vecteurs Download PDF

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WO2017189901A1
WO2017189901A1 PCT/US2017/029933 US2017029933W WO2017189901A1 WO 2017189901 A1 WO2017189901 A1 WO 2017189901A1 US 2017029933 W US2017029933 W US 2017029933W WO 2017189901 A1 WO2017189901 A1 WO 2017189901A1
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Philip Ng
Donna J. PALMER
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Baylor College of Medicine
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16051—Methods of production or purification of viral material
    • C12N2740/16052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • Embodiments of the disclosure concern at least the fields of cell biology, molecular biology, virology, and recombinant nucleic acid technology, for example.
  • Helper-dependent adenoviral vectors are devoid of all viral coding sequences and have proven to be excellent vectors for many gene and cell therapy applications because they can mediate high efficiency transduction of many different cells types from many different species in vivo and in vitro independent of the cell cycle, they have an enormous cloning capacity of 36 kb, do not integrate into the host genome and, provide long-term transgene expression with reduced toxicity 1 .
  • HDAd do not contain any viral genes, they must be produced using a helper virus (HV) 2 .
  • HV helper virus
  • the HV is an El-deleted adenovirus whose packaging signal is flanked by loxP sites.
  • El -complementing cells expressing Cre are co-infected with the HDAd and the HV wherein the packaging signal of the HV is excised by Cre-mediated site-specific recombination rendering it unpackagable but still able to undergo DNA replication and trans-complement HDAd production 2 .
  • HDAds expressing certain transgenes cannot be produced because the transgene product is toxic to the producer cells, especially when present in large quantities, such as during vector production.
  • 10 5 to 10 6 progeny adenoviral genomes are produced post-infection, only about 20% of which are packaged into virions 3 .
  • transgene products that may not normally be toxic may have toxic effects when present in such overwhelming quantities.
  • this problem is further exacerbated by the very strong promoters/enhancers that are normally employed in gene transfer vectors. Therefore, development of strategies to overcome this obstacle is important and necessary for the production of these recalcitrant HDAds.
  • VA RNAI virus- associated non-coding RNAs
  • VA RNAI functions to ensure high level adenoviral protein synthesis by binding to and inhibiting protein kinase R (PKR), part of the antiviral interferon response, while the role of VA RNAII is not well known.
  • PSR protein kinase R
  • These short RNA transcripts (-160 nucleotides) have a stem-loop structure similar to pre- miRNA and are expressed throughout the virus life cycle, reaching very high concentrations during the late phase of infection (10 8 molecules of VA RNA I/cell and 10 7 molecules of VA RNAII/cell).
  • VA RNAI and VA RNAII are exported to the cytosol by Exportin 5 where they are processed by Dicer into functional microRNAs, called mivaRNAs, which are incorporated into the RNA-induced silencing complex (RISC) 6 ' 7 ' 8 .
  • mivaRNAI derived from VA RNAI has been shown to target the mRNA from reporter transgenes engineered to contain the complementary target sequence in their 3' untranslated region (UTR), thereby inhibiting their expression 7 ' 9 10 11 .
  • UTR 3' untranslated region
  • host cellular mRNAs targeted by VA RNAI-derived mivaRNAI resulting in downregulated expression have been
  • the present disclosure satisfies a long-felt need in the art by providing simple strategies of exploiting expression of VA RNAI from the HV to downregulate transgene expression from the HDAd during its production. In this way, recalcitrant HDAds can be easily produced.
  • composition comprising a vector polynucleotide comprising an expression cassette, the expression cassette comprising: a) a coding sequence; and b) one or more untranslated sequences operably linked to the coding sequence, wherein the untranslated sequences comprise a target sequence for a microRNA produced from the adenoviral viral associated RNA I (VA RNAI) (mivaRNAI) and/or a microRNA produced from the adenoviral VA RNAII (mivaRNAII).
  • the vector may be a viral vector, such as a helper- dependent adenoviral vector (HDAd), adeno-associated viral vector, lentiviral vector, or retroviral vector.
  • the untranslated sequence is 5 '-untranslated sequence, 3 '-untranslated sequence, or both.
  • the coding sequence may encode a therapeutic gene product and/or a diagnostic gene product. In certain aspects, the coding sequence encodes a gene product that is not a reporter gene product.
  • a mivaRNAI target sequence comprises a) the sequence 5 ' -AAAGGAGC ACTCCCCCGTTGTCTG-3 ' (SEQ ID NO: l); b) a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to 5 '-AAAGGAGC ACTCCCCCGTTGTCTG-3' (SEQ ID NO:2); or c) a sequence that is complementary to the sequence in a) or b).
  • the 5'-terminal end of the sequence further comprises 1, 2, 3, 4, 5, or more nucleotides, and in specific examples the 1, 2, 3, 4, 5, or more nucleotides are A.
  • the 5'- terminal end of the sequence lacks 1, 2, 3, 4, 5, or more nucleotides of the sequence.
  • the 3'-terminal end of the sequence further comprises 1, 2, 3, 4, 5, or more nucleotides.
  • the 3'-terminal end of the sequence may lack 1, 2, 3, 4, 5, or more nucleotides of the sequence.
  • the mivaRNAI target sequence comprises: a) the sequence 5'-AAAAGGAGCACTCCCCCGTTGTCTG-3' (SEQ ID NO:3); b) a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to 5'- AAAAGGAGC ACTCCCCCGTTGTCTG-3' (SEQ ID NO:4); or c) a sequence that is complementary to the sequence in a) or b).
  • the 5'-terminal end of the sequence further comprises 1, 2, 3, 4, 5, or more nucleotides, such as the 1, 2, 3, 4, 5, or more nucleotides being A.
  • the 5'-terminal end of the sequence may lack 1, 2, 3, 4, 5, or more nucleotides of the sequence.
  • the 3 '-terminal end of the sequence further comprises 1, 2, 3, 4, 5, or more nucleotides. In some cases, the 3'-terminal end of the sequence lacks 1, 2, 3, 4, 5, or more nucleotides of the sequence.
  • the mivaRNAII target sequence comprises: a) the sequence 5'-AGGGGCTCGTCCCTGTTTCCG-3' (SEQ ID NO:5); b) a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to 5'- AGGGGCTCGTCCCTGTTTCCG-3 ' (SEQ ID NO:6); or c) a sequence that is complementary to the sequence in a) or b).
  • the 5'-terminal end of the sequence further comprises 1, 2, 3, 4, 5, or more nucleotides, such as the 1, 2, 3, 4, 5, or more nucleotides being A.
  • the 5'- terminal end of the sequence lacks 1, 2, 3, 4, 5, or more nucleotides of the sequence.
  • the 3'-terminal end of the sequence further comprises 1, 2, 3, or more nucleotides.
  • the 3'- terminal end of the sequence may lack 1, 2, 3, 4, 5, or more nucleotides of the sequence.
  • the vector is a helper-dependent adenoviral vector that derives from adenovirus serotype 1, adenovirus serotype 2, adenovirus serotype 5, adenovirus serotype 6, adenovirus serotype 35, or a chimera thereof.
  • the cell comprises a helper virus that encodes VA RNAI, VA RNAII, or both.
  • the helper virus is an El-deleted adenovirus comprising a packaging signal flanked by loxP sites; 2) the HV comprises a packaging signal flanked by firt sites; 3) the HV comprises a pIX deletion so that only HDAd of a certain size can be packaged, 3) the HV comprises a mutated, inefficient packaging signal; or 4) the HV comprises an insertion of cpC31 phage into it to delay its life cycle relative to the HDAd.
  • the cell may or may not express Cre.
  • a method of producing a composition of the disclosure comprising the steps of: a) providing, obtaining, or generating an expression cassette comprising a coding sequence operably linked to a 3' untranslated region; and b) modifying the 3' untranslated sequence to comprise a target sequence for a microRNA produced from the adenoviral viral associated RNA I (VA RNAI) (mivaRNAI) and/or a microRNA produced from the adenoviral VA RNAII (mivaRNAII).
  • VA RNAI adenoviral viral associated RNA I
  • mivaRNAII adenoviral viral associated RNA I
  • the method may further comprise the step of incorporating the expression cassette into a vector, for example the expression cassette may be comprised in a helper-dependent adenoviral vector, an adenoviral associated vector, a lentiviral vector, or a retroviral vector.
  • the method comprises the step of transducing a cell with the vector, such as a helper-dependent adenoviral vector or adeno-associated viral vector, the cell comprises a helper virus.
  • the method further comprises the step of transducing the cell with a helper virus.
  • the helper virus may encode VA RNAI, VA RNAII, or both.
  • the helper virus is an El-deleted adenovirus comprising a packaging signal flanked by loxP sites.
  • a method of producing a vector in a cell comprising the steps of: a) providing, obtaining, or generating in the cell a vector that includes an expression cassette that comprises a coding sequence operably linked to at least one untranslated region that comprises a target sequence for mivaRNAI and/or a target sequence for mivaRNAII; and b) exposing the vector to the mivaRNAI and/or mivaRNAII under suitable conditions such that expression of the coding sequence is inhibited upon binding of the mivaRNAI and/or
  • the vector is a helper-dependent adenoviral vector or adeno-associated viral vector.
  • the mivaRNAI and/or mivaRNAII are expressed from a vector in the cell.
  • the mivaRNAI and/or mivaRNAII may be expressed from a helper virus.
  • Total cellular DNA extracted from serial passages was digested with ApaLI and analyzed by agarose gel electrophesis to identify those passages containing peak vector titer which is indicated by the visible presence of vector-specific bands in the presence of the background cellular DNA smear.
  • the lanes labeled pHV contain the HV plasmid digested with ApaLI and Pad to serve as a control for HV specific bands.
  • the lanes labelled pHDAd contain the corresponding HDAd plasmid digested with ApaLI and Pmel to serve as a control for HDAd specific bands.
  • Passage 0 the initial transfection with pHDAd and infection with HV of the producer cells
  • FIG. 2 Schematic of amplification and large-scale HDAd production.
  • the earliest passage that contains peak vector titer during vector amplification was passage 2 for HDAd-CAG-LacZ, passage 6 for HDAd-CAG- hyPB, and passage 3 for HDAd-CAG-hyPB-VAI.
  • crude viral lysate from these passages were used to coinfect a single 150 mm dish of producer cells along with HV.
  • the resulting crude viral lysate from this single 150 mm dish was used to co-infect 2 L of producer cells along with HV and the HDAd was subsequently purified by CsCl ultracentrifugation.
  • FIGS 3A-3L Appearance of virus band in CsCl gradient and vector genomic structure.
  • (3 A to 31) The first continuous CsCl gradient for the indicated vector preparation is shown.
  • (3 J to 3L) The result of restriction analyses of virion DNA extracted from the indicated vector preparation is shown.
  • the lanes labelled pHV contain HV plasmid digested with ApaLI and Pad to serve as a control for HV specific bands.
  • the lanes labelled pHDAd-CAG-LacZ, pHDAd-CAG-hyPB, and pHDAd-CAG-hyPB-VAI contain the plasmid form of the indicated HDAd digested with ApaLI and Pmel to serve as a control for the HDAd-specific bands.
  • Total viral particles (vp) obtained from each preparation is indicated.
  • FIG. 4 Strategy to downregulate transgene expression from HDAd during vector production.
  • VA RNAI is expressed from the HV which is processed into mivaRNAI by Dicer and incorporated into the RISC and can cleave mRNA expressed from the HDAd which contains the mivaRNAI target sequence in the 3' UTR.
  • the HV genome cannot be packaged into virions because of excision of the floxed packaging signal ( ⁇ ) by Cre.
  • FIG. 5 Modification of hyPBase expression cassette to permit downregulation by the HV.
  • a sequence corresponding to nucleotide 119 to 159 from VA RNAI is inserted into the 3' UTR of the HDAd expression cassette (SEQ ID NO:7-8).
  • the mivaRNAI target sequence is boxed.
  • the 3' UTR is part of the multiple cloning site of the expression plasmid (SEQ ID NO:9).
  • FIGS. 6A and 6B HDAd-CAG-hyPB-VAI expresses functional hyPBase.
  • 6A HDAd-PB-TR contains a 2.3 kb segment of DNA flanked by PB TRs. hyPB-mediated excision of this DNA segment converts a 4.1 kb PCR product to a 1.8 kb PCR product.
  • (6B) PCR analyses of DNA extracted from human iPS cells infected with the indicated vectors. Lane 5 contains the PCR product obtained from uninfected human iPS cells to provide a control for nonspecific amplification products. Lane 6 contains the PCR product from the plasmid used to make HDAd-PB-TR to provide a control for the 4.1 kb unexcised PCR product.
  • embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method or
  • composition described herein can be implemented with respect to any other method or composition described herein.
  • the disclosure concerns compositions and methods related to efficient expression of a vector.
  • a transgene capable of being expressed in a vector is in need of inhibition of its expression.
  • the disclosure concerns vector or expression construct configurations that allow for silencing (in part or to undetectable levels) of a transgene.
  • compositions and method(s) of using the composition(s) concern a vector polynucleotide comprising an expression cassette, the expression cassette comprising: a) a coding sequence; and b) one or more untranslated sequences operably linked to the coding sequence, wherein the untranslated sequences comprise a target sequence for a microRNA produced from the adenoviral viral associated RNA I (VA RNAI) (mivaRNAI) and/or a microRNA produced from the adenoviral VA RNAII (mivaRNAII).
  • VA RNAI adenoviral viral associated RNA I
  • mivaRNAI adenoviral viral associated RNA I
  • the vector may be of any kind, including adenoviral vectors, lentiviral vectors, helper-dependent adenoviral vectors (HDAd), adeno-associated viral vector, and so forth.
  • helper-dependent adenoviral vectors that express certain transgene products are impossible to produce because the transgene product is toxic to the producer cells, especially when made in large amounts during vector production.
  • Downregulating transgene expression from the HDAd during vector production is a way to solve this problem.
  • the inventors show that this can be accomplished by inserting the target sequence for the adenoviral VA RNAI into the 3' untranslated region of the expression cassette in the HDAd.
  • the VA RNAI produced by the HV will target the transgene mRNA from the HDAd via the endogenous cellular RNAi pathway. Once the HDAd is produced and purified, transduction of the target cells results in unimpeded transgene expression because of the absence of HV. This simple and universal strategy permits for the robust production of otherwise recalcitrant HDAds.
  • Embodiments of the disclosure encompass methods and compositions related to downregulation of transgene expression including for production of a certain vector, such as helper virus-mediated downregulation of transgene expression that permits production of a particular vector, such as a recalcitrant helper-dependent adenoviral vector.
  • a certain vector such as helper virus-mediated downregulation of transgene expression that permits production of a particular vector, such as a recalcitrant helper-dependent adenoviral vector.
  • embodiments concern production of recalcitrant helper-dependent Ads.
  • PiggyBac is a transposon isolated from cabbage looper moth Tichoplusia ni and encodes a transposase that catalyzes PB transposition 12 .
  • Hyperactive PB transposase (hyPBase) bears 7 amino acid substitutions resulting in a 17-fold and 9-fold increase in excision and integration, respectively, compared to wildtype 13 .
  • the inventors sought to produce a HDAd expressing hyPBase from the strong CAG promoter (HDAd-CAG-hyPB) using a well- established protocol 14 15 that entails serial coinfections (called passages) of the producer cells with the HDAd and the HV to increase the HDAd titer, followed by identification of the earliest passage containing peak HDAd titer which would then be used to initiate large-scale vector production. To determine the passages that comprise peak HDAd titers, total cellular DNA was extracted from the producer cells at each serial passage, and examined by agarose gel electrophoresis after ApaLI digestion.
  • Serial passages with peak HDAd titers can be identified by the visible presence of HD Ad-specific bands in the presence of the background cellular DNA smear, and is typically first attained at serial passage 2 or 3 14 ' 15 .
  • vector-specific bands were not visible until serial passage 5 or 6 (FIGS. IB to IE).
  • the resulting crude viral lysates from this 150 mm dish was used to co-infect 2 L of producer cells along with HV for large scale production, and the vectors were purified by CsCl centrifugation (FIG. 2). Following CsCl ultracentrifugation, a single band was observed in the gradient for HDAd-CAG-LacZ as expected (FIG. 3A). In contrast, multiple bands were observed in the CsCl gradient for the four preparations of HDAd-CAG-hyPB (FIGS. 3B to 3E) which is indicative of genomic rearrangement of the HDAd and/or HV.
  • DNA was extracted from the virions obtained from the CsCl gradients, digested with ApaLI and analyzed by agarose gel electrophoresis.
  • the DNA pattern of HDAd-CAG-LacZ was identical to the plasmid from which it was derived (except for the expected absence of the 2.5 kb fragment containing the bacterial plasmid DNA) indicating no DNA rearrangements (FIG. 3J).
  • the four preparations of HD Ad-C AG-hyPB revealed different DNA patterns with some bands corresponding to the HDAd and some to the HV, as well as novel bands not expected of either, all of which is consistent with a mixed population of various rearranged HDAd and HV DNA (FIG. 3K). Because these preparations are useless for their intended purpose, their precise DNA rearrangements were not investigated further.
  • VA RNAI is expressed from the HV which is processed into mivaRNAs by Dicer and incorporated into the RISC and, importantly, this has been shown to downregulate expression cassettes bearing the complementary mivaRNAI sequence in the 3 'UTR 7 ' 9 10 11 .
  • downregulating transgene expression from the HDAd should be achievable during vector production by simply inserting the mivaRNAI target sequence into the 3 'UTR of the HDAd's expression cassette.
  • this modification would not inhibit transgene expression by the HDAd in the transduced target cell because of the absence of the HV.
  • HDAd-CAG-hyPB-VAI was created which bears, within the 3' UTR, nucleotides 119 to 159 from VA RNAI within which resides the mivaRNAI target sequence (FIG. 5).
  • peak vector titers were first reached by passage 2 to 3 (FIG. IF to II).
  • passage 3 was chosen to initiate large scale vector production (FIG. 2), and in all 4 cases a single virus band was obtained in the CsCl gradient (FIGS. 3F to 31).
  • HDAd-CAG-hyPB-VAI expresses functional hyPBase
  • human induced pluriopotent stem (iPS) cells 16 were coinfected with HDAd-CAG-hyPB-VAI and HDAd-PB-TR.
  • HDAd-PB-TR contains a 2.3 kb segment of DNA flanked by PB terminal repeats (TRs) and is thus excisable in the presence of hyPBase (FIG. 6A).
  • TRs PB terminal repeats
  • iPS cells were also infected with each vector alone, or mock infected. The next day, total DNA was extracted from the treated cells and subjected to PCR analyses.
  • a 4.1 kb PCR product is expected, and this is converted to a 1.8 kb PCR product following hyPBase-mediated excision (FIG. 6A).
  • the plasmid pHDAd-PB-TR (used to make HDAd-PB-TR), was included in the PCR assay as a control and, as expected, yielded only the unexcised 4.1 kb PCR product (FIGS. 6B, lane 6).
  • infection with HDAd-PB-TR alone does not result in excision as evident by the presence of the 4.1 kb PCR product and the absence of the 1.8 kb PCR product (FIG. 6B, lane 4).
  • the 1.8 kb PCR product indicative of hyPBase-mediated excision, is only present following co-infection of cells with HDAd-CAG-hyPB-VAI and HDAd-PB-TR (FIG. 6B, lane 2).
  • the unexcised 4.1 kb PCR product remains visible following co-infection with HDAd-CAG-hyPB-VAI and HDAd- PB-TR (FIG.
  • the HDAd genome along with its transgene expression cassette, is replicated to very high copy numbers in the producer cell.
  • the extraordinarily high transgene copy numbers exacerbated by the use of strong promoter/enhancers, result in very high quantities of transgene product in the producer cells during vector production.
  • a transgene product that is otherwise benign may have toxic effects on the producer cells and this could lead to a selection for rearranged vectors with no or reduced transgene expression.
  • HDAd expressing hyPBase from the strong CAG promoter is an example of such a vector; hyBPase is not toxic to mammalian cells but our repeated attempts to produce this vector were unsuccessful resulting in HDAd and HV genome rearrangements.
  • the HV expresses VA RNAI, a short non-coding RNA that is processed in functional miRNAs, called mivaRNA, by the endogenous cellular RNAi pathway in the producer cells to downregulate transgene expression from the HDAd.
  • mivaRNA a short non-coding RNA that is processed in functional miRNAs
  • This simple modification allowed for repeated and robust production of an HDAd expressing the functional hyPBase from the strong CAG promoter.
  • This strategy is straightforward and universal; it does not require the use of a special producer cell line, or drugs to suppress or induce transgene expression, and no special DNA expression control elements need be included in the expression cassette.
  • Saydaminova et al 17 reported a miRNA-mediated method of downregulating transgene expression from a HDAd during its production.
  • miRNA expression profiling By miRNA expression profiling, Saydaminova et al. identified two endogenous cellular miRNAs, hsa-miR183-5p and hsa-miR218-5p, that were strongly expressed in the producer 293-Cre cells but not in their intended human CD34+ target cells.
  • transgene expression was suppressed during vector production by hsa-miR183-5p and hsa-miR218-5p present in the 293-Cre producer cells.
  • transgene expression was unimpeded following HDAd transduction of CD34+ cells due to the absence of hsa-miR183-5p and hsa-miR218-5p in these cells.
  • this strategy would be ineffective for target cells, unlike CD34+ cells, that expressed either hsa-miR183-5p or hsa-miR218-5p.
  • the inventors have developed a simple and universal strategy to downregulate transgene expression from HDAd during vector production. This permits production of HDAd that otherwise could not be produced because of transgene product- mediated cellular toxicity. Indeed, the inventors have subsequently used this strategy to produce other recalcitrant HDAds that could not previously produce despite multiple attempts (not shown). Examples of Materials and Methods
  • An expression cassette containing the CAG promoter, hyPBase coding sequence 13 and the SV40 polyadenylation signal was inserted into the Ascl site of the HDAd genomic plasmid ⁇ 28 ⁇ 4 18 and the resulting plasmid was used to produce HDAd-CAG-hyPB as described below.
  • the VA RNAI target sequence created by annealing two oligonucleotide (sequence shown in Figure 5), was inserted into the Notl site in the 3' UTR of hyPBase expression cassette which was then inserted into the Ascl site of ⁇ 28 ⁇ 4 and the resulting plasmid was used to produce HDAd-CAG-hyPB-VAI as described below.
  • the expression cassette in HDAd-CAG-LacZ is identical to the one in HDAd-CAG-hyPB except that the hyPB coding sequence was replaced with the E. coli beta-galactosidase coding sequence.
  • HDAd Amplification of HDAd was performed as described in detail elsewhere 14 15 . Briefly, 20 ⁇ g of the plasmid form of the HDAd was digested with Pmel and transfected into a confluent 60 mm dish of 116 cells 14 by calcium phosphate co-precipitation (Promega, Madison, WI) and then the cells were infected with the HV AdNG163 19 at an MOI of 2000 vp/cell (serial passage 0).
  • the HDAd titer was increased by serial coinfections as follows; for each serial coinfection (called a passage), a confluent 60 mm dish of 116 cells was coinfected with HV (200 vp/cell) and 20% of the crude viral lysate containing the HDAd from the previous passage. Total DNA was extracted from each serial passage, digested with ApaLI and visualized by ethidium bromide staining following agarose gel electrophoresis. Serial passages containing peak titers of HDAd were identified by the visible presence of HDAd-specific bands in the agarose gel.
  • the earliest serial passage containing the most intensely visible HDAd-specific bands was used to initial large-scale vector production as follows; 20% of the crude viral lysate from the aforementioned passage was used to co-infect a single 150 mm dish of 116 cells along with AdNG163. 48 hours later, 100%) of the crude viral lysate from this single 150 mm dish was used to co-infect 2 L of 116 cells (lxlO 9 cells total) along with AdNG163 at an MOI of 200 vp/cell.
  • HDAd was purified from the co-infected 116 cells 48 later by triple CsCl ultracentrifugation; one step gradient followed by two continuous gradients.
  • hyPBase-mediated DNA excision was analyzed as follows; feeder free human iPS cells 16 were maintained in mTeSR 1 (STEMCELL Technologies, Vancouver, Canada) on Matrigel (Corning, Tewksbury, MA) coated plates. The iPS cells were infected as follows;
  • 2xl0 6 cells were resuspended in 1 ml mTeSR 1 supplemented with Y27632 (Reagents Direct, Encinitas, CA) to 10 ⁇ in a 1.5 ml microfuge tube and infected with HDAd at an MOI of 350 vp/cell for 1 hour at 37°C with gentle rocking. Following infection, cells were washed twice with 1 ml mTeSR 1 supplemented with Y27632 to 10 ⁇ and plated in a single Matrigel coated well of a 6 well plate in mTeSR 1 supplemented with Y27632 to 10 ⁇ . The next day, DNA was extracted from the infected cells for PCR analysis. PCR was performed with PrimeStar GXL (Takara-Clonetech, Mountain View, CA) using primers 5'
  • CTCAGTTTTCCTGGATTATGCCTGGCACC (SEQ ID NO: 10) and 5'
  • GCCTGACCAACATGGAGAAACCCCATCTC (SEQ ID NO: 11). Thermocycling conditions were as follows; 1 min at 94°C, followed by 30 cycles of 98°C for 10 sec and 72°C for 10 min, and a final extension of 10 min at 72°C.
  • RNAII-derived small RNAs are efficiently incorporated into the rna-induced silencing complex and associate with polyribosomes.
  • Adenovirus VA RNA-derived miRNAs target cellular genes involved in cell growth, gene expression and DNA repair. Nucleic Acids Res. 38: 750-763. Bellutti, F, Kauer, M, Kneidinger, D, Lion, T, Klein, R (2015). Identification of RISC- associated adenoviral microRNAs, a subset of their direct targets, and global changes in the targetome upon lytic adenovirus 5 infection. J Virol. 89: 1608-1627 Cary, LC, Goebel, M, Corsaro, BG, Wang, HG, Rosen, E, Fraser, ML (1989).

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Abstract

Selon des modes de réalisation, la présente divulgation concerne des vecteurs particuliers dans lesquels des transgènes sont réduits au silence par une ou plusieurs séquences non traduites comprenant une séquence cible pour un microARN produit à partir d'un ARN I adéno-associé (VA ARN I) (miARN I VA) et/ou un microARN produit à partir d'un ARN II adénoviral (miARN II VA); et leur procédé de production. Des exemples de séquences spécifiques sont en outre décrits.
PCT/US2017/029933 2016-04-27 2017-04-27 Silençage de l'expression des transgènes lors de la production de vecteurs Ceased WO2017189901A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021003432A1 (fr) * 2019-07-02 2021-01-07 Fred Hutchinson Cancer Research Center Vecteurs ad35 recombinants et améliorations de thérapie génique associées
EP3621982A4 (fr) * 2017-05-12 2021-05-19 University of Massachusetts Système de production de vecteurs viraux
WO2026073244A1 (fr) 2024-09-30 2026-04-02 Ensoma, Inc. Constructions pour l'expression multi-lignée d'agents thérapeutiques

Non-Patent Citations (3)

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Title
APARICIO, O ET AL.: "Adenovirus Virus-Associated RNA Is Processed to Functional Interfering RNAs Involved in Virus Production", JOURNAL OF VIROLOGY, vol. 80, no. 3, February 2006 (2006-02-01), pages 1376 - 1384, XP055181585 *
BELLUTTI, F ET AL.: "Identification of RISC-associated adenoviral microRNAs, a subset of their direct targets, and global changes in the targetome upon lytic adenovirus 5 infection", JOURNAL OF VIROLOGY, vol. 89, no. 3, February 2015 (2015-02-01), pages 1608 - 1627, XP055437018 *
PALMER, DJ ET AL.: "Helper virus-mediated downregulation of transgene expression permits production of recalcitrant helper-dependent adenoviral vector", METHODS & CLINICAL DEVELOPMENT., vol. 3, 8 June 2016 (2016-06-08), pages 16039, XP055437022 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3621982A4 (fr) * 2017-05-12 2021-05-19 University of Massachusetts Système de production de vecteurs viraux
US11767539B2 (en) 2017-05-12 2023-09-26 University Of Massachusetts Viral vector production
US12344857B2 (en) 2017-05-12 2025-07-01 University Of Massachusetts Viral vector production
WO2021003432A1 (fr) * 2019-07-02 2021-01-07 Fred Hutchinson Cancer Research Center Vecteurs ad35 recombinants et améliorations de thérapie génique associées
WO2026073244A1 (fr) 2024-09-30 2026-04-02 Ensoma, Inc. Constructions pour l'expression multi-lignée d'agents thérapeutiques

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