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  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
  • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
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  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10041—Use of virus, viral particle or viral elements as a vector
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  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
  • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10351—Methods of production or purification of viral material
  • C12N2710/10352—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
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  • 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
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
• • C—CHEMISTRY; METALLURGY
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal
• • C—CHEMISTRY; METALLURGY
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  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
  • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

• This application relates generally to adenoviral vectors and gene therapy, and, more specifically, to non-replicating adenovirus expression vectors, methods of synthesis, and modes of replication.
• Adenoviral vector nucleotide sequences are often many kilobases long, which makes in vitro manipulation less convenient and harder to achieve. Manipulation of adenoviral vectors, if done improperly, can also leaded to unchecked replication of the vector. Moreover, common mammalian cell lines used to produce adenovirus often possess endogenous copies of adenoviral genes, which can recombine with mutant adenoviral vectors and create infectious replicating viruses. Thus, there exists a need for safe, non-infectious, recombinant, non-replicating adenoviral vectors with effective expression cassettes for therapeutic use.
• the present disclosure provides an adenovirus vector possessing a) one or more mutations that render it replication incompetent, and b) carrying a nucleotide expression cassette that allows expression of therapeutics when transduced into host cells. Sequences of the engineered adenovirus vectors are disclosed herein. Protocols and methods for more efficient production in bacteria of the synthesized adenovirus vectors are also provided herein. Finally, creation of an engineered mammalian cell line for replication of the engineered adenoviral vector to eliminate possible contamination of replication competent adenovirus is produced herewith.
• the non-replicating adenovirus expression vector may include a) one or more mutations that render the adenovirus replication incompetent and b) at least one nucleotide sequence encoding a protein or an RNA.
• the vector comprises a nucleotide sequence having at least 80% sequence identity to any of SEQ ID NOs: 1-2.
• SEQ ID NO: 1 encodes a full vector with plasmid backbone.
• SEQ ID NO: 2 encodes a full adenoviral vector of the present disclosure without a plasmid backbone.
• the vector is an adenovirus serotype 5 vector.
• the mutations may comprise deletion of either an El gene or an E3 gene or both.
• At least one nucleotide sequence in the non-replicating adenovirus expression vector may be a transgene.
• the transgene expresses human IL10.
• the transgene further comprises at least one of an enhancer/promoter region including CAG, human ILIO cDNA, or a polyadenylation signal, or any combination thereof.
• the polyadenylation signal further may be an SV40 region and/or a full poly A signal.
• the polyadenylation signal may be inserted, in some embodiments, between nucleotides 440 and 3515 in SEQ ID NO: 3.
• SEQ ID NO: 3 may be derived from accession number AY3339865 in the GenBank database.
• the recombinant vector can include a nucleic acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than 99% sequence identity to SEQ ID NOs: 1-2.
• a method of synthesizing an adenovirus vector is also disclosed herein.
• the method may include: a) producing a plurality of overlapping adenovirus sub-fragments, each sub- fragment including a portion of the full genome of the adenovirus; b) circularizing the sub- fragments to form plasmid structures; c) assembling the circularized sub-fragments into a linear structure, wherein the vector includes a combination of two or more sub-fragments.
• the adenovirus vector may be adenovirus serotype 5.
• the vector may replicate at high efficiency.
• a first sub-fragment may include at least one sequence as set forth in SEQ ID NOs: 4-5.
• a second sub-fragment may include at least one sequence as set forth in SEQ ID NOs: 6-7.
• each sub-fragment may include about 50% of the full adenovirus genome.
• At least one fragment may include a transgene that expresses human IL-10.
• the transgene further may include at least one of an enhancer/promoter region, human IL10 cDNA, or a polyadenylation signal, or any combination thereof.
• the enhancer or promoter region may be a CAG promoter.
• the transgene further may include at least a polyadenylation signal comprising an SV40 promoter or a full poly(A) signal.
• the vector sequence also may have a linear cloning vector and or a backbone linear cloning vector. In certain embodiment, the linear cloning vector may be pJazz-OK.
• the human IL10 cDNA sequence may be optimized and encoded from SEQ ID NO: 8.
• a mammalian cell configured to replicate adenoviral vectors, wherein the cell line comprises nucleotide sequences expressing El A and E1B gene products but is devoid of other adenovirus sequences is also provided herein.
• the cell line further may comprise a lentiviral vector engineered to express the El A and E1B gene products, wherein the lentiviral vector inhibits reconstitution of functional adenovirus when a mutant adenoviral viral vector is introduced into the cell line.
• the cell line may comprise HeLa cells.
• the cell line is produced by transfecting HEK293 cells with a lentiviral genome plasmid comprising at least an El A encoding sequence and an ElB-encoding sequence.
• the plasmid further may comprise at least an El A encoding sequence and an ElB- encoding sequence contains El A and E1B sequences arranged in a bidirectional expression unit with a bidirectional promoter to prevent recombination with a mutant adenoviral vector.
• the bidirectional promoter may be a combination of truncated CMV and PGK promoters.
• the cell line further can include a puromycin drug resistance gene in the lentiviral genome sequence to differentiate cells which have integrated the lentiviral sequence when the puromycin drug resistance gene is expressed.
• a final vector of SEQ ID NO: 9 is used to produce lentivirus and HeLa cells, transduced and selected for puromycin resistance.
• a method of producing and/or expanding the synthetic non-replicating adenoviral vector disclosed above is also provided herein.
• the method may include transforming a mammalian cell with an adenovirus vector comprising: a) one or more mutations that render the adenovirus replication incompetent and b) at least one nucleotide sequence encoding human IL- 10, wherein the mammalian cell comprises nucleotide sequences expressing E1A and E1B gene products but is otherwise devoid of other adenovirus sequences, culturing the transformed mammalian cell in a cell line containing an inserted lentiviral El A and E1B vector, and isolating the adenovirus vector.
• a method of treating a subject in need of a gene product may comprise, consist essentially of, or further, consist of administering to the subject a vector comprising (a) one or more mutations that render the adenovirus replication incompetent and (b) at least one nucleotide sequence encoding the gene product.
• the vector comprises a nucleotide sequence having at least 80% sequence identity over the entire sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a complementary sequence thereto.
• the vector may be an adenovirus serotype 5 vector.
• the one or more mutations may be a deletion of either an El gene or an E3 gene or both.
• the at least one nucleotide sequence further may include a transgene.
• the transgene expresses human ILIO.
• the subject may be a human.
• the gene product is a peptide, including human peptides.
• the gene product can be RNA.
• FIG. l is a schematic diagram of the genomic organization of a full non-replicating, adenoviral vector of the present disclosure, without a plasmid backbone.
• FIG. 2 is a schematic diagram of the genome organization of a non-replicating, adenoviral vector of the present disclosure, with a plasmid backbone.
• FIG. 3 is a schematic diagram of the genomic organization of an exemplary first sub- fragment in linear form.
• FIG. 4 depicts a schematic diagram of the genomic organization of an exemplary second sub-fragment in linear form.
• FIG. 5 shows a schematic diagram of the genomic organization of an exemplary first sub-fragment in circularized form.
• FIG. 6 shows a schematic diagram of the genomic organization of an exemplary first second-fragment in circularized form.
• FIG. 7 shows a schematic diagram of genomic organization of the final vector combining a first sub-fragment and a second sub-fragment.
• FIG. 8 is a schematic diagram of genomic organization of a vector used to produce lentivirus and HeLa cell lines transduced and selected for puromycin resistance.
• FIG. 9 is a flowchart of a disclosed production process of safe, replication incompetent adenoviral serotype 5 vectors.
• FIG. 10 shows a schematic diagram of the full El cell creation vector with a plasmid backbone.
• adenovirus means a medium-sized (90-100 nm), nonenveloped polyhedral virus that may include a capsid and a double-stranded linear DNA genome.
• the adenovirus can be a naturally occurring, but isolated, adenovirus or a recombinant adenovirus, or a chimeric variant thereof.
• administering means a method of giving a dosage of a pharmaceutical composition (e.g ., a recombinant adenovirus of the invention) to a subject.
• a pharmaceutical composition e.g ., a recombinant adenovirus of the invention
• the compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by
• protein includes peptides, polypeptides, or proteins of any length and any secondary or tertiary structure.
• gene product is meant to include mRNAs, other nucleic acids (e.g, microRNAs) transcribed from a gene as well as proteins, including polypeptides, translated from those mRNAs.
• a gene product can be a soluble protein, chemokine, cytokine, soluble receptor, an antibody, antibody fragments, an antibody -like molecule, or enzyme.
• the gene product can be an antigen-binding fragment of an antibody, or the gene product can be some or all of the variable portion of IgG or IgM.
• the gene product can be an antibody fragment lacking the Fc domain.
• the gene product can comprise or consist of a complementarity-determining region (CDR).
• CDR complementarity-determining region
• the gene product can be an anti-IL6 antibody or antibody fragment.
• the gene product is from a virus.
• the gene product is a therapeutic gene product, including, but not limited to, the interleukins (e.g ., human IL-10), interferon proteins, Factor VIII, Factor IX, erythropoietin, alpha- 1 antitrypsin, calcitonin, glucocerebrosidase, growth hormone, low density lipoprotein (LDL), receptor IL-2 receptor and its antagonists, insulin, globin, immunoglobulins, catalytic antibodies, insulin-like growth factors, superoxide dismutase, immune responder modifiers, parathyroid hormone and interferon, nerve growth factors, tissue plasminogen activators, and colony stimulating factors.
• the interleukins e.g ., human IL-10
• interferon proteins e.g IL-10
• Factor VIII Factor VIII
• Factor IX erythropoietin
• portion or “fragment” is meant a part of a whole.
• a portion may comprise at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the entire length of an polynucleotide or polypeptide sequence region.
• a portion may include at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000 or more contiguous nucleotides of a reference polynucleotide molecule.
• a portion may include at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, or 350 or more contiguous amino acids of a reference polypeptide molecule.
• sequence identity or “sequence similarity” means that the identity or similarity between two or more amino acid sequences, or two or more nucleotide sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of “percentage (%) identity,” wherein the higher the percentage, the more identity shared between the sequences. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similarity shared between the sequences. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. Sequence identity may be measured using sequence analysis software on the default setting. Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
• a “subject” is a vertebrate, such as a mammal ( e.g ., primates and humans). Mammals also include, but are not limited to, farm animals (such as cows), sport animals (e.g., horses), pets (such as cats and dogs), mice, and rats.
• a subject to be treated according to the methods described herein e.g, a subject having a disease such as cancer and/or a disease caused by an infective agent, e.g, a bacterium, virus, fungus, or parasite
• an infective agent e.g, a bacterium, virus, fungus, or parasite
• Diagnosis may be performed by any suitable means.
• a subject in whom the development of an infection or disease (or disease symptoms) is being prevented may or may not have received such a diagnosis.
• a subject to be treated according to the present invention may have been subjected to standard tests or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors (e.g, exposure to a biological agent, such as a virus).
• vector refers to a composition that includes one or more genes (non- structural or structural), or fragments thereof, from a viral species, such as an adenoviral species (e.g, sAd5, sAdl, etc.), that may be used to transmit one or more heterologous genes from a viral or non-viral source to a host or subject.
• the nucleic acid material of the viral vector may be encapsulated, e.g, in a lipid membrane or by structural proteins (e.g, capsid proteins), that may include one or more viral polypeptides (e.g, a glycoprotein).
• the viral vector can be used to infect cells of a subject, which, in turn, promotes the translation of the heterologous gene(s) of the viral vector into a protein product.
• virus is defined as an infectious agent that is unable to grow or reproduce outside a host cell and that infects mammals (e.g, humans) or birds.
• the complete genome sequences of novel adenoviral vectors are disclosed below.
• the adenovirus 5 genome is over thirty-five kilobases in size, making in vitro manipulation less convenient. Thus, synthetic DNA methods were used to generate two or more small fragments to allow for higher efficiency molecular cloning using bacterial hosts.
• the final, non-replicating adenovirus expression vector may include a) one or more mutations that render the adenovirus replication incompetent and b) at least one nucleotide sequence encoding a protein or an RNA.
• FIG. l is a schematic diagram of the genome organization of a non-replicating, adenoviral vector of the present disclosure, without a plasmid backbone.
• FIG. 2 is a schematic diagram of the genome organization of a non-replicating, adenoviral vector of the present disclosure, with a plasmid backbone.
• the vector comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NOs: 1-2.
• the recombinant vector can include a nucleic acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, greater than 99%, or any value therebetween sequence identity to SEQ ID NOs: 1-2.
• the vector is an adenovirus serotype 5 vector.
• the mutations may comprise deletion of either an El gene or an E3 gene or both, to prevent adenoviral replication in vivo.
• At least one nucleotide sequence in the non-replicating adenovirus expression vector may be a transgene.
• the transgene expresses human ILIO.
• the transgene further comprises at least one of an enhancer/promoter region including CAG, human ILIO cDNA, or a polyadenylation signal, or any combination thereof.
• the polyadenylation signal further may be an SV40 region and/or a full poly A signal.
• the polyadenylation signal may be inserted, in some embodiments, between nucleotides 440 and 3515 in SEQ ID NO: 3.
• SEQ ID NO: 3 may be derived from accession number AY3339865 in the GenBank database.
• a transgene for expression of human ILIO is included in the adenoviral sequence, which further includes an enhancer/promoter region (CAG), human ILIO cDNA, and a polyadenylation signal (SV40, full polyA signal), which is inserted between nucleotide 440 and 3515 relative to Adenovirus sequence (GenBank accession number AY339865), replacing the El genes but leaving the pIX gene intact.
• CAG enhancer/promoter region
• SV40 full polyA signal
• Sequences encoding an inserted transgene in the adenoviral vector may include a variety of gene products, and RNA or DNA transcribed therapeutics, including, but not limited to, interferon (IFN) proteins, Factor VIII, Factor IX, erythropoietin, alpha-1 antitrypsin, calcitonin, glucocerebrosidase, growth hormone, low density lipoprotein (LDL), receptor IL-2 receptor and its antagonists, insulin, globin, immunoglobulins, catalytic antibodies, the interleukins, insulin-like growth factors, superoxide dismutase, immune responder modifiers, parathyroid hormone and interferon, nerve growth factors, tissue plasminogen activators, and/or colony stimulating factors, or fragments thereof.
• IFN interferon
• Factor VIII Factor VIII
• Factor IX erythropoietin
• alpha-1 antitrypsin alpha-1 antitrypsin
• the engineered adenoviral vector may include any nucleotide (DNA or RNA) sequence configured for inclusion with the expression cassette.
• exemplary transgenes that may be inserted into the adenoviral construct include, but are not limited to, Angiotensin-converting enzyme 2 precursor (ACE2) antibodies (SEQ ID NO: 14 [no plasmid backbone], SEQ ID NO: 15 [with plasmid backbone]), chimeric monoclonal antibodies such as Unitixin scFv (SEQ ID NO: 16 [no plasmid backbone], SEQ ID NO: 17 [with plasmid backbone]), transforming growth factor (TGF) beta res IL-10 (SEQ ID NO: 18 [with plasmid backbone], SEQ ID NO: 19 [no plasmid backbone]), interleukin 13 (SEQ ID NO: 20 [no plasmid backbone], SEQ ID NO: 21 [with plasmid backbone]), interleukin 8 with short hair
• a method of synthesizing an adenovirus vector is also disclosed herein.
• the method may include: a) producing a plurality of overlapping adenovirus sub-fragments, each sub- fragment including a portion of the full genome of the adenovirus; b) circularizing the sub- fragments to form plasmid structures; c) assembling the circularized sub-fragments into a linear structure, wherein the vector includes a combination of two or more sub-fragments.
• the adenovirus vector may be adenovirus serotype 5.
• the vector may replicate at high efficiency.
• a first sub-fragment may include at least one sequence as set forth in SEQ ID NOs: 4-5.
• a second sub-fragment may include at least one sequence as set forth in SEQ ID NOs: 6-7.
• FIG. 3 depicts a schematic diagram of the genomic organization of an exemplary first sub-fragment in linear form.
• FIG. 4 depicts a schematic diagram of the genomic organization of an exemplary second sub-fragment in linear form.
• FIG. 5 shows a schematic diagram of the genomic organization of an exemplary first sub-fragment in circularized form and
• FIG. 6 shows a schematic diagram of the genomic organization of an exemplary first second- fragment in circularized form.
• FIG. 7 shows a schematic diagram of genomic organization of the final vector combining a first sub -fragment and a second sub -fragment.
• the final adenovirus serotype 5 genome may be assembled from the plurality of circularized fragments, which creates a linear structure for high level DNA replication in bacteria used in viral production.
• the high level of DNA replication and design allow for greater amounts of DNA to use for transfection and first round viral production. This is particularly useful when manipulating larger adenoviral vectors, such as the serotype 5 adenoviral vector, which is over 35 kilobases in size.
• each sub-fragment may include about 50% of the full adenovirus genome.
• At least one fragment may include a transgene that expresses human IL-10.
• the transgene further may include at least one of an enhancer/promoter region, human IL10 cDNA, or a polyadenylation signal, or any combination thereof.
• the enhancer or promoter region may be a CAG promoter.
• the transgene further may include at least a polyadenylation signal comprising an SV40 promoter or a full poly(A) signal.
• the vector sequence also may have a linear cloning vector and or a backbone linear cloning vector.
• the linear cloning vector may be pJazz-OK (see, e.g., BigEasy® v2.0: Linear Cloning Kits, LUCIGEN CORPORATION, Jan. 1, 2018), hereby incorporated by reference).
• the teachings of the human IL10 cDNA sequence may be optimized and encoded from SEQ ID NO: 8.
• a mammalian cell configured to replicate adenoviral vectors, wherein the cell line comprises nucleotide sequences expressing El A and E1B gene products but is devoid of other adenovirus sequences is also provided herein.
• the adenoviral vectors disclosed above may have been mutated to remove the viral El and E3 genes in order to prevent the viruses from replicating and thus being infectious.
• Mammalian cell lines supplied with the two El gene products, El A and E1B can support replication of the mutant virus.
• a common mammalian cell line used to produce adenovirus, HEK293 possesses an endogenous copy of the adenoviral El gene, which can recombine with mutant adenoviral vector and recreate infectious replicating virus. Therefore, a novel cell line, devoid of adenoviral sequences, is provided herein to express the El A and E1B gene products using a vector which does not allow reconstitution of functional adenovirus when the mutant viral vector is introduced.
• the cell line may comprise a lentiviral vector engineered to express the El A and E1B gene products, wherein the lentiviral vector inhibits reconstitution of functional adenovirus when a mutant adenoviral viral vector is introduced into the cell line.
• the cell line may comprise HeLa cells.
• the cell line is produced by transfecting HEK293 cells with a lentiviral genome plasmid comprising at least an El A encoding sequence and an ElB-encoding sequence.
• the plasmid further may comprise at least an El A encoding sequence and an ElB- encoding sequence contains El A and E1B sequences arranged in a bidirectional expression unit with a bidirectional promoter to prevent recombination with a mutant adenoviral vector.
• the bidirectional promoter may be a combination of truncated CMV and PGK promoters.
• the cell line further can include a puromycin drug resistance gene in the lentiviral genome sequence to differentiate cells which have integrated the lentiviral sequence when the puromycin drug resistance gene is expressed.
• a final El cell creation vector of SEQ ID NO: 9 is used to produce lentivirus and HeLa cells, transduced and selected for puromycin resistance.
• FIG. 8 is a schematic diagram of genomic organization of a vector used to produce lentivirus and HeLa cell lines transduced and selected for puromycin resistance.
• polynucleotide sequence of SEQ ID NO: 9 does not include a plasmid backbone.
• SEQ ID NO: 10 encodes a full El cell creation vector with a plasmid backbone, as depicted in the schematic diagram of the plasmid genome in FIG. 10.
• a method of producing and/or expanding the synthetic non-replicating adenoviral vector disclosed above is also provided herein.
• the method may include transforming a mammalian cell with an adenovirus vector comprising: a) one or more mutations that render the adenovirus replication incompetent and b) at least one nucleotide sequence encoding human IL- 10, wherein the mammalian cell comprises nucleotide sequences expressing E1A and E1B gene products but is otherwise devoid of other adenovirus sequences, culturing the transformed mammalian cell in a cell line containing an inserted lentiviral El A and E1B vector, and isolating the adenovirus vector.
• the engineered adenoviral serotype 5 vector of the disclosure is introduced into engineered cells in order to generate mutant adenovirus which expresses human IL-10.
• FIG. 9 is a flowchart, wherein it can be seen that in this mode of production a lentiviral E1A/E1B vector is first introduced into HeLa cells. The modified HeLa cell lines may then be tested for vector production to be sure there is no contamination of the cell line. Assuming there is no infection contamination or unexpected replication, one can then proceed with small scale vector production and amplification.
• mutant adenoviral vector of the present disclosure and the associated production cell line allow for the expansion of non-infectious therapeutic agents expressing adenovirus without the risk of recombination and restoration of replication competence.
• Potential applications may include, but are not limited to, adenoviral expression of human IL-10, which in turn can inhibit immune activity in autoimmune disorders as well as reduce rejection risk in allo-transplant or xenotransplant.
• a method of treating a subject in need of a gene product may comprise, consist essentially of, or further, consist of administering to the subject a vector comprising (a) one or more mutations that render the adenovirus replication incompetent and (b) at least one nucleotide sequence encoding the gene product.
• the vector comprises a nucleotide sequence having at least 80% sequence identity over the entire sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a complementary sequence thereto.
• the vector may be an adenovirus serotype 5 vector.
• the one or more mutations may be a deletion of either an El gene or an E3 gene or both.
• the at least one nucleotide sequence further may include a transgene.
• the transgene expresses human ILIO.
• the subject may be a human.
• the gene product is a peptide, including human peptides.
• the gene product can be RNA.
• Example 1 Synthesis and description of an engineered adenovirus serotype 5 vector is described in Example 1.
• the adenovirus sequence used for synthesis was derived from public domain sources, notably sequence AY339865, available in GenBank, worldwideweb.ncbi.nlm.nih.gov/genbank/. Two smaller fragments were generated using synthetic DNA methods to allow for higher efficiency cloning using bacterial hosts. The synthetic fragments were designed to include deletions of the El and E3 genes to prevent adenoviral replication in vivo.
• a transgene for expression of human IL-10 was included in the adenoviral sequence, which includes an enhancer/promoter region (CAG), human ILIO cDNA, and a polyadenylation signal (SV40, full polyA signal), which was inserted between nucleotides 440 and 3515 relative to Adenovirus sequence AY339865, which replaces the El gene, but leaves the pIX gene intact.
• CAG enhancer/promoter region
• SV40 full polyA signal
• adenovirus 5 genome was synthesized as several overlapping DNA fragments. The overlapping DNA fragments were assembled into two linear sub -fragments, each bearing approximately one half of the full genome.
• a first sub-fragment may include at least one sequence as set forth in SEQ ID NOs: 4-5.
• a second sub-fragment may include at least one sequence as set forth in SEQ ID NOs: 6-7.
• FIG. 3 depicts a schematic diagram of the genomic organization of an exemplary first sub-fragment in linear form.
• FIG. 4 depicts a schematic diagram of the genomic organization of an exemplary second sub-fragment in linear form.
• adenovirus serotype 5 genomic sub-fragments were circularized to form traditional plasmid structures to enable efficient replication in bacterial hosts.
• FIG. 5 shows a schematic diagram of the genomic organization of an exemplary first sub-fragment in circularized form
• FIG. 6 shows a schematic diagram of the genomic organization of an exemplary first second-fragment in circularized form.
• the final adenovirus serotype 5 genome was assembled from the two circularized fragments, creating a linear structure for high level DNA replication in bacteria use in viral production.
• the high level of DNA replication and design allow for greater amounts of DNA to use for transfection and first round viral production.
• the final vector may comprise additive combination of the first sub-fragment and the second sub-fragment.
• Vector sequences to allow for high efficiency bacterial replication can include pJazz-OK from Lucigen Catalog No. 43036.
• pJazz-OK allows for less DNA loss due to replication stress and much more convenient cloning. See , e.g. , training manual available atworldwideweb.lucigen.com/docs/manuals/MA033- BigEasy-v20-Linear-Cloning-Kit.pdf.
• the sequence also may include human ILIO complementary DNA sequences, as a non-limiting example.
• the human IL-10 cDNA sequence may be sequence codon optimized from NM 00572.3 using the codon optimization tool in Geneious version 9.0, as disclosed in SEQ ID NO: 8.
• adenoviral genome sequence listings may be derived from public sources.
• an engineered mammalian cell line for replication of the engineered adenoviral vector to eliminate possible contamination of the replication competent adenovirus is described. Described above, the adenoviral vector was mutated to remove the viral El and E3 genes in order to prevent the virus from replicating and thus being infectious. Mammalian cells supplied with the two El gene products, El A and E1B, can support replication of the mutant virus.
• a common mammalian cell line used to produce adenovirus, HEK293 possesses an endogenous copy of the adenoviral El gene, which can recombine with mutant adenoviral vectors and recreate infectious replicating virus.
• a novel cell line devoid of adenoviral sequences is engineered here to express the El A and E1B gene products using a lentiviral vector which does not allow reconstitution of functional adenovirus when the mutant adenoviral vector is introduced.
• the adenoviral El gene products, El A and E1B were inserted into a plasmid backbone comprising the backbone viral elements and Puromycin resistance from Systems Biosciences Catalog Number CD510B-1 and the bidirectional promoter from ABM Catalog Number LV039.
• the bidirectional promoter was situated such that the El A and E1B were in different orientations and driven by separate parts of the promoter element.
• the lentiviral genome plasmid and accessory plasmids from CellBioLabs Catalog Number VPK-206 were transfected into HEK293T (ATCC Catalog Number CRL-3216) cells to create a lentiviral vector.
• the El A and E1B sequences were arranged in a bidirectional expression unit, to prevent recombination with the mutant adenoviral vector created in Examples 1 and 2, discussed above.
• the bidirectional promoter is a combination of truncated CMV and PGK promoters.
• a puromycin drug resistance gene is included in the lentiviral sequence to allow selection of cells which have integrated the lentiviral sequence.
• the final vector is diagrammed and mapped in FIG. 8, and was used to produce lentivirus, and HeLa cells (ATCC Catalog Number CCL2) transduced and selected for puromycin resistance.
• FIG. 9 is a brief flowchart of the process of this Example. As illustrated, the lentiviral E1A/E1B vector is inserted into the HeLa cells. The modified cells are then tested for vector production. Finally, a user may proceed with vector production and amplification of the cell line.
• SEQ ID NO: 9 SEQ encodes the full El cell creation vector without the plasmid backbone.
• SEQ ID NO: 10 encodes the full El cell creation vector and includes the plasmid backbone.
• FIG. 10 shows a schematic diagram of the full El cell creation vector with the plasmid backbone.
• a method of producing a vector as in Examples 1 and 2 may include transforming a mammalian cell with an adenovirus vector comprising: a) one or more mutations that render the adenovirus replication incompetent and b) at least one nucleotide sequence encoding a transgene, wherein the mammalian cell comprises nucleotide sequences expressing El A and E1B gene products but is otherwise devoid of other adenovirus sequences, culturing the transformed mammalian cell in a cell line containing an inserted lentiviral El A and E1B vector, and isolating the adenovirus vector.
• mutant adenoviral vector and associated production cells allow the production of non-infectious human IL-10 expressing adenovirus without the risk of recombination and restoration of replication competence.
• a potential application of the disclosed vectors, methods, and cells include adenoviral expression of human IL-10 to inhibit immune activity in autoimmune disorders.
• the aspects of the disclosure may also be used to reduce rejection risk in allotransplant and xerotransplant procedures.
• a range includes each individual member.
• a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
• a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

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