Adenoviral expression vector and methods and cell lines for production

Abstract

To provide an adenovirus expression vector.SOLUTION: Provided is an adenovirus expression vector, including: a) one or more mutations that render the adenovirus replication incompetent; and b) at least one nucleotide sequence encoding a protein or an RNA. A method of synthesizing the adenovirus vector is also provided. The synthesis includes: a) producing a plurality of overlapping adenovirus sub-fragments, each sub-fragment comprising a portion of the full genome of the adenovirus; b) circularizing the sub-fragments to form plasmid structures; and c) assembling the circularized sub-fragments into a linear structure, the vector comprising a combination of two or more sub-fragments. Also provided is a mammalian cell line configured to replicate adenoviral vectors, the cell line comprising nucleotide sequences expressing ElA and E1B gene products, but being devoid of other adenovirus sequences.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 006,266, filed April 7, 2020, which is incorporated herein in its entirety for all purposes.

[0003] Fields of use

[0004] This application relates generally to adenoviral vectors and gene therapy, and more specifically to non-replicating adenoviral expression vectors, methods of synthesis, and modes of replication. [Background technology]

[0005] Adenoviral vector sequences are often several kilobases long, making in vitro manipulation inconvenient and difficult to achieve. Improper handling of adenoviral vectors can also lead to unchecked replication of the vector. Furthermore, common mammalian cell lines used to produce adenovirus often harbor endogenous copies of adenoviral genes, which can recombine with mutant adenoviral vectors to create infectious, replicating viruses. Therefore, there is a need for safe, non-infectious, recombinant, non-replicating adenoviral vectors carrying effective expression cassettes for therapeutic use. It would also be desirable to provide protocols for more efficient production of these recombinant vectors in bacteria, overcoming the difficulties of manipulating numerous adenoviral sequences. Finally, it would be further desirable to provide mammalian cell lines for the replication of engineered adenoviral vectors that eliminate the possibility of contaminating replication-competent adenovirus. Summary of the Invention

[0006] The present disclosure provides adenoviral vectors having a) one or more mutations that render them replication-disabled, and b) a nucleotide expression cassette that allows for the expression of a therapeutic agent when transduced into a host cell. The sequences of the engineered adenoviral vectors are disclosed herein. Also provided herein are protocols and methods for more efficient bacterial production of synthetic adenoviral vectors. Finally, the creation of engineered mammalian cell lines for the replication of engineered adenoviral vectors to eliminate the possibility of contamination with replication-competent adenovirus is provided herein.

[0007] A non-replicating adenoviral expression vector may contain a) one or more mutations that render adenoviral replication dysfunctional and b) at least one nucleotide sequence encoding a protein or RNA. In some embodiments, the vector contains a nucleotide sequence having at least 80% sequence identity to any of SEQ ID NOS: 1-2. SEQ ID NOS: 1 encodes a full-length vector having a plasmid backbone. SEQ ID NOS: 2 encodes a full-length adenoviral vector of the present disclosure without a plasmid backbone. In certain preferred embodiments, the vector is an adenovirus serotype 5 vector. The mutations may include deletions of either or both of the E1 and E3 genes. At least one nucleotide sequence in the non-replicating adenoviral expression vector may be a transgene. In certain embodiments, the transgene expresses human IL10. In some embodiments, the transgene further comprises at least one of an enhancer / promoter region comprising CAG, a human IL10 cDNA, or a polyadenylation signal, or any combination thereof. The polyadenylation signal may further comprise an SV40 region and / or a full-length polyA signal. A polyadenylation signal may, in some embodiments, be inserted between nucleotides 440 and 3515 in SEQ ID NO: 3. SEQ ID NO: 3 may be derived from Accession No. AY3339865 in the GenBank database.

[0008] Insertion of a transgene into an adenoviral vector can replace the E1 gene but leave the pIX gene intact. In some embodiments of the present disclosure, the recombinant vector can comprise a nucleic acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than 99% sequence identity to SEQ ID NOs: 1-2.

[0009] Also disclosed herein is a method for synthesizing an adenoviral vector. This method may include: a) producing multiple overlapping adenoviral subfragments, each subfragment comprising a portion of the entire adenoviral genome; b) circularizing the subfragments to form a plasmid structure; and c) assembling the circularized subfragments into a linear structure, wherein the vector comprises a combination of two or more subfragments. In some embodiments of this method, the adenoviral vector may be adenovirus serotype 5. In certain embodiments, the vector may replicate with high efficiency.

[0010] The first subfragment may comprise at least one sequence set forth in SEQ ID NOs: 4-5. In some embodiments, the second subfragment may comprise at least one sequence set forth in SEQ ID NOs: 6-7.

[0011] In some embodiments, each subfragment may comprise approximately 50% of the full-length adenoviral genome. At least one fragment may comprise a transgene expressing human IL-10. The transgene may further comprise at least one of an enhancer / promoter region, a human IL10 cDNA, or a polyadenylation signal, or any combination thereof. The enhancer or promoter region may be a CAG promoter. The transgene may further comprise at least an SV40 promoter or a polyadenylation signal containing a full-length poly(A) signal. The vector sequence may also comprise a linear cloning vector and / or a backbone linear cloning vector. In a specific embodiment, the linear cloning vector may be pJazz-OK. The human IL10 cDNA sequence may be optimized and encoded by SEQ ID NO:8.

[0012] Also provided herein are mammalian cells adapted to replicate adenoviral vectors, wherein the cell line comprises nucleotide sequences expressing E1A and E1B gene products but lacks other adenoviral sequences. The cell line may further comprise a lentiviral vector engineered to express E1A and E1B gene products, which inhibits reconstitution of functional adenovirus when a mutant adenoviral vector is introduced into the cell line. In some embodiments, the cell line may comprise HeLa cells. In some embodiments, the cell line is produced by transfecting HEK293 cells with a lentiviral genome plasmid comprising at least E1A and E1B coding sequences.

[0013] The plasmid may further comprise at least E1A and E1B coding sequences, including E1A and E1B sequences arranged in a bidirectional expression unit with a bidirectional promoter to prevent recombination with the mutant adenoviral vector. In some embodiments of the cell line, the bidirectional promoter may be a combination of a truncated CMV and PGK promoter.

[0014] The cell line may further comprise a puromycin drug resistance gene in the lentiviral genome sequence, and cells that have integrated the lentiviral sequence can be identified when the puromycin drug resistance gene is expressed. In some embodiments, the final vector of SEQ ID NO: 9 is used to produce lentivirus and HeLa cells are transduced and selected for puromycin resistance.

[0015] Also provided herein are methods for producing and / or propagating the synthetic, non-replicating adenoviral vectors disclosed above. The methods may include transforming mammalian cells with an adenoviral vector containing a) one or more mutations that render adenoviral replication dysfunctional, and b) at least one nucleotide sequence encoding human IL-10. The mammalian cells contain nucleotide sequences that express the E1A and E1B gene products but lack other adenoviral sequences, culturing the transformed mammalian cells in a cell line containing the inserted lentiviral E1A and E1B vectors, and isolating the adenoviral vector.

[0016] In a further aspect of the present disclosure, also provided herein are methods for treating a subject in need of a gene product. In some embodiments, the method may consist essentially of, or additionally comprise, administering to the subject a vector comprising (a) one or more mutations that render adenoviral replication dysfunctional, and (b) at least one nucleotide sequence encoding the gene product. In some embodiments of the method, 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 sequence complementary thereto. In certain embodiments, the vector may be an adenovirus serotype 5 vector. Additionally or alternatively, the one or more mutations may be a deletion of either or both of the E1 gene and the E3 gene. The at least one nucleotide sequence may further comprise a transgene. In some embodiments, the transgene expresses human IL10.

[0017] In some embodiments of the method, the subject may be a human. In certain embodiments, the gene product is a peptide, including a human peptide. In some embodiments, the gene product may be RNA. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram of the genomic organization of a full-length non-replicating adenoviral vector of the present disclosure, without a plasmid backbone. [Figure 2] FIG. 2 is a schematic diagram of the genomic organization of a non-replicating adenoviral vector of the present disclosure having a plasmid backbone. [Figure 3] FIG. 3 is a schematic representation of an exemplary first sub-fragment genomic organization in linear form. [Figure 4] FIG. 4 shows a schematic representation of the genomic organization of an exemplary second sub-fragment in linear form. [Figure 5] FIG. 5 shows a schematic representation of the genomic organization of an exemplary first sub-fragment in circular form. [Figure 6] FIG. 6 shows a schematic diagram of the genomic organization of an exemplary first second fragment in circular form. [Figure 7] FIG. 7 shows a schematic representation of the genomic organization of the final vector combining the first and second sub-fragments. [Figure 8] Figure 8 is a schematic representation of the genomic organization of the vectors used to produce lentivirus and HeLa cell lines transduced and selected for puromycin resistance. [Figure 9] FIG. 9 is a flow chart of the disclosed manufacturing process for safe, replication-deficient adenoviral serotype 5 vectors. [Figure 10] FIG. 10 shows a schematic diagram of the full-length E1 cell generation vector with a plasmid backbone. DETAILED DESCRIPTION OF THE INVENTION

[0019] definition

[0020] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, etc.

[0021] As used herein, the term "about" encompasses the range of experimental error that can occur in measurements, and would be apparent to one of ordinary skill in the art.

[0022] As used herein, "adenovirus" refers to a medium-sized (90-100 nm), non-enveloped, polyhedral virus that may contain a capsid and a double-stranded, linear DNA genome. Adenoviruses may be naturally occurring, isolated, or recombinant adenoviruses, or chimeric variants thereof.

[0023] As used herein, "administering" refers to a method of providing a subject with a dosage of 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, transcutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, intramucosally, intrapericardially, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion directly bathing target cells, by catheter, by lavage, by tube feeding, in a cream, or in a lipid composition. The preferred method of administration can vary depending on various factors (e.g., the components of the composition being administered and the severity of the condition being treated).

[0024] Throughout this specification and the claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0025] As used herein, "protein" includes peptides, polypeptides, or proteins of any length and any secondary or tertiary structure.

[0026] As used herein, "gene product" is meant to include mRNAs transcribed from a gene, other nucleic acids (e.g., microRNA), and proteins, including polypeptides, transcribed from those mRNAs. A gene product can be a soluble protein, chemokine, cytokine, soluble receptor, antibody, antibody fragment, antibody-like molecule, or enzyme. For example, a gene product can be an antigen-binding fragment of an antibody, or a gene product can be part or all of the variable portion of an IgG or IgM. In some embodiments, a gene product can be an antibody fragment lacking the Fc domain. A gene product can comprise or consist of a complementarity-determining region (CDR). In some embodiments, a gene product can be an anti-IL6 antibody or antibody fragment. In some embodiments, a gene product is derived from a virus. In some embodiments, the gene product is a therapeutic gene product, including, but not limited to, interleukins (e.g., human IL-10), interferon proteins, Factor VIII, Factor IX, erythropoietin, alpha-1 antitrypsin, calcitonin, glucocerebrosidase, growth hormone, low density lipoprotein (LDL), the receptor IL-2 receptor and its antagonists, insulin, globins, immunoglobulins, catalytic antibodies, insulin-like growth factors, superoxide dismutase, immune response modifiers, parathyroid hormone and interferons, nerve growth factor, tissue plasminogen activator, and colony stimulating factors.

[0027] By "portion" or "fragment" is meant a portion of a whole. A portion may include at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the entire length of a polynucleotide or polypeptide sequence region. In the case of polynucleotides, for example, a portion can 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. In the case of polypeptides, for example, a portion can 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 more consecutive amino acids of the reference polypeptide molecule.

[0028] As used herein, "sequence identity" or "sequence similarity" refers to the identity or similarity between two or more amino acid sequences or two or more base sequences, expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of "percentage (%) identity," with higher percentages indicating greater shared identity between the sequences. Sequence similarity can be measured in terms of percentage similarity (taking into account conservative amino acid substitutions). Higher percentages indicate greater shared similarity between the sequences. Homologs or orthologs of nucleic acid or amino acid sequences have relatively high levels of sequence identity / similarity when aligned using standard methods. Sequence identity can be measured using sequence analysis software with default settings. Such software can match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

[0029] A "subject" is a vertebrate such as a mammal (e.g., a primate or human). Mammals also include, but are not limited to, farm animals (e.g., cattle), sport animals (e.g., horses), pets (such as cats and dogs), mice, and rats. A subject treated by the methods described herein (e.g., a subject having a disease such as cancer and / or a disease caused by an infectious agent, e.g., a bacterium, virus, fungus, or parasite) may have been diagnosed by a physician as having such a condition. Diagnosis may be made by any appropriate means. A subject in whom the onset of an infection or disease (or disease symptoms) is being prevented may or may not have received such a diagnosis. One of skill in the art will understand that a subject treated according to the present invention may have been subjected to standard testing, or may have been identified 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) without testing.

[0030] As used herein, the term "vector" refers to a composition comprising one or more genes (nonstructural or structural) or fragments thereof from a viral species, such as an adenovirus species (e.g., sAd5, sAd1, etc.), and can be used to transfer one or more heterologous genes from a viral or nonviral source to a host or subject. The nucleic acid material of a viral vector may be encapsulated, for example, in a lipid membrane or by structural proteins (e.g., capsid proteins), and may contain one or more viral polypeptides (e.g., glycoproteins). The viral vector can be used to infect the cells of a subject, thereby facilitating translation of the heterologous gene(s) of the viral vector into a protein product.

[0031] The term "virus" as used herein is defined as an infectious agent that is unable to grow or reproduce outside a host cell and that infects mammals (eg, humans) or birds.

[0032] Other features and advantages of the invention will be apparent from the following detailed description, drawings, and claims.

[0033] Adenovirus species polypeptides of the present disclosure

[0034] The complete genome sequence of the novel adenovirus vector is disclosed below. The Adenovirus 5 genome is over 35 kilobases, making it less convenient for in vitro manipulation. Therefore, synthetic DNA techniques were used to generate two or more smaller fragments, allowing for more efficient molecular cloning using bacterial hosts. The final, non-replicating adenovirus expression vector may contain a) one or more mutations that render adenovirus replication dysfunctional, and b) at least one nucleotide sequence encoding a protein or RNA.

[0035] Figure 1 is a schematic diagram of the genomic organization of a non-replicating adenoviral vector of the present disclosure without a plasmid backbone. Figure 2 is a schematic diagram of the genomic organization of a non-replicating adenoviral vector of the present disclosure with a plasmid backbone. In some embodiments of the present disclosure, the vector comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NOs: 1-2. In some embodiments of the present disclosure, the recombinant vector may comprise a nucleic acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 95%, about 96%, about 96%, about 97%, about 98%, about 99%, greater than 99%, or any value therebetween, sequence identity to SEQ ID NOs: 1-2.

[0036] In certain preferred embodiments, the vector is an adenovirus serotype 5 vector. The mutations may include deletion of either or both of the E1 and E3 genes to prevent adenovirus replication in vivo. At least one nucleotide sequence in the non-replicating adenovirus expression vector may be a transgene. In certain embodiments, the transgene expresses human IL10. In some embodiments, the transgene further comprises at least one of an enhancer / promoter region comprising CAG, a human IL10 cDNA, or a polyadenylation signal, or any combination thereof. The polyadenylation signal may further comprise an SV40 region and / or a full-length polyA signal. In some embodiments, the polyadenylation signal may be inserted between nucleotides 440 and 3515 in SEQ ID NO:3. SEQ ID NO:3 may be derived from the GenBank database under accession number AY3339865. In a particularly preferred embodiment, a transgene for expressing human IL10 is contained in the adenoviral sequence, which further comprises an enhancer / promoter region (CAG), human IL10 cDNA, and a polyadenylation signal (SV40, full-length polyA signal), which is inserted between nucleotides 440 and 3515 relative to the adenoviral sequence (GenBank accession number AY339865), replacing the E1 gene but leaving the pIX gene intact.

[0037] The transgene coding sequence inserted into the adenoviral vector can include a variety of gene products and can include RNA or DNA transcription 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), IL-2 receptor and its antagonists, insulin, globins, immunoglobulins, catalytic antibodies, interleukins, insulin-like growth factors, superoxide dismutase, immune response modifiers, parathyroid hormone and interferons, nerve growth factor, tissue plasminogen activator, and / or colony stimulating factors, or fragments thereof.

[0038] The engineered adenoviral vector can include any nucleotide (DNA or RNA) sequence that is configured to be included with an expression cassette. Exemplary transgenes that can be inserted into the adenoviral construct include, but are not limited to, chimeric monoclonal antibodies such as angiotensin-converting enzyme 2 precursor (ACE2) antibody (SEQ ID NO: 14 [without plasmid backbone], SEQ ID NO: 15 [with plasmid backbone]), unitixin scFv (SEQ ID NO: 16 [without plasmid backbone], SEQ ID NO: 17 [with plasmid backbone]), transforming growth factor (TGF) beta-less IL-10 (SEQ ID NO: 18 [with plasmid backbone], SEQ ID NO: 19 [without plasmid backbone]), interleukin-13 (SEQ ID NO: 20 [without plasmid backbone], SEQ ID NO: 21 [with plasmid backbone]), interleukin-8 with short hairpin RNA (shRNA) (SEQ ID NO: 22 [with plasmid backbone], SEQ ID NO: 23 [with plasmid backbone]), and the like. 23 [no plasmid backbone]), IL-1 receptor agonist 2 and IL-10 (SEQ ID NO:24 [with plasmid backbone], SEQ ID NO:25 [no plasmid backbone]), IL-1 receptor agonist (SEQ ID NO:26 [with plasmid backbone], SEQ ID NO:27 [no plasmid backbone]), endothelial nitric oxide synthase (eNOS) (SEQ ID NO:28 [with plasmid backbone], SEQ ID NO:29 [no plasmid backbone]), cystic fibrosis transmembrane conductance regulator (CFTR) (SEQ ID NO:30 [with plasmid backbone], SEQ ID NO:31 [no plasmid backbone]), beta-1,4-galactosyltransferase 1 (B4GALT1) enzyme (SEQ ID NO:32 [with plasmid backbone], SEQ ID NO:33 [no plasmid backbone]).

[0039] A protocol for more efficient adenovirus production in bacteria

[0040] Also disclosed herein is a method for synthesizing an adenoviral vector. The method may include: a) producing multiple overlapping adenoviral subfragments, each subfragment comprising a portion of the entire adenoviral genome; b) circularizing the subfragments to form a plasmid structure; and c) assembling the circularized subfragments into a linear structure, wherein the vector comprises a combination of two or more subfragments. In some embodiments of the method, the adenoviral vector may be adenovirus serotype 5. In certain embodiments, the vector may replicate with high efficiency.

[0041] The first sub-fragment may comprise at least one sequence set forth in SEQ ID NOs:4-5. In some embodiments, the second sub-fragment may comprise at least one sequence set forth in SEQ ID NOs:6-7. Figure 3 shows a schematic diagram of the genomic organization of an exemplary first sub-fragment in linear form. Figure 4 shows a schematic diagram of the genomic organization of an exemplary second sub-fragment in linear form. Figure 5 shows a schematic diagram of the genomic organization of an exemplary first sub-fragment in circular form, and Figure 6 shows a schematic diagram of the genomic organization of an exemplary first second sub-fragment in circular form.

[0042] Figure 7 shows a schematic diagram of the genome organization of the final vector, combining the first and second subfragments. Here, we demonstrate that the final adenovirus serotype 5 genome can be assembled from multiple circular fragments, creating a linear structure for high-level DNA replication in bacteria used for virus production. High-level DNA replication and engineering allows for increased amounts of DNA to be used for gene transfer and first-round virus production. This is particularly useful when engineering larger adenovirus vectors, such as serotype 5 adenovirus vectors exceeding 35 kilobases in size.

[0043] In some embodiments, each subfragment may comprise approximately 50% of the entire adenoviral genome. At least one fragment may comprise a transgene expressing human IL-10. The transgene may further comprise at least one of an enhancer / promoter region, a human IL10 cDNA, a polyadenylation signal, or any combination thereof. The enhancer or promoter region may be a CAG promoter. The transgene may further comprise at least a polyadenylation signal, including an SV40 promoter or a full-length poly(A) signal. The vector sequence may also comprise a linear cloning vector and / or a backbone linear cloning vector. In certain embodiments, the linear cloning vector may be pJazz-OK (see, e.g., BigEasy® v2.0: Linear Cloning Kits, LUCIGEN CORPORATION, January 1, 2018, incorporated herein by reference). The teachings of the human IL10 cDNA sequence may be optimized and encoded from SEQ ID NO:8.

[0044] Engineered mammalian cell lines for the replication of engineered adenoviral vectors

[0045] Also provided herein are mammalian cell lines that have been adapted to replicate adenoviral vectors, said cell lines comprising nucleotide sequences that express the E1A and E1B gene products but lacking other adenoviral sequences.

[0046] The adenoviral vectors disclosed above may be mutated to remove the viral E1 and E3 genes to prevent the virus from replicating and thus becoming infectious. Mammalian cell lines supplied with the two E1 gene products, E1A and E1B, can support the replication of the mutant virus. However, HEK293, a common mammalian cell line used to produce adenovirus, contains an endogenous copy of the adenoviral E1 gene and can recombine with a mutant adenoviral vector to produce infectious, replicating virus. Therefore, novel cell lines lacking adenoviral sequences are provided herein to express the E1A and E1B gene products using a vector that is unable to reconstitute functional adenovirus when the mutant viral vector is introduced.

[0047] The cell line may comprise a lentiviral vector engineered to express E1A and E1B gene products, wherein the lentiviral vector inhibits reconstitution of functional adenovirus when a mutant adenovirus vector is introduced into the cell line. In some embodiments, the cell line may comprise HeLa cells. In some embodiments, the cell line is produced by transfecting HEK293 cells with a lentiviral genome plasmid containing at least E1A and E1B coding sequences.

[0048] The plasmid may further comprise at least E1A and E1B coding sequences, including E1A and E1B sequences arranged in a bidirectional expression unit with a bidirectional promoter to prevent recombination with the mutant adenoviral vector. In some embodiments of the cell line, the bidirectional promoter may be a combination of a truncated CMV and PGK promoter.

[0049] The cell line further comprises a puromycin drug resistance gene in the lentiviral genomic sequence, allowing cells that have integrated the lentiviral sequence to be identified when the puromycin drug resistance gene is expressed. In some embodiments, the final E1 cell production vector of SEQ ID NO: 9 is used to produce lentivirus and HeLa cells, transduced and selected for puromycin resistance. Figure 8 is a schematic diagram of the genomic organization of the vector used to produce lentivirus and HeLa cell lines transduced and selected for puromycin resistance.

[0050] The polynucleotide sequence of SEQ ID NO: 9 does not include the plasmid backbone. SEQ ID NO: 10, however, encodes the full-length E1 cell generation vector with the plasmid backbone, as shown in the schematic diagram of the plasmid genome in Figure 10.

[0051] Also provided herein are methods for producing and / or propagating the synthetic non-replicating adenoviral vectors disclosed above, which may include transforming mammalian cells with an adenoviral vector comprising a) one or more mutations that render adenoviral replication dysfunctional, and b) at least one nucleotide sequence encoding human IL-10, wherein the mammalian cells comprise nucleotide sequences that express E1A and E1B gene products but lack other adenoviral sequences, culturing the transformed mammalian cells in a cell line containing the inserted lentiviral E1A and E1B vectors, and isolating the adenoviral vector.

[0052] In some embodiments, the engineered adenovirus serotype 5 vectors of the present disclosure are introduced into engineered cells to generate mutant adenoviruses expressing human IL-10. Figure 9 is a flow chart illustrating that in this production mode, the lentiviral E1A / E1B vector is first introduced into HeLa cells. The modified HeLa cell line may be tested for vector production to ensure the cell line is free of contamination. Assuming there is no infectious contamination or unexpected replication, small-scale vector production and amplification can proceed.

[0053] Together, the mutant adenoviral vectors and associated producer cell lines of the present disclosure enable the expansion of non-infectious therapeutic agents expressing adenovirus without the risk of recombination and replication recovery.

[0054] Potential uses may include, but are not limited to, adenoviral expression of human IL-10, which in turn may inhibit immune activity in autoimmune disorders as well as reduce the risk of rejection in allo- or xenotransplants.

[0055] In a further aspect of the present disclosure, methods of treating a subject in need of a gene product are also provided herein. In some embodiments, the methods may consist essentially of, or additionally comprise, administering to the subject a vector comprising (a) one or more mutations that render adenoviral replication dysfunctional, and (b) at least one nucleotide sequence encoding the gene product. In some embodiments of the methods, 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 sequence complementary thereto. In certain embodiments, the vector may be an adenovirus serotype 5 vector. Additionally or alternatively, the one or more mutations may be a deletion of either or both of the E1 gene and the E3 gene. The at least one nucleotide sequence may further comprise a transgene. In some embodiments, the transgene expresses human IL10.

[0056] In some embodiments of the method, the subject may be a human. In certain embodiments, the gene product is a peptide, including a human peptide. In some embodiments, the gene product may be RNA. [Example]

[0057] The following examples are intended to illustrate the present invention and are not intended to limit the invention in any way.

[0058] The practice of the present invention may employ, unless otherwise indicated, conventional techniques of molecular biology, cell biology, and recombinant DNA, which are within the skill of those in the art (see, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manuel, 4th edition, 2012; Ausubel, et al. Current Protocols in Molecular Biology, 1987).

[0059] Example 1: Sequences of engineered adenovirus serotype 5 vectors

[0060] The synthesis and description of the engineered adenovirus serotype 5 vector are described in Example 1. The adenovirus sequence used for synthesis was derived from public domain sources, specifically GenBank, sequence AY339865, available at worldwideweb.ncbi.nlm.nih.gov / genbank / . Synthetic DNA methods were used to generate two smaller fragments, allowing for more efficient cloning using bacterial hosts. The synthetic fragment was designed to contain deletions of the E1 and E3 genes to prevent adenovirus replication in vivo. The transgene for human IL-10 expression was included in the adenovirus sequence and contained an enhancer / promoter region (CAG), human IL10 cDNA, and a polyadenylation signal (SV40, full polyA signal), which was inserted between nucleotides 440 and 3515 relative to the adenovirus sequence AY339865, replacing the E1 gene but leaving the pIX gene intact.

[0061] Example 2: A more efficient AdVector production protocol in bacteria

[0062] This example describes the construction of a plasmid-based system for generating recombinant sAd5 vectors and a protocol for their mass production in bacteria. In one embodiment of this disclosure, in the first step, the adenovirus 5 genome was synthesized as several overlapping DNA fragments. The overlapping DNA fragments were assembled into two linear subfragments, each containing approximately half of the entire genome.

[0063] The first subfragment can comprise at least one sequence set forth in SEQ ID NOs: 4-5. In some embodiments, the second subfragment can comprise at least one sequence set forth in SEQ ID NOs: 6-7. Figure 3 shows a schematic representation of the genomic organization of an exemplary first subfragment in linear form. Figure 4 shows a schematic representation of the genomic organization of an exemplary second subfragment in linear form.

[0064] In the second step of the protocol, the adenovirus serotype 5 genome subfragments were circularized to form conventional plasmid structures, allowing efficient replication in the bacterial host.

[0065] FIG. 5 shows a schematic representation of the genomic organization of an exemplary first sub-fragment in circular form, and FIG. 6 shows a schematic representation of the genomic organization of an exemplary first second fragment in circular form.

[0066] In the third step of the protocol, the final adenovirus serotype 5 genome was assembled from the two circular fragments, creating a linear structure for high-level DNA replication in bacteria for virus production. High-level DNA replication and design allow for increased amounts of DNA to be used for gene transfer and first-round virus production. The final vector may contain an additive combination of the first and second subfragments.

[0067] Engineering a DNA sequence encoding an adenovirus 5 vector for efficient replication in bacteria allows for better initial vector construction. A vector sequence that allows for efficient bacterial replication can include pJazz-OK from Lucigen catalog number 43036. pJazz-OK reduces DNA loss due to replication stress, making cloning more convenient. See, for example, the training manual available at worldwideweb.lucigen.com / docs / manuals / MA033-BigEasy-v20-Linear-Cloning-Kit.pdf. Sequences can also include, as a non-limiting example, a human IL-10 complementary DNA sequence. The human IL-10 cDNA sequence can be codon-optimized from NM_00572.3 using the codon optimization tool in Geneious version 9.0, as disclosed in SEQ ID NO:8.

[0068] Other adenovirus genome sequence listings can be derived from public sources.

[0069] Example 3: Creation of engineered mammalian cell lines for replication of engineered adenoviral vectors

[0070] This example describes the creation of an engineered mammalian cell line for the replication of adenoviral vectors engineered to eliminate potential contamination with replication-competent adenovirus. As described above, the adenoviral vector was mutated to remove the viral E1 and E3 genes to prevent the virus from replicating and thus becoming infectious. Mammalian cells supplied with two E1 gene products, E1A and E1B, can support the replication of the mutant virus. HEK293, a common mammalian cell line used to produce adenovirus, contains an endogenous copy of the adenoviral E1 gene and can recombine with a mutant adenoviral vector to recreate infectious, replicating virus. Therefore, a novel cell line lacking adenoviral sequences is now engineered to express the E1A and E1B gene products using a lentiviral vector that does not allow for the reconstitution of functional adenovirus when a mutant adenoviral vector is introduced.

[0071] The adenovirus E1 gene products E1A and E1B were inserted into a plasmid backbone containing backbone viral components and puromycin resistance from Systems Biosciences catalog number CD510B-1, and a bidirectional promoter from ABM catalog number LV039. The bidirectional promoter was positioned so that E1A and E1B were in different orientations and driven by separate portions of the promoter component.

[0072] The lentiviral genome plasmid and accessory plasmids from CellBioLabs catalog number VPK-206 were transfected into HEK293T (ATCC catalog number CRL-3216) cells to generate the lentiviral vector. The E1A and E1B sequences were placed in a bidirectional expression unit to prevent recombination with the mutant adenoviral vectors generated in Examples 1 and 2 discussed above. The bidirectional promoter is a combination of the truncated CMV and PGK promoters. A puromycin drug resistance gene was included in the lentiviral sequence to allow for selection of cells that had integrated the lentiviral sequences. The final vector is depicted and mapped in Figure 8 and was used to produce lentivirus and transduce HeLa cells (ATCC catalog number CCL2) for selection for puromycin resistance.

[0073] The vectors described in Examples 1 and 2 were introduced into the cells generated in Example 3 to generate mutant adenoviruses capable of expressing human IL-10. Figure 9 is a simplified flowchart of the process in this example. As shown, the lentiviral E1A / E1B vector is inserted into HeLa cells. The modified cells are then tested for vector production. Finally, the user may proceed with vector production and amplification in cell lines. The sequence of SEQ ID NO: 9 encodes the full-length E1 cell generation vector, without the plasmid backbone. SEQ ID NO: 10 encodes the full-length E1 cell generation vector, including the plasmid backbone. Figure 10 shows a schematic diagram of the full-length E1 cell generation vector with the plasmid backbone.

[0074] In summary, disclosed herein are methods for producing vectors such as those of Examples 1 and 2. The methods may include transforming mammalian cells with an adenoviral vector comprising a) one or more mutations that render adenoviral replication dysfunctional, and b) at least one nucleotide sequence encoding a transgene, the mammalian cells comprising nucleotide sequences that express E1A and E1B gene products but lacking other adenoviral sequences, culturing the transformed mammalian cells in a cell line containing the inserted lentiviral E1A and E1B vectors, and isolating the adenoviral vector.

[0075] One advantage of the above-described methods, vectors, and cell lines is that the mutant adenoviral vectors and associated producer cells enable the production of non-infectious human IL-10 expressing adenovirus without the risk of recombination and replication recovery. Potential uses of the disclosed vectors, methods, and cells include adenoviral expression of human IL-10 to inhibit immune activity in autoimmune disorders. Embodiments of the present disclosure may also be used to reduce the risk of rejection in allotransplantation and xenotransplantation procedures.

[0076] Doctrine of Equivalents

[0077] The present technology is not limited by the specific embodiments described in this application, which are intended as single illustrations of individual aspects of the technology. Many modifications and variations of the present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those recited herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. The present technology is limited only by the terms of the appended claims, and the full scope of equivalents to which such claims are entitled. It is to be understood that the present technology is not limited to particular methods, reagents, compounds, compositions, or biological systems, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0078] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual members or subgroups of members in the Markush group.

[0079] As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein encompass any and all possible subranges and combinations thereof. Any listed range can be readily recognized as fully descriptive and allowing for the same range to be equally divided into at least one half, one third, one quarter, one fifth, one tenth, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. As will also be understood by those skilled in the art, all language such as "up to," "at least," "greater than," "less than," etc., refers to a range that is inclusive of the recited number and can then be divided into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells.

[0080] All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent not inconsistent with the explicit teachings of this specification.

[0081] Other embodiments are within the scope of the following claims.

[0082] Sequence Listing

[0083] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10

[0084] Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6 Table 2-7 Table 2-8 Table 2-9 Table 2-10 Table 2-11 Table 2-12 Table 2-13

[0085] Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10

[0086] Table 4

[0087] Table 5-1 Table 5-2

[0088] Table 6

[0089] Table 7-1 Table 7-2 Table 7-3 Table 7-4 Table 7-5 Table 7-6 Table 7-7

[0090] Table 8

[0091] Table 9-1 Table 9-2 Table 9-3

[0092] Table 10-1 Table 10-2 Table 10-3

[0093] Table 11-1 Table 11-2

[0094] Table 12-1 Table 12-2 Table 12-3 Table 12-4 Table 12-5 Table 12-6 Table 12-7 Table 12-8

[0095] Table 13

[0096] Table 14-1 Table 14-2 Table 14-3 Table 14-4 Table 14-5 Table 14-6 Table 14-7 Table 14-8 Table 14-9 Table 14-10

[0097] Table 15-1 Table 15-2 Table 15-3 Table 15-4 Table 15-5 Table 15-6 Table 15-7 Table 15-8 Table 15-9 Table 15-10 Table 15-11 Table 15-12 Table 15-13

[0098] Table 16-1 Table 16-2 Table 16-3 Table 16-4 Table 16-5 Table 16-6 Table 16-7 Table 16-8 Table 16-9 Table 16-10 Table 16-11 Table 16-12 Table 16-13

[0099] Table 17-1 Table 17-2 Table 17-3 Table 17-4 Table 17-5 Table 17-6 Table 17-7 Table 17-8 Table 17-9 Table 17-10

[0100] Table 18-1 Table 18-2 Table 18-3 Table 18-4 Table 18-5 Table 18-6 Table 18-7 Table 18-8 Table 18-9 Table 18-10 Table 18-11 Table 18-12 Table 18-13

[0101] Table 19-1 Table 19-2 Table 19-3 Table 19-4 Table 19-5 Table 19-6 Table 19-7 Table 19-8 Table 19-9 Table 19-10

[0102] Table 20-1 Table 20-2 Table 20-3 Table 20-4 Table 20-5 Table 20-6 Table 20-7 Table 20-8 Table 20-9 Table 20-10 Table 20-11 Table 20-12 Table 20-13

[0103] Table 21-1 Table 21-2 Table 21-3 Table 21-4 Table 21-5 Table 21-6 Table 21-7 Table 21-8 Table 21-9

[0104] Table 22-1 Table 22-2 Table 22-3 Table 22-4 Table 22-5 Table 22-6 Table 22-7 Table 22-8 Table 22-9 Table 22-10 Table 22-11 Table 22-12

[0105] Table 23-1 Table 23-2 Table 23-3 Table 23-4 Table 23-5 Table 23-6 Table 23-7 Table 23-8 Table 23-9

[0106] Table 24-1 Table 24-2 Table 24-3 Table 24-4 Table 24-5 Table 24-6 Table 24-7 Table 24-8 Table 24-9 Table 24-10 Table 24-11 Table 24-12 Table 24-13

[0107] Table 25-1 Table 25-2 Table 25-3 Table 25-4 Table 25-5 Table 25-6 Table 25-7 Table 25-8 Table 25-9 Table 25-10

[0108] Table 26-1 Table 26-2 Table 26-3 Table 26-4 Table 26-5 Table 26-6 Table 26-7 Table 26-8 Table 26-9 Table 26-10 Table 26-11 Table 26-12 Table 26-13

[0109] Table 27-1 Table 27-2 Table 27-3 Table 27-4 Table 27-5 Table 27-6 Table 27-7 Table 27-8 Table 27-9 Table 27-10

[0110] Table 28-1 Table 28-2 Table 28-3 Table 28-4 Table 28-5 Table 28-6 Table 28-7 Table 28-8 Table 28-9 Table 28-10 Table 28-11 Table 28-12 Table 28-13 Table 28-14

[0111] Table 29-1 Table 29-2 Table 29-3 Table 29-4 Table 29-5 Table 29-6 Table 29-7 Table 29-8 Table 29-9 Table 29-10

[0112] Table 30-1 Table 30-2 Table 30-3 Table 30-4 Table 30-5 Table 30-6 Table 30-7 Table 30-8 Table 30-9 Table 30-10 Table 30-11 Table 30-12 Table 30-13 Table 30-14

[0113] Table 31-1 Table 31-2 Table 31-3 Table 31-4 Table 31-5 Table 31-6 Table 31-7 Table 31-8 Table 31-9 Table 31-10 Table 31-11

[0114] Table 32-1 Table 32-2 Table 32-3 Table 32-4 Table 32-5 Table 32-6 Table 32-7 Table 32-8 Table 32-9 Table 32-10 Table 32-11 Table 32-12 Table 32-13 Table 32-14

[0115] Table 33-1 Table 33-2 Table 33-3 Table 33-4 Table 33-5 Table 33-6 Table 33-7 Table 33-8 Table 33-9 Table 33-10

Claims

1. a) one or more mutations that render adenoviral replication dysfunctional, and b) at least one nucleotide sequence encoding a gene product:

1. A non-replicating adenoviral expression vector comprising: The vector comprising a nucleotide sequence having at least 95% sequence identity to the entire sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or the entire sequence complementary thereto.

2. The vector of claim 1 , wherein the vector comprises a nucleic acid sequence having at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 or 2.

3. The vector of claim 1 , wherein the vector comprises an adenovirus serotype 5 vector.

4. 2. The vector of claim 1, wherein the one or more mutations comprise a deletion of either or both of the E1 gene or the E3 gene.

5. The vector of claim 1 , wherein at least one nucleotide sequence encoding the gene product further comprises a polyadenylation signal.

6. The vector of claim 5 , wherein the polyadenylation signal comprises an SV40 region, a full-length polyA signal, or both.

7. 6. The vector of claim 5, wherein the insertion of at least one nucleotide sequence encoding a gene product replaces the E1 gene but leaves the pIX gene intact.

8. 1. A non-replicating adenoviral expression vector for use in treating a subject in need of a gene product, comprising: a) one or more mutations that render adenoviral replication dysfunctional, and b) at least one nucleotide sequence encoding a gene product: Including, The vector comprising a nucleotide sequence having at least 95% sequence identity to the entire sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or the entire sequence complementary thereto.

9. 9. The vector of claim 8, wherein the vector comprises a nucleotide sequence having at least 97% sequence identity to the entire sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence complementary thereto.

10. The vector of claim 8 , wherein the vector comprises an adenovirus serotype 5 vector.

11. 9. The vector of claim 8, wherein the one or more mutations comprise a deletion of either or both of the E1 gene or the E3 gene.