Patent Application
20250019667
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 5, 2022, is named 2013906-0023_SL.txt and is 722,042 bytes in size.
Adeno-associated virus (AAV) technology has quickly become a dominant form of gene therapy for genetic diseases. AAVs can be produced in large scale in a variety of host cell systems, including mammalian cells, such as HEK293 cells. Traditionally, AAV production in mammalian cells involves the introduction of multiple plasmids to the host cells, the plasmids encoding, for example, a human gene or genes of interest, and various viral genes critical for viral replication and packaging. Due to the number of genes required for proper replication, these are traditionally delivered on two or three separate plasmids.
One such plasmid, termed an “adenoviral helper” plasmid, contains genes critical for AAV production from a host cell. Adenoviral helper plasmids containing E2a, VA RNA, and E4 genes have been shown to be critical to promoting AAV production in mammalian host cell systems.
Despite much advancement over the last two decades, concerns regarding the cost and safety of AAV production continue to limit the therapeutic potential of AAV technology. These concerns are due in part to the large size of many helper plasmids, which is due to the provision of a large number of genes on a single helper plasmid to support AAV production. The safety concerns are due in part to the production, albeit at low levels, of potentially cytotoxic and/or inflammatory viral proteins that are not necessary for AAV replication.
In some embodiments, the present disclosure, provides, among other things, an adenoviral helper plasmid. In some embodiments, the present disclosure provides an adenoviral helper plasmid with reduced size relative to those known in the art. In some embodiments, the present disclosure provides an adenoviral helper plasmid comprising nucleotide sequences encoding E2a, VA RNA, E4; and an L4 region. In some embodiments, an adenoviral helper plasmid as described herein comprises nucleotide sequences encoding proteins from other viruses. In some embodiments, an adenoviral helper plasmid as described herein comprises nucleotide sequences encoding proteins from other viruses, including HSV-1 UL30, HSV-1 UL42, and/or HSV-1 UL29.
In some embodiments, the present disclosure provides an adenoviral helper plasmid that does not comprise one or more nucleotide sequences encoding one or more of fiber protein; L1-52/55K (Packaging Protein 3), peripentonal Hexon-Associated protein, and an L4 region. In some embodiments, the present disclosure provides an adenoviral helper plasmid comprising a fragment, portion, or partial form of E2a protein, VA RNA, E4, L1-52/55K (Packaging Protein 3), peripentonal Hexon-Associated protein, and an L4 region. In some embodiments, the present disclosure provides an adenoviral helper plasmid that does not comprise one or more nucleotide sequences encoding one or more of Hexon Associated Precursor (L4 pVIII) protein, DNA Terminal Protein, and 23 kDa endoprotease. In some embodiments, the present disclosure provides an adenoviral helper plasmid that does not comprise one or more nucleotide sequences encoding one or more of E4orf1 and E4orf2. In some embodiments, an adenoviral helper plasmid provides herein comprises a kanamycin resistance gene.
In some embodiments, the present disclosure provides an adenoviral helper plasmid in which expression of E2a protein is under the control of one or more of an E2a promoter, chicken β-actin promoter, and SV40 promoter. In some embodiments, the present disclosure provides an adenoviral helper plasmid in which expression of E4 open reading frame (orf) is under the control of one or more of a chicken β-actin promoter and SV40 promoter.
In some embodiments, the present disclosure provides an adenoviral helper plasmid comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 1-3, 5, 7, 9, 11-12, 14-20, 22, 24, 26-29, 31, 33, 35-37, 39-70, 72, 74, 76, 78, or 80. In some embodiments, the present disclosure provides an adenoviral helper plasmid comprising a nucleotide sequence that encodes for an amino acid sequence that is at least 80% identical to SEQ ID NO: 4, 6, 8, 10, 13, 21, 23, 25, 30, 32, 34, 38, 71, 73, 75, 77, 79, or 81. In some embodiments, the present disclosure provides an adenoviral helper plasmid comprising a nucleotide sequence that is at least 80% identical to any one of SEQ ID NO: 41-66.
Agent: In general, the term “agent”, as used herein, is used to refer to an entity (e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc, or complex, combination, mixture or system [e.g., cell, tissue, organism] thereof), or phenomenon (e.g., heat, electric current or field, magnetic force or field, etc). In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and/or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. In some cases, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and/or is substantially free of any polymer and/or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.
Approximately/about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.
Corresponding to: As used herein, the term “corresponding to” may be used to designate the position/identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid “corresponding to” a residue at position 190, for example, need not actually be the 190th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify “corresponding” amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and/or nucleic acids in accordance with the present disclosure.
Downstream: As used herein, the term “downstream” refers to the location or position of a nucleic acid sequence relative to a reference nucleic acid sequence, particularly a position that, during RNA transcription, is closer to the 3′ end of the transcribed RNA molecule encoded by the reference sequence. For example, for two sequences, A and B, such that sequence A is downstream of sequence B, transcription of sequence B proceeds toward sequence A.
Nucleic acid: As used herein, in its broadest sense, the term “nucleic acid” refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, “nucleic acid” refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a “nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more “peptide nucleic acids”, which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and/or 5′-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.
Operably linked: As used herein, the term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control element “operably linked” to a functional element is associated in such a way that expression and/or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, “operably linked” control elements are contiguous (e.g., covalently linked) with the coding elements of interest; in some embodiments, control elements act in trans to or otherwise at a distance from the functional element of interest.
Producer cell: As used herein, the term “producer cell” refers to any cell used to produce recombinant AAV (rAAV). In some embodiments, a producer cell is a mammalian cell. In some embodiments, a producer cell is a transformed mammalian cell. In some embodiments, a producer cell is a Vero, HeLa, HEK293, HEK293T cell or derivative thereof.
Transformation: As used herein, the term “transformation” refers to any process by which exogenous DNA is introduced into a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. In some embodiments, a particular transformation methodology is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, mating, lipofection. In some embodiments, a “transformed” cell is stably transformed in that the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. In some embodiments, a transformed cell transiently expresses introduced nucleic acid for limited periods of time.
Upstream: As used herein, the term “upstream” refers to the location or position of a nucleic acid sequence relative to a reference nucleic acid sequence, particularly a position that, during RNA transcription, is closer to the 5′ end of the transcribed RNA molecule encoded by the reference sequence. For example, for two sequences, A and B, such that sequence A is upstream of sequence B, transcription of sequence B proceeds away from sequence A.
Vector: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
The helper functions adenoviruses provide for AAV replication have been previously described. Without wishing to be bound by any particular hypothesis, adenoviral E1A protein has been described to activate AAV gene expression by binding and activating the AAV P5 rep promoter. Similarly, E2A, another adenoviral protein, has been described to activate AAV P5 promoter transcription. E2A has also been described to cooperate with virus associated RNA I (VA RNAI) to enhance the translation of AAV RNAs. Adenoviral E4orf4 has been shown to induce cell-cycle arrest at the G2/M border, as well as to aid in AAV production. Adenoviral E4orf6 has been described to enhance the conversion of single-stranded recombinant AAV genomes into double-stranded genomes, a rate-limiting step of viral DNA-replication both in vitro and in vivo. VA RNAI has also been described to support AAV replication. It has been described that VA RNAI physically interacts with the double-stranded RNA-activated protein kinase (PKR), which would otherwise elicit an antiviral immune response blocking viral protein production.
Prior studies have suggested that in HEK293 cells, which provide the E1 gene, the minimal set of genes in trans for efficient recombinant AAV production is E2a, E4orf6 and the VA RNAI gene. A helper plasmid named pXX6, containing this set of genes, is used for the production of adenovirus-free recombinant AAV.
One major ongoing challenge in the development and optimization of AAV vectors for clinical applications is to increase the amount of viruses being produced. Due to their non-proliferative nature, their production depends solely on the transfection efficiency of the parvoviral genomic components into the packaging cell lines (for example human embryonic kidney cells, HEK293 or HEK293T, or insect cells e.g., Sf9). Thus, it remains of high importance to develop means to increase recombinant AAV (rAAV) production.
Other major challenges related to the production of rAAVs for clinical applications are those related to the cost to produce such rAAVs in large quantities, and also to the safety of the final products themselves. For example, commercially available helper plasmids, such as pXX6-80, appear to transcribe low levels of the Ad fiber protein. Importantly, the fiber protein is not required for AAV production, and could be immunogenic in humans. In addition, the size of pXX6-80 is rather large, at over 18 kb. This large plasmid size increases the difficulty and cost of its manufacturing, which can be highly impactful when sourcing GMP plasmids for the manufacturing of clinical-grade AAV.
Different versions of adenoviral helper plasmids have been derived by others, including, for example, pFAdDeltaF6 (derived at the University of Pennsylvania) and pHelper (Agilent). The pFAdDeltaF6 plasmid is about 3 kb smaller than pXX6-80, but retains the fiber gene sequence. The pHelper plasmid, which is available from Agilent, is smaller than pXX6-80, at about 11.6 kb. However, it contains an ampicillin resistance gene, which is generally discouraged for plasmids used in AAV production.
The present disclosure addresses the above-described technical challenges by providing compositions and methods described herein.
In some embodiments, the present disclosure relates to adenovirus derived helper plasmids (an adenoviral helper plasmid) comprising adenoviral DNA sequences encoding viral helper proteins. In some embodiments, adenoviral helper plasmids of the present invention are used in methods of production of recombinant adeno-associated viruses (rAAVs). In some embodiments, adenoviral helper plasmids of the present disclosure increase production of rAAVs.
In some embodiments, the present disclosure provides an adenoviral helper plasmid comprising nucleotide sequences encoding proteins derived from sources that are not adenovirus. In some embodiments, the present disclosure provides an adenoviral helper plasmid comprising nucleotide sequences encoding proteins derived from viruses other than adenovirus. In some embodiments, an adenoviral helper plasmid comprises all or a portion of an adenoviral nucleotide sequence encoding adenoviral proteins E2a and E4, as well as non-coding RNA VA RNA. In some embodiments, present disclosure describes improved adenoviral helper plasmids that are smaller than the leading commercially available adenoviral helper plasmids, and that allow for safer and less costly production of rAAVs in producer cell expression systems.
In some embodiments, the present disclosure provides an adenoviral helper plasmid that has reduced overall size relative to presently available adenoviral helper plasmids (e.g., pXX6-80 at 18.932 kbp; pALD-X80 at 18.876 kbp; pHelper at 11.635 kbp; pFAdDeltaF6 at 15.420 kbp).
In some embodiments, the present disclosure provides adenoviral helper plasmids having a smaller size. In some embodiments, an adenoviral helper plasmid of the present disclosure is approximately between 6.5 kb and 15.5 kb. In some embodiments, an adenoviral helper plasmid of the present disclosure has a size that is approximately 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 16 kb. In some embodiments, an adenoviral helper plasmid of the present disclosure has a size that is approximately 6-7 kb; 6.5-7.5 kb; 7-8 kb; 7.5-8.5 kb; 8-9 kb; 8.5-9.5 kb; 9-10 kb; 9.5-10.5 kb; 10-11 kb; 10.5-11.5 kb; 11-12 kb; 11.5-12.5 kb; 12-13 kb; 12.5-13.5 kb; 13-14 kb; 13.5-14.5 kb; 14-15 kb; 14.5-15.5 kb; 15-16 kb. The smaller size of the adenoviral helper plasmids of the present disclosure enables the simpler and less costly production of AAV at the quantities necessary for large-scale manufacturing of AAV. In some embodiments, removing genes and/or portions of genes makes an adenoviral helper plasmid of the present disclosure safer, since the producing cells would not produce the adenovirus structural proteins (e.g., fiber), that could co-purify with AAV during downstream processing and would therefore present a lower risk of inadvertently introducing adenovirus structural proteins to patients.
In some embodiments, removal of adenoviral helper genes resulting in a smaller adenoviral helper plasmid enables addition of supplementary genes to further improve AAV quality and yield. Although these supplementary genes increase the size of the plasmid relative to the smallest versions, they enable comparable or higher AAV productivity and are therefore worth the additional cost to produce. Importantly, these plasmids are still smaller than commercially available helper plasmids such as, for example, pALD-X80.
In some embodiments, an adenoviral helper plasmid of the present disclosure comprises one or more nucleotide sequence(s) encoding proteins selected from the group consisting of E2b, E2a, E4orf4, E1B55K, E1b19K, E1a, E4orf6, VA RNA, and combinations thereof.
In some embodiments, an adenoviral helper plasmid comprises a nucleotide sequence encoding an E2a protein, an E4 region, and a VA RNA region. In some embodiments, an E4 region comprises one or more of E4orf1, E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, and E4orf7. In some embodiments, E4orf1 has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 70. In some embodiments, E4orf1 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 71. In some embodiments, E4orf2 has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 72. In some embodiments, E4orf2 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 73. In some embodiments, E4orf3 has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 74. In some embodiments, E4orf3 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 75. In some embodiments, E4orf4 has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 76. In some embodiments, E4orf4 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 77. In some embodiments, E4orf6 has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 78. In some embodiments, E4orf6 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 79. In some embodiments, E4orf7 has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 80. In some embodiments, E4orf7 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 81. In some embodiments, an adenoviral helper plasmid does not comprise a nucleotide sequence comprising E4orf1. In some embodiments, an adenoviral helper plasmid does not comprise a nucleotide sequence comprising E4orf2. In some embodiments, an adenoviral helper plasmid does not comprise a nucleotide sequence comprising E4orf1 and does not comprise a nucleotide sequence comprising E4orf2. In some embodiments, expression of the E4 region is under the control of an E4 mini promoter. In some embodiments, an E4 region is operably linked to an E4 mini promoter. In some embodiments, an E4 mini promoter has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, an E4 region is operably linked to an SV40 promoter. In some embodiments, expression of the E4 region is under the control of an SV40 promoter. In some embodiments, an SV40 promoter has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 2.
In some embodiments, an adenoviral helper plasmid of the present invention comprises a resistance gene. In some embodiments, an adenoviral helper plasmid of the present invention comprises an ampicillin resistance gene (e.g., a nucleotide sequence encoding a protein conferring resistance to ampicillin). In some embodiments, an adenoviral helper plasmid of the present invention does not comprise an ampicillin resistance gene. In some embodiments, an adenoviral helper plasmid of the present invention comprises a kanamycin resistance gene (e.g., a nucleotide sequence encoding a protein conferring resistance to kanamycin). In some embodiments, an adenoviral helper plasmid of the present invention does not comprise a kanamycin resistance gene.
In some embodiments, an adenoviral helper plasmid of the present disclosure does not comprise a nucleotide sequence encoding adenoviral fiber protein. In some embodiments, an adenoviral helper plasmid does not comprise a nucleotide sequence encoding a full-length adenoviral fiber protein. In some embodiments, an adenoviral helper plasmid comprises a nucleotide sequence encoding a portion or fragment of adenoviral fiber protein. In some embodiments, an adenoviral helper plasmid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to that of pXX6-80, excluding the nucleotide sequence encoding an adenoviral fiber protein.
In some embodiments, an adenoviral helper plasmid of the present disclosure does not comprise a nucleotide sequence encoding a L1-52/55K (Packaging Protein 3) protein. In some embodiments, an adenoviral helper plasmid of the present invention does not comprise a nucleotide sequence encoding Peripentonal Hexon-Associated genes.
In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a complete L4 (hexon assembly) gene. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding a complete L4 (hexon assembly). In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, an adenoviral helper plasmid of the present invention comprises a complete L4 (33 kDa Ex2) gene. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding a complete L4 (33 kDa Ex2). In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 6.
In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a complete L4 Encapsidation Protein gene. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding a complete L4 Encapsidation Protein. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8.
In some embodiments, an adenoviral helper plasmid of the present disclosure does not comprise an L4 (hexon assembly) gene. In some embodiments, an adenoviral helper plasmid does not comprise an L4 Encapsidation Protein gene. In some embodiments, an adenoviral helper plasmid does not comprise an L4 (hexon assembly) gene and does not comprise an L4 Encapsidation Protein gene. In some embodiments, an adenoviral helper plasmid of the present disclosure does not comprise a nucleotide sequence encoding L4 (hexon assembly). In some embodiments, an adenoviral helper plasmid does not comprise a nucleotide sequence encoding L4 Encapsidation Protein. In some embodiments, an adenoviral helper plasmid does not comprise a nucleotide sequence encoding L4 (hexon assembly) and does not comprise a nucleotide sequence encoding L4 Encapsidation Protein gene. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding a fragment of L4 33 kDa Ex2. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 9. In some embodiments, an adenoviral helper plasmid comprises a nucleotide sequence encoding a fragment of L4 33 kDa Ex2. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 10. In some embodiments, a nucleotide sequence encoding a fragment of L4 33 kDa Ex2 comprises an E2a promoter region (see, for example, Casper et al., “Identification of an adeno-associated virus Rep protein binding site in the adenovirus E2a promoter.” Journal of virology 79.1 (2005)). In some embodiments, an E2a promoter region has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 11. In some embodiments, an adenoviral helper plasmid does not comprise a nucleotide sequence encoding a fragment of L4 33 kDa Ex2. In some embodiments, an adenoviral helper plasmid does not comprise an E2a promoter region.
In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding a fragment of hexon-associated precursor (L4 pVIII). In some embodiments, an adenoviral helper plasmid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 12. In some embodiments, an adenoviral helper plasmid comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 13. In some embodiments, an adenoviral helper plasmid does not comprise a nucleotide sequence encoding hexon-associated precursor (L4 pVIII). In some embodiments, an adenoviral helper plasmid does not comprise a nucleotide sequence encoding a fragment of partial hexon-associated precursor (L4 pVIII).
In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a VA RNA region having a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 14. In some embodiments, an adenoviral helper plasmid comprises a VA RNA region having a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 15. In some embodiments, a VA RNA region comprises a VA RNAI gene having a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 16. In some embodiments, a VA RNA region comprises a VA RNAI gene having a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 17. In some embodiments, a VA RNA region comprises a VA RNAII gene having a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 18. In some embodiments, a VA RNA region comprises a VA RNAII gene having a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 19.
In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding a fragment of DNA Terminal Protein. In some embodiments, a nucleotide sequence encoding a fragment of DNA Terminal Protein is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 20. In some embodiments, a fragment of DNA Terminal Protein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 21. In some embodiments, an adenoviral helper plasmid does not comprise a nucleotide sequence encoding DNA Terminal Protein. In some embodiments, an adenoviral helper plasmid comprises a nucleotide sequence encoding a fragment of 23 kDa endoprotease. In some embodiments, an adenoviral helper plasmid comprise a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 22. In some embodiments, a fragment of 23 kDa endoprotease region has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 23. In some embodiments, an adenoviral helper plasmid does not comprise a nucleotide sequence encoding 23 kDa endoprotease region.
In some embodiments, an adenoviral helper plasmid of the present disclosure comprises an E2a gene. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding E2a. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 24. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 25. In some embodiments, expression of E2a is under the control of a promoter. In some embodiments, a nucleotide sequence encoding E2a is operably linked to a promoter. In some embodiments, a promoter is, for example, a CMV promoter, a PGK promoter, an SV40 promoter, an EF-1α promoter, a Ubc promoter, a CAG promoter, or a β-actin promoter. In some embodiments, a nucleotide sequence encoding E2a is operably linked to a transcriptional enhancer. In some embodiments, a transcriptional enhancer is, for example, a CMV enhancer. In some embodiments, a nucleotide sequence encoding E2a is operably linked to a regulatory intron. In some embodiments, expression of E2a is under the control of a chicken β-actin promoter. In some embodiments, a nucleotide sequence encoding E2a is operably linked to a chicken β-actin promoter. In some embodiments, a chicken β-actin promoter has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 26. In some embodiments, a chicken β-actin promoter is positioned upstream of a nucleotide sequence encoding E2a. In some embodiments, expression of E2a is under the control of an E2a promoter and a chicken β-actin promoter. In some embodiments, a nucleotide sequence encoding E2a is operably linked to an E2a promoter and a chicken β-actin promoter. In some embodiments, a chicken β-actin promoter is positioned upstream of an E2a promoter. In some embodiments, expression of E2a is under the control of chicken β-actin promoter and a CMV enhancer. In some embodiments, a nucleotide sequence encoding E2a is operably linked to a chicken β-actin promoter and a CMV enhancer. In some embodiments, a chicken β-actin promoter and a CMV enhancer are positioned upstream of an E2a promoter. In some embodiments, an adenoviral helper plasmid comprises an E2a polyadenylation signal. In some embodiments, an E2a polyadenylation signal is positioned downstream of a nucleotide sequence encoding E2a. In some embodiments, an E2a polyadenylation signal has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 27. In some embodiments, an adenoviral helper plasmid comprises an SV40 polyadenylation signal. In some embodiments, an SV40 polyadenylation signal is positioned downstream of a nucleotide sequence encoding E2a. In some embodiments, an SV40 polyadenylation signal is positioned downstream of an E2a polyadenylation signal. In some embodiments, an SV40 polyadenylation signal has a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 28.
In some embodiments, an adenoviral helper plasmid comprises a nucleotide sequence encoding UL30 derived from HSV-1. In some embodiments, a nucleotide sequence encoding UL30 has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 29. In some embodiments, an amino acid sequence UL30 is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 30. In some embodiments, an adenoviral helper plasmid comprises a nucleotide sequence encoding UL42 derived from HSV-1. In some embodiments, a nucleotide sequence encoding UL42 has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 31. In some embodiments, an amino acid sequence of UL42 is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 32. In some embodiments, an adenoviral helper plasmid comprises a nucleotide sequence encoding UL30 derived from HSV-1, and a nucleotide sequence encoding UL42 derived from HSV-1. In some embodiments, a nucleotide sequence encoding UL30 and a nucleotide sequence encoding UL42 are separated by a P2a cleavage site. In some embodiments, a P2a cleavage site has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 33. In some embodiments, a P2a cleavage site has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 34. In some embodiments, expression of UL30 and/or UL42 gene is/are under the control of an EF-1α promoter. In some embodiments, a nucleotide sequence encoding UL30 is operably linked to a promoter. In some embodiments, a nucleotide sequence encoding UL30 is operably linked to a CMV promoter, a PGK promoter, an SV40 promoter, an EF-1α promoter, a Ubc promoter, a CAG promoter, or a β-actin promoter. In some embodiments, a nucleotide sequence encoding UL30 is operably linked to a transcriptional enhancer. In some embodiments, a transcriptional enhancer is, for example, a CMV enhancer. In some embodiments, a nucleotide sequence encoding UL30 is operably linked to a regulatory intron. In some embodiments a nucleotide sequence encoding UL42 and/or a nucleotide sequence encoding UL30 are operably linked to an EF-1α promoter. In some embodiments, an EF-1α promoter has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 35. In some embodiments, expression of UL30 and/or UL42 is/are under the control of an SV40 promoter. In some embodiments, a nucleotide sequence encoding UL42 and/or a nucleotide sequence encoding UL30 are operably linked to an SV40 promoter. In some embodiments, an SV40 promoter has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 68.
In some embodiments, an adenoviral helper plasmid comprises a polyadenylation signal. In some embodiments. a polyadenylation signal is a β-globin polyadenylation signal, SV40 polyadenylation signal, or a Bovine Growth Hormone (bGH) polyadenylation signal. In some embodiments, an adenoviral helper plasmid comprises a polyadenylation signal downstream of a nucleotide sequence encoding UL42. In some embodiments, an adenoviral helper plasmid comprises a β-globin polyadenylation signal downstream of a nucleotide sequence encoding UL42. In some embodiments, a β-globin polyadenylation signal has a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 36. In some embodiments, an adenoviral helper plasmid comprises a Bovine Growth Hormone (bGH) polyadenylation signal downstream of a nucleotide sequence encoding UL42. In some embodiments, a bGH polyadenylation signal has a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 69.
In some embodiments, an adenoviral helper plasmid comprises a nucleotide sequence encoding UL29 derived from HSV-1. In some embodiments, a nucleotide sequence encoding UL29 is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 37. In some embodiments, an amino acid sequence of UL29 is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 38. In some embodiments, a nucleotide sequence encoding UL29 is operably linked to a promoter. In some embodiments, a nucleotide sequence encoding UL30 is operably linked to a CMV promoter, a PGK promoter, an SV40 promoter, an EF-1α promoter, a Ubc promoter, a CAG promoter, or a β-actin promoter. In some embodiments, a nucleotide sequence encoding UL29 is operably linked to a transcriptional enhancer. In some embodiments, a transcriptional enhancer is, for example, a CMV enhancer. In some embodiments, a nucleotide sequence encoding UL29 is operably linked to a regulatory intron. In some embodiments, expression of UL29 is under the control of an HSV TK promoter. In some embodiments, a nucleotide sequence encoding UL29 is operably linked to an HSV TK promoter. In some embodiments, an HSV TK promoter has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 39.
In some embodiments, an adenoviral helper plasmid comprises a polyadenylation signal downstream of a nucleotide sequence encoding UL29. In some embodiments, a polyadenylation signal is a β-globin polyadenylation signal, SV40 polyadenylation signal, or a Bovine Growth Hormone (bGH) polyadenylation signal. In some embodiments, an adenoviral helper plasmid comprises an HSV TK polyadenylation signal downstream of a nucleotide sequence encoding UL29. In some embodiments, an HSV TK polyadenylation signal has a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 40.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 41. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (Hexon Assembly) (SEQ ID NO: 3: SEQ ID NO: 4), L4 (33 kDa Ex2) (SEQ ID NO: 5; SEQ ID NO: 6), L4 Encapsidation Protein (22 kDa) (SEQ ID NO: 7; SEQ ID NO: 8), L4 pVIII Hexon-Associated Precursor (SEQ ID NO: 12; SEQ ID NO: 13), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA RNAII-A (SEQ ID NO: 18), partial DNA Terminal Protein (SEQ ID NO: 20; SEQ ID NO: 21), 23 kDa endoprotease fragment region (SEQ ID NO: 22; SEQ ID NO: 23), and E2a (SEQ ID NO: 24; SEQ ID NO: 25), and does not comprise the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, and Peripentonal Hexon-Associated genes.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 42. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA RNAII-A (SEQ ID NO: 18), partial DNA Terminal Protein (SEQ ID NO: 20; SEQ ID NO: 21), 23 kDa endoprotease fragment region (SEQ ID NO: 22; SEQ ID NO: 23), and E2a (SEQ ID NO: 24; SEQ ID NO: 25), and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, and L4 pVIII Hexon-Associated Precursor.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 43. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), and E2a (SEQ ID NO: 24; SEQ ID NO: 25), and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, L4 pVIII Hexon-Associated Precursor, DNA Terminal Protein, and 23 kDa endoprotease fragment region.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 44. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), and SV40 polyadenylation signal downstream of E2a (SEQ ID NO: 28), and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, L4 pVIII Hexon-Associated Precursor, DNA Terminal Protein, and 23 kDa endoprotease fragment region.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 45. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA RNAII-A (SEQ ID NO: 18), partial DNA Terminal Protein (SEQ ID NO: 20; SEQ ID NO: 21), 23 kDa endoprotease fragment region (SEQ ID NO: 22; SEQ ID NO: 23), and E2a (SEQ ID NO: 24; SEQ ID NO: 25), and SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 67), and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene. Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, and L4 pVIII Hexon-Associated Precursor, and SV40 polyadenylation signal downstream of E2a.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 46. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), and SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 67), and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, L4 pVIII Hexon-Associated Precursor, DNA Terminal Protein, and 23 kDa endoprotease fragment region, and SV40 polyadenylation signal downstream of E2a.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 47. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: SV40 promoter upstream of E4 region (SEQ ID NO: 2), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA RNAII-A (SEQ ID NO: 18), partial DNA Terminal Protein (SEQ ID NO: 20; SEQ ID NO: 21), 23 kDa endoprotease fragment region (SEQ ID NO: 22; SEQ ID NO: 23), and E2a (SEQ ID NO: 24; SEQ ID NO: 25), and SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 67), and does not comprise the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, and L4 pVIII Hexon-Associated Precursor, SV40 polyadenylation signal downstream of E2a, and E4 mini promoter upstream of E4 region.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 48. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: SV40 promoter upstream of E4 region (SEQ ID NO: 2), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VARNA region B (SEQ ID NO: 15) , VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), and SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 67), and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, L4 pVIII Hexon-Associated Precursor, DNA Terminal Protein, and 23 kDa endoprotease fragment region, and SV40 polyadenylation signal downstream of E2a, and E4 mini promoter upstream of E4 region.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 49. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA RNAII-A (SEQ ID NO: 18), partial DNA Terminal Protein (SEQ ID NO: 20; SEQ ID NO: 21), 23 kDa endoprotease fragment region (SEQ ID NO: 22; SEQ ID NO: 23), E2a (SEQ ID NO: 24; SEQ ID NO: 25), and a chicken β-actin promoter upstream of E2a, and does not comprise the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, and L4 pVIII Hexon-Associated Precursor.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 50. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), and a chicken β-actin promoter upstream of E2a, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, L4 pVIII Hexon-Associated Precursor, DNA Terminal Protein, and 23 kDa endoprotease fragment region.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 51. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), SV40 polyadenylation signal downstream of E2a (SEQ ID NO: 28), and a chicken β-actin promoter upstream of E2a, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, L4 pVIII Hexon-Associated Precursor, DNA Terminal Protein, and 23 kDa endoprotease fragment region.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 52. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA RNAII-A (SEQ ID NO: 18), partial DNA Terminal Protein (SEQ ID NO: 20; SEQ ID NO: 21), 23 kDa endoprotease fragment region (SEQ ID NO: 22; SEQ ID NO: 23), and E2a (SEQ ID NO: 24; SEQ ID NO: 25). SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 67), and a chicken β-actin promoter upstream of E2a, and does not comprise the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, and L4 pVIII Hexon-Associated Precursor, and SV40 polyadenylation signal downstream of E2a.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 53. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 67), and a chicken β-actin promoter upstream of E2a, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, L4 pVIII Hexon-Associated Precursor, DNA Terminal Protein, and 23 kDa endoprotease fragment region, and SV40 polyadenylation signal downstream of E2a.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 54. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: SV40 promoter upstream of E4 region (SEQ ID NO: 2), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA RNAII-A (SEQ ID NO: 18), partial DNA Terminal Protein (SEQ ID NO: 20; SEQ ID NO: 21), 23 kDa endoprotease fragment region (SEQ ID NO: 22; SEQ ID NO: 23), and E2a (SEQ ID NO: 24; SEQ ID NO: 25), SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 67), and a chicken-actin promoter upstream of E2a, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, and L4 pVIII Hexon-Associated Precursor, SV40 polyadenylation signal downstream of E2a, and E4 mini promoter upstream of E4 region.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 55. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: SV40 promoter upstream of E4 region (SEQ ID NO: 2), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), SV40 polyadenylation signal downstream of E2a (SEQ ID NO: 28), SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 67), and a chicken β-actin promoter upstream of E2a, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, L4 pVIII Hexon-Associated Precursor, DNA Terminal Protein, and 23 kDa endoprotease fragment region, and E4 mini promoter upstream of E4 region.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 56. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: SV40 promoter upstream of E4 region (SEQ ID NO: 2), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), SV40 polyadenylation signal downstream of E2a (SEQ ID NO: 28), SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 67), and a chicken β-actin promoter upstream of E2a, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, L4 pVIII Hexon-Associated Precursor, DNA Terminal Protein, and 23 kDa endoprotease fragment region, E4 mini promoter upstream of E4 region, a gene encoding E4orf1, a gene encoding E4orf2, and a gene encoding E4orf3.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 57. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA RNAII-A (SEQ ID NO: 18), partial DNA Terminal Protein (SEQ ID NO: 20; SEQ ID NO: 21), 23 kDa endoprotease fragment region (SEQ ID NO: 22; SEQ ID NO: 23), E2a (SEQ ID NO: 24; SEQ ID NO: 25), a chicken β-actin promoter upstream of E2a, an HSV-1-derived UL30 gene (SEQ ID NO: 29; SEQ ID NO: 30), an HSV-1-derived UL42 gene (SEQ ID NO: 31; SEQ ID NO: 32), EF-1α promoter (SEQ ID NO: 35) upstream of UL30, and a β-globin polyadenylation signal (SEQ ID NO: 36) downstream of UL42, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, and L4 pVIII Hexon-Associated Precursor.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 58. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA RNAII-A (SEQ ID NO: 18), partial DNA Terminal Protein (SEQ ID NO: 20; SEQ ID NO: 21), 23 kDa endoprotease fragment region (SEQ ID NO: 22; SEQ ID NO: 23), E2a (SEQ ID NO: 24; SEQ ID NO: 25), a chicken β-actin promoter upstream of E2a, an HSV-1-derived UL30 gene (SEQ ID NO: 29; SEQ ID NO: 30), an HSV-1-derived UL42 gene (SEQ ID NO: 31; SEQ ID NO: 32), SV40 promoter (SEQ ID NO: 68) upstream of UL30, and a Bovine Growth Hormone (bGH) polyadenylation signal (SEQ ID NO: 69) downstream of UL42, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, and L4 pVIII Hexon-Associated Precursor.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 59. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), a chicken β-actin promoter upstream of E2a, an HSV-1-derived UL30 gene (SEQ ID NO: 29; SEQ ID NO: 30), an HSV-1-derived UL42 gene (SEQ ID NO: 31; SEQ ID NO: 32), SV40 promoter (SEQ ID NO: 68) upstream of UL30, and a Bovine Growth Hormone (bGH) polyadenylation signal (SEQ ID NO: 69) downstream of UL42, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, L4 pVIII Hexon-Associated Precursor, DNA Terminal Protein, and 23 kDa endoprotease fragment region.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 60. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), SV40 polyadenylation signal downstream of E2a (SEQ ID NO: 28), a chicken β-actin promoter upstream of E2a, an HSV-1-derived UL30 gene (SEQ ID NO: 29; SEQ ID NO: 30), an HSV-1-derived UL42 gene (SEQ ID NO: 31; SEQ ID NO: 32), SV40 promoter (SEQ ID NO: 68) upstream of UL30, and a Bovine Growth Hormone (bGH) polyadenylation signal (SEQ ID NO: 69) downstream of UL42, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, L4 pVIII Hexon-Associated Precursor, DNA Terminal Protein, and 23 kDa endoprotease fragment region.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 61. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA RNAII-A (SEQ ID NO: 18), partial DNA Terminal Protein (SEQ ID NO: 20; SEQ ID NO: 21), 23 kDa endoprotease fragment region (SEQ ID NO: 22; SEQ ID NO: 23), and E2a (SEQ ID NO: 24; SEQ ID NO: 25), SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 67), a chicken β-actin promoter upstream of E2a, an HSV-1-derived UL30 gene (SEQ ID NO: 29; SEQ ID NO: 30), an HSV-1-derived UL42 gene (SEQ ID NO: 31; SEQ ID NO: 32), SV40 promoter (SEQ ID NO: 68) upstream of UL30, and a Bovine Growth Hormone (bGH) polyadenylation signal (SEQ ID NO: 69) downstream of UL42, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, and L4 pVIII Hexon-Associated Precursor, and SV40 polyadenylation signal downstream of E2a.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 62. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1). L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 67), a chicken β-actin promoter upstream of E2a, an HSV-1-derived UL30 gene (SEQ ID NO: 29; SEQ ID NO: 30), an HSV-1-derived UL42 gene (SEQ ID NO: 31; SEQ ID NO: 32), SV40 promoter (SEQ ID NO: 68) upstream of UL30, and a Bovine Growth Hormone (bGH) polyadenylation signal (SEQ ID NO: 69) downstream of UL42, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, L4 pVIII Hexon-Associated Precursor, DNA Terminal Protein, and 23 kDa endoprotease fragment region, and SV40 polyadenylation signal downstream of E2a.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 63. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: SV40 promoter upstream of E4 region (SEQ ID NO: 2), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA RNAII-A (SEQ ID NO: 18), partial DNA Terminal Protein (SEQ ID NO: 20; SEQ ID NO: 21), 23 kDa endoprotease fragment region (SEQ ID NO: 22; SEQ ID NO: 23), and E2a (SEQ ID NO: 24; SEQ ID NO: 25), SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 67), a chicken β-actin promoter upstream of E2a, an HSV-1-derived UL30 gene (SEQ ID NO: 29; SEQ ID NO: 30), an HSV-1-derived UL42 gene (SEQ ID NO: 31; SEQ ID NO: 32), SV40 promoter (SEQ ID NO: 68) upstream of UL30, and a Bovine Growth Hormone (bGH) polyadenylation signal (SEQ ID NO: 69) downstream of UL42, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene. Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, and L4 pVIII Hexon-Associated Precursor, SV40 polyadenylation signal downstream of E2a, and E4 mini promoter upstream of E4 region.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 64. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: SV40 promoter upstream of E4 region (SEQ ID NO: 2), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VARNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), SV40 polyadenylation signal downstream of E2a (SEQ ID NO: 28), SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 67), a chicken β-actin promoter upstream of E2a, an HSV-1-derived UL30 gene (SEQ ID NO: 29; SEQ ID NO: 30), an HSV-1-derived UL42 gene (SEQ ID NO: 31; SEQ ID NO: 32), SV40 promoter (SEQ ID NO: 68) upstream of UL30, and a Bovine Growth Hormone (bGH) polyadenylation signal (SEQ ID NO: 69) downstream of UL42, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, L4 pVIII Hexon-Associated Precursor, DNA Terminal Protein, and 23 kDa endoprotease fragment region, and E4 mini promoter upstream of E4 region.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 65. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: E4 mini promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA RNAII-A (SEQ ID NO: 18), partial DNA Terminal Protein (SEQ ID NO: 20; SEQ ID NO: 21), 23 kDa endoprotease fragment region (SEQ ID NO: 22; SEQ ID NO: 23), E2a (SEQ ID NO: 24; SEQ ID NO: 25), a chicken β-actin promoter upstream of E2a, an HSV-1-derived UL29 gene (SEQ ID NO: 37; SEQ ID NO: 38), HSV TK promoter (SEQ ID NO: 39) upstream of UL29, and an HSV TK polyadenylation signal (SEQ ID NO: 40) downstream of UL29, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, and L4 pVIII Hexon-Associated Precursor.
In some embodiments, an adenoviral helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 66. In some embodiments, an adenoviral helper plasmid of the present disclosure comprises the following components having nucleotide sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the indicated sequences: SV40 promoter upstream of E4 region (SEQ ID NO: 2), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), SV40 polyadenylation signal downstream of E2a (SEQ ID NO: 28), SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 67), and a chicken β-actin promoter upstream of E2a, and does not comprise or encode the following components: a fiber gene, an L1-52/55K (Packaging Protein 3) gene, Peripentonal Hexon-Associated genes, full-length L4 (Hexon Assembly) gene, L4 Encapsidation Protein, L4 pVIII Hexon-Associated Precursor, L4 (33 kDa Ex2), DNA Terminal Protein, and 23 kDa endoprotease fragment region, E4 mini promoter upstream of E4 region, a gene encoding E4orf1, a gene encoding E4orf2, and a gene encoding E4orf3.
In some embodiments, an adenoviral helper plasmid of the present disclosure is useful in methods of producing rAAV. In some embodiments, rAAV is produced by transfection of a producer cell. In some embodiments, a producer cell is a mammalian cell. In some embodiments, a producer cell is a transformed mammalian cell. In some embodiments, a producer cell is a Vero, HeLa, HEK293, HEK293T cell or derivative thereof.
In some embodiments, a method of producing a rAAV comprises transfection of a producer cell with an AAV vector plasmid, an AAV Rep-Cap expressing plasmid, and an adenoviral helper plasmid. In some embodiments, an AAV vector plasmid comprises AAV inverted terminal repeats (ITRs) and a transgene of interest. In some embodiments, an adenoviral helper plasmid is any adenoviral helper plasmid described herein.
In some embodiments, a method of producing a rAAV comprises transfection of a producer cell stably expressing Rep-Cap. In some embodiments, a method of producing a rAAV comprises transfection of a producer cell stably expressing Rep-Cap with an AAV vector plasmid and an adenoviral helper plasmid. In some embodiments, an AAV vector plasmid comprises AAV inverted terminal repeats (ITRs) and a transgene of interest. In some embodiments, an adenoviral helper plasmid is any adenoviral helper plasmid described herein.
The main purpose of the work described in this disclosure is to develop novel adenoviral helper plasmids for rAAV production that are smaller, contain fewer non-necessary adenoviral genes, and that function as well or better than the most commonly used adenoviral helper plasmids.
The plasmids provided in this disclosure were synthesized de novo, were sequence-verified, and were scaled up for use in large-scale rAAV manufacturing. Production of rAAV studies were performed to compare vector yields when using the provided plasmids versus other commercially available adenoviral helper plasmids. Vector quality and activity were also assessed from rAAV produced with the different adenoviral helper plasmids to confirm that rAAV produced with the provided plasmids is at least equivalent, if not superior in quality. Taken together, these following examples demonstrate that provided adenoviral helper plasmids generate rAAV of high yield and quality, in a potentially safer and more cost-effective design.
HEK293 cells were transfected with a control adenoviral helper plasmid (e.g., a commercially available plasmid, such as pALD-X80, or an adenoviral helper plasmid described in herein. The adenoviral helper plasmid was co-transfected along with pAAVrep2cap9 and pAAV-CMV-GFP plasmids using PEI transfection in order to generate AAV9/ssCMV-GFP. Four days post-transfection, the HEK293 cells were harvested via 0.5% Triton X-100 lysis and nuclease addition (to degrade RNA, cellular genomic DNA, and remaining plasmid DNA). After 3 hours of lysis/nuclease treatment, the cell lysate was sampled and submitted for qPCR titer analysis. Samples were treated with another nuclease, then EDTA and heat-treated, followed by qPCR of diluted samples to determine vector genome copy number per sample. As a metric of transfection efficiency, cells positive for GFP were quantified using fluorescence microscopy.
To reduce the size of the adenoviral helper plasmid, an adenoviral helper plasmid (pEMBR-1.2: SEQ ID NO: 41) was designed which lacks the fiber gene, the L1-52/55K (Packaging Protein 3) gene, and most of the Hexon Associated Precursor, as well as the Peripentonal Hexon-Associated protein. These deletions were made relative to commercially available helper plasmids, such as pXX6-80. The Adenoviral helper genes were synthesized and assembled into a kanamycin-resistant plasmid backbone. The resulting plasmid is approximately 6.7 kb smaller than pXX6-80.
The adenoviral helper plasmid described above enabled the production of AAV in HEK293 cells. No major difference in AAV vector yield was observed between cells transfected with pALD-X80, and cells transfected with pEMBR-1.2 as measured by qPCR (See
In order to further reduce the size of the adenoviral helper plasmid, an adenoviral helper plasmid was designed which lacks the fiber gene, the L1-52/55K (Packaging Protein 3) gene, and most of the Hexon Associated Precursor, as well as the Peripentonal Hexon-Associated protein (as in pEMBR-1.2—See Example 2), and which further lacks a complete L4 (Hexon Assembly) region (pEMBR-1.3: SEQ ID NO: 42; See
In order to further optimize pEMBR-1.3, the VA RNA region of pEMBR-1.3 was replaced with a VA RNA region derived from AAV-2 (VA RNA-B: SEQ ID NO: 15). This version is named pEMBR-1.3B (SEQ ID NO: 43; See
In order to enhance the viral productivity of the pEMBR-1.3 plasmids, adenoviral helper plasmids were designed that contain the features of pEMBR-1.3, and that further includes a chicken β-actin promoter (SEQ ID NO: 26) upstream of the E2a gene in order to enhance expression of the E2a protein (pEMBR-1.4: SEQ ID NO: 49; See
Another version of pEMBR-1.4 was constructed to include the AAV-2 derived VA RNA region, as in pEMBR-1.3B. This version is named pEMBR-1.4B (SEQ ID NO: 50; See
Another version of pEMBR-1.4 was constructed to include an SV40 polyadenylation signal in order to further enhance the expression of E2A. This version is named pEMBR-1.4B2 (SEQ ID NO: 51).
In order to further promote AAV production using the disclosed adenoviral helper plasmids, several supplementary accessory genes were added to the minimalized plasmids while ensuring that the size of the plasmids does not exceed that of currently commercially available adenoviral helper plasmids (such as pALD-X80).
The pEMBR-1.5 (SEQ ID NO: 57; See
Similarly to the other “B” designs, additional versions of pEMBR-1.5B were constructed to include the smaller AAV-2-derived VA RNA I and II, without the flanking the DNA Terminal Protein or Endoprotease gene sequences (pEMBR-1.5B: SEQ ID NO: 59).
Similarly to other “B2” designs, additional versions of pEMBR-1.5B2 were constructed to include the SV40 polyadenylation signal for higher E2A expression (pEMBR-1.5B2: SEQ ID NO: 60).
This Example further confirms that removal of adenoviral helper genes resulting in a smaller adenoviral helper plasmid enables addition of supplementary genes to further improve AAV quality and yield. Specifically, various pEMBR plasmids of various sizes and comprising various supplementary genes (e.g., UL30, UL42, etc.) were designed from pEMBR-1.2 and pEMBR-1.5a backbone plasmids and tested for the production of AAV.
The pEMBR-1.2B2 (SEQ ID NO: 94) adenoviral helper plasmid was designed to include “B2” designs comprising a SV40 poly A site to potentially increase expression of E2A and a synthesized sequence of a smaller VA region (contains Ad2 VA RNA I and VA RNA II) that does not contain the flanking Ad Terminal Protein nor Endoprotease gene sequences. This region was synthesized with flanking StuI and BsrGI sites and the insert was cloned into pEMBR-1.2 to make pEMBR-1.2B2.
The pEMBR-1.2B2C (SEQ ID NO: 95) adenoviral helper plasmid (See
The pEMBR-1.2B2D (SEQ ID NO: 96) adenoviral helper plasmid (See
Vector yields for AAV (e.g., AAV9) in clarified lysate as measured by qPCR are shown in
The pEMBR-1.2C (SEQ ID: NO. 97) adenoviral helper plasmid was designed to include “C” designs, similarly to the other “C” designs as described above. Furthermore, the pEMBR-1.2D (SEQ ID: NO. 98) adenoviral helper plasmid was designed to include “D” designs, similarly to the other “D” designs as described above.
Vector yields for AAV (e.g., AAV9) in clarified lysate as measured by qPCR are shown in
The pEMBR-1.5A (SEQ ID NO: 58) adenoviral helper plasmid (See
Seeing as both pEMBR-1.5A and pEMBR-1.4 produced AAV at a substantially lower titer relative to pEMBR-1.2 (See
The pEMBR-1.55B2 (SEQ ID NO: 99) adenoviral helper plasmid (See
The pEMBR-1.55B2 OO (SEQ ID NO: 100) adenoviral helper plasmid (See
The pEMBR-1.55B2C (SEQ ID NO: 101) adenoviral helper plasmid (See
The pEMBR-1.55B2C OO (SEQ ID NO: 102) adenoviral helper plasmid (See
The pEMBR-1.55B2D (SEQ ID NO: 103) adenoviral helper plasmid (See
The pEMBR-1.55B2D OO (SEQ ID NO: 104) adenoviral helper plasmid (See
Vector yields for AAV (e.g., AAV9) in clarified lysate as measured by qPCR are shown in
The below Table of Sequences lists and describes the various sequences discussed herein. Unless stated otherwise, all sequences are recited with 5′ to 3′ directionality of the positive strand of a plasmid. This directionality is preserved irrespective of the orientation of a gene or element described to be associated with a sequence. Asterisks as used herein indicate a stop codon.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:
The present application claims the benefit of U.S. Provisional Application No. 63/188,294 filed May 13, 2021, the content of which is hereby incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/29193 | 5/13/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63188294 | May 2021 | US |
