WO2023143063A1 - 一种提高杆状病毒系统生产腺相关病毒的方法及应用 - Google Patents

一种提高杆状病毒系统生产腺相关病毒的方法及应用 Download PDF

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WO2023143063A1
WO2023143063A1 PCT/CN2023/071631 CN2023071631W WO2023143063A1 WO 2023143063 A1 WO2023143063 A1 WO 2023143063A1 CN 2023071631 W CN2023071631 W CN 2023071631W WO 2023143063 A1 WO2023143063 A1 WO 2023143063A1
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expression cassette
nucleic acid
baculovirus
sequence
acid molecule
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李华鹏
陈君霖
钟育健
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广州派真生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/866—Baculoviral vectors
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the invention relates to a baculovirus expression vector, especially a baculovirus expression vector with an adeno-associated virus AD sequence.
  • the invention also relates to the application of the baculovirus expression vector to prepare recombinant adeno-associated virus.
  • Adeno-associated viral vector is a reliable tool for gene transfer in vitro and in vivo. It has good targeting, high safety, and strong transduction ability, and has been widely used in many gene therapy clinical trials. Including familial lipoprotein lipase deficiency, hemophilia, ophthalmology, musculospinal and other genetic diseases, all of which have clinical reports under research.
  • adeno-associated virus production systems mainly include HEK293 cell system, insect cell baculovirus system, and herpes virus system.
  • the HEK293 system package can be used for adherent culture and suspension culture, and also includes two-plasmid or three-plasmid transfection methods. Its production cycle is short, but it involves relatively complicated aspects such as plasmids and cells, which makes it difficult to carry out large-scale cultivation in the actual production process and is expensive.
  • the herpes virus system produces adeno-associated virus, and there may be sensitized herpes virus in the process, and its safety has certain potential risks.
  • Insect cell baculovirus system because of its high cell density, large-scale culture, and good safety, is considered to be one of the potential breakthroughs to overcome the technical barriers that are difficult for large-scale culture of adeno-associated virus.
  • Optimizing and improving the production efficiency of adeno-associated virus mediated by the baculovirus system is the key work of scientists.
  • Adeno-associated virus inverted terminal repeat contains 145 nucleotides, divided into A, B, C, D four components. Among them, A-A', BB', and C-C' are three parts of 125 nucleotides to form a T-shaped palindromic hairpin structure, which is used as a viral DNA replication primer to initiate the replication of the adeno-associated virus.
  • the A sequence contains the RBE (rep binding element) structure, which is the REP protein recognition binding site 1 .
  • the D sequence is independent of the T- shaped hairpin structure and is critical for the length of the AAV packaging gene, viral DNA replication, and viral infectivity3-4.
  • AD sequence also contains transcription initiation signal (A sequence 88 to 95, D sequence 126 to 133) which can be used as a promoter to initiate gene transcription 5 .
  • George Aslanidi et al. used the baculovirus homologous recombination sequence (HR) and the RBE element to propose that the baculovirus immediate early trans regulator 1 (IE-1) could induce the binding of the HR element, enhance the expression of Rep and Cap genes, and accumulate The Rep protein binds to the RBE element, further promoting the accumulation of Rep protein 6 .
  • HR baculovirus homologous recombination sequence
  • IE-1 immediate early trans regulator 1
  • the use of AD sequences to promote baculovirus packaging and the effect of AD sequences on the efficiency of AAV produced by baculovirus systems have not been reported.
  • isolated nucleic acid molecules comprising:
  • AAV adeno-associated virus
  • Cap an AAV capsid protein gene
  • the AD sequence or functional variant thereof is located within or within 5 kb of the Rep expression cassette.
  • the AD sequence or functional variant thereof is located within or within 5 kb of the Cap expression cassette.
  • the AD sequence or its functional variant in the nucleic acid molecule: 1) the AD sequence or its functional variant is located in the Rep expression cassette, and the distance from the Cap expression cassette is no more than 5kb; 2) the AD sequence or Its functional variant is located in the Cap expression cassette, and the distance from the Rep expression cassette is no more than 5kb; or 3) the AD sequence or its functional variant is located between the Rep expression cassette and the Cap expression cassette outside, and the distance from both the Rep expression cassette and the Cap expression cassette is no more than 5kb.
  • the AD sequence or a functional variant thereof is located within the Rep expression cassette after a stop codon, and/or the AD sequence or a functional variant thereof is located within the Cap expression cassette and located after the stop codon.
  • the Rep expression cassette and the Cap expression cassette are transcribed in different directions, and the AD sequence or a functional variant thereof is located at the promoter of the Rep expression cassette and the promoter of the Cap expression cassette between sons.
  • the promoter of the Rep expression cassette and/or the Cap expression cassette is an insect cell promoter
  • the promoters of the Rep expression cassette and the Cap expression cassette are independently selected from P10 and Polh promoters, respectively.
  • the AD sequence includes the nucleotide sequence shown in SEQ ID NO:56.
  • the functional variant of the AD sequence comprises a nucleotide sequence having at least 60%, 70%, 80% or 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:56 , and, when the nucleic acid molecule is introduced into a suitable host cell, the expression of the AAV replication gene (Rep) expression cassette and/or the AAV capsid protein gene (Cap) expression cassette in the host cell can be increased of expression.
  • Rep AAV replication gene
  • Cap AAV capsid protein gene
  • the Rep expression cassette is used to transcribe and express Rep78/Rep68 and Rep52/Rep40 proteins.
  • the Cap expression cassette is used to transcribe and express VP1 and VP2/VP3 proteins.
  • the Rep expression cassette and/or the Cap expression cassette comprises an intron sequence comprising a promoter sequence.
  • said promoter contained by said intron sequence is a polh promoter.
  • the intron sequence includes the nucleotide sequence shown in SEQ ID NO: 57 or a functional variant thereof.
  • the intron sequence is located between Rep coding nucleotide sequence 530 and 531 in the Rep expression cassette to drive transcription of the Rep52/Rep40 subunit coding sequence.
  • the intron sequence is located between Cap coding nucleotide sequences 25 and 26 in the Rep expression cassette to drive the transcription of the VP2 and VP3 subunit coding sequences.
  • expression vectors comprising the nucleic acid molecules described above.
  • the expression vector is an expression vector with pFastBac TM Dual as the backbone.
  • the expression vector is a baculovirus expression vector.
  • the baculovirus vector is produced by the Bac-to-Bac TM baculovirus expression system.
  • the expression vector further includes an additional expression cassette.
  • the additional expression cassette comprises from 5' to 3':
  • the promoter in the additional expression cassette is a mammalian cell promoter.
  • the gene of interest includes a protein or RNA coding sequence.
  • host cells comprising the nucleic acid molecules or expression vectors described above.
  • the nucleic acid molecule is integrated into the genome of the host cell.
  • the host cell is an insect cell.
  • the host cell is an insect cell SF9, SF21 or high five.
  • baculovirus expression vectors comprising
  • a first baculovirus expression vector comprising the above-mentioned nucleic acid molecule, wherein the nucleic acid molecule comprises the Rep expression cassette and the Cap expression cassette;
  • the second baculovirus expression vector which includes the target gene expression cassette and the AAV ITRs located on both sides of the target gene expression cassette.
  • baculovirus expression vectors comprising
  • a first baculovirus expression vector which includes the above-mentioned nucleic acid molecule, wherein the nucleic acid molecule includes the Rep expression cassette and does not include the Cap expression cassette;
  • a second baculovirus expression vector which includes the above-mentioned nucleic acid molecule, wherein the nucleic acid molecule includes the Cap expression cassette and does not include the Rep expression cassette;
  • a third baculovirus expression vector which includes a target gene expression cassette and AAV ITRs located on both sides of the target gene expression cassette.
  • host cells comprising a combination of the above-mentioned baculovirus expression vectors.
  • host cells comprising
  • An expression vector which includes a target gene expression cassette and AAV ITRs located on both sides of the target gene expression cassette.
  • rAAV in insect cells comprising:
  • the second baculovirus includes the gene expression cassette of interest and the AAV ITRs located on both sides of the gene expression cassette of the gene of interest;
  • rAAV in insect cells comprising:
  • the first baculovirus includes the above nucleic acid molecule, wherein the nucleic acid molecule includes the Rep expression cassette and does not include the Cap expression cassette;
  • the second baculovirus includes the above-mentioned nucleic acid molecule, wherein the nucleic acid molecule includes the Cap expression cassette and does not include the Rep expression cassette;
  • the third baculovirus includes the target gene expression cassette and is located at The AAV ITRs on both sides of the target gene gene expression cassette;
  • methods of producing AAV in insect cells comprising:
  • nucleic acid molecule 1) integrating the above-mentioned nucleic acid molecule into the genome of the insect line, wherein the nucleic acid molecule includes the Rep expression cassette and the Cap expression cassette;
  • the serotype of the aforementioned AAV or rAAV is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13.
  • This paper provides a method to improve the production of adeno-associated virus by the baculovirus system.
  • the titer of the baculovirus and the efficiency of the production of the adeno-associated virus by the baculovirus are significantly improved. It reduces the number of passages of baculovirus in the actual production process, solves the disadvantages of long time for building the baculovirus seed bank and poor genetic stability caused by high algebra, and shortens the cycle of producing adeno-associated virus by the baculovirus system.
  • Figure 1 shows the map of the expression vector containing the AD sequence designed in Example 1, which contains the AD sequence, Rep expression cassette and Cap expression cassette in the order from 5' to 3'.
  • Figure 2 shows the map of the expression vector containing the AD sequence designed in Example 2, which contains the Rep expression cassette, AD sequence and Cap expression cassette in the order from 5' to 3'.
  • Figure 3 shows the map of the expression vector containing the AD sequence designed in Example 3, which contains the Rep expression cassette, Cap expression cassette and AD sequence in the order from 5' to 3'.
  • Figure 4 shows the effects of different AD sequences, Rep expression cassettes and Cap expression cassette positional combinations in Example 4 on the packaging titer of baculovirus.
  • Figure 5 shows the effects of different AD sequences, Rep expression cassettes and Cap expression cassette positional combinations in Example 5 on the yield of adeno-associated virus produced by baculovirus.
  • nucleic acid molecule refers to a polymer of nucleotides.
  • nucleotide polymers may contain natural and/or unnatural nucleotides and include, but are not limited to, DNA, RNA and PNA.
  • Nucleic acid sequence refers to the linear sequence of nucleotides comprised in a nucleic acid molecule or polynucleotide.
  • isolated nucleic acid molecule means a nucleic acid molecule that has been removed from its natural environment (e.g., a cellular environment) in which it exists and is substantially free of one or more substances with which it is normally associated in nature, e.g., proteins, nucleic acids, lipids , carbohydrates, cell membranes, etc., or artificially prepared (such as artificially synthesized) nucleic acid molecules.
  • Adeno-associated virus is a non-enveloped icosahedral capsid virus of the family Parvoviridae, comprising a single-stranded DNA viral genome.
  • the family Parvoviridae includes the genus Dependovirus, which includes AAV, which relies on the presence of a helper virus, such as adenovirus, for its replication. Owing to its relatively simple structure, ability to infect a variety of cells (including quiescent and dividing cells) without integration into the host genome, and its relatively mild immunogenic profile, AAV has proven useful as a biological tool. Also contemplated herein are AAV-based expression vectors, including recombinant AAV (rAAV) with a gene of interest for therapeutic purposes.
  • rAAV recombinant AAV
  • the wild-type AAV viral genome is a linear, single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) in length.
  • ITRs Inverted terminal repeats
  • AAV viral genomes generally include two ITR sequences. These ITRs have a characteristic T-shaped hairpin structure.
  • the double-stranded hairpin structure includes multiple functions including, but not limited to, serving as an origin of DNA replication by serving as a primer for the endogenous DNA polymerase complex of the host virus-replicating cell.
  • the wild-type AAV viral genome also includes the nucleotide sequences of two open reading frames, one for the four nonstructural Rep proteins (Rep78, Rep68, Rep52, Rep40, encoded by the Rep gene), and the other for the three capsids Or structural proteins (VP1, VP2, VP3, encoded by capsid genes or Cap genes).
  • Rep proteins are important for replication and packaging, while capsid proteins assemble to form the protein coat of AAV, or AAV capsid. Alternate splicing and alternate start codons and promoters result in the generation of four different Rep proteins from a single open reading frame and three capsid proteins from a single open reading frame.
  • viral capsid protein or "capsid protein” as used in reference to AAV refers to the protein of AAV that is capable of self-assembly to produce AAV particles, also known as coat protein or VP protein. It consists of three subunits, VP1, VP2 and VP3, which are usually expressed from a single nucleic acid molecule and interact together to form an icosahedral symmetric capsid.
  • serotype as used in reference to AAV is used to refer to the distinction of AAV's capsid protein serologically from other AAV serotypes. Serological uniqueness was determined based on the reactivity of an antibody with one AAV and the lack of cross-reactivity with the other or another AAV. Such differences in cross-reactivity are often due to differences in capsid protein sequences/epitopes (eg, due to differences in VP1, VP2 and/or VP3 sequences of AAV serotypes).
  • Recombinant AAV vector refers to removing part of wild-type genes (such as Rep gene and Cap gene) from the AAV genome by using molecular biology methods, and replacing them with heterologous nucleic acid sequences (such as protein or RNA for therapeutic purposes) coding sequence) replacement derived AAV genome.
  • wild-type genes such as Rep gene and Cap gene
  • heterologous nucleic acid sequences such as protein or RNA for therapeutic purposes
  • rAAV vectors typically, one or two inverted terminal repeat (ITR) sequences of the AAV genome are retained.
  • ITR inverted terminal repeat
  • rAAV is replication deficient, lacking sequences encoding functional Rep and Cap proteins in its viral genome.
  • These replication-deficient AAV particles may lack most of the parental coding sequences and carry essentially only one or two AAV ITR sequences and the target nucleic acid for delivery to a cell, tissue, organ or organism.
  • expression cassette refers to a coding sequence (such as a protein coding sequence or an RNA coding sequence) that can be transcribed in a suitable expression environment (such as an intracellular environment) and further translated as appropriate (such as a product A nucleic acid sequence that is a protein).
  • An expression cassette typically includes a transcriptional regulatory sequence (e.g., promoter, enhancer), a coding sequence operably linked to the transcriptional regulatory sequence, and a polyadenylation signal (or transcriptional termination signal), wherein the transcriptional regulatory sequence is usually located upstream of the coding sequence .
  • the expression cassette usually also includes a 5' untranslated region (5'-UTR) or a 3' untranslated region (3'-UTR).
  • promoter refers to a segment involved in the recognition and binding of DNA-dependent RNA polymerase and other proteins (trans-acting transcription factors) to initiate and control the transcription of one or more coding sequences DNA sequence. It is usually located upstream of the transcription initiation site (i.e., the 5' end), and includes some conserved sequences, such as TATA box, etc. In some examples herein, some promoters were used to drive the transcription of the Rep expression cassette and the Cap expression cassette in insect cells. These promoters include P10 promoter or polh promoter (or called PH promoter).
  • enhancer refers to a DNA sequence on an expression vector that enhances the transcriptional activity of a promoter.
  • the activity of an enhancer to enhance transcription is usually non-directional (that is, it is located upstream, downstream, or even inside the target protein-coding gene), and in some cases it can act at a distance (such as several Kb from the transcription start site).
  • operably linked refers to a linkage between polynucleotide elements in a functional relationship.
  • a nucleic acid or polynucleotide sequence is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a transcriptional regulatory sequence such as a promoter, enhancer, or other expression control element known in the art will be operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • polyadenylation signal refers to the mechanism that terminates the transcription process and adds a polyA tail to the 3' end of the transcribed mRNA. This tailing process is catalyzed by polyadenylate polymerase to form mature mRNA.
  • sequence related to the polyadenylation on the expression cassette is called “polyadenylation signal sequence” or “PolyA signal region (PolyA signal)”.
  • untranslated region refers to the fragments located at both ends of the coding region of mRNA that are not translated into protein.
  • the mRNA segment located upstream of the coding region is called the 5' untranslated region (5'-UTR), and the segment located downstream of the coding region is called the 3' untranslated region (3'-UTR).
  • the untranslated region can regulate gene expression at the level of gene transcription and post-transcriptional translation.
  • the 5' untranslated region can affect the transcription efficiency, stability and nuclear transport of the gene's mRNA, resulting in changes in protein expression levels; 3'
  • the untranslated region mainly affects the stability and translation efficiency of mRNA.
  • the DNA fragment corresponding to the above-mentioned untranslated region in the expression vector or gene sequence is also called 5' untranslated region (5'-UTR) or 3' untranslated region (3'-UTR).
  • expression vector refers to a nucleic acid molecule comprising various expression elements for expressing a protein of interest or RNA of interest in a host cell.
  • these expression elements generally include promoters, enhancers, polyadenylation signal sequences, and the like.
  • the expression vector usually also includes an E. coli replicon sequence.
  • the expression vector may also include an antibiotic resistance gene or a selectable marker gene for selection (such as ampicillin resistance gene (AmpR), thymidine kinase gene (TK), kanamycin resistance gene (KanR), new Mycin resistance gene (NeoR), etc.) and multiple cloning site (MCS) for gene insertion of interest.
  • an antibiotic resistance gene such as ampicillin resistance gene (AmpR), thymidine kinase gene (TK), kanamycin resistance gene (KanR), new Mycin resistance gene (NeoR), etc.
  • MCS multiple cloning site
  • the expression vector is a baculovirus expression vector or an rAAV expression vector.
  • Baculovirus is a double-stranded DNA virus that specifically infects arthropods.
  • Autographa californica nucleopolyhedrovirus (AcMNPV) is the type species of baculovirus. Since Smith GE and others first used baculovirus to express human ⁇ -interferon gene in insect cells in 1983, due to the advantages of low cost, high yield, and various post-translational modification systems, baculovirus expression vectors have been widely used in scientific research. and are widely used in production.
  • the target gene can be placed under the regulation of the baculovirus overexpression promoter, so that the target gene can be overexpressed at the end of infection.
  • polyhedrin polyhedrin
  • p10 protein proteins that can be overexpressed in the very late stage of infection. They are not necessary for the replication of the virus in cultured cells in vitro, so the promoters of these two genes can be used to promote the expression of the target gene, and the required protein can be produced at a high level at the end of the infection.
  • the baculovirus genome is used as an expression vector, in order to accommodate more foreign genes or increase the expression of the target protein, non-essential sequences in the genome can be removed.
  • Baculovirus is an excellent protein expression vector, especially suitable for large-scale production of bioengineering enzymes, drugs and other active proteins.
  • AD sequence refers to the A and D sequences of the ITR from AAV linked to each other.
  • the AD sequence is shown in SEQ ID NO:56.
  • SEQ ID NO: 56 gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct.
  • the "functional variant" of the AD sequence refers to basically retaining the ability of the AD sequence to promote the production of baculovirus vectors and rAAV, but the nucleotide sequence is different from the sequence shown in SEQ ID NO:56.
  • the functional variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 relative to the sequence shown in SEQ ID NO:56 One or more nucleotide substitutions, additions or deletions.
  • the functional variant has at least 60%, such as at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.
  • the functional variant retains the function of the sequence shown in SEQ ID NO: 56 relative to the sequence shown in SEQ ID NO: 56, in terms of retention in the production of baculovirus vectors and/or rAAV At least 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100%.
  • the functional variant retains at least 80% of the function of the sequence set forth in SEQ ID NO: 56 relative to the sequence set forth in SEQ ID NO: 56, with respect to retention in the production of baculovirus vectors and/or rAAV , 90%, or even 100%.
  • Those skilled in the art can obtain these functional variants by performing various substitution, addition or deletion operations on the sequence shown in SEQ ID NO:56, and verifying the function of the obtained sequence.
  • not more than 5 kb from the expression cassette refers to the terminal core of the AD sequence (or its functional variant) in the same nucleic acid molecule (linear or circular molecule).
  • the distance between the nucleotide and the terminal nucleotide of the expression cassette expressed in the number of nucleotides, not exceeding 5000 nucleotides, such as not exceeding 4000 nucleotides, not exceeding 3000 nucleotides, not exceeding 2000 nucleotides Nucleotides, even up to 1000 nucleotides.
  • the AD sequence (or a functional variant thereof) is no more than 5 kb in nucleotides from the two proximal ends of the expression cassette.
  • the distance between them is considered to be 0 nucleotides.
  • stop codon refers to a trinucleotide sequence that terminates peptide chain elongation during protein synthesis using mRNA as a template.
  • the common stop codons are UAG, UAA and UGA, and the corresponding stop codon sequences on the coding DNA are TAG, TAA and TGA.
  • references to an AD sequence (or a functional variant thereof) being located “after the stop codon” means that the AD sequence (or a functional variant thereof) is located downstream of a stop codon (such as TAG, TAA or TGA) in the expression cassette in which it is located (ie 3' direction), especially downstream and immediately following the stop codon.
  • a stop codon such as TAG, TAA or TGA
  • the AD sequence (or its functional variant) is "located between two promoters"
  • the distance between the two promoters is relatively close (for example, tens to hundreds of nucleotides apart), and is not identified by other promoters.
  • Functional long sequences (such as other expression cassettes) are separated.
  • the two promoters can be considered to exist "back-to-back” and drive transcription in different directions, such as the P10 promoter and the Polh promoter in the pFastBac TM Dual vector.
  • the term "host cell” refers to a cell in which an expression vector can be maintained and/or replicated, including prokaryotic and eukaryotic cells, such as bacteria (such as E. coli), fungi (yeast), insect cells, and mammalian cells.
  • prokaryotic and eukaryotic cells such as bacteria (such as E. coli), fungi (yeast), insect cells, and mammalian cells.
  • the host cells used are preferably insect cells, such as SF9, SF21 or high five cell lines.
  • intron refers to the non-coding sequence of the gene sequence, which can be transcribed, but is cut out during the process of mRNA processing and maturation, so its sequence is not reflected in the mature mRNA.
  • the coding DNA sequence may be separated by one or more introns, but all may be transcribed and translated into the same protein of interest.
  • promoter-containing intron means that the promoter contained in the intron can initiate the transcription of its downstream coding sequence, but does not substantially affect the coding sequence driven by its upstream promoter (which is interrupted by the intron). ) transcription.
  • promoter-containing introns are particularly useful for driving transcription of overlapping genes of interest. In a specific example, it can be used to drive the transcription of Rep proteins (including Rep78, Rep68, Rep52 and Rep40 subunits) or VP proteins (including VP1, VP2 and VP3 subunits).
  • functional variants of the above-mentioned intron sequences can also be used, as long as they can realize or substantially realize the above-mentioned functions.
  • Functional variants of the intron retain or substantially retain the ability to drive transcription of its downstream coding sequence (while not affecting or substantially not affecting its upstream promoter-driven coding sequence (which is interrupted by the intron) transcription), but differs from the sequence shown in SEQ ID NO:57 in the nucleotide sequence.
  • the functional variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 relative to the sequence shown in SEQ ID NO:57 One or more nucleotide substitutions, additions or deletions.
  • the functional variant has at least 60%, such as at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.
  • Those skilled in the art can obtain these functional variants by performing various substitutions, additions or deletions on the sequence shown in SEQ ID NO:57, and verifying the function of the obtained sequence.
  • sequence identity when referring to nucleotide sequences, refers to the amount of degree of identity between two nucleotide sequences (e.g., a query sequence and a reference sequence) , usually expressed as a percentage. Typically, prior to calculating the percent identity between two nucleotide sequences, the sequences are aligned and gaps, if any, are introduced. If the bases in the two sequences are the same at a certain alignment position, the two sequences are considered to be consistent or match at this position; if the bases in the two sequences are different, they are considered to be inconsistent or mismatched at this position.
  • sequence identity is obtained by dividing the number of matching positions by the total number of positions in the alignment window. In other algorithms, the number of gaps and/or gap lengths are also taken into account.
  • sequence comparison algorithms or software include DANMAN, CLUSTALW, MAFFT, BLAST, MUSCLE, etc.
  • the published alignment software BLAST available from https://www.ncbi.nlm.nih.gov/) can be used to obtain the optimal sequence alignment by using default settings and calculating Sequence identity between two nucleotide sequences.
  • the Bac-to-Bac TM Baculovirus Expression System provides Thermo Fisher with an insect baculovirus expression system that relies on a site-specific transposition to place a DNA sequence of interest (such as one or more multiple expression cassettes) onto a bacmid.
  • the system includes the following elements: pFastBac TM vector or similar vector (e.g. pFastBac TM Dual), containing transposon elements Tn7-L and Tn7R, DNA sequence between them (e.g. one or more expression cassettes, resistance gene, etc.
  • DH10Bac strain is a competent Escherichia coli, and it is also the host bacteria of vectors such as pFastBac TM , which contains Escherichia coli-baculovirus shuttle Plasmid (bacmid) and helper plasmid, bacmid contains mini-attTn7 site, helper plasmid encodes transposable protein.
  • the DNA sequence between Tn7-L and Tn7R is transposed into the mini-attTn7 site of the bacmid to form a recombinant bacmid.
  • Recombinant bacmid infects insect cells to form recombinant baculovirus, which can be followed by multiple generations of infection and large-scale recombinant protein expression.
  • Bac-to-Bac TM baculovirus expression system see the manufacturer's instructions.
  • the pFastBac TM Dual expression vector is similar to the pFastBac TM vector, but it contains two multiple cloning sites for introducing foreign genes, which are located downstream of the promoters P10 and Polh respectively, so it can be used to express two foreign proteins simultaneously in insect cells .
  • This paper is based, at least in part, on the unexpected discovery that when AD sequences are introduced into expression vectors such as baculovirus expression, the production of baculoviruses can be facilitated, and when the baculoviruses are used to produce rAAV, the Yield of rAAV.
  • nucleic acid molecules comprising AD sequences.
  • the nucleic acid molecule may include one or more expression cassettes for expression of the protein of interest.
  • the AD sequence can be located upstream, downstream or within the expression cassette.
  • the AD sequence is not located in the expression cassette, it is preferably not more than 5 kb away from the expression cassette, for example 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, 0.4 kb, 0.3 kb, 0.2 kb, 0.1 kb or closer to the expression cassette .
  • the AD sequence can be in the 5' untranslated region (5'-UTR) or the 3' untranslated region (3'-UTR), or it can be placed in an intron and the Introns are located between coding sequences.
  • the AD sequence is located in the expression cassette, it is located after the stop codon; more preferably, it is located after and immediately after the stop codon.
  • the AD sequence can be located upstream, downstream or within either expression cassette.
  • the AD sequence is not located in either expression cassette, it is preferably not more than 5kb from both expression cassettes, such as 4kb, 3kb, 2kb, 1kb, 0.5kb, 0.4kb, 0.3kb, 0.2kb, 0.1kb or closer.
  • the AD sequence may be located between the two promoters.
  • the nucleic acid molecule contains a Rep expression cassette for expression of Rep proteins (Rep78, Rep68, Rep52, and Rep40). In other embodiments, the nucleic acid molecule contains a Cap expression cassette for expression of VP proteins (VP1, VP2 and VP3). In yet other embodiments, the nucleic acid molecule contains a Rep expression cassette for expressing Rep proteins (Rep78, Rep68, Rep52, and Rep40) and a Cap expression cassette for expressing VP proteins (VP1, VP2, and VP3).
  • expression vectors especially baculovirus expression vectors, comprising the above nucleic acid molecules are provided herein.
  • the baculovirus expression vector can be produced by the Bac-to-Bac TM baculovirus expression system.
  • the baculovirus vector comprises the combination of the AD sequence, the Cap expression cassette and the Rep expression cassette in the order of 5' to 3'.
  • the baculovirus vector comprises the AD sequence, a combination of a Rep expression cassette and a Cap expression cassette in the order of 5' to 3'.
  • the baculovirus vector comprises the combination of the Rep expression cassette, the AD sequence and the Cap expression cassette in the order of 5' to 3'.
  • the baculovirus vector comprises the combination of the Cap expression cassette, the AD sequence and the Rep expression cassette in the order of 5' to 3'.
  • the baculovirus vector comprises the combination of the Cap expression cassette, the Rep expression cassette and the AD sequence in the order of 5' to 3'.
  • the baculovirus vector comprises the combination of the Rep expression cassette, the Cap expression cassette and the AD sequence in the order of 5' to 3'.
  • the present invention also provides a method for preparing recombinant adeno-associated virus (rAAV) through the baculovirus vector.
  • rAAV recombinant adeno-associated virus
  • rAAV is produced using a baculovirus system.
  • a nucleic acid molecule containing a Rep expression cassette, a Cap expression cassette and an AD sequence (or a functional variant thereof) can be integrated into a host cell, such as an insect cell, and an ITR with AAV is introduced into the host cell.
  • the expression vector of the target gene (such as the coding sequence of the therapeutic target protein) can generate and obtain the rAAV which can be used to contain the target gene.
  • a nucleic acid molecule containing a Rep expression cassette, a Cap expression cassette, and an AD sequence (or a functional variant thereof) and a gene expression cassette of interest flanked by AAV ITR sequences can be introduced into the same baculovirus expression vector, and use the baculovirus to infect host cells (such as insect cells), and rAAV that can be used to contain the gene of interest can be produced and obtained.
  • rAAV is produced using a two-baculovirus system.
  • a nucleic acid molecule containing a Rep expression cassette, a Cap expression cassette, and an AD sequence can be constructed in a baculovirus expression vector, and at the same time, the target gene with the ITR sequence of AAV on both sides can be expressed
  • the cassette is constructed in another baculovirus expression vector, and when the two baculoviruses are infected to the same host cell (such as an insect cell), rAAV that can be used to contain the gene of interest can be generated and obtained.
  • a triple baculovirus system is used in the production of rAAV.
  • the Rep expression cassette and the Cap expression cassette are respectively constructed in two different baculovirus expression vectors, either or both of these two baculovirus expression vectors may contain AD sequences;
  • the target gene expression cassette of the ITR sequence is constructed in another baculovirus expression vector.
  • the obtained rAAV can then be used to infect mammals or mammalian cells, such as human or human cells, and express the gene of interest.
  • the target gene can be any nucleotide sequence, as long as the formed construct remains within the packaging capacity of the AAV viral genome.
  • the target gene can encode target protein or target RNA (such as various interfering small RNAs), and all of them can be therapeutic gene products.
  • a therapeutic gene product may be a polypeptide or RNA molecule, or other gene product that, when expressed in a target cell, provides a desired therapeutic effect. Examples of therapeutic effects include elimination of undesired activity, such as elimination of enzyme activity, or compensation of genetic defects, among others.
  • the protein of interest encoded by the polynucleotide may be used as a marker protein for assessing cellular transformation and expression. Suitable marker proteins for this purpose are eg the fluorescent protein GFP or firefly luciferase.
  • baculovirus vectors are used to prepare rAAv
  • two or three baculovirus vector combinations are two or three baculovirus vector combinations.
  • this paper actually also provides a baculovirus vector containing a Rep expression cassette, a Cap expression cassette, or a Rep expression cassette and a Cap expression cassette, which can be used with users
  • the self-constructed target gene expression cassette is used together to produce rAAV with the desired target gene.
  • the method for generating rAAV comprises the following steps:
  • Baculovirus packaging comprising REP, CAP and AD sequences
  • the pFastBac TM Dual shuttle plasmid is used in this method to produce baculovirus vectors based on the Bac-to-Bac system.
  • the present invention has the following beneficial effects: a method for improving the production of adeno-associated virus by the baculovirus system disclosed in this paper, through the arrangement and combination of the AD sequence and Rep and Cap at different positions, significantly improves the baculovirus
  • the packaging titer is about 3-33 times, thereby reducing the number of baculovirus passages and avoiding the instability of passages caused by excessive passages.
  • the invention improves the production efficiency of the adeno-associated virus by the baculovirus system.
  • the yield of the adeno-associated virus produced by the baculovirus vector carrying the AD sequence is increased by 2-5 times. Therefore, the production cost of the adeno-associated virus is reduced, the clinical application of the adeno-associated virus is increased, and research and development costs are reduced.
  • This article provides methods for improving the production of adeno-associated virus in baculovirus systems. Through the combination of AD sequence and Rep and Cap proteins at different positions, the titer of baculovirus and the packaging efficiency of adeno-associated virus are improved. It can be applied to produce adeno-associated virus with different baculovirus systems, such as one bac system, two bac system or three bac system.
  • the following takes the patent document (CN200780037031.2) as an example to illustrate the production of AAV2 by two baculovirus systems with introns embedded in the promoter.
  • Insect cell growth was maintained at a cell density of 5E+6cell/ml at 28°C.
  • the medium used was ESF921 medium serum-free medium (Expression Systems), containing 100 units/ml of penicillin and 100 ug/ml of streptomycin.
  • the Bac-to-bac TM system was used, and the pFastBac TM Dual expression plasmid was from Thermofisher (Product No. 10712024).
  • the Rep and Cap protein gene expression cassettes and AD sequences were constructed into pFastBac TM Dual to form the first plasmid.
  • the second plasmid containing the ITRs on both sides and the EGFP gene expression cassette was constructed. According to the instructions, the constructed plasmid was diluted to a concentration of 2ng/ul, and 2ul of plasmid DNA was used to transform DH10Bac competent cells.
  • SF9 cells in the logarithmic growth phase were collected, collected by centrifugation, and subpackaged and cultured at 1E+8cell/ml, with a total volume of 15ml.
  • the first baculovirus BAC-CAP-REP-(REP-AD) comprises an AD sequence located behind the stop codon TAA of the AAV2 Rep gene expression cassette, the Rep gene expression cassette and the Cap gene expression cassette, respectively at P10, Transcription under the action of the PH promoter to produce AAV2 in insect cells, as shown in Figure 1.
  • the pFastBac TM Dual expression plasmid was obtained from Thermofisher (Cat. No. 10712024), and pRep2Cap2 containing Rep2 and Cap2 was obtained from our company.
  • pRep2Cap2 containing Rep2 and Cap2 was obtained from our company.
  • sequence information please refer to the AAV genome (NCBI accession number: AF043303).
  • the pfast-CAP-REP control plasmid was constructed by gene synthesis and seamless cloning.
  • the intron sequence containing the PH promoter was inserted between nucleotides 530 and 531 of the Rep coding sequence, and its sequence information can refer to the AAV genome (NCBI accession number: AF043303) for Rep52/Rep40 expression.
  • the Rep sequence is driven by the P10 promoter to express Rep78/Rep68.
  • the sequence information can refer to the AAV genome (NCBI accession No.: AF043303).
  • the constructed pfast-CAP-REP control plasmid was transformed into DH10BAC (Thermofisher) competent to construct the corresponding bacmid, and the bacmid was transfected into SF9 cells to package BAC-CAP-REP baculovirus. See Table 1 for relevant primers.
  • the AD sequence is placed at the TAA stop codon position of the nucleotide of the REP coding sequence, so as to construct the pfast-CAP-REP-(REP-AD) plasmid and package the baculovirus . See Table 1 for relevant primers.
  • This example uses a two baculovirus system to produce AAV2 adeno-associated virus.
  • the first baculovirus BAC-CAP-REP-(PH-AD) contains the AD sequence between the AAV2 Rep gene expression cassette and the Cap gene expression cassette to produce AAV2 in insect cells, as shown in Figure 2 .
  • FIG. 1 Refer to Example 1 for the construction of the pfast-CAP-REP-(PH-AD) plasmid. See Table 1 for relevant primers.
  • This example uses a two baculovirus system to produce AAV2 adeno-associated virus.
  • the first baculovirus BAC-CAP-REP-(CAP-AD) contained the AD sequence behind the stop codon of the Cap gene expression cassette to produce AAV2 in insect cells, as shown in FIG. 3 .
  • FIG. 3 Refer to Example 1 for the construction of the pfast-CAP-REP-(CAP-AD) plasmid. See Table 1 for relevant primers.
  • Example 1 the first baculoviruses constructed in Example 1, Example 2, and Example 3 containing AD sequences at different positions were packaged. Baculovirus titer determinations were performed as described. By comparing with the control group BAC-CAP-REP containing Rep gene expression cassette and Cap gene expression cassette, but without AD sequence. It was found that different permutations and combinations containing AD sequences had different effects on increasing the titer of baculovirus, and the results are shown in Figure 4 and Table 2. Among them, the AD sequence is located behind the CAP gene expression frame, namely BAC-CAP-REP-(CAP-AD), which is increased by 3.3 times.
  • the AD sequence is located between the REP gene expression box and the CAP gene, that is, BAC-CAP-REP-(PH-AD), which is increased by 33.1 times.
  • AD is located behind the expression box of REP gene, namely BAC-CAP-REP-(REP-AD), which is increased by 21.3 times.
  • the first baculovirus expression vector containing the Rep and Cap protein gene expression cassettes and the AD sequence packaged in Implementation 4 was used to carry out AAV2 adeno-association with the second baculovirus expression vector containing ITR and GFP gene expression cassettes on both sides.
  • the results show that different AD sequence positions have different elevating effects on baculovirus-mediated adeno-associated virus packaging.
  • the results are shown in Figure 5 and Table 3.
  • the AD sequence is located behind the CAP gene, that is, AAV-CAP-REP-(CAP-AD), which is increased by 1.34 times.
  • the AD sequence is located behind the REP gene, that is, AAV-CAP-REP-(REP-AD) is increased by 3.77 times.
  • the AD sequence is located between the REP gene and the CAP gene, that is, AAV-CAP-REP-(PH-AD), which increases 2.58 times.

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Abstract

本文公开了提高杆状病毒系统生产腺相关病毒的方法及应用。所述重组杆状病毒载体包含AAV衣壳蛋白基因表达盒（Cap）、AAV Rep基因表达盒（Rep）和腺相关病毒反向末端重复序列（ITR）的AD序列（AD），所述ITR的AD序列可位于Rep基因表达盒后面，或者Rep基因表达盒以及Cap基因表达盒之间，及Cap基因表达盒后面。与包含Cap和Rep但没有AD序列的对照相比，携带包含Cap、Rep和AD序列的重组杆状病毒的昆虫细胞中杆状病毒和AAV的产生水平更高。本发明的方法应用于制备重组腺相关病毒，通用性强，效率高。

Description

一种提高杆状病毒系统生产腺相关病毒的方法及应用
本申请要求于2022年1月25日提交至中国专利局、申请号为202210087849.1、发明名称为“一种提高杆状病毒系统生产腺相关病毒的方法及应用”的中国专利申请的优先权，在此通过引用将其全文并入本文。
技术领域
本发明涉及杆状病毒表达载体，尤其是带有腺相关病毒AD序列的杆状病毒表达载体。本发明还涉及该杆状病毒表达载体制备重组腺相关病毒的应用。
背景技术
腺相关病毒载体(AAV)是体外体内基因转移可靠的工具。其靶向性好，安全性高，转导能力强，在许多基因治疗临床试验中得到广泛的使用。包括家族性脂蛋白脂肪酶缺乏症，血友病，眼科以及肌肉脊髓等遗传疾病，均有在研临床报道。
腺相关病毒难以大规模培养导致其药物价格昂贵是限制其临床运用的主要障碍之一。目前腺相关病毒生产系统主要包括HEK293细胞系统、昆虫细胞杆状病毒系统、疱疹病毒系统。HEK293系统包装可贴壁培养以及悬浮培养，同时也包括两质粒或者三质粒转染的方式。其生产周期短，但涉及质粒，细胞等方面较为复杂，导致在实际生产过程中很难进行大规模培养，价格昂贵。疱疹病毒系统生产腺相关病毒，过程可能存在致敏性疱疹病毒，其安全性存在一定的潜在风险。昆虫细胞杆状病毒系统，由于其具有细胞密度高，可规模化培养，且安全性较好等优点，被认为是潜在克服腺相关病毒难以大规模培养的技术壁垒突破口之一。优化提高杆状病毒系统介导的腺相关病毒生产效率，是科学家的重点工作。
腺相关病毒反向末端重复序列(inverted terminal repeat，ITR)包含145个核苷酸，分为A、B、C、D四个组成部分。其中A-A’,B-B’,C-C’三个部分125核苷酸组成T型回文发夹结构，作为病毒DNA复制引物起始腺相关病毒复制。A序列含有RBE(rep binding element)结构，是REP蛋白识别绑定位点 1。B、C序列作为发夹结构臂，一旦突变或缺失，AAV包装能力显著下降 2。D序列独立于T型发夹结构，对于腺相关病毒包装基因长度，病毒DNA复制以及病毒的感染能力至关重要 3-4。AD序列还包含转录起始信号(A序列88 to 95，D序列126to 133)可以作为启动子起始基因转录 5。George Aslanidi等人通过杆状病毒同源重组序列(HR)与RBE元件，提出通过杆状病毒即刻早期反式调节因子1(IE-1)诱导结合HR元件，增强Rep，Cap基因表达，同时积累的Rep蛋白结合RBE元件，进一步促进Rep蛋白的积累 6。然而使用AD序列促进杆状病毒包装，以及AD序列对杆状病毒系统生产的腺相关病毒效率的影响并未有相关报道。
发明内容
在一方面，本文提供了分离的核酸分子，其包括：
1)腺相关病毒(AAV)复制基因(Rep)表达盒和/或AAV衣壳蛋白基因(Cap)表达盒；以及
2)来自AAV反向末端重复序列(ITR)的AD序列或其功能性变体。
在一些实施方案中，所述AD序列或其功能性变体位于所述Rep表达盒内或者距离所述Rep表达盒不超过5kb。
在一些实施方案中，所述AD序列或其功能性变体位于所述Cap表达盒内或者距离所述Cap表达盒不超过5kb。
在一些实施方案中，所述核酸分子中：1)所述AD序列或其功能性变体位于所述Rep表达盒内，并且距离所述Cap表达盒不超过5kb；2)所述AD序列或其功能性变体位于所述Cap表达盒内，并且距离所述Rep表达盒不超过5kb；或者3)所述AD序列或其功能性变体位于所述Rep表达盒和所述Cap表达盒之外，并且距离所述Rep表达盒和所述Cap表达盒均不超过5kb。
在一些实施方案中，所述AD序列或其功能性变体位于所述Rep表达盒内且位于终止密码子之后，和/或所述AD序列或其功能性变体位于所述Cap表达盒内且位于终止密码子之后。
在一些实施方案中，所述Rep表达盒和所述Cap表达盒以不同方向转录，并且所述AD序列或其功能性变体位于所述Rep表达盒的启动子和所述Cap表达盒的启动子之间。
在一些实施方案中，所述Rep表达盒和/或所述Cap表达盒的启动子为昆虫细胞启动子
在一些实施方案中，所述Rep表达盒和所述Cap表达盒的启动子分别独立地选自P10和Polh启动子。
在一些实施方案中，所述AD序列包括SEQ ID NO:56所示的核苷酸序列。
在一些实施方案中，所述AD序列的功能性变体包括与SEQ ID NO:56所示的核苷酸序列有至少60％、70％、80％或90％序列一致性的核苷酸序列，并且，当将所述核酸分子引入适合的宿主细胞后，能够提高所述AAV复制基因(Rep)表达盒和/或所述AAV衣壳蛋白基因(Cap)表达盒的在所述宿主细胞中的表达量。
在一些实施方案中，所述Rep表达盒用于转录和表达Rep78/Rep68和Rep52/Rep40蛋白。
在一些实施方案中，所述Cap表达盒用于转录和表达VP1以及VP2/VP3蛋白。
在一些实施方案中，所述Rep表达盒和/或所述Cap表达盒包括含有启动子序列的内含子序列。
在一些实施方案中，所述内含子序列含有的所述启动子为polh启动子。
在一些实施方案中，所述内含子序列包括SEQ ID NO:57所示核苷酸序列或其功能性变体。
在一些实施方案中，所述内含子序列位于Rep表达盒中Rep编码核苷酸序列530和531之间，以驱动Rep52/Rep40亚基编码序列的转录。
在一些实施方案中，所述其中所述内含子序列位于Rep表达盒中Cap编码核苷酸序列25和26之间，以驱动VP2和VP3亚基编码序列的转录。
另一方面，本文提供了包括上述核酸分子的表达载体。
在一些实施方案中，所述表达载体为以pFastBac TM Dual为骨架的表达载体。
在一些实施方案中，所述表达载体为杆状病毒表达载体。
在一些实施方案中，所述杆状病毒载体通过Bac-to-Bac TM杆状病毒表达系统制备。
在一些实施方案中，所述表达载体还包括另外的表达盒。
在一些实施方案中，所述另外的表达盒从5'至3'包括：
1)AAV的ITR；
2)启动子；
3)与所述启动子可操作地连接的目的基因；
4)多聚腺苷酸化信号；以及
5)AAV的ITR。
在一些实施方案中，所述另外的表达盒中的启动子为哺乳动物细胞启动子。
在一些实施方案中，所述目的基因包括蛋白或RNA编码序列。
另一方面，本文提供了宿主细胞，其包括上述核酸分子或表达载体。
在一些实施方案中，所述核酸分子整合在所述宿主细胞的基因组中。
在一些实施方案中，所述宿主细胞为昆虫细胞。
在一些实施方案中，所述宿主细胞为昆虫细胞SF9、SF21或high five。
另一方面，本文提供了杆状病毒表达载体组合，其包括
1)第一杆状病毒表达载体，其包括上述核酸分子，其中该核酸分子包括所述Rep表达盒和所述Cap表达盒；以及
2)第二杆状病毒表达载体，其包括目的基因表达盒和位于所述目的基因基因表达盒两侧的AAV的ITR。
另一方面，本文提供了杆状病毒表达载体组合，其包括
1)第一杆状病毒表达载体，其包括上述核酸分子，其中该核酸分子包括所述Rep表达盒并且不包括所述Cap表达盒；
2)第二杆状病毒表达载体，其包括上述核酸分子，其中该核酸分子包括所述Cap表达盒并且不包括所述Rep表达盒；
3)第三杆状病毒表达载体，其包括目的基因表达盒和位于所述目的基因基因表达盒两侧的AAV的ITR。
另一方面，本文提供了包括上述杆状病毒表达载体组合的宿主细胞。
另一方面，本文提供了宿主细胞，其包括
1)整合进所述宿主细胞的基因组中的上述核酸分子；以及
2)表达载体，其包括目的基因表达盒和位于所述目的基因表达盒两侧的AAV的ITR。
另一方面，本文提供了产生rAAV的方法，其包括
1)在培养基中培养包括上述杆状病毒表达载体组合的昆虫细胞；
2)从所述培养基和/或所述昆虫细胞中回收所述rAAV。
另一方面，本文提供了在昆虫细胞中产生rAAV的方法，其包括：
1)用第一杆状病毒和第二杆状病毒共感染所述宿主细胞，其中所述第一杆状病毒包括上述核酸分子，其中该核酸分子包括所述Rep表达盒和所述Cap表达盒；第二杆状病毒包括目的基因表达盒和位于所述目的基因基因表达盒两侧的AAV的ITR；
2)在培养基中培养所述昆虫细胞；以及
3)从所述培养基和/或所述昆虫细胞中回收所述rAAV。
另一方面，本文提供了在昆虫细胞中产生rAAV的方法，包括：
1)用第一杆状病毒、第二杆状病毒和第三杆状病毒共感染所述宿主细胞，其中所述第一杆状病毒包括上述核酸分子，其中该核酸分子包括所述Rep表达盒且不包括所述Cap表达盒；第二杆状病毒包括上述核酸分子，其中该核酸分子包括所述Cap表达盒且不包括所述Rep表达盒；第三杆状病毒包括目的基因表达盒和位于所述目的基因基因表达盒两侧的AAV的ITR；
2)在培养基中培养所述昆虫细胞；以及
3)从所述培养基和/或所述昆虫细胞中回收所述rAAV。
另一方面，本文提供了在昆虫细胞中产生AAV的方法，其包括：
1)将上述核酸分子整合进所述昆虫系的基因组，其中该核酸分子包括所述Rep表达盒和所述Cap表达盒；
2)以杆状病毒载体感染所述昆虫细胞，所述杆状病毒载体包括目的基因表达盒和位于所述目的基因基因表达盒两侧的AAV的ITR；
3)在培养基中培养所述昆虫细胞；以及
4)从所述培养基和/或所述昆虫细胞中回收所述rAAV。
在一些实施方案中，上述AAV或rAAV的血清型为1、2、3、4、5、6、7、8、9、10、11、12或13。
本文提供了提高杆状病毒系统生产腺相关病毒的方法，通过AD序列与不同位置的Rep，Cap组合，显著提高了杆状病毒的滴度，以及杆状病毒生产腺相关病毒的效率。减少了实际生产过程中，杆状病毒的传代次数，解决了杆状病毒种子库建库时间长，代数高导致的遗传稳定性差等缺点，缩短了杆状病毒系统生产腺相关病毒的周期。
附图说明
图1显示了实施例1中设计的含AD序列的表达载体图谱，其按5'至3'的顺序包含所述AD序列，Rep表达盒及Cap表达盒。
图2显示了实施例2中设计的含AD序列的表达载体图谱，其按5'至3'的顺序包含所述Rep表达盒，AD序列及Cap表达盒。
图3显示了实施例3中设计的含AD序列的表达载体图谱，其按5'至3'的顺序包含所述Rep表达盒，Cap表达盒及AD序列。
图4显示了实施例4中不同AD序列、Rep表达盒及Cap表达盒位置排列组合对杆状病毒包装滴度的影响。
图5显示了实施例5中不同AD序列、Rep表达盒及Cap表达盒位置排列组合对杆状病毒生产腺相关病毒产量的影响。
具体实施方式
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。
“和/或”，指其前后两要素仅存在其中之一，或者二者都存在。例如，“X和/或Y”，应被理解为意指“X”、“Y”或者“X和Y”。
本文中，术语“核酸分子”、“核酸”和“多核苷酸”可互换使用，指核苷酸聚合物。此类核苷酸聚合物可含有天然和/或非天然核苷酸且包括(但不限于)DNA、RNA和PNA。“核酸序列”指包含于核酸分子或多核苷酸中的核苷酸线性序列。“分离的核酸分子”指该核酸分子脱离了其所存在的天然环境(如细胞内环境)，基本上不含通常与其天然相关联的一种或更多种物质，例如蛋白质、核酸、脂质、碳水化合物、细胞膜等，或者为人工制备的(如人工合成的)核酸分子。
“腺相关病毒(AAV)”是细小病毒科的非包膜二十面体衣壳病毒，包括单链DNA病毒基因组。细小病毒科包括依赖病毒属，其包括AAV，依赖于辅助病毒如腺病毒的存在来进行其复制。归因于结构相对简单，能够感染多种细胞(包括静止和分裂细胞)而不整合到宿主基因组中以及其相对温和的免疫原性特征，AAV已被证明可用作生物学工具。本文还考虑了基于AAV的表达载体，包括带有目的基因而用于治疗目的的重组AAV(rAAV)。
野生型AAV病毒基因组是一个线性、单链DNA(ssDNA)分子，长度为约5,000个核苷酸(nt)。反向末端重复序列(ITR)通常在5'和3'末端对病毒基因组封端，为病毒基因组提供复制起点。AAV病毒基因组通常包括两个ITR序列。这些ITR具有特征性T形发夹结构。双链发夹结构包括多种功能，包括但不限于通过充当宿主病毒复制细胞的内源DNA聚合酶复合物的引物来充当DNA复制的起点。
野生型AAV病毒基因组还包括两个开放阅读框的核苷酸序列，一个为四个非结构性Rep蛋白(Rep78、Rep68、Rep52、Rep40，由Rep基因编码)，并且另一个为三个衣壳或结构性蛋白(VP1、VP2、VP3，由衣壳基因或Cap基因编码)。Rep蛋白对于复制和包装很重要，而衣壳蛋白则组装以形成AAV的蛋白外壳或AAV衣壳。交替的剪接和交替的起始密码子和启动子导致从单个开放阅读框产生四种不同的Rep蛋白，并从单个开放阅读框产生三种衣壳蛋白。
提及AAV情况下，所用的术语“病毒衣壳蛋白”或“衣壳蛋白”指AAV的能够自装配以产生AAV颗粒的蛋白质，也称为外壳蛋白或VP蛋白。其由三种亚基VP1、VP2和 VP3组成，它们通常由单一核酸分子表达，并且在一起相互作用形成二十面体对称的衣壳。
提及AAV情况下所用的术语“血清型”用于指AAV的衣壳蛋白在血清学上不同于其它AAV血清型的区别。血清学独特性的确定是基于一种抗体与一种AAV之间具有反应性，而与其他或另一种AAV缺乏交叉反应性。这种交叉反应性差异通常是由于衣壳蛋白序列/抗原决定簇的差异(例如，由于AAV血清型的VP1、VP2和/或VP3序列差异)所致。
“重组AAV载体(rAAV)”指通过使用分子生物学方法从AAV基因组中去除部分野生型基因(例如Rep基因和Cap基因)，并以异源核酸序列(例如用于治疗目的的蛋白或RNA的编码序列)替换而衍生的AAV基因组。通常，对于rAAV载体来说，AAV基因组的一个或两个反向末端重复(ITR)序列保留其中。大多数情况下，rAAV是复制缺陷的，在其病毒基因组中缺乏编码功能性Rep和Cap蛋白的序列。这些复制缺陷的AAV颗粒可能缺乏大多数亲本编码序列，并且基本上仅携带一个或两个AAV ITR序列和用于递送至细胞、组织、器官或生物体的目标核酸。
术语“表达盒(expression cassette)"指在适合的表达环境(如细胞内环境)下能够使其中的编码序列(例如蛋白编码序列或RNA编码序列)进行转录，并视情况进一步进行翻译(例如产物为蛋白)的一段核酸序列。表达盒通常包括转录调控序列(例如启动子，增强子)、与转录调控序列可操作地连接的编码序列和多聚腺苷酸化信号(或转录终止信号)，其中转录调控序列通常位于编码序列上游。另外，表达盒通常还包括为5’非翻译区(5'-UTR)或3’非翻译区(3’-UTR)。
术语“启动子(promoter)”或“启动子序列”指参与DNA依赖性RNA聚合酶及其它蛋白质(反式作用转录因子)的识别和结合以启动和控制一个或多个编码序列的转录的一段DNA序列。其通常位于转录起始位点的上游(即5’端)，并包括一些保守序列，例如TATA盒等。在本文的一些实例中，一些启动子用于在昆虫细胞中启动Rep表达盒及Cap表达盒的转录。这些启动子包括P10启动子或polh启动子(或称为PH启动子)。
术语“增强子(enhancer)”或“增强子序列”指表达载体上可增强启动子转录活性的DNA序列。增强子增强转录的活性通常无方向性(即可位于目的蛋白编码基因的上游、下游，甚至内部)，并且在一些情况下可以远距离起作用(例如距转录起始位点数个Kb)。
术语“可操作地连接”指多核苷酸元件之间具有功能关系的连接。当核酸或多核苷酸序列被置于与另一核酸序列具有功能关系时，它们之间是“可操作地连接的”。比如，转录调控序列例如启动子、增强子或本领域中公知的其它表达控制元件如果影响编码序列的转录，则其与该编码序列将是可操作地连接的。
术语“多聚腺苷酸化信号(Polyadenylation signal)”指终止转录过程并把一段多聚A尾巴加到转录生成的mRNA 3’末端的机制。该加尾过程由多聚腺苷酸聚合酶催化进行，进而形成成熟的mRNA。相应地，表达盒上与该多聚腺苷酸化相关的序列称为“多聚腺苷酸化信号序列”或“PolyA信号区(PolyA signal)”。
术语“非翻译区(Untranslated Region,UTR)”指位于mRNA上编码区两端不翻译为蛋白质的片段。其中位于编码区上游的mRNA片段被称为5’非翻译区(5'-UTR)，位于编码区下游的片段则被称为3’非翻译区(3’-UTR)。非翻译区可以在基因转录和转录后翻译水平上调控基因表达，例如，5'非翻译区能够影响基因的mRNA的转录效率、稳定性以及出核转运，从而导致蛋白表达水平的改变；3'非翻译区则主要影响mRNA的稳定性和翻译效率。为方便阐述，将表达载体或基因序列中分别与上述非翻译区对应的DNA片段也称为5’非翻译区(5'-UTR)或3’非翻译区(3’-UTR)。
术语“表达载体(expression vector)”指包含各种表达元件以用于在宿主细胞中表达目的蛋白或目的RNA的核酸分子。对于用于在真核细胞中表达目的蛋白的表达载体，这些表达元件通常包括启动子、增强子、多腺苷酸化信号序列等。为了方便在大肠杆菌中扩增，该表达载体通常还包括大肠杆菌复制子序列。此外，表达载体还可包括用于筛选的抗生素抗性基因或选择标记基因(例如氨苄青霉素抗性基因(AmpR)，胸苷激酶基因(TK)，卡那霉素抗性基因(KanR)，新霉素抗性基因(NeoR)等)和用于目的基因插入的多克隆位点(MCS)。在本发明的一些实施方案中，表达载体为杆状病毒表达载体或rAAV表达载体。
杆状病毒是特异感染节肢动物的双链DNA病毒，苜蓿银纹夜蛾核型多角体病毒(Autographa californica nucleopolyhedrovirus，AcMNPV)是杆状病毒的模式种。自从1983年Smith GE等首次用杆状病毒在昆虫细胞中表达人β-干扰素基因以来，由于具有低成本、高产量，而且具备各种翻译后修饰系统等优点，杆状病毒表达载体在科研和生产中被广泛应用。将杆状病毒基因组用作表达载体时，可以将目的基因置于杆状病毒超量表达启动子的调控下，使目的基因在感染末期超量表达。杆状病毒感染昆虫细胞时，在感染的极晚期有两个蛋白质可以超量表达：多角体蛋白和p10蛋白。它们对病毒在体外培养细胞中复制都是非必须的，所以可以利用这两个基因的启动子启动目的基因的表达，可以在感染末期高水平地生产所需的蛋白。通常，杆状病毒基因组用作表达载体时，为了容纳更多的外源基因或增加目的蛋白的表达量，可将其基因组中非必须序列去除。杆状病毒表达载体的优点包括：病毒遗传背景清楚；基因容量大，能承载较大目的基因；生物安全性好，天然情况下不能感染人类；可对表达的蛋白进行糖基化修饰等。杆状病毒是一种优良的蛋白表达载体，尤其适合于生物工程的酶类，药物类等活性蛋白的大规模生产。
术语“AD序列”指相互连接的来自AAV的ITR的A序列和D序列。在一个具体实例中，AD序列的如SEQ ID NO:56所示。
SEQ ID NO:56：gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct。
AD序列的“功能性变体”指基本上保留了AD序列促进杆状病毒载体以及rAAV的生成的能力，但在核苷酸序列上与SEQ ID NO:56所示序列有差异。在一些实施方案中，该功能性变体相对于SEQ ID NO:56所示序列有1个、2个、3个、4个、5个、6个、7个、8个、9个、10个或更多个核苷酸替换、添加或删除。在另一些实施方案中，该功能性变体相对于SEQ ID NO:56所示序列有至少60％，例如至少70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列一致性。在一些实 施方案中，该功能性变体相对于SEQ ID NO:56所示序列，在保留存进杆状病毒载体和/或rAAV的生成方面，保留了SEQ ID NO:56所示序列功能的至少30％、40％、50％、60％、70％、80％、90％或甚至100％。优选地，该功能性变体相对于SEQ ID NO:56所示序列，在保留存进杆状病毒载体和/或rAAV的生成方面，保留了SEQ ID NO:56所示序列功能的至少80％、90％或甚至100％。本领域技术人员可通过对SEQ ID NO:56所示序列进行各种替换、添加或删除操作，并验证所获得序列的功能，从而获得这些功能性变体。
提及AD序列(或其功能性变体)的位置时，“距离表达盒不超过5kb”指同一核酸分子(线性或环状分子)中，AD序列(或其功能性变体)的末端核苷酸与表达盒末端核苷酸之间的距离，以核苷酸数量表示，不超过5000个核苷酸，例如不超过4000个核苷酸，不超过3000个核苷酸，不超过2000个核苷酸，甚至不超过1000个核苷酸。在一些实施方案中，AD序列(或其功能性变体)与表达盒的两相近末端的核苷酸距离不超过5kb。当AD序列或其功能性变体处于表达盒内部时，认为它们之间的距离为0个核苷酸。
术语“终止密码子”指以mRNA为模板合成蛋白质的过程中终止肽链延长的三核苷酸序列。常见的终止密码子为UAG、UAA和UGA，相应的编码DNA上的终止密码子序列是TAG、TAA和TGA。
提及AD序列(或其功能性变体)位于“终止密码子之后”，指AD序列(或其功能性变体)位于所在的表达盒中终止密码子(如TAG、TAA或TGA)的下游(即3'方向)，尤其是位于下游并紧接该终止密码子。
当AD序列(或其功能性变体)“位于两启动子之间”时，指这两个启动子之间距离较近(例如相隔几十个至几百个核苷酸)，不被其他功能性长序列(例如其他表达盒)隔开。这种情况下，这两个启动子可以认为是“背靠背”存在，并且以不同方向驱动转录，如pFastBac TM Dual载体中的P10启动子和Polh启动子。
术语“宿主细胞”指表达载体可在其中维持和/或复制的细胞，包括原核细胞和真核细胞，例如细菌(如大肠杆菌)、真菌(酵母菌)、昆虫细胞以及哺乳动物细胞。在本文提供的制备rAAv的方法中，所用的宿主细胞优选昆虫细胞，例如SF9、SF21或high five细胞系。
术语“内含子”指基因序列的非编码序列，其可被转录，但在mRNA加工成熟过程中被剪切掉，故其序列不体现在成熟mRNA上。因此，在一个用于产生目的蛋白的表达盒中，编码DNA序列可被一个或更多个内含子隔开，但均可转录和翻译为相同的目的蛋白。
术语“含启动子的内含子”指该内含子中含有的启动子可启动其下游编码序列的转录，但是基本上不影响其上游启动子驱动的编码序列(其被该内含子隔断)的转录。这种含启动子的内含子尤其可用于驱动相互间有重叠的目的基因的转录。在一个具体实例中，可将其用于驱动Rep蛋白(包括Rep78、Rep68、Rep52和Rep40亚基)或VP蛋白(包括VP1、VP2和VP3亚基)的转录。
专利文献(CN200780037031.2)中描述了可用于同时表达Rep蛋白或VP蛋白各亚基的含Polh启动子的内含子，其内含子序列为：
GTAAGTACTCCCTATCAGTGATAGAGATCTATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGAAGGGGGAGACCTGTAGTCAGAGCCCCCGGGCAGCACACACTGACATCCACTCCCTTCCTATTGTTTCAG(SEQ ID NO:57)。该内含子内的polh启动子序列为：
Figure PCTCN2023071631-appb-000001
类似地，除了上述内含子序列之外，也可以使用上述内含子序列的功能性变体，只要它们可以实现或基本上实现上述功能。该内含子的功能性变体保留或基本上保留了驱动其下游编码序列的转录的能力(同时不影响或基本上不影响其上游启动子驱动的编码序列(其被该内含子隔断)的转录)，但在核苷酸序列上与SEQ ID NO:57所示序列有差异。在一些实施方案中，该功能性变体相对于SEQ ID NO:57所示序列有1个、2个、3个、4个、5个、6个、7个、8个、9个、10个或更多个核苷酸替换、添加或删除。在另一些实施方案中，该功能性变体相对于SEQ ID NO:57所示序列有至少60％，例如至少70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列一致性。本领域技术人员可通过对SEQ ID NO:57所示序列进行各种替换、添加或删除操作，并验证所获得序列的功能，从而获得这些功能性变体。
当提及核苷酸序列时，术语“序列一致性(sequence identity)”(也称为“序列同一性”)指两核苷酸序列(例如查询序列和参照序列)之间一致性程度的量，一般以百分比表示。通常，在计算两核苷酸序列之间的一致性百分比之前，先进行序列比对(alignment)并引入缺口(gap)(如果有的话)。如果在某个比对位置，两序列中的碱基相同，则认为两序列在该位置一致或匹配；两序列中的碱基不同，则认为在该位置不一致或错配。在一些算法中，用匹配位置数除以比对窗口中的位置总数以获得序列一致性。在另一些算法中，还将缺口数量和/或缺口长度考虑在内。常用的序列对比算法或软件包括DANMAN、CLUSTALW、MAFFT、BLAST、MUSCLE等。出于本发明的目的，可以采用公开的比对软件BLAST(可从https://www.ncbi.nlm.nih.gov/获得)，通过使用缺省设置来获得最佳序列比对并计算出两核苷酸序列之间的序列一致性。
Bac-to-Bac TM杆状病毒表达系统为Thermo Fisher公司提供了一种昆虫杆状病毒表达系统，该系统依赖一个位点特异性的转座可将感兴趣的一段DNA序列(例如一个或更多个表达盒)重组到杆状病毒穿梭质粒(bacmid)上。该系统包括如下元件：pFastBac TM载体或类似载体(例如pFastBac TM Dual)，含有转座子元件Tn7-L和Tn7R，它们之间的DNA序列(例如一个或更多个表达盒，抗性基因等)在转座蛋白作用下可被剪切并插入目标位点(如mini-attTn7)；DH10Bac菌株，为感受态大肠杆菌，也是pFastBac TM等载体的宿主菌，其含有大肠杆菌-杆状病毒穿梭质粒(bacmid)和辅助质粒，bacmid中含有mini-attTn7位点， 辅助质粒编码转座蛋白。一旦pFastBac TM等载体转化进入DH10Bac菌株中，Tn7-L和Tn7R之间的DNA序列转座插入bacmid的mini-attTn7位点，形成重组bacmid。重组bacmid在感染昆虫细胞，形成重组杆状病毒，随后可进行多代感染，并进行大规模重组蛋白表达。关于Bac-to-Bac TM杆状病毒表达系统的更多描述，可参见厂商说明书。
pFastBac TM Dual表达载体与pFastBac TM载体类似，但其含有两个可引入外源基因的多克隆位点，分别位于启动子P10和Polh下游，因此可用于在昆虫细胞中同时表达两种外源蛋白。
关于Bac-to-Bac TM杆状病毒表达系统和pFastBac TM Dual表达载体的更多描述，可参见Thermo Fisher公司提供的说明书。
本文至少部分地基于意外地发现，当将AD序列引入表达载体(如杆状病毒表达)中时，可促进杆状病毒的产生，并且当将该杆状病毒用于生产rAAV时，可大大提高rAAV的产量。
因此，在一些实施方案中，本文提供了包括AD序列的核酸分子。该核酸分子可包括一个或更多个用于目的蛋白表达的表达盒。
当所述核酸分子包括一个表达盒时，AD序列可位于该表达盒的上游、下游或其中。当AD序列不位于表达盒中时，优选其距离该表达盒不超过5kb，例如距离该表达盒4kb、3kb、2kb、1kb、0.5kb、0.4kb、0.3kb、0.2kb、0.1kb或更近。当AD序列位于表达盒中时，其可以位于5’非翻译区(5'-UTR)或3’非翻译区(3’-UTR)中，或者可将其至于内含子中，并将该内含子至于编码序列之间。优选地，当AD序列位于表达盒中时，其位于终止密码子之后；更优选地，其位于终止密码子之后并紧接终止密码子。
当所述核酸分子包括两个表达盒时，AD序列可位于任一表达盒的上游、下游或其中。当AD序列不位于任一表达盒中时，优选其距离两个表达盒都不超过5kb，例如距离两个表达盒4kb、3kb、2kb、1kb、0.5kb、0.4kb、0.3kb、0.2kb、0.1kb或更近。尤其是，当该两个表达盒通过“背靠背”的两个启动子表达时，AD序列可位于该两个启动子之间。
在一些实施方案中，所述核酸分子含有用于表达Rep蛋白(Rep78、Rep68、Rep52和Rep40)的Rep表达盒。在另一些实施方案中，所述核酸分子含有用于表达VP蛋白(VP1、VP2和VP3)的Cap表达盒。在又一些实施方案中，所述核酸分子含有用于表达Rep蛋白(Rep78、Rep68、Rep52和Rep40)的Rep表达盒和用于表达VP蛋白(VP1、VP2和VP3)的Cap表达盒。
相应地，本文提供了含有上述核酸分子的表达载体，尤其是杆状病毒表达载体。在一些实施方案中，该杆状病毒表达载体可通过Bac-to-Bac TM杆状病毒表达系统产生。
在一些实施方案中，所述杆状病毒载体，按5’至3’的顺序包含所述AD序列，Cap表达盒及所述Rep表达盒组合。
在一些实施方案中，所述杆状病毒载体按5’至3’的顺序包含所述AD序列，Rep表达盒及Cap表达盒组合。
在一些实施方案中，所述杆状病毒载体按5’至3’的顺序包含所述Rep表达盒，AD序列及Cap表达盒组合。
在一些实施方案中，所述杆状病毒载体按5’至3’的顺序包含所述Cap表达盒，AD序列及Rep表达盒组合。
在一些实施方案中，所述杆状病毒载体按5’至3’的顺序包含所述Cap表达盒，Rep表达盒及AD序列组合。
在一些实施方案中，所述杆状病毒载体按5’至3’的顺序包含所述Rep表达盒，Cap表达盒及AD序列组合。
另外，本文还提供了通过杆状病毒载体制备重组腺相关病毒(rAAV)的方法。
在一些实施方案中，采用一杆状病毒系统在制备rAAV。在一些实施方案，可将含有Rep表达盒、Cap表达盒和AD序列(或其功能性变体)的核酸分子整合进宿主细胞，例如昆虫细胞，并向该宿主细胞中引入带有AAV的ITR序列的目的基因(例如治疗性目标蛋白编码序列)表达载体，可生成并获得可用于含有目的基因的rAAV。在另一些实施方案中，可将含有Rep表达盒、Cap表达盒和AD序列(或其功能性变体)的核酸分子以及两侧带有AAV的ITR序列的目的基因表达盒引入同一杆状病毒表达载体中，并用该杆状病毒感染宿主细胞(例如昆虫细胞)，可生成并获得可用于含有目的基因的rAAV。
在一些实施方案中，采用二杆状病毒系统在制备rAAV。例如，可将含有Rep表达盒、Cap表达盒和AD序列(或其功能性变体)的核酸分子构建在一个杆状病毒表达载体中，同时将两侧带有AAV的ITR序列的目的基因表达盒构建在另一杆状病毒表达载体中，当将这两种杆状病毒感染同一宿主细胞(例如昆虫细胞)，可生成并获得可用于含有目的基因的rAAV。
在另一些实施方案中，采用三杆状病毒系统在制备rAAV。例如，Rep表达盒和Cap表达盒分别构建在不同的两个杆状病毒表达载体中，这两个杆状病毒表达载体的任一个或二者可含有AD序列；同时将两侧带有AAV的ITR序列的目的基因表达盒构建在另一杆状病毒表达载体中，当将这三种杆状病毒感染同一宿主细胞(例如昆虫细胞)，可生成并获得可用于含有目的基因的rAAV。
所获得的rAAV随后可用于感染哺乳动物或哺乳动物细胞，如人或人细胞，并表达目的基因。该目的基因可为任意核苷酸序列，只要所形成的构建体保持在AAV病毒基因组的包装能力以内即可。
该目的基因可编码目的蛋白或目的RNA(如各种干扰小RNA)，并且它们都可以是治疗性基因产物。治疗性基因产物可以是多肽或RNA分子，或者是当在靶细胞中表达时提供所需治疗效果的其它基因产物。治疗效果的实例包括消除非所需活性，例如消除酶活性，或者弥补遗传缺陷等。可选地或者另外地，由所述多核苷酸编码的目的蛋白质可用作评估细胞转化和表达的标记蛋白。用于此目的的合适标记蛋白有例如荧光蛋白GFP或萤火虫荧光素酶。
当采用二杆状病毒载体或三杆状病毒载体来制备rAAv时，本文提供了二杆状病毒载体组合或三杆状病毒载体组合。另外，由于目的基因表达盒可根据使用需要而独立制备，本文实际上也提供了含Rep表达盒、含Cap表达盒或者含Rep表达盒和Cap表达盒的杆状病毒载体，它们可与使用者自己构建的目的基因表达盒配合使用而生产带有期望的目的基因的rAAV。
在一个具体实例中，本文提供的生成rAAV的方法包括以下步骤：
1)包含有REP、CAP及AD序列的杆状病毒包装；
2)杆状病毒载体扩增；
3)杆状病毒载体感染昆虫细胞，收集腺相关病毒，进行分析纯化。
优选地，在该方法中利用pFastBac TM Dual穿梭质粒，基于Bac-to-Bac系统生产杆状病毒载体。
本发明与现有的技术相比取得的下列有益效果：本文公开的一种提高杆状病毒系统生产腺相关病毒方法，通过AD序列与不同位置的Rep，Cap排列组合，显著提高了杆状病毒的包装滴度，约3-33倍，从而减少了杆状病毒传代次数，避免了传代次数过多，所导致的传代不稳定性。同时将本发明提高了杆状病毒系统生产腺相关病毒效率，与没有携带AD序列的组合对比，携带AD序列的杆状病毒载体所生产的腺相关病毒产量提高2-5倍。从而降低了腺相关病毒生产的成本，增加了腺相关病毒的临床运用并降低研发费用。
为了使本发明的目的、技术方案以及优点更加清楚明白，以下结合附图以及实施例，对本发明进一步详细的说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。此处，下面所描述的本发明各个实施方式中所涉及特征只要彼此之间未构成冲突就可以相互组合。
本文提供了提高杆状病毒系统生产腺相关病毒的方法。通过AD序列与不同的位置的Rep、Cap蛋白相互组合，提高杆状病毒滴度及腺相关病毒包装效率。可适用于把不同的杆状病毒系统生产腺相关病毒，如一杆状病毒系统(one bac system)，两杆状病毒系统(two bac system)或三杆状病毒系统(three bac system)。下面以专利文献(CN200780037031.2)内含子嵌入启动子的两杆状病毒系统生产AAV2为例进行说明。
昆虫细胞培养
在28℃条件下，以5E+6cell/ml细胞密度维持昆虫细胞生长。所用培养基为ESF921培养基无血清培养基(Expression Systems)，含有100单位/ml的青霉素以及100ug/ml的链霉素。
制备目的质粒以及Bacmid
采用Bac-to-bac TM系统，pFastBac TM Dual表达质粒来源于Thermofisher公司(货号10712024)。通过PCR扩增等分子操作，将Rep与Cap蛋白基因表达框以及AD序列构建进pFastBac TM Dual中形成第一质粒。同理构建含有两侧ITR以及EGFP基因表达框的第二质粒。根据说明书，将构建好的质粒稀释至2ng/ul的浓度，并且使用2ul的质粒DNA 来转化DH10Bac感受态细胞。孵育2天后，挑取白色菌落并且制备小量制备的杆状病毒DNA Bacmid。将小量制备的杆状病毒DNA Bacmid用于转染Sf9细胞以产生重组杆状病毒。
制备杆状病毒
将提取好的Bacmid与转染试剂，按Bacmid：转染试剂＝1ug：3ul的比例转染SF9细胞。将转染好的细胞置于28℃培养箱，培养5-7天，仔细观察细胞生长状态。5-7天后，收集上清液，离心。作为P0代病毒液。吸取10-100ul进行P1代扩增。简单步骤如下：吸取P0代液体100ul，加入15cm皿SF9细胞中，培养5-6天，观察细胞形态。6天之后，收集上清液，作为P1代。重复以上步骤，吸取100ul，继续感染SF9细胞，作为P2代。
杆状病毒滴度测定
首先利用DNAase I消化以去除病毒液内所含核酸污染。简单说明：将样品5ul样品加入15ul配制好的DNAase I消化体系中，37℃孵育30分钟，100℃灭活10分钟。离心吸取上清进行QPCR反应测定，引物见表1(SEQ ID NO:1和2)。
腺相关病毒包装及滴度测定
取处于对数生长期的SF9细胞，离心收集细胞，按1E+8cell/ml进行分装培养，其中总体积为15ml。含Rep与Cap蛋白基因表达框以及AD序列的第一杆状病毒表达载体，含有两侧ITR以及EGFP基因表达框第二杆状病毒表达载体 ，按MOI比例第一杆状病毒表达载体：第二杆状病毒表达载体＝10：5进行感染，置于28℃细胞摇床进行培养3天。收集细胞以及培养基。加入适量的氯仿以及氯化钠对细胞进行裂解。对所得裂解液进行滴度测定，以反映腺相关病毒包装包装滴度，引物见表1(SEQ ID NO:3和4)。
表1构建杆状病毒载体及其检测所使用的扩增引物
Figure PCTCN2023071631-appb-000002
Figure PCTCN2023071631-appb-000003
Figure PCTCN2023071631-appb-000004
Figure PCTCN2023071631-appb-000005
实施例1
本实施例采用两杆状病毒系统生产AAV2腺相关病毒。在该实施例中，第一杆状病毒BAC-CAP-REP-(REP-AD)包含AD序列位于AAV2Rep基因表达盒终止密码子TAA后面，Rep基因表达盒与Cap基因表达盒，分别在P10，PH启动子作用下进行转录，以在昆虫细胞中产生AAV2，如图1所示。
pFastBac TM Dual表达质粒来源于Thermofisher公司(货号10712024)，含有Rep2及Cap2的pRep2Cap2来自本公司，其序列信息可参考AAV基因组(NCBI登录号：AF043303)。通过基因合成及无缝克隆等方式构建pfast-CAP-REP对照质粒。其中含有PH启动子的内含子序列参考专利200780037031.2。简单说明如下。将含有PH启动子的内含子序列插入Rep编码序列核苷酸530和531之间，其序列信息可参考AAV基因组(NCBI登录号：AF043303)以进行Rep52/Rep40表达。同时Rep序列由P10启动子驱动，以表达Rep78/Rep68。利用PH启动子表达VP1，同时将含有PH启动子的内含子置于CAP编码序列核苷酸25和26之间，以驱动转录表达VP2和VP3蛋白，其序列信息可参考AAV基因组(NCBI登录号：AF043303)。构建好的pfast-CAP-REP对照质粒转化DH10BAC(Thermofisher)感受态，以此构建对应bacmid，将该bacmid转染SF9细胞包装BAC-CAP-REP杆状病毒。相关引物见表1。
在构建好pfast-CAP-REP对照质粒基础上将AD序列置于REP编码序列核苷酸TAA终止密码子位置，以此构建pfast-CAP-REP-(REP-AD)质粒，并包装杆状病毒。相关引物见表1。
实施例2
本实例采用两杆状病毒系统生产AAV2腺相关病毒。在该实例中，第一杆状病毒BAC-CAP-REP-(PH-AD)包含AD序列位于AAV2Rep基因表达盒与Cap基因表达盒之间，以在昆虫细胞中产生AAV2，如图2所示。pfast-CAP-REP-(PH-AD)质粒构建参考实施例1。相关引物见表1。
实施例3
本实例采用两杆状病毒系统生产AAV2腺相关病毒。在该实例中，第一杆状病毒BAC-CAP-REP-(CAP-AD)包含AD序列位于Cap基因表达盒终止密码子后面，以在昆虫细胞中产生AAV2，如图3所示。pfast-CAP-REP-(CAP-AD)质粒构建参考实施例1。相关引物见表1。
实施例4
本实例中，将实施例1，实施例2，实施例3所构建的含有不同位置的AD序列的第一杆状病毒进行包装。根据所述方法进行杆状病毒滴度测定。通过与含有Rep基因表达盒与Cap基因表达盒，但不含AD序列的对照组BAC-CAP-REP相比。发现含有AD序列的不同排列组合对于杆状病毒的滴度提升效果不同，结果如图4和表2所示。其中AD序列位于CAP基因表达框后面，即BAC-CAP-REP-(CAP-AD)，提高3.3倍。AD序列位于REP基因表达框与CAP基因之间，即BAC-CAP-REP-(PH-AD)，提高33.1倍。AD位于REP基因表达框后面，即BAC-CAP-REP-(REP-AD)，提高21.3倍。
表2杆状病毒滴度检测结果
Figure PCTCN2023071631-appb-000006
实施例5
本实施例利用实施4包装好的含Rep与Cap蛋白基因表达框以及AD序列的第一杆状病毒表达载体，与含有两侧ITR以及GFP基因表达框第二杆状病毒表达载体进行AAV2腺相关病毒包装。结果显示不同的AD序列位置，对于杆状病毒介导的腺相关病毒包装具有不同的提升效果结果如图5和表3所示。其中AD序列位于CAP基因后面，即AAV-CAP-REP-(CAP-AD)，提高1.34倍。AD序列位于REP基因后面，即AAV-CAP-REP-(REP-AD)提高3.77倍。AD序列位于REP基因与CAP基因之间，即AAV-CAP-REP-(PH-AD)，提高2.58倍。
表3腺相关病毒滴度检测结果
Figure PCTCN2023071631-appb-000007
参考文献：
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[2]Zhou Q,Tian W,Liu C,et al.Deletion of the B-B'and C-C'regions of inverted terminal repeats reduces rAAV productivity but increases transgene expression[J].Rep,2017,7(1):5432.
[3]Ling C,Wang Y,Lu Y,et al.Enhanced Transgene Expression from Recombinant Single-Stranded D-Sequence-Substituted Adeno-Associated Virus Vectors in Human Cell Lines In Vitro and in Murine Hepatocytes In Vivo[J].Journal of Virology,2015,89(2):952-61.
[4]Wang X S,Ponnazhagan S,Srivastava A.Rescue and Replication Signals of the Adeno-associated Virus 2Genome[J].Journal of Molecular Biology,1995,250(5):573-580.
[5]Haberman R P,Mccown T J,Samulski R J.Novel Transcriptional Regulatory Signals in the Adeno-Associated Virus Terminal Repeat A/D Junction Element[J].Journal of Virology,2000,74(18):8732-8739.
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Claims (38)

  1. 分离的核酸分子，包括：
    1)腺相关病毒(AAV)复制基因(Rep)表达盒和/或AAV衣壳蛋白基因(Cap)表达盒；以及
    2)来自AAV反向末端重复序列(ITR)的AD序列或其功能性变体。
  2. 如权利要求1所述的核酸分子，其中所述AD序列或其功能性变体位于所述Rep表达盒内或者距离所述Rep表达盒不超过5kb。
  3. 如权利要求1或2所述的核酸分子，其中所述AD序列或其功能性变体位于所述Cap表达盒内或者距离所述Cap表达盒不超过5kb。
  4. 如权利要求1-3任一项所述的核酸分子，其中：
    1)所述AD序列或其功能性变体位于所述Rep表达盒内，并且距离所述Cap表达盒不超过5kb；
    2)所述AD序列或其功能性变体位于所述Cap表达盒内，并且距离所述Rep表达盒不超过5kb；或者
    3)所述AD序列或其功能性变体位于所述Rep表达盒和所述Cap表达盒之外，并且距离所述Rep表达盒和所述Cap表达盒均不超过5kb。
  5. 如权利要求1-4任一项所述的核酸分子，其中所述AD序列或其功能性变体位于所述Rep表达盒内且位于终止密码子之后，和/或所述AD序列或其功能性变体位于所述Cap表达盒内且位于终止密码子之后。
  6. 如权利要求1-5任一项所述的核酸分子，其中所述Rep表达盒和所述Cap表达盒以不同方向转录，并且所述AD序列或其功能性变体位于所述Rep表达盒的启动子和所述Cap表达盒的启动子之间。
  7. 如权利要求1-6任一项所述的核酸分子，其中所述Rep表达盒和/或所述Cap表达盒的启动子为昆虫细胞启动子
  8. 如权利要求1-7任一项所述的核酸分子，其中所述Rep表达盒和所述Cap表达盒的启动子分别独立地选自P10和Polh启动子。
  9. 如权利要求1-8任一项所述的核酸分子，其中所述AD序列包括SEQ ID NO:56所示的核苷酸序列。
  10. 如权利要求1-9任一项所述的核酸分子，其中所述AD序列的功能性变体包括与SEQ ID NO:56所示的核苷酸序列有至少60％、70％、80％或90％序列一致性的核苷酸序列，并且，当将所述核酸分子引入适合的宿主细胞后，能够提高所述AAV复制基因(Rep)表达盒和/或所述AAV衣壳蛋白基因(Cap)表达盒的在所述宿主细胞中的表达量。
  11. 如权利要求1-10任一项所述的核酸分子，其中所述Rep表达盒用于转录和表达Rep78/Rep68和Rep52/Rep40蛋白。
  12. 如权利要求1-11任一项所述的核酸分子，其中所述Cap表达盒用于转录和表达VP1以及VP2/VP3蛋白。
  13. 如权利要求1-12任一项所述的核酸分子，其中所述Rep表达盒和/或所述Cap表达盒包括含有启动子序列的内含子序列。
  14. 如权利要求1-13任一项所述的核酸分子，其中所述内含子序列含有的所述启动子为polh启动子。
  15. 如权利要求1-14任一项所述的核酸分子，其中所述内含子序列包括SEQ ID NO:57所示核苷酸序列或其功能性变体。
  16. 如权利要求1-15任一项所述的核酸分子，其中所述内含子序列位于Rep表达盒中Rep编码核苷酸序列530和531之间，以驱动Rep52/Rep40亚基编码序列的转录。
  17. 如权利要求1-16任一项所述的核酸分子，其中所述其中所述内含子序列位于Rep表达盒中Cap编码核苷酸序列25和26之间，以驱动VP2和VP3亚基编码序列的转录。
  18. 包括权利要求1-17任一项所述的核酸分子的表达载体。
  19. 如权利要求18所述的表达载体，其中所述表达载体为以pFastBac TM Dual为骨架的表达载体。
  20. 如权利要求18或19所述的表达载体，其中所述表达载体为杆状病毒表达载体。
  21. 如权利要求18-20任一项所述的表达载体，其中所述杆状病毒载体通过Bac-to-Bac  TM杆状病毒表达系统制备。
  22. 如权利要求18-21任一项所述的表达载体，其中所述表达载体还包括另外的表达盒。
  23. 如权利要求18-22任一项所述的表达载体，其中所述另外的表达盒从5'至3'包括：
    1)AAV的ITR；
    2)启动子；
    3)与所述启动子可操作地连接的目的基因；
    4)多聚腺苷酸化信号；以及
    5)AAV的ITR。
  24. 如权利要求18-23任一项所述的表达载体，其中所述另外的表达盒中的启动子为哺乳动物细胞启动子。
  25. 如权利要求18-24任一项所述的表达载体，其中所述目的基因包括蛋白或RNA编码序列。
  26. 宿主细胞，包括权利要求1-17任一项所述的核酸分子或权利要求18-25任一项所述的表达载体。
  27. 如权利要求26所述的宿主细胞，其中所述核酸分子整合在所述宿主细胞的基因组中。
  28. 如权利要求26或27所述的宿主细胞，其中所述宿主细胞为昆虫细胞。
  29. 如权利要求26-28任一项所述的宿主细胞，其中所述宿主细胞为昆虫细胞SF9、SF21或high five。
  30. 杆状病毒表达载体组合，包括
    1)第一杆状病毒表达载体，其包括权利要求1-17任一项所述的核酸分子，其中所述核酸分子包括所述Rep表达盒和所述Cap表达盒；以及
    2)第二杆状病毒表达载体，其包括目的基因表达盒和位于所述目的基因基因表达盒两侧的AAV的ITR。
  31. 杆状病毒表达载体组合，包括
    1)第一杆状病毒表达载体，其包括权利要求1-17任一项所述的核酸分子，其中所述核酸分子包括所述Rep表达盒并且不包括所述Cap表达盒；
    2)第二杆状病毒表达载体，其包括权利要求1-17任一项所述的核酸分子，其中所述核酸分子包括所述Cap表达盒并且不包括所述Rep表达盒；
    3)第三杆状病毒表达载体，其包括目的基因表达盒和位于所述目的基因基因表达盒两侧的AAV的ITR。
  32. 包括权利要求30或31的杆状病毒表达载体组合的宿主细胞。
  33. 宿主细胞，包括
    1)整合进所述宿主细胞的基因组中的权利要求1-17任一项所述的核酸分子；以及
    2)表达载体，其包括目的基因表达盒和位于所述目的基因表达盒两侧的AAV的ITR。
  34. 产生rAAV的方法，包括
    1)在培养基中培养权利要求32或33任一项所述的昆虫细胞；
    2)从所述培养基和/或所述昆虫细胞中回收所述rAAV。
  35. 在昆虫细胞中产生rAAV的方法，包括：
    1)用第一杆状病毒和第二杆状病毒共感染所述宿主细胞，其中所述第一杆状病毒包括权利要求1-17任一项所述的核酸分子，其中所述核酸分子包括所述Rep表达盒和所述Cap表达盒；第二杆状病毒包括目的基因表达盒和位于所述目的基因基因表达盒两侧的AAV的ITR；
    2)在培养基中培养所述昆虫细胞；以及
    3)从所述培养基和/或所述昆虫细胞中回收所述rAAV。
  36. 在昆虫细胞中产生rAAV的方法，包括：
    1)用第一杆状病毒、第二杆状病毒和第三杆状病毒共感染所述宿主细胞，其中所述第一杆状病毒包括权利要求1-17任一项所述的核酸分子，其中所述核酸分子包括所述Rep表达盒且不包括所述Cap表达盒；第二杆状病毒包括权利要求1-17任一项所述的核酸分子，其中所述核酸分子包括所述Cap表达盒且不包括所述Rep表达盒；第三杆状病毒包括目的基因表达盒和位于所述目的基因基因表达盒两侧的AAV的ITR；
    2)在培养基中培养所述昆虫细胞；以及
    3)从所述培养基和/或所述昆虫细胞中回收所述rAAV。
  37. 在昆虫细胞中产生AAV的方法，包括：
    1)将权利要求1-17任一项所述的核酸分子整合进所述昆虫系的基因组，其中所述核酸分子包括所述Rep表达盒和所述Cap表达盒；
    2)以杆状病毒载体感染所述昆虫细胞，所述杆状病毒载体包括目的基因表达盒和位于所述目的基因基因表达盒两侧的AAV的ITR；
    3)在培养基中培养所述昆虫细胞；以及
    4)从所述培养基和/或所述昆虫细胞中回收所述rAAV。
  38. 如权利要求1-17任一项所述的核酸分子，权利要求18-25任一项所述的表达载体，权利要求26-29、32、33任一项所述的宿主细胞，权利要求30或31所述的宿主细胞，或权利要求34-37所述的方法，其中所述AAV的血清型为1、2、3、4、5、6、7、8、9、10、11、12或13。
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