Virus vector for reducing replicative adenovirus pollution and construction method

Abstract

The invention discloses a virus vector for reducing replication type adenovirus pollution and a construction method. According to the present invention, the homologous recombination probability of the virus vector and the adenovirus E1 region gene integrated in the packaging cell is low, the replicative adenovirus pollution is reduced, and the safety and the effectiveness of the adenovirus gene treatment product are improved.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a virus vector for reducing replication-type adenovirus contamination and a construction method thereof. Background Art

[0002] Adenovirus is a non-enveloped linear double-stranded DNA virus that is widely distributed in nature, with at least 100 serotypes. The mammalian adenovirus genome is about 36 kb long. Figure 1The following is a schematic diagram of the genome structure of adenovirus (Francesco Vetrini, et al. Gene Therapy with Helper-Dependent Adenoviral Vectors: Current Advances and Future Perspectives. Viruses 2010, 2, 1886-1917; doi: 10.3390 / v2091886). There is an inverted terminal repeat (ITR) at each end of the genome. The inner side of the ITR is the viral packaging signal (ψ), which is a cis-acting element required for adenovirus packaging. Adenovirus genes can be divided into two categories: early genes (E) and late genes (L) according to whether they are expressed before or after DNA replication. The four early transcription elements (E1, E2, E3, and E4) have regulatory functions. Among them, the proteins expressed by the early genes E1a and E1b are responsible for transactivating the expression of other viral genes, the E2 protein is responsible for the replication of viral DNA, the E3 protein is responsible for the immune response of host cells, and the E4 protein inhibits the apoptosis of host cells. Late genes encode the structural genes of the virus and some gene regulatory proteins. The genes in the E1 region of adenovirus are activated immediately after the virus enters the cell nucleus. The encoded products are the main transcription regulators that regulate the functions of all early genes. The E1 region is an essential region for viral replication. Adenoviruses that lack the E1 region are a type of virus that must be replicated and proliferated in special engineered cells or with the assistance of non-deleted adenoviruses. Due to the lack of the essential region for replication, adenoviruses can only replicate in auxiliary cells that express the E1 gene product. The most commonly used auxiliary cell is the 293 cell line, which is transfected with the left terminal fragment of Ad5 by human embryonic kidney cells. A fragment containing the adenovirus E1 region is integrated into its genome, and the E1 region protein can be continuously expressed. The genes in the E2 region encode DNA binding protein (DBP, DNA Binding Protein), terminal protein precursor (pTP) and viral DNA polymerase (pol). The three proteins interact with at least three intracellular factors to initiate adenovirus DNA replication and the transcription and translation of viral late genes. E3 is a non-essential region for replication, and its deletion can greatly expand the insertion capacity. The expression of the IVa2 gene is between the expression of early genes and late genes in the life cycle of adenovirus, so it is called an intermediate gene. It is also called a delayed early gene because its expression is immediately after the early gene. The E4 region is mainly related to the metabolism of viral messenger RNA, and also has the function of promoting viral DNA replication and shutting down host protein synthesis. In addition, IVa2 is a transcriptional activator of the major late promoter (MLP) of adenovirus, and plays an important role in the activation of MLP.The expression of the main adenovirus coat proteins Hexon, Penton, Fiber and other late proteins are all regulated by MLP. At the same time, IVa2 plays an important role in the assembly of viral coat proteins and the encapsidation of the viral genome. Therefore, the recombinant adenovirus lacking the IVa2 gene is theoretically blocked in the expression of structural proteins, the assembly of viral particles and the encapsidation of the genome, and thus cannot produce progeny viruses.

[0003] Gene therapy products have become a hot topic in drug research and development at home and abroad in recent years. In the research and development of gene therapy products and the large-scale production process, how to control the quality of the products and ensure their safety and effectiveness is a key link in the successful application of such products. The biological functions of adenoviruses have been studied very clearly. They have low pathogenicity to humans and have high transduction efficiency for both dividing and non-dividing cells. In addition, they have the advantages of broad tissue affinity and non-integration into the host genome. Adenoviruses can be transformed into replication-defective viral vectors and selectively replicating oncolytic viral vectors.

[0004] The first generation of adenovirus vectors removed the E1 and (or) E3 regions of the adenovirus genome. The loss of E1 makes it unable to replicate itself, and can only rely on trans-complementation provided by packaging cells such as HEK293A cells for replication and amplification, thereby ensuring the safety of adenovirus. HEK293 cells commonly used for adenovirus packaging are immortalized cells formed by human embryonic kidney cells transfected with sheared human adenovirus 5 (Ad5). The cells contain and express the transfected Ad5 gene. The linear nucleotides 1-4344 of Ad5 are integrated into chromosome 19 (19q13.2) of HEK293 cells, and there are sequences identical to those of serotype 5 adenovirus, including the left terminal repeat sequence (ITR), packaging signal sequence (ψ), E1 gene, IX gene, and about 250bp of the C-terminus of IVa2 gene. Even if the E1 gene is deleted from the adenovirus vector, the packaging signal sequence upstream of the E1 gene and the 3' ends of the IX and IVa2 genes downstream of the E1 gene still have the risk of homologous recombination with the Ad5 1-4344 gene integrated into the packaging cell genome. The overlapping sequences of the recombinant adenovirus and 293 cells undergo homologous recombination, resulting in the replacement of the target gene by the E1 gene, generating a recombinant adenovirus that can replicate and amplify autonomously - a replication-competent adenovirus, namely RCA virus (Replication Competent Adenoviruses, RCAs). Experiments have shown that the number of RCAs increases with the number of passages of the adenovirus vector in 293 cells. RCAs can replicate and amplify autonomously in their host cells, and possible consequences include increased local inflammatory responses and tissue damage, as well as uncontrolled systemic replication in immunocompromised individuals. Therefore, the generation of RCAs will seriously interfere with the efficacy of the recombinant adenovirus vector and bring safety risks. In gene therapy drugs using non-replicating adenovirus as a vector, the control standard for RCA is less than 1RCA / 3×10 10 VP. How to reduce replication-competent adenovirus contamination and improve the safety and efficacy of adenovirus gene therapy products has become a topic that needs urgent research. Summary of the invention

[0005] In order to reduce the probability of homologous recombination between a recombinant adenovirus and a host cell, the present invention provides an adenovirus vector for reducing contamination of replicative adenovirus and a construction method thereof.

[0006] In one aspect, the present disclosure provides a recombinant adenovirus vector with reduced contamination by a replicative adenovirus. Compared with an adenovirus vector or a wild-type adenovirus, the recombinant adenovirus vector with reduced contamination by a replicative adenovirus lacks all or part of the sequences of the E1B region gene and the E3 region gene, wherein the fragment containing the packaging signal (ψ) and the fragment containing the IX gene located downstream of the 3' side of the left ITR of the adenovirus vector or the wild-type adenovirus are deleted; and the fragment containing the packaging signal (ψ) and the fragment containing the IX gene are inserted between the downstream of the E4 region gene of the adenovirus vector or the wild-type adenovirus and the 5' side of the right ITR. The invention relates to a fragment of a gene, wherein the inserted fragment containing a packaging signal (ψ) is located upstream of the 5' side of the right ITR, the inserted fragment containing the IX gene is located upstream of the inserted fragment containing the packaging signal (ψ), and the directions of the inserted fragment containing the packaging signal (ψ) and the inserted fragment containing the IX gene are opposite, wherein the inserted fragment containing the packaging signal (ψ) at least has a nucleotide sequence necessary for the incorporation of the viral genome into the viral capsid during viral assembly, and the inserted fragment containing the IX gene at least has a nucleotide sequence capable of expressing the IX protein.

[0007] In another aspect, the present disclosure provides a host cell comprising the aforementioned recombinant adenovirus vector with reduced contamination by replicating adenovirus.

[0008] In another aspect, the present disclosure provides a composition comprising the aforementioned recombinant adenovirus vector with reduced replication-competent adenovirus contamination, and a pharmaceutically acceptable carrier.

[0009] In another aspect, the present disclosure provides a method for producing a recombinant adenovirus, comprising:

[0010] (1) mixing the aforementioned recombinant adenovirus vector with reduced replication-competent adenovirus contamination with host cells, so that the recombinant adenovirus vector with reduced replication-competent adenovirus contamination is introduced into the host cells;

[0011] (2) Growing the host cell under conditions for producing the recombinant adenovirus.

[0012] In another aspect, the present disclosure provides use of the aforementioned recombinant adenovirus vector and / or composition with reduced replication-competent adenovirus contamination in preparing a recombinant adenovirus.

[0013] Genetic recombination is a common phenomenon in the biological world. Homologous recombination is the exchange between any two gene DNA sequences with a homologous sequence. The condition for homologous recombination to occur is the presence of homologous sequences between two DNA molecules, which has nothing to do with the actual base sequence order. As long as they are similar, and the longer the similar segment, the higher the frequency of recombination. If the homologous sequence is too short, it is difficult for recombination to occur. In the present disclosure, in order to reduce the probability of homologous recombination, the packaging signal and IX gene in the adenovirus vector that are homologous to the Ad5 gene integrated into the cell genome are moved to the E4 region; further, the 3' end sequence of IVa2 is subjected to multi-site synonymous mutations, and the sequences in the E1 region that are homologous to the Ad5 sequence transfected by the packaging cell are transferred or subjected to synonymous mutations, and there are no large fragments of homologous sequences, which minimizes the probability of homologous recombination; the IX gene is inserted forward in the E4 region, and the packaging signal sequence is inserted reversely at the same position. The arrangement direction of the IX gene sequence and the packaging signal sequence is inconsistent with the Ad5 sequence transfected in the packaging cell, thereby also avoiding the possibility of recombination in the E4 region. The experiments disclosed in the present invention prove that the above modifications to the adenovirus vector greatly reduce the probability of homologous recombination, thereby reducing the contamination of replicative adenovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The diagram is a schematic diagram of the genome structure of adenovirus. The arrow in the figure indicates the direction of gene transcription.

[0015] Figure 2 This is a map of the pAd / PL-DEST plasmid.

[0016] Figure 3 Schematic diagram of the structure of CR-rAd5 (Conditionally Replicative recombinant human Ad5) virus vector; In the figure: Insert: inserted target protein gene.

[0017] Figure 4 Schematic diagram of the L-RCA-rAd5 (Lower RCA recombinant human Ad5) virus vector structure; In the figure: Insert: inserted target protein gene.

[0018] Figure 5 1 is the electrophoresis result diagram of the CR-rAd5 control virus plasmid after restriction digestion, and 2 is the result diagram of agarose gel electrophoresis detection of the digestion product after the virus plasmid was digested with restriction endonuclease XbaI.

[0019] Figure 6Figures 1 and 2 show normal HEK293A cells and HEK293A cells with cytopathic effect during the packaging of CR-rAd5 control virus. A is a normal HEK293A cell; B is a HEK293A cell with cytopathic effect. The magnifications are all 10×10.

[0020] Figure 7 This is the result of agarose gel electrophoresis detection of the digested products after genomic DNA was extracted from the low CR-rAd5 control virus and digested with restriction endonuclease BstZ17I-HF.

[0021] Figure 8 This is the electrophoresis diagram of PCR identification using CR-rAd5 control virus as template.

[0022] Fig. 9 The figure is a comparison diagram of the IVa2 C-terminal nucleotide sequence of the synonymous mutation and the wild-type nucleotide sequence.

[0023] Fig.10 This is a diagram showing the electrophoresis result after restriction digestion of the L-RCA-rAd5 viral plasmid, and is a diagram showing the result of agarose gel electrophoresis detection of the digestion product after the viral plasmid was digested with the restriction endonuclease XbaI.

[0024] Fig.11 Figure 2 shows normal HEK293A cells and HEK293A cells with cytopathic effect during L-RCA-rAd5 virus packaging. A is a normal HEK293A cell; B is a HEK293A cell with cytopathic effect. The magnification is 10×10.

[0025] Fig.12 This is a graph showing the results of agarose gel electrophoresis detection of the digested products after genomic DNA of low RCA recombinant human adenovirus type 5 (L-RCA-rAd5) was extracted and digested with restriction endonuclease SpeI-HF.

[0026] Fig.13 This is the electrophoresis diagram of PCR identification using low RCA recombinant human adenovirus type 5 (L-RCA-rAd5) as template.

[0027] Fig.14 The figure shows the changes in the RCA levels of conditionally replicative recombinant human adenovirus type 5 (CR-rAd5) and low RCA recombinant human adenovirus type 5 (L-RCA-rAd5) after serial passaging. DETAILED DESCRIPTION

[0028] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. If no specific conditions are specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer shall be followed.

[0029] The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market.

[0030] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0031] As used herein, the term "a" or "an" refers to one or more entities; for example, "a nucleotide sequence" is understood to mean one or more nucleotide sequences. Therefore, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0032] As used herein, the term "about" or "consisting essentially of" refers to a value or composition within an acceptable error range of the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" or "consisting essentially of" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "consisting essentially of" can mean a range of up to 20%. In addition, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times of a value. When a particular value or composition is provided in the present application and claims, unless otherwise stated, it should be assumed that the meaning of "about" or "consisting essentially of" is within an acceptable error range for the particular value or composition.

[0033] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer values ​​within the recited range and fractions of the stated value (such as tenths and hundredths of integers) where appropriate.

[0034] As used herein, the terms "adenovirus" and "adenoviral particle" include any and all viruses that can be classified as adenoviruses, including any adenovirus that infects humans or animals, including all groups, subgroups, and serotypes. Thus, as used herein, "adenovirus" and "adenoviral particle" refer to the virus itself or its derivatives, and encompass all serotypes and subtypes and naturally occurring and recombinant forms. In one embodiment, such adenovirus infects human cells.

[0035] As used herein, the term "adenoviral vector" refers to an adenovirus that has been genetically modified to behave differently from a natural wild-type virus, wherein the adenoviral genome has been manipulated to accommodate a non-natural nucleic acid sequence relative to the adenoviral genome. Typically, an adenoviral vector is produced by introducing one or more mutations (e.g., deletions, insertions, or substitutions) into the adenoviral genome of an adenovirus to accommodate the insertion of a non-natural nucleic acid sequence into the adenovirus. For example, an adenoviral vector can be modified so that it cannot replicate outside a specific packaging cell line. In some aspects, an adenoviral vector is genetically modified to carry one or more genes encoding a protein of interest. The protein of interest can be a non-adenoviral protein. Examples of adenoviruses that can be used as viral vectors of the present invention include those having or derived from serotypes Ad2, Ad4, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (e.g., RhAd52), and Pan9 (also known as AdC68); these vectors can be derived from, for example, humans, chimpanzees (e.g., ChAd1, ChAd3, ChAd7, ChAd8, ChAd21, ChAd22, ChAd23, ChAd24, ChAd25, ChAd26, ChAd 27.1, ChAd28.1, ChAd29, ChAd30, ChAd31.1, ChAd32, ChAd33, ChAd34, ChAd35.1, ChAd36, ChAd37.2, ChAd39, ChAd40.1, ChAd41.1, ChAd42.1, ChAd43, ChAd44, ChAd45, ChAd46, ChAd48, ChAd49, ChAd49, ChAd50, ChAd67, or SA7P) or a rhesus adenovirus (e.g., rhAd51, rhAd52, or rhAd53).

[0036] Preferably, the adenoviral vector is a recombinant human adenoviral vector, such as recombinant human adenovirus serotype 5, or any of recombinant human adenovirus serotypes 26, 4, 35, 7, 48, etc. Recombinant viral vectors that can be used in the present application can be prepared using methods known in the art according to the present disclosure. For example, taking into account the degeneracy of the genetic code, several nucleic acid sequences encoding the same polypeptide can be designed. The polynucleotide encoding the protein of interest can be optionally codon-optimized to ensure correct expression in a host cell (e.g., a bacterial or mammalian cell). Codon optimization is a widely used technique in the art, and methods for obtaining codon-optimized polynucleotides will be well known to those skilled in the art according to the present disclosure.

[0037] As used herein, the term "E1 region" refers to a group of genes present in the adenovirus genome. These genes (such as but not limited to E1A and E1B) are expressed in the early stages of viral replication and activate the expression of other viral genes. In one embodiment, the adenovirus packaging cell line of the present disclosure includes all coding sequences that constitute the E1 region.

[0038] In one embodiment, the adenovirus packaging cell line of the present disclosure includes some coding sequences that constitute the E1 region (e.g., E1A or E1B). As used herein, the term "E1A" refers to all gene products of the adenovirus E1A region, including the expression products of two major RNAs: 13S and 12S. The expression products are translated into polypeptides of 289 and 243 amino acids, respectively. These two proteins differ in 46 amino acids and are spliced ​​from 12S mRNA. For the purposes of the present invention, the packaging cell line can express 289 polypeptides, 243 polypeptides, or both 289 and 243 polypeptides. As used herein, the term "E1B" refers to all gene products of the adenovirus E1B region, including 3 major polypeptides of 19kd and 55kd. E1B19kd and 55kd proteins are important in cell transformation. For the purposes of the present invention, the packaging cell line can express 19Kd polypeptides, 55Kd polypeptides, or both 19Kd and 55Kd polypeptides.

[0039] As used herein, the term "E2" refers to a cistron having at least 3 ORFs, all of which are involved in DNA replication, including polymerase. The E2 late promoter of adenovirus has been described by, for example, Swaminathan, S. and Thimmapaya, 8. (1995) Gurr. Top. Microbial. Immunol., 199, 177-194. In the adenovirus system, the E2 late promoter, together with the E2 early promoter, has the function of controlling the adenovirus E2 region and / or genes E2A and E2B. In this case, the synthesis of E2 mRNA initially starts from the E2 early promoter. About five to seven hours after cell infection, a switch to the E2 late promoter occurs.

[0040] As used herein, the term "E3 region" refers to a group of genes present in the adenovirus genome and expressed in the early stages of the viral replication cycle. These genes express proteins that interact with the host immune system. These genes are not essential for viral replication in vitro and therefore may be deleted in adenovirus vectors.

[0041] As used herein, the term "E4 region" refers to a group of genes present in the adenovirus genome near the right terminal repeat (ITR) and expressed in the early stages of the viral replication cycle. The E4 region includes at least 7 ORFs. The products of the E4 region promote viral gene expression and replication, interact with host cell components, and participate in lytic infection and tumor formation.

[0042] As used herein, the term "packaging signal" refers to a nucleotide sequence present in the viral genome and necessary for the incorporation of the viral genome into the viral capsid during viral assembly. The packaging signal of adenovirus is naturally located at the left end, downstream of the left inverted terminal repeat (ITR). The packaging signal can be represented as "ψ".

[0043] The term "target protein" refers in a broad sense to any protein of particular relevance in an industrial protein production process. Target proteins include, but are not limited to, heterologous therapeutic proteins, marker proteins of host cells, or proteins with functions such as protein secretion, post-translational protein modification, translation, transcription, cell cycle regulation, or nutrient metabolism. Such target proteins include, but are not limited to, enzymes, hormones, growth factors, cytokines, immunoglobulins (e.g., antibodies), and / or any fusion proteins.

[0044] As used herein, the term "vector" refers to any recombinant polynucleotide construct (such as a DNA construct) that can be used for transformation (i.e., introducing heterologous DNA into a host cell) purposes. An exemplary type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be connected. Another exemplary type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced. The term "expression vector" refers to a vector with the ability to incorporate heterologous nucleic acid fragments (such as DNA) into foreign cells and to express them in foreign cells. In other words, an expression vector includes a nucleic acid sequence / fragment (such as DNA, mRNA) that can be transcribed or expressed in a target cell. Many viral, prokaryotic and eukaryotic expression vectors are known and / or commercially available. Selecting an appropriate expression vector is within the knowledge of those skilled in the art. The expression vector may include one or more regulatory sequences.

[0045] The terms "viral vector" and "virus" are used interchangeably herein to refer to any obligate intracellular parasite without protein synthesis or energy production machinery. The viral genome can be RNA or DNA of a protein-coated structure containing a lipid membrane. The terms virus and viral vector are used interchangeably herein. Useful viruses in the practice of the present invention include recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably selected from baculoviridiae, parvoviridiae, picornoviridiae, herpesviridiae, poxviridae or adenoviridae. The virus is modified by DNA recombination technology to contain an exogenous transgene (e.g., a nucleic acid sequence encoding a target gene) and can be engineered to be replication-deficient, conditionally replicative or replication-competent. A minimal vector system can also be used, in which the viral backbone contains only the sequences required for viral vector packaging, optionally including a transgenic expression cassette. The term "replication-deficient" refers to a vector whose replication is highly attenuated in wild-type mammalian cells. In order to mass produce this vector, the missing function is usually supplemented by co-transfection with a helper virus or by genome modification, thereby establishing a producer cell line. The term "replication-competent viral vector" refers to a viral vector capable of infecting, DNA replication, packaging and lysing infected cells. The term "conditional replication-competent viral vector" or "conditional replication-defective virus" used herein refers to a replication-competent vector that is designed to achieve selective expression in a specific cell type. This conditional replication can be achieved by operably connecting a tissue-specific, tumor-specific or cell-type-specific or other selectively induced regulatory control sequence to an early gene (such as the E1 gene of an adenovirus vector). Infecting an object with a recombinant virus or a non-viral vector can provide long-term expression of a target gene in an object. In one embodiment, the nucleic acid sequence encoding the target gene in a viral vector system is under the control of a constitutive promoter, a regulatable promoter, an inducible promoter, a tissue-specific or tumor-specific promoter or a time-regulated promoter.

[0046] As used herein, the term "construct" refers to an artificially assembled or isolated nucleic acid molecule that may include one or more nucleic acid sequences, wherein the nucleic acid sequence may be a coding sequence (i.e., a sequence encoding an end product), a regulatory sequence, a non-coding sequence, or any combination thereof. The term construct includes, for example, a vector, a plasmid, but should not be considered to be limited thereto. In some embodiments, the term "regulatory sequence" refers to a DNA sequence that is necessary for affecting the expression of a coding sequence that is operably connected (linked) (connected / ligated) thereto. The nature of the regulatory sequence varies according to the host cell. For example, in prokaryotes, a regulatory / control sequence may include a promoter, a ribosome binding site, and / or a terminator. For example, in eukaryotes, a regulatory / control sequence may include a promoter (e.g., constitutive or inducible), a terminator, an enhancer, a transactivator, and / or a transcription factor. The regulatory sequence that is "operably connected" to a coding sequence is connected in such a way that the expression of the coding sequence is achieved under suitable conditions. In some embodiments, "construct" or "DNA construct" refers to an artificially assembled or isolated nucleic acid molecule comprising a coding region of interest and optionally additional regulatory or non-coding sequences.

[0047] For autonomous replication, the vector may further include an origin of replication that enables the vector to autonomously replicate in a host cell. The term "origin of replication" or "plasmid replicator" or "replicon" is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo. In order to maintain the vector of the present invention in a host cell, an origin of replication (ORI) (also referred to as an autonomous replication sequence (ARS) or replicon) is necessary. An autonomous replication sequence can be any suitable sequence available to those skilled in the art that allows plasmid replication independent of chromosomal replication. The origin of replication can be any plasmid replication factor that mediates autonomous replication that works in a cell. Examples of origins of replication include, but are not limited to, ColE1 origin of replication, pMB1 origin of replication, pBR322 origin of replication, pUC origin of replication (e.g., pUC57 origin of replication), pSC101 origin of replication, and R6Kγ origin of replication. The origin of replication can be high copy or low copy. When present in a vector, a high copy origin of replication can result in a vector with a high copy number (e.g., 150 to 200) per cell. When present in a vector, a medium copy origin of replication can result in a vector having a medium copy number per cell (e.g., 25 to 50). When present in a vector, a low copy origin of replication can result in a vector having a low copy number per cell (e.g., 1 to 3).

[0048] "Selectable marker gene" is a gene that allows easy selection of transformed cells, and its product provides antibiotic resistance or virus resistance, heavy metal resistance, auxotrophic prototrophy, etc. Selectable markers can be introduced into cells on a vector as an expression cassette, or can be introduced on a separate vector. Some antibiotic resistance proteins can be used as selectable markers, such as ampicillin resistance (AmpR), chloramphenicol resistance (CmR), neomycin resistance, G418 resistance, puromycin resistance, hygromycin resistance, bleomycin resistance, kanamycin resistance, methotrexate resistance, phleomycin resistance or benomyl resistance. Some chromogenic proteins or fluorescent proteins can be used as selectable markers. There are many various chromogenic proteins or fluorescent proteins, such as red fluorescent protein (RFP), green fluorescent protein (GFP), mCherry, dsRed, etc. Nutrient deficiency markers such as URA3, ADE2, HIS3, LEU2, TRP1 or LYS2, auxotrophic markers allow screening of the expression of selectable marker genes.

[0049] As used herein, the term "pharmaceutically acceptable" means that the carrier or excipient will not cause any unnecessary or adverse effects in the subject to which they are administered at the dosage and concentration adopted. Such pharmaceutically acceptable carriers and excipients are well known in the art. The preferred formulation of the pharmaceutical composition depends on the intended mode of administration and therapeutic application. The composition may include a pharmaceutically acceptable, non-toxic carrier or diluent, which is defined as a vehicle commonly used to formulate a pharmaceutical composition for administration to animals or humans. The diluent should be selected to avoid affecting the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. It should be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a specific application.

[0050] In one aspect, the present disclosure provides a recombinant adenovirus vector with reduced contamination by a replicative adenovirus. Compared with an adenovirus vector or a wild-type adenovirus, the recombinant adenovirus vector with reduced contamination by a replicative adenovirus lacks all or part of the sequences of the E1B region gene and the E3 region gene, wherein the fragment containing the packaging signal (ψ) and the fragment containing the IX gene located downstream of the 3' side of the left ITR of the adenovirus vector or the wild-type adenovirus are deleted; and the fragment containing the packaging signal (ψ) and the fragment containing the IX gene are inserted between the downstream of the E4 region gene of the adenovirus vector or the wild-type adenovirus and the 5' side of the right ITR. The invention relates to a fragment of a gene, wherein the inserted fragment containing a packaging signal (ψ) is located upstream of the 5' side of the right ITR, the inserted fragment containing the IX gene is located upstream of the inserted fragment containing the packaging signal (ψ), and the directions of the inserted fragment containing the packaging signal (ψ) and the inserted fragment containing the IX gene are opposite, wherein the inserted fragment containing the packaging signal (ψ) at least has a nucleotide sequence necessary for the incorporation of the viral genome into the viral capsid during viral assembly, and the inserted fragment containing the IX gene at least has a nucleotide sequence capable of expressing the IX protein.

[0051] In some embodiments, the IVa2 protein encoding gene of the recombinant adenovirus vector for reducing replication-competent adenovirus contamination has a synonymous mutation.

[0052] In some embodiments, the target protein gene is inserted between the 3' side of the left ITR of the recombinant adenovirus vector that reduces replication-competent adenovirus contamination and the upstream of the E1A gene. In some embodiments, a tumor-specific promoter is operably linked to the E1A gene, preferably, the tumor-specific promoter is selected from hTERT promoter, PEG-3 promoter or Survivin gene promoter. In some embodiments, a constitutive promoter or an inducible promoter is operably linked to the target gene, preferably, the constitutive promoter is selected from CMV promoter, CAG promoter, PGK promoter or ACTB promoter.

[0053] In some embodiments, the insertion direction of the inserted fragment containing the packaging signal (ψ) is 3'→5', and the insertion direction of the inserted fragment containing the IX gene is 5'→3'.

[0054] In some embodiments, the adenoviral vector is of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 2, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 70, 71, 72, 73, 74 and 75 adenovirus vectors, preferably adenovirus serotype 4, 5, 7, 26, 35 or 48 adenovirus vectors, preferably human adenovirus vectors.

[0055] In some embodiments, the adenoviral vector is an adenoviral type 5 vector or an adenoviral type 35 vector.

[0056] In some embodiments, the adenoviral vector is a human adenoviral type 5 vector.

[0057] In some embodiments, the adenoviral vector is pAd / PL-DEST, pAdEasy-1, or pAd / CMV / V5-DEST.

[0058] In some embodiments, the wild-type adenovirus is selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 1, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 70, 71, 72, 73, 74 and 75 wild-type adenovirus, preferably serotype 4, 5, 7, 26, 35 or 48 wild-type adenovirus, preferably human adenovirus.

[0059] In some embodiments, the wild-type adenovirus is a wild-type adenovirus type 5 or adenovirus type 35.

[0060] In some embodiments, the wild-type adenovirus is a human wild-type adenovirus type 5.

[0061] In some embodiments, the recombinant adenoviral vector with reduced replication-competent adenovirus contamination is a recombinant type 5 adenoviral vector.

[0062] In some embodiments, the recombinant adenovirus vector with reduced replication-competent adenovirus contamination is a recombinant human adenovirus type 5 vector.

[0063] In some embodiments, the target gene encodes an active protein molecule. In some embodiments, the active protein molecule is selected from an immunomodulatory molecule, an antigen binding molecule, an antigen or a ligand of the antigen, a toxin, an enzyme or other active protein molecule.

[0064] In some embodiments, the target gene encodes an immunomodulatory molecule. In some embodiments, the immunomodulatory molecule is a cytokine, a cytokine receptor, or a co-stimulatory ligand.

[0065] In some embodiments, the immunomodulatory molecule is selected from the group consisting of TNF-α, IFN-α, IFN-β, IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-7, IL-10, IL-10R, IL-12A, IL-12B, IL-15, IL-23A / p19, IL24, IL-27, IL-33, IL-35, IL-1 5. IL-15 receptor fusion protein, TGF-β, TGF-βtrap, IL-10trap, VEGF, CD80, CD137L, GM-CSF, GITR ligand, OX40 ligand, CD40 ligand, CD154, CD70, CD86, CD137, CD137L, BORIS / CTCFL, bone morphogenetic protein (BMP), TNFSF9, FGF, ICAM, their functional fragments, and their derivatives.

[0066] In some embodiments, the target gene encodes an antigen binding molecule. In some embodiments, the antigen binding molecule is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TGF-β antibody, and an anti-CTLA-4 antibody or a functional fragment thereof.

[0067] In some embodiments, the target gene encodes an antigen or a ligand of the antigen. In some embodiments, the antigen is selected from the group consisting of CEA, HER-2 / neu, EGFRvIII, EpCAM, erbB2, erbB3, erbB4, CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD80, CD133, CD135 (Flt3), Flt3I, CD138, NY-ESO-1, GD2, GD3, MAGE-A1, MAGE-A3, MART1, MART2, MUC1, mesothelin, NKG2D ligand, BRAF, p53, T cell receptor, carcinoembryonic antigen, PSMA, functional fragments thereof, and derivatives thereof.

[0068] In some embodiments, the gene of interest encodes a toxin. In some preferred embodiments, the toxin is diphtheria toxin, ricin toxin, or Pseudomonas exotoxin.

[0069] In some embodiments, the target gene encodes an enzyme. In some preferred embodiments, the enzyme is selected from the group consisting of esterase, carboxypeptidase, metalloproteinase, streptokinase, β-glucosidase, β-glucuronidase, β-galactosidase, β-lactamase, relaxin, collagenase, arginase, fragments thereof, and derivatives thereof.

[0070] In some embodiments, the target gene encodes a tumor suppressor gene, a growth factor, a coagulation factor, an apoptosis gene, a fragment thereof, or a derivative thereof.

[0071] In some embodiments, the target gene encodes angiostatin, endostatin, thymidine kinase, a functional fragment thereof, or a derivative thereof.

[0072] In some embodiments, the recombinant adenoviral vector with reduced contamination by replicating adenovirus further comprises an origin of replication and / or a selectable marker gene.

[0073] In some embodiments, the replication origin is selected from the group consisting of a pMB1 replication origin (pMB1 ori), a ColE1 replication origin (ColE1ori), a pBR322 replication origin (pBR322 ori), a pUC replication origin ((pUC ori)) (e.g., a pUC57 replication origin ((pUC57 ori))), a pSC101 replication origin (pSC101ori) and a R6Kγ replication origin (R6Kγori).

[0074] In some embodiments, the selectable marker gene is selected from one or more of an antibiotic or virus resistance gene, a heavy metal resistance gene, and an auxotrophic gene.

[0075] In some embodiments, the antibiotic or viral resistance gene is selected from one or more of an ampicillin resistance gene, a chloramphenicol resistance gene, a neomycin resistance gene, a G418 resistance gene, a puromycin resistance gene, a hygromycin resistance gene, a bleomycin resistance gene, a kanamycin resistance gene, a methotrexate resistance gene, a phleomycin resistance gene, and a benomyl resistance gene.

[0076] In some embodiments, the auxotrophic gene is selected from one or more of ura3, ade2, his3, leu2, trpl and lys2.

[0077] In some embodiments, the method comprises the steps of:

[0078] (1) taking an adenoviral vector or a wild-type adenovirus and retaining the left and right ITRs of the adenoviral vector or the wild-type adenovirus;

[0079] (2) deleting the fragment containing the packaging signal (ψ) and the fragment containing the IX gene located downstream of the 3' side of the left ITR of the adenoviral vector or wild-type adenovirus;

[0080] (3) inserting a fragment containing a packaging signal (ψ) and a fragment containing the IX gene into the adenovirus vector or wild-type adenovirus between the downstream of the E4 region gene and the 5' side of the right ITR, wherein the inserted fragment containing the packaging signal (ψ) is located upstream of the 5' side of the right ITR, the inserted fragment containing the IX gene is located upstream of the inserted fragment containing the packaging signal (ψ), and the directions of the inserted fragment containing the packaging signal (ψ) and the inserted fragment containing the IX gene are opposite;

[0081] (4) deleting all or part of the sequence of the E1B gene of the adenovirus vector or wild-type adenovirus so that the E1B gene cannot express the gene product of the active E1B region; retaining the entire E1A gene; wherein:

[0082] (i) If the adenovirus vector lacks the E1B gene and cannot express the gene product of the active E1B region, step (4) is not performed;

[0083] (ii) if the adenoviral vector lacks the E1A gene, inserting the E1A gene downstream of the 3' side of the left ITR of the adenoviral vector and upstream of the IX gene, so that the adenoviral vector can express all gene products of the E1A gene after the insertion of the E1A gene;

[0084] (5) deleting all or part of the sequence of the E3 region gene of the adenovirus vector or wild-type adenovirus so that the E3 gene cannot express the gene product of the active E3 region; wherein:

[0085] If the adenovirus vector lacks all or part of the sequence of the E3 region gene and cannot express the gene product of the active E3 region, step (5) is not performed;

[0086] The order of steps (2)-(5) can be arbitrarily changed, or steps (2)-(5) can be performed simultaneously to obtain the recombinant adenovirus vector with reduced replication-competent adenovirus contamination.

[0087] In some embodiments, the method further comprises: (6) performing a synonymous mutation on the IVa2 protein encoding gene of the adenovirus vector or wild-type adenovirus; wherein the order of steps (2)-(6) can be arbitrarily changed, or steps (2)-(6) can be performed simultaneously.

[0088] In some embodiments, the method further comprises: (7) virus packaging: introducing the obtained recombinant adenovirus vector with reduced replication-competent adenovirus contamination into packaging cells (such as HEK293 cells) to obtain virus particles.

[0089] In some embodiments, the adenoviral vector is pAd / PL-DEST, and the method comprises the steps of:

[0090] (i) taking a pAd / PL-DEST plasmid and retaining the left and right ITRs of the pAd / PL-DEST plasmid;

[0091] (ii) deleting the fragment containing the packaging signal (ψ) and the fragment containing the IX gene located 3′ downstream of the left ITR of the pAd / PL-DEST plasmid;

[0092] (iii) inserting a fragment containing a packaging signal (ψ) and a fragment containing the IX gene into the region downstream of the gene in the E4 region of the pAd / PL-DEST plasmid and between the 5' side of the right ITR, wherein the inserted fragment containing the packaging signal (ψ) is located upstream of the 5' side of the right ITR, the inserted fragment containing the IX gene is located upstream of the inserted fragment containing the packaging signal (ψ), and the directions of the inserted fragment containing the packaging signal (ψ) and the inserted fragment containing the IX gene are opposite;

[0093] (iv) inserting the E1A gene downstream of the 3' side of the left ITR of the pAd / PL-DEST plasmid and upstream of the IX gene, so that the pAd / PL-DEST plasmid can express all gene products of the E1A gene after the E1A gene is inserted;

[0094] The order of steps (ii)-(iv) can be arbitrarily changed, or steps (ii)-(iv) can be performed simultaneously to obtain the recombinant adenovirus vector with reduced contamination by replicating adenovirus.

[0095] In some embodiments, the adenoviral vector is pAd / PL-DEST, and the method comprises the steps of:

[0096] ① Designing a first sgRNA near the insertion site of the E4 region of the pAd / PL-DEST plasmid, mixing the first sgRNA, Cas9 nuclease and the pAd / PL-DEST plasmid, and the first sgRNA guides the Cas9 nuclease to cut the pAd / PL-DEST plasmid to obtain the pAd / PL-DEST plasmid treated with the first sgRNA and Cas9 nuclease;

[0097] ② Using the pAd / PL-DEST plasmid as a template, PCR amplification was performed to obtain fragment A containing the IX gene and fragment B containing the packaging signal (ψ), respectively, wherein the fragment A and the 5' end of the cut of the pAd / PL-DEST plasmid treated with the first sgRNA and Cas9 nuclease have an overlapping sequence I of at least 10 bp, and the fragment B and the 3' end of the cut of the pAd / PL-DEST plasmid treated with the first sgRNA and Cas9 nuclease have an overlapping sequence II of at least 10 bp, and at the same time, there is an overlapping sequence III of at least 10 bp between the fragment A and the fragment B; wherein the fragment A maintains a forward direction, and the fragment B is in a reverse direction;

[0098] ③ The pAd / PL-DEST plasmid obtained in step ① after being treated with the first sgRNA and Cas9 nuclease is mixed with fragment A and fragment B obtained in step ②, and recombined and connected to obtain an intermediate vector;

[0099] ④ Design the second sgRNA and the third sgRNA, mix the second sgRNA and the third sgRNA, Cas9 nuclease and the intermediate vector, and the second sgRNA and the third sgRNA guide the Cas9 nuclease to cut the intermediate vector, and obtain the intermediate vector treated with the second sgRNA, the third sgRNA and the Cas9 nuclease:

[0100] ⑤ Perform PCR amplification with the template synthesized by gene, and respectively amplify fragment C containing pAd / PL-DEST (downstream of the 3' side of the left ITR - at least 10 bp sequence upstream of the packaging signal (ψ)) - (downstream of the packaging signal (ψ) - at least 20 bp sequence upstream of attR1) - promoter - target gene - polyadenylation signal, and fragment D containing the E1A gene and the IVa2 C-terminal gene fragment with synonymous mutation, fragment C and the nick upstream of the intermediate vector treated with the second sgRNA, the third sgRNA and the Cas9 nuclease have at least 10 bp overlapping sequence IV, fragment D and the nick downstream of the intermediate vector treated with the second sgRNA, the third sgRNA and the Cas9 nuclease have at least 10 bp overlapping sequence V, and at the same time, fragment C and fragment D have at least 10 bp overlapping sequence VI; preferably, the promoter is CMV promoter, CAG promoter, PGK promoter or ACTB promoter; the polyadenylation signal is SV40 PA, BGH PA, hGH PA or rbGlob PA;

[0101] ⑥ The intermediate vector obtained in step ④ after being treated with the second sgRNA and the third sgRNA and the Cas9 nuclease is mixed with the fragment C and the fragment D obtained in step ⑤, and recombined and connected to obtain a recombinant adenovirus vector with reduced contamination of replicative adenovirus; wherein, after the fragment C and the fragment D are connected, they replace the IVa2 C-terminal gene without synonymous mutation at the 3' downstream of the left ITR of pAd / PL-DEST.

[0102] In some preferred embodiments, in step ①, the insertion site in the E4 region of the pAd / PL-DEST plasmid is a site corresponding to 35773 bp of wild-type Ad5; the first sgRNA has a sequence as shown in SEQ ID NO:11.

[0103] In some preferred embodiments, in step ②, fragment A contains a 3525-4080 bp fragment corresponding to the wild-type Ad5 IX gene; fragment B contains an Ad5 129-392 bp fragment of a packaging signal (ψ); the overlapping sequence I sequence is shown in SEQ ID NO: 31; the overlapping sequence II sequence is shown in SEQ ID NO: 32; and the overlapping sequence III sequence is shown in SEQ ID NO: 33.

[0104] In some preferred embodiments, in step ③, the intermediate vector sequence is as shown in SEQ ID NO:13.

[0105] In some preferred embodiments, in step ⑤, the second sgRNA sequence is as shown in SEQ ID NO: 14; the third sgRNA sequence is as shown in SEQ ID NO: 15; the fragment containing pAd / PL-DEST (downstream of the 3' side of the left ITR-at least 10 bp sequence upstream of the packaging signal (ψ))-(downstream of the packaging signal (ψ)-at least 20 bp sequence upstream of attR1)-promoter-target gene-polyadenylation signal is a fragment containing pAd / PL-DEST (104-128 bp)-(393-504 bp)-CMV-hXGENE-SV40 PA, and its sequence corresponds to the sequence shown in nucleotides 104 to 2374 of SEQ ID NO: 8; the fragment containing the E1A gene and the IVa2 C-terminal gene with synonymous mutations is a fragment containing pSurvivin-E1A-E1A terminal-IVa2 C-terminal gene with synonymous mutations, and its sequence corresponds to SEQ ID NO:8 has the sequence shown at nucleotides 2375 to 4233; the overlapping sequence IV is shown in SEQ ID NO:34; the overlapping sequence V is shown in SEQ ID NO:35; and the overlapping sequence VI is shown in SEQ ID NO:36.

[0106] The packaging signal (ψ) sequence was deleted from the pAd / PL-DEST(104-128bp)-(393-504bp)-CMV-hXGENE-SV40 PA fragment.

[0107] pSurvivin-E1A-E1A terminal-The IX gene segment was deleted from the IVa2 C-terminal gene segment by synonymous mutation.

[0108] In some preferred embodiments, in step ⑥, fragment C and fragment D are connected to replace the nucleotide sequence of positions 104 to 3168 of pAd / PL-DEST shown in SEQ ID NO:1; the sequence of the recombinant adenovirus vector with reduced replication-competent adenovirus contamination is shown in SEQ ID NO:8.

[0109] In some preferred embodiments, 29 mutations in total are made to the IVa2 C-terminus; the sequence of the IVa2 C-terminal gene fragment after synonymous mutations is shown in SEQ ID NO:9.

[0110] In another aspect, the present disclosure provides a host cell comprising the aforementioned recombinant adenovirus vector with reduced contamination by replicating adenovirus.

[0111] In another aspect, the present disclosure provides a composition comprising the aforementioned recombinant adenovirus vector with reduced replication-competent adenovirus contamination, and a pharmaceutically acceptable carrier.

[0112] In another aspect, the present disclosure provides a method for producing a recombinant adenovirus, comprising:

[0113] (1) mixing the aforementioned recombinant adenovirus vector with reduced replication-competent adenovirus contamination with host cells, so that the recombinant adenovirus vector with reduced replication-competent adenovirus contamination is introduced into the host cells;

[0114] (2) growing the host cell under conditions for producing the recombinant adenovirus;

[0115] In some embodiments, the host cell is a HEK293 cell, preferably a HEK293A cell.

[0116] In some embodiments, the method further comprises:

[0117] (3) Collecting the cell suspension, freezing and thawing it, and collecting the supernatant by centrifugation to obtain the recombinant virus suspension.

[0118] In some embodiments, the method further comprises:

[0119] (4) Purifying the recombinant virus suspension described in step (3).

[0120] In another aspect, the present disclosure provides use of the aforementioned recombinant adenovirus vector and / or composition with reduced replication-competent adenovirus contamination in preparing a recombinant adenovirus.

[0121] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, materials, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0122] The features and performance of the present invention are further described in detail below in conjunction with the embodiments. Obviously, the present disclosure is not limited to the following embodiments, and many variations are possible. Therefore, modifications or improvements made by those skilled in the art based on the disclosure of the present disclosure should all fall within the scope of protection claimed in the present disclosure.

[0123] Example 1: Preparation of conditionally replicative recombinant human adenovirus type 5 vector

[0124] In this example, a conditionally replicative recombinant human adenovirus type 5 vector (CR-rAd5) was prepared, which was subsequently used as a control virus vector. The pAd / PL-DEST plasmid (purchased from Invitrogen, catalog number V49420, Figure 2, https: / / www.snapgene.com / plasmids / viral_expression_and_packaging_vectors / pAd_PL-DEST) as the basic framework, the pAd / PL-DEST plasmid lacks the E1 region and E3 region genes of Ad5, wherein the E1 gene region (including E1A and E1B genes) is replaced by Gateway conversion components such as the chloramphenicol resistance gene (CmR), the toxic protein ccdB gene, and the attR1 and attR2 sites, and the entire E1A and E1B genes are missing; the E3 region lacks part of the E3 CR1-alpha0 and E3 14.7K genes, as well as all the E3 gp19K, ADP, E3RID-alpha and E3 RID-beta genes. The full-length nucleotide sequence of the pAd / PL-DEST vector is shown in SEQ ID NO: 1.

[0125] The conditionally replicative recombinant human adenovirus type 5 vector (CR-rAd5) uses the pAd / PL-DEST plasmid as the basic framework, and replaces the chloramphenicol resistance gene, the toxic protein ccdB gene, and the Gateway conversion components such as the attR1 and attR2 sites with the E1A gene regulated by the tumor-specific Survivin gene promoter and the target protein gene regulated by the CMV promoter to enhance tumor killing. The tumor-specific promoter regulates the expression of the E1A gene, so that the conditionally replicative recombinant human adenovirus type 5 (CR-rAd5) can only proliferate in tumor cells. The conditionally replicative recombinant human adenovirus type 5 vector constructed by the above method is the control virus vector, and the full-length nucleotide sequence is shown in SEQ ID NO: 2, and the schematic diagram of the virus structure is shown in Figure 3 .

[0126] The preparation, packaging and identification steps of the conditionally replicative recombinant human adenovirus type 5 vector (control virus vector) are as follows:

[0127] 1.1 Preparation of control viral vector

[0128] The pAd / PL-DEST plasmid was transformed into JM110 chemical competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd., catalog number DL1025S), wherein JM110 is a strain lacking methyltransferases dam and dcm, and the extracted pAd / PL-DEST plasmid DNA can be cleaved by endonucleases sensitive to dam and dcm methylation. The single clone obtained after transformation was picked to extract the plasmid, and then the demethylated pAd / PL-DEST was digested with ClaI, and then the fragment 1 containing the CMV-XGENE-SV40 PA fragment (corresponding to 505-2632bp of SEQ ID NO: 2) and the fragment 2 containing the pSurvivin-E1A-E1Aterminal fragment (corresponding to 2633-4068bp of SEQ ID NO: 2) synthesized by gene were connected with the pAd / PL-DEST vector digested with ClaI by in vitro recombination. The successfully constructed vector is a conditionally replicative recombinant human adenovirus type 5 vector (CR-rAd5).

[0129] The preparation process of the control viral vector is as follows:

[0130] (a) 100 ng of pAd / PL-DEST plasmid was transferred into 100 μl JM110 competent cells and heat-shocked at 42°C for 45 seconds for transformation. A single clone colony was picked for culture and then the pAd / PL-DEST plasmid was extracted.

[0131] (b) The pAd / PL-DEST plasmid extracted from the JM110 strain was digested with ClaI restriction endonuclease (NEB, R0197S) using rCutsmart digestion buffer at 37°C for 60 min. The large fragment (33151 bp) after digestion was recovered by gel recovery for later use. The reaction system is shown in Table 1:

[0132] Table 1 Reaction system

[0133] Components Volume (μl) pAd / PL-DEST plasmid (JM110) 24 10×rCutsmart Buffer 5 ClaI 1 <![CDATA[ddH2O]]> 20 total 50

[0134] (c) PCR amplification was performed using the gene-synthesized fragment 1 containing the CMV-XGENE-SV40 PA fragment (corresponding to 505-2632 bp of SEQ ID NO: 2) and the fragment 2 containing the pSurvivin-E1A-E1A terminal fragment (corresponding to 2633-4068 bp of SEQ ID NO: 2) as templates. The 5' end of the amplified fragment 1 containing the CMV-XGENE-SV40 PA fragment had a 20 bp overlapping sequence 1 (SEQ ID NO: 28: 5'-CTCGAGCGGCCGCTAGCGAC-3') with the upstream of the first ClaI restriction site. The 3' end of the amplified fragment 2 containing the pSurvivin-E1A-E1A terminal fragment had a 20 bp overlapping sequence 1 (SEQ ID NO: 29: 5'-GGGCGTGGCTTAAGGGTGGG-3') with the downstream of the second ClaI restriction site. There was also a 20 bp overlapping sequence 3 (SEQ ID NO: 21: 5'-GGGCGTGGCTTAAGGGTGGG-3') between the two fragments. NO:30: 5'-GTATCTTACTCACTCAGACC-3'). The PCR amplification reaction system is shown in Table 2; the reaction program is 98°C, 10s; 58°C, 20s; 72°C, 90s; 30 cycles.

[0135] Table 2 Reaction system

[0136] Components Volume (μl) Q5 High-Fidelity 2X Master Mix(NEB,M0494S) 25 10μM Forward Primer 2.5 10μM Reverse Primer 2.5 Template DNA 1 <![CDATA[ddH2O]]> 19 total 50

[0137] (d) The pAd / PL-DEST plasmid digested with ClaI was recombined and ligated with the amplified fragment 1 containing the CMV-XGENE-SV40 PA fragment and the fragment 2 containing the pSurvivin-E1A-E1A terminal fragment by the Gibson in vitro recombination method. The ligation reaction system is shown in Table 3. The reaction procedure was 50°C for 30 min.

[0138] Table 3 Reaction system

[0139] Components Volume (μl) pAd / PL-DEST (ClaI digestion) 3 CMV-XGENE-SV40PA fragment 0.5 pSurvivin-E1A-E1A terminal fragment 0.5 2×Gibson Assembly Master Mix(NEB,E2611S) 5 <![CDATA[ddH2O]]> 1 total 10

[0140] (e) Take 5 μl of the reaction system after in vitro recombination and transform Mach1-T1 Escherichia coli competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd., product number DL1015), and heat shock at 42°C for 45 seconds. Screen positive clones by colony PCR method, extract plasmids, and use restriction endonuclease XbaI (NEB, product number R0145S) for enzyme digestion identification, use rCutsmart enzyme digestion buffer, and digest at 37°C for 30 minutes. Run the enzyme digestion product on 1% agarose gel electrophoresis to observe the bands. The enzyme digestion identification results are shown in Figure 5, four bands of 18003-bp, 8102-bp, 7395-bp, and 3262-bp were observed, which was in line with expectations. The plasmid verified by enzyme digestion was sent for sequencing, and the plasmid verified by sequencing was the CR-rAd5 control virus vector. The complete nucleotide sequence of the vector constructed by the above method is shown in SEQ ID NO: 2. The colony PCR reaction system is shown in Table 4; the reaction program is 98°C, 10s; 56°C, 10s; 72°C, 20s; 30 cycles.

[0141] Table 4 Reaction system

[0142] Components Volume (μl) 1.1×Gold Medal Mix Ver.2 (TSINGKE, TSE102) 22 10μM Forward Primer 1 10μM Reverse Primer 1 Template DNA 1 total 25

[0143] 1.2 Control virus packaging

[0144] The constructed control virus vector was extracted in large quantities and endotoxin was removed. It was linearized with PacI (NEB, catalog number R0547S), digested at 37°C for 1h, and then incubated at 65°C for 20min to inactivate the endonuclease Pac I. It was then purified using a DNA purification recovery kit to recover the linearized virus plasmid DNA. When the degree of polymerization of HEK293A cells (Invitrogen, catalog number R70507) was 80%-90%, the transfection reagent Lipofectamine 2000 (Thermo Fisher Scientific, catalog number 11668019) was used to form a Lipofectamine 2000-DNA complex with the linearized virus plasmid DNA for transfection, and the changes in cell status were observed daily. The steps are as follows:

[0145] (1) Prepare transfection solutions A and B: Add 4 μg of the recombinant plasmid linearized and recovered by PacI to 500 μl Opti-MEM TM I reduced serum medium (purchased from Thermo Fisher Scientific , Catalog No. 31985070) was pipetted and mixed to obtain transfection solution A; 10 μl Lipofectamine 2000 transfection reagent was added to 490 μl Opti-MEM TM Mix well by pipetting in the serum-minus medium to obtain transfection solution B.

[0146] (2) Transfer all of Transfection Solution B to Transfection Solution A and mix thoroughly by pipetting to obtain Transfection Solution C. Leave the mixture at room temperature for 25 min.

[0147] (3) Add all of the transfection solution C to the T25 flask with HEK293A cells, shake it crosswise to mix, and place it in a 37°C, 5% CO2 constant temperature incubator for 6 hours, then discard the supernatant.

[0148] (4) Add 10 ml of DMEM medium containing 10% FBS and no antibiotics (purchased from Cytiva, catalog number SH30022.01) to the culture flask and culture for 10-14 days, and observe the cell status every day. HEK293A cells will show cytopathic effect (CPE) 10-14 days after transfection. When more than 50% of the cells fall off the bottom of the culture flask, collect the cell suspension, freeze and thaw three times, centrifuge at 500×g for 10 minutes, and collect the supernatant to obtain the recombinant virus suspension. Normal HEK293A cells (A) and HEK293A cells with cytopathic effect (B) are shown in Figure 2. Figure 6 shown.

[0149] 1.3 Identification of control virus genome by restriction digestion

[0150] The viral genomic DNA was extracted from the recombinant virus suspension and digested with restriction endonuclease BstZ17I-HF (NEB, R3594S) using rCutsmart digestion buffer at 37°C for 0.5 h. The BstZ17I-HF digestion product was subjected to 1% agarose gel electrophoresis to observe the bands. The results are shown in Figure 7 As shown, two bands of 28375-bp and 6313-bp were observed, which was in line with expectations.

[0151] 1.4 Control virus PCR identification

[0152] Using viral genomic DNA as template, use identification primers:

[0153] CR-rAd5-F:5'-GACCGTTTACGTGGAGACTC-3' (SEQ ID NO:3);

[0154] CR-rAd5-R:5'-GAAATCCCCGTGAGTCAAAC-3' (SEQ ID NO:4).

[0155] The identification reaction system is shown in Table 5, and the reaction procedure is shown in Table 6.

[0156] Table 5 Identification reaction system

[0157] Components Volume (μl) Q5 High-Fidelity 2X Master Mix 10 10μM Forward Primer 1 10μM Reverse Primer 1 Viral genomic DNA 1(about 150ng) <![CDATA[ddH2O]]> 7 total 20

[0158] Table 6 Identification reaction PCR program

[0159]

[0160] The PCR products were analyzed by 1% agarose gel electrophoresis. Figure 8 A 579-bp band was observed, which was in line with expectations.

[0161] The control virus genome was subsequently sequenced to ensure the integrity of the virus sequence. The samples were sent to a sequencing company and the virus genome was sequenced by NGS sequencing. The results were completely consistent with the designed sequence, indicating that the virus was successfully constructed. The virus that was sequenced correctly was the conditionally replicative recombinant human adenovirus type 5 (CR-rAd5).

[0162] Example 2: Preparation of a viral vector with reduced contamination by replicating adenovirus

[0163] In this example, a viral vector (L-RCA-rAd5) with reduced contamination of replicative adenovirus was prepared. The L-RCA-rAd5 viral vector was constructed in two steps: (1) using pAd / PL-DEST plasmid (purchased from Invitrogen, catalog number V49420) as a template, fragment 4 containing the packaging signal sequence Ad5 129-392 fragment (SEQ ID NO: 5) was amplified and reversely inserted into the E4 region, and fragment 3 containing the IX gene Ad5 3525-4080 fragment (SEQ ID NO: 6) was amplified and forwardly inserted upstream of the reverse complementary fragment of the reversely inserted Ad5 129-392bp fragment containing the packaging signal sequence. The pAd / PL-DEST plasmid treated with sgRNA was mixed with fragments 3 and 4, and recombined and connected by the Gibson in vitro recombination method to obtain the intermediate vector IX-phi-E4-DEST plasmid. (2) PCR amplification was performed using the template synthesized by gene synthesis to amplify fragment 5 containing pAd / PL-DEST (104-128 bp)-(393-504 bp)-CMV-hXGENE-SV40 PA fragment (corresponding to 104-2374 bp of SEQ ID NO: 8) and fragment 6 containing pSurvivin-E1A-E1A terminal-IVa2 C-terminal gene fragment with synonymous mutation (corresponding to 2375-4233 bp of SEQ ID NO: 8). These two fragments were connected to replace pAd / PL-DEST 104-3168 bp. The IX-phi-E4-DEST plasmid treated with Cas9 nuclease and sgRNA was mixed with fragments 5 and 6 for recombination and connection to obtain the L-RCA-rAd5 viral vector, the schematic diagram of which is shown in FIG. Figure 4 The packaging signal (ψ) sequence was deleted from fragment 5 containing the pAd / PL-DEST (104-128 bp)-(393-504 bp)-CMV-hXGENE-SV40 PA fragment. The IX gene fragment was deleted from fragment 6 containing the pSurvivin-E1A-E1A terminal-IVa2 C-terminal gene fragment with synonymous mutations.

[0164] The Ad5 129-392 bp fragment (SEQ ID NO:5) containing a packaging signal (ψ) sequence (SEQ ID NO:26) located on the 3' side of the left ITR (5'ITR, SEQ ID NO:24) and the Ad5 3525-4080 bp fragment containing the IX gene (SEQ ID NO:6, containing the IX gene promoter, the IX gene coding sequence (SEQ ID NO:27) and the transcription termination sequence) were deleted, and the two deleted fragments were inserted between the downstream of the E4 gene and the 5' side of the right ITR (3'ITR, SEQ ID NO:25), wherein the deleted Ad5 129-392 bp fragment containing the packaging signal sequence was reversely inserted into the wild-type Ad5 35773 bp (the wild-type Ad5 genomic nucleotide sequence is shown in SEQ ID NO:7 (GenBank: AC_000008)), and the deleted Ad5 3525-4080 bp fragment containing the IX gene (SEQ ID NO:6, containing the IX gene promoter, the IX gene coding sequence (SEQ ID NO:27) and the transcription termination sequence) were inserted into the wild-type Ad5 35773 bp (the wild-type Ad5 genomic nucleotide sequence is shown in SEQ ID NO:7 (GenBank: AC_000008)). NO:6) was inserted forward into the upstream of the reverse inserted Ad5 129-392bp fragment containing the packaging signal sequence. At the same time, a synonymous mutation was made every 3-5 amino acids in the 333-450aa interval of the IVa2 protein, and a total of 24 mutations were made in the base sequence corresponding to the amino acids in this segment. The full-length nucleotide sequence of the vector constructed by the above method is shown in SEQ ID NO:8; the IVa2 C-terminal nucleotide sequence (SEQ ID NO:9) after the synonymous mutation was compared with the wild-type nucleotide sequence (SEQ ID NO:10) as shown in FIG. Fig. 9 , the nucleotides of synonymous mutations are underlined.

[0165] The steps for the preparation, identification, passage, purification and analysis of the viral vector (L-RCA-rAd5) with reduced replication-competent adenovirus contamination are as follows:

[0166] 2.1 Preparation of viral vector (L-RCA-rAd5) with reduced contamination of replicative adenovirus:

[0167] (a) Designing 0718-sgRNA near 35773bp of wild-type Ad5, the sequence of which is shown in SEQ ID NO: 11; synthesizing sgRNA in vitro transcription forward primer 0718-sgT7F, the sequence of which is shown in SEQ ID NO: 12, using a one-step sgRNA in vitro transcription kit (Jianan protein, catalog number E369) to transcribe 0718-sgRNA, and then cutting the pAd / PL-DEST plasmid with NLS-Cas9 Nuclease protein (Jianan protein, catalog number E365-01A) to obtain the pAd / PL-DEST plasmid treated with sgRNA and Cas9 nuclease, wherein the reaction system is shown in Table 7:

[0168] Table 7 Reaction system

[0169] Components Volume (μl) pAd / PL-DEST plasmid 24 10×Cas9 Buffer 5 NLS-Cas9 Nuclease 2 0718-sgRNA 1 <![CDATA[ddH2O]]> 18 total 50

[0170] (b) PCR amplification was performed using the pAd / PL-DEST plasmid as a template to obtain fragment 3 of the Ad5 3525-4080 bp fragment containing the IX gene and fragment 4 of the Ad5 129-392 bp fragment containing the packaging signal (ψ), respectively. Fragment 3 and the 5' end of the cut after sgRNA cutting have a 28 bp overlapping sequence 4 (SEQ ID NO:31: 5'-AATCGTCACTTCCGTTTTCCCACGTTAC-3'), and fragment 4 and the 3' end of the cut after sgRNA cutting have a 26 bp overlapping sequence 5 (SEQ ID NO:32: 5'-GTCACTTCCCATTTTAAGAAAACTAC-3'), and there is a 20 bp overlapping sequence 6 (SEQ ID NO:33: 5'-AAGCAAGTGTCTGGGCGAGT-3') between the two fragments (fragment 3 and fragment 4); in addition, Ad5 containing the IX gene The fragment 3 of 3525-4080 bp fragment was kept in the forward direction, while the fragment 4 of Ad5 129-392 bp fragment containing the packaging signal (ψ) was in the reverse direction. The two fragments (fragment 3 and fragment 4) obtained by amplification were recovered by gel cutting and used for later use. The PCR reaction system is shown in Table 2; the reaction program is 98℃, 10s; 58℃, 20s; 72℃, 30s; 30 cycles.

[0171] (c) The pAd / PL-DEST plasmid treated with sgRNA obtained in step (a) was mixed with fragment 3 of the Ad5 3525-4080 bp fragment containing the IX gene and fragment 4 of the Ad5 129-392 bp fragment containing the packaging signal (ψ) obtained in step (b), and recombined and ligated by the Gibson in vitro recombination method. The ligation reaction system is shown in Table 8; the reaction procedure is 50° C., 30 min.

[0172] Table 8 Reaction system

[0173] Components Volume (μl) pAd / PL-DEST (after sgRNA and Cas9 treatment) 3 Fragment 3 of the Ad5 3525-4080 bp fragment containing the IX gene 0.5 Fragment 4 of the Ad5 129-392 bp fragment containing the packaging signal (ψ) 0.5 2×Gibson Assembly Master Mix(NEB,E2611S) 5 <![CDATA[ddH2O]]> 1 total 10

[0174] (d) Take 5 μl of the reaction system after in vitro recombination in step (c) to transform Mach1-T1 Escherichia coli competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd., catalog number DL1015), and heat shock at 42°C for 45 seconds. Screen positive clones by colony PCR method, extract plasmids and send them for sequencing. The vector with correct sequencing is the intermediate vector IX-phi-E4-DEST plasmid (SEQ ID NO: 13). The colony PCR reaction system is shown in Table 4; the reaction program is 98°C, 10 seconds; 56°C, 10 seconds; 72°C, 20 seconds; 30 cycles.

[0175] (e) 0816-5sg1 (SEQ ID NO: 14) and 0816-3sg1 (SEQ ID NO: 15) were designed, and sgRNA in vitro transcription forward primers 0816-5sg1-T7F (SEQ ID NO: 16) and 0816-3sg1-T7F (SEQ ID NO: 17) were synthesized, and 0816-5sg1 and 0816-3sg1 were transcribed using a one-step sgRNA in vitro transcription kit, and then the intermediate vector IX-phi-E4-DEST plasmid was cut together with the NLS-Cas9Nuclease protein. After the reaction was completed, the large fragment was cut and recovered for standby use; the reaction system is shown in Table 9:

[0176] Table 9 Reaction system

[0177] Components Volume (μl) IX-phi-E4-DEST plasmid 20 10×Cas9 Buffer 5 NLS-Cas9 Nuclease 2 0816-5sg1 1 0816-3sg1 1 <![CDATA[ddH2O]]> 21 total 50

[0178] (f) PCR amplification was performed using the gene synthesized template to amplify fragment 5 containing the pAd / PL-DEST (104-128 bp)-(393-504 bp)-CMV-hXGENE-SV40 PA fragment (corresponding to 104-2374 bp of SEQ ID NO: 8) and fragment 6 containing the pSurvivin-E1A-E1A terminal-IVa2 C-terminal gene fragment with synonymous mutation (corresponding to 2375-4233 bp of SEQ ID NO: 8). Fragment 5 had a 25 bp overlapping sequence 7 (SEQ ID NO: 34: 5'-TAGTGTGGCGGAAGTGTGATGTTGC-3') upstream of the 0816-5sg1 cut, and fragment 6 had a 30 bp overlapping sequence 8 (SEQ ID NO: 35: 5'-TAGTGTGGCGGAAGTGTGATGTTGC-3') downstream of the 0816-3sg1 cut. NO:35: 5'-CCATATCCCTCCGGGGATTCATGTTGTGCA-3'). At the same time, there is also a 20 bp overlapping sequence 9 (SEQ ID NO:36: 5'-TCAATGTATCTTACTCACTC-3') between the two fragments (fragment 5 and fragment 6). The two amplified fragments (fragment 5 and fragment 6) were recovered by gel cutting and used for later use. The PCR reaction system is shown in Table 2; the reaction program is 98℃, 10s; 60℃, 20s; 72℃, 120s; 30 cycles.

[0179] (g) mixing the IX-phi-E4-DEST plasmid treated with Cas9 nuclease and sgRNA in step (e) with fragment 5 containing the pAd / PL-DEST (104-128 bp)-(393-504 bp)-CMV-hXGENE-SV40 PA fragment (corresponding to 104-2374 bp of SEQ ID NO: 8) amplified in step (f) and fragment 6 containing the pSurvivin-E1A-E1A terminal-IVa2 C-terminal gene fragment with synonymous mutation (corresponding to 2375-4233 bp of SEQ ID NO: 8), and recombining and ligating them by the Gibson in vitro recombination method. The fragment 6 of the IVa2 C-terminal gene fragment (corresponding to 2375-4233 bp of SEQ ID NO: 8) with synonymous mutation was ligated to replace pAd / PL-DEST104-3168 bp. The ligation reaction system is shown in Table 10; the reaction procedure is 50°C, 30 min.

[0180] Table 10 Reaction system

[0181] Components Volume (μl) IX-phi-E4-DEST plasmid (after Cas9 and sgRNA treatment) 3 Fragment 5 containing pAd / PL-DEST(104-128bp)-(393-504bp)-CMV-hXGENE-SV40PA fragment 0.5 pSurvivin-E1A-E1A terminal-IVa2 C-terminal gene fragment with synonymous mutation 6 0.5 2×Gibson Assembly Master Mix(NEB,E2611S) 5 <![CDATA[ddH2O]]> 1 total 10

[0182] (h) Take 5 μl of the reaction system after in vitro recombination in step (g) and transform Mach1-T1 Escherichia coli competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd., product number DL1015), and heat shock at 42°C for 45 seconds. Screen positive clones by colony PCR method, extract plasmids, and use restriction endonuclease XbaI (NEB, product number R0145S) for enzyme digestion identification, use rCutsmart enzyme digestion buffer, and digest at 37°C for 30 minutes. Run the enzyme digestion product on 1% agarose gel electrophoresis to observe the bands. The enzyme digestion identification results are shown in Fig.10 , four bands of 18003-bp, 8583-bp, 6895-bp, and 3268-bp were observed, which was in line with expectations. The plasmid verified by enzyme digestion was sent for sequencing, and the constructed vector was sequenced using the Sanger sequencing method. The sequencing results were consistent with the map, indicating that the viral vector was successfully constructed. The vector that was sequenced correctly was the final target viral vector L-RCA-rAd5, and the full-length nucleotide sequence was shown in SEQ ID NO:8. The colony PCR reaction system is shown in Table 4; the reaction program is 98°C, 10s; 56°C, 10s; 72°C, 20s; 30 cycles.

[0183] The successfully constructed L-RCA-rAd5 viral vector was subjected to large-scale plasmid extraction and endotoxin removal, linearized with PacI, digested at 37°C for 1h, and then incubated at 65°C for 20min to inactivate the endonuclease Pac I, and then purified using a DNA purification recovery kit to recover the linearized viral plasmid DNA. At the same time, HEK293A cells were cultured in DMEM medium (purchased from Cytiva, catalog number SH30022.01), 10% fetal bovine serum was added to the culture medium, and the cells were cultured in an incubator at 37°C and 5% CO2 concentration. When the degree of polymerization of HEK293A cells was 80%-90%, the transfection reagent Lipofectamine 2000 (purchased from Thermo Fisher Scientific, catalog number 11668019) was used to form a Lipofectamine2000-DNA complex with the linearized viral plasmid DNA for transfection, and the changes in cell status were observed daily. The steps are as follows:

[0184] (1) Prepare transfection solutions A and B: Add 4 μg of the recombinant plasmid linearized and recovered by PacI to 500 μl Opti-MEM TMI reduced serum medium (purchased from Thermo Fisher Scientific, catalog number 31985070) was pipetted and mixed to obtain transfection solution A; 10 μl Lipofectamine 2000 transfection reagent was added to 490 μl Opti-MEM TM Mix well by pipetting in the serum-minus medium to obtain transfection solution B.

[0185] (2) Transfer all of Transfection Solution B to Transfection Solution A and mix thoroughly by pipetting to obtain Transfection Solution C. Leave the mixture at room temperature for 25 min.

[0186] (3) Add all of the transfection solution C to the T25 flask with HEK293A cells, shake it crosswise to mix, and place it in a 37°C, 5% CO2 constant temperature incubator for 6 hours, then discard the supernatant.

[0187] (4) Add 10 ml of DMEM medium containing 10% FBS and no antibiotics to the culture flask and culture for 10-14 days, and observe the cell status every day. HEK293A cells will show cytopathic effect 10-14 days after transfection. When more than 50% of the cells fall off the bottom of the culture flask, the cell suspension is collected, and after freezing and thawing three times, the supernatant is collected by centrifugation at 500×g for 10 minutes to obtain the recombinant virus suspension. Normal HEK293A cells (A) and HEK293A cells with cytopathic effect (B) are shown in Figure 2. Fig.11 shown.

[0188] 2.2 Viral genome digestion identification

[0189] The viral genomic DNA was extracted from the recombinant virus suspension and digested with restriction endonuclease SpeI-HF (NEB, R3133V) using rCutsmart digestion buffer at 37°C for 0.5 h. The SpeI-HF digestion product was electrophoresed on a 1% agarose gel to observe the bands. The results are shown in Fig.12 As shown, three bands of 26607-bp, 7804-bp, and 264-bp were observed, which was in line with expectations.

[0190] 2.3 Virus PCR identification

[0191] Using viral genomic DNA as template, use identification primers:

[0192] F: 5'-ACTGACTTTGCTTTCCTGAG-3' (SEQ ID NO: 18);

[0193] R: 5'-TTGTGTTACTCATAGCGCGT-3' (SEQ ID NO: 19).

[0194] The identification reaction system is shown in Table 5, and the reaction procedure is shown in Table 6.

[0195] The PCR products were analyzed by 1% agarose gel electrophoresis. Fig.13 A 318-bp band was observed, which was in line with expectations.

[0196] The viral genome was subsequently sequenced to ensure that the viral sequence was complete. The samples were sent to a sequencing company and the viral genome was sequenced by NGS sequencing. The results were completely consistent with the designed sequence, indicating that the virus was successfully constructed. The virus that was sequenced correctly was the low RCA recombinant adenovirus type 5 (L-RCA-rAd5).

[0197] 2.4 Virus Passaging

[0198] The harvested virus suspension was inoculated into HEK293A cells with a polymerization degree of 80%-90%. After 48-72h, HEK293 cells showed cytopathic effect, and more than 50% fell off from the bottom of the culture flask, and the cell suspension was harvested. The cell suspension was centrifuged at 500×g, 4℃ for 10min, the supernatant was discarded, and the precipitate was resuspended in 0.5ml DMEM medium. After freezing and thawing three times, the harvested virus suspension was inoculated into HEK293A cells with a polymerization degree of 80%-90%, and one virus passage was completed.

[0199] Similarly, the harvested low RCA recombinant adenovirus type 5 (L-RCA-rAd5) and control virus were passaged 14 times in HEK293A cells. The control virus was Figure 3 The conditionally replicative recombinant human adenovirus type 5 (CR-rAd5) with E1B and E3 regions deleted is shown in FIG.

[0200] 2.5 Virus purification

[0201] Since adenovirus is packaged and amplified in HEK293A, the genomic DNA of HEK239A cells may be mixed in when extracting viral genomic DNA, which contains some E1 region genes, which will affect the subsequent quantitative detection of reverse mutations. Therefore, SuperNuclease (purchased from Sino Biological, catalog number GMP-SSNP01) was first added to the harvested virus liquid, and incubated at 37°C for 60 minutes to remove some residual nucleic acids. The harvested virus suspension was then purified using the VivaPure AdenoPACK 20RT adenovirus purification kit (purchased from Sartorius, catalog number VS-AVPQ022) to remove residual host proteins and nucleic acids and obtain virus particles.

[0202] 2.6 Quantitative analysis of reverse mutations in viruses

[0203] The number of virus and reverse mutation copies was quantified by the dye method fluorescence quantification method. The 2nd, 4th, 6th, 8th, 10th, 12th, and 14th generations of viruses were taken for quantitative analysis of the number of virus particles and reverse mutations. 100 μl of the harvested virus suspension was taken to extract the viral genomic DNA, and then the number of virus particles and the number of reverse mutations were quantified by fluorescence quantitative PCR. The viral genomic DNA was diluted 100 times when the number of virus particles was quantified, and no dilution was done when the reverse mutation was quantified. The fluorescence quantitative PCR system is shown in Table 11; the fluorescence quantitative PCR program is shown in Table 12.

[0204] Table 11 Fluorescence quantitative PCR system

[0205]

[0206] Table 12 Fluorescence quantitative PCR program

[0207]

[0208] Primers for quantification of viral particle numbers are:

[0209] F:5'-AGTGACGTTTTTGGTGTGCG-3'(SEQ ID NO:20),

[0210] R: 5'-TACTCGGTTACGCCCAAATTTAC-3' (SEQ ID NO: 21);

[0211] The primers used to quantify the number of reverse mutations are:

[0212] mut-F:5'-AGATCAAGGATAATTGCGCT-3'(SEQ ID NO:22),

[0213] mut-R: 5'-ACTTGCAATCTGGCCTAAGT-3' (SEQ ID NO: 23).

[0214] The result judgment standard is 1×10 9 The number of reverse mutations in the number of virus particles is quantitatively calculated as follows: Fig.14 As shown, the results showed that: after continuous passaging to the 14th generation of the virus, the number of reversion mutations of the low RCA recombinant adenovirus type 5 (L-RCA-rAd5) remained at a low level during the continuous passaging; while the number of reversion mutations of the control group conditionally replicative recombinant human adenovirus type 5 (CR-rAd5) remained at a high level during the continuous passaging, and the RCA number increased with the increase in the number of passages of the adenovirus vector on 293 cells.

[0215] According to the requirements of the U.S. Food and Drug Administration (FDA), 10 The result judgment standard of the present invention is that every 1×10 9 The number of reverse mutations in the number of virus particles. The experimental results show that the low RCA recombinant adenovirus type 5 constructed by the present invention will not produce more reverse mutations during continuous passage, and can achieve the effect of reducing the contamination of replicative adenovirus.

Claims

1. A recombinant adenovirus vector with reduced contamination by adenoviruses, wherein, relative to adenovirus vectors or wild-type adenoviruses, the recombinant adenovirus vector with reduced contamination by adenoviruses lacks all or part of the sequences of the E1B region gene and the E3 region gene, wherein: The fragment containing the packaging signal (ψ) and the fragment containing the IX gene located downstream of the 3' side of the left ITR of the adenovirus vector or wild-type adenovirus are deleted; the fragment containing the packaging signal (ψ) and the fragment containing the IX gene are inserted between the downstream of the E4 region gene of the adenovirus vector or wild-type adenovirus and the 5' side of the right ITR, wherein the inserted fragment containing the packaging signal (ψ) is located upstream of the 5' side of the right ITR, the inserted fragment containing the IX gene is located upstream of the inserted fragment containing the packaging signal (ψ), and the directions of the inserted fragment containing the packaging signal (ψ) and the inserted fragment containing the IX gene are opposite, wherein the inserted fragment containing the packaging signal (ψ) at least has a nucleotide sequence necessary for the incorporation of the viral genome into the viral capsid during viral assembly, and the inserted fragment containing the IX gene at least has a nucleotide sequence capable of expressing the IX protein.

2. The recombinant adenovirus vector with reduced replication-competent adenovirus contamination according to claim 1, wherein: The IVa2 protein encoding gene of the recombinant adenovirus vector for reducing replication-type adenovirus contamination has a synonymous mutation.

3. The recombinant adenovirus vector with reduced replication-competent adenovirus contamination according to claim 1 or 2, wherein: The insertion direction of the inserted fragment containing the packaging signal (ψ) is 3'→5', and the insertion direction of the inserted fragment containing the IX gene is 5'→3'; Preferably, the target protein gene is inserted between the 3' side of the left ITR of the recombinant adenovirus vector for reducing replication-competent adenovirus contamination and the upstream of the E1A gene; preferably, a tumor-specific promoter is operably linked to the E1A gene, preferably, the tumor-specific promoter is selected from hTERT promoter, PEG-3 promoter or Survivin gene promoter; preferably, a constitutive promoter or an inducible promoter is operably linked to the target gene, preferably, the constitutive promoter is selected from CMV promoter, CAG promoter, PGK promoter or ACTB promoter; Preferably, the adenoviral vector is selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 , 60, 62, 63, 64, 65, 67, 69, 70, 71, 72, 73, 74 and 75 adenovirus vectors, preferably adenovirus serotype 4, 5, 7, 26, 35 or 48 adenovirus vectors, preferably human adenovirus vectors; more preferably, the adenovirus vector is adenovirus 5, 26 or 35 vectors; more preferably, the adenovirus vector is a human adenovirus 5 vector; preferably, the adenovirus vector is pAd / PL-DEST, pAdEasy-1 or pAd / CMV / V5-DEST; Preferably, the wild-type adenovirus is selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 7, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 70, 71, 72, 73, 74 and 75 wild-type adenovirus, preferably a human wild-type adenovirus; more preferably, the wild-type adenovirus is a wild-type adenovirus of type 5, 26 or 35; more preferably, the wild-type adenovirus is a human wild-type adenovirus of type 5; Preferably, the recombinant adenovirus vector with reduced contamination by replication-competent adenovirus is a recombinant adenovirus type 5 vector; more preferably, the recombinant adenovirus vector with reduced contamination by replication-competent adenovirus is a recombinant human adenovirus type 5 vector; Preferably, the target gene encodes an active protein molecule; preferably, the active protein molecule is selected from an immunomodulatory molecule, an antigen binding molecule, an antigen or a ligand of the antigen, a toxin, an enzyme or other active protein molecule; Preferably, the target gene encodes an immunomodulatory molecule; preferably, the immunomodulatory molecule is a co-stimulatory ligand, a cytokine or a cytokine receptor; more preferably, the immunomodulatory molecule is selected from the group consisting of TNF-α, IFN-α, IFN-β, IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-7, IL-10, IL-10R, IL-12A, IL-12B, IL-15, IL-23A / p19, IL24 , IL-27, IL-33, IL-35, IL-15, IL-15 receptor fusion protein, TGF-β, TGF-βtrap, IL-10trap, VEGF, CD80, CD137L, GM-CSF, GITR ligand, OX40 ligand, CD40 ligand, CD154, CD70, CD86, CD137, CD137L, BORIS / CTCFL, bone morphogenetic protein (BMP), TNFSF9, FGF, ICAM, their functional fragments, and derivatives thereof; Preferably, the target gene encodes an antigen binding molecule; preferably, the antigen binding molecule is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TGF-β antibody and an anti-CTLA-4 antibody or a functional fragment thereof; Preferably, the target gene encodes an antigen or a ligand of the antigen; preferably, the antigen is selected from the group consisting of CEA, HER-2 / neu, EGFRvIII, EpCAM, erbB2, erbB3, erbB4, CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD80, CD133, CD135 (Flt3), Flt3I, CD138, NY-ESO-1, GD2, GD3, MAGE-A1, MAGE-A3, MART1, MART2, MUC1, mesothelin, NKG2D ligand, BRAF, p53, T cell receptor, carcinoembryonic antigen, PSMA, functional fragments thereof, and derivatives thereof; Preferably, the target gene encodes a toxin; preferably, the toxin is diphtheria toxin, ricin toxin or Pseudomonas exotoxin; Preferably, the target gene encodes an enzyme; preferably, the enzyme is selected from the group consisting of esterase, carboxypeptidase, metalloproteinase, streptokinase, β-glucosidase, β-glucuronidase, β-galactosidase, β-lactamase, relaxin, collagenase, arginase, fragments thereof, and derivatives thereof; Preferably, the target gene encodes a tumor suppressor gene, a growth factor, a coagulation factor, an apoptosis gene, a fragment thereof, or a derivative thereof; Preferably, the target gene encodes angiostatin, endostatin, thymidine kinase, functional fragments thereof, or derivatives thereof; Preferably, the recombinant adenovirus vector with reduced replication-type adenovirus contamination further comprises a replication origin and / or a selection marker gene; Preferably, the replication origin is selected from the group consisting of pMB1 replication origin (pMB1 ori), ColE1 replication origin (ColE1ori), pBR322 replication origin (pBR322ori), pUC replication origin ((pUCori)) (e.g., pUC57 replication origin ((pUC57ori))), pSC101 replication origin (pSC101ori) and R6Kγ replication origin (R6Kγori); Preferably, the selection marker gene is selected from one or more of antibiotic or virus resistance genes, heavy metal resistance genes, and auxotrophic genes; Preferably, the antibiotic or virus resistance gene is selected from one or more of an ampicillin resistance gene, a chloramphenicol resistance gene, a neomycin resistance gene, a G418 resistance gene, a puromycin resistance gene, a hygromycin resistance gene, a bleomycin resistance gene, a kanamycin resistance gene, a methotrexate resistance gene, a phleomycin resistance gene and a benomyl resistance gene; Preferably, the nutritional deficiency gene is selected from one or more of ura3, ade2, his3, leu2, trpl and lys2.

4. A method for preparing a recombinant adenovirus vector with reduced contamination by a replicative adenovirus according to any one of claims 1 to 3, comprising the following steps: (1) taking an adenoviral vector or a wild-type adenovirus and retaining the left and right ITRs of the adenoviral vector or the wild-type adenovirus; (2) deleting the fragment containing the packaging signal (ψ) and the fragment containing the IX gene located downstream of the 3' side of the left ITR of the adenoviral vector or wild-type adenovirus; (3) inserting a fragment containing a packaging signal (ψ) and a fragment containing the IX gene into the adenovirus vector or between the downstream of the E4 region gene of the wild-type adenovirus and the 5' side of the right ITR, wherein: The inserted fragment containing the packaging signal (ψ) is located upstream of the 5' side of the right ITR, the inserted fragment containing the IX gene is located upstream of the inserted fragment containing the packaging signal (ψ), and the directions of the inserted fragment containing the packaging signal (ψ) and the inserted fragment containing the IX gene are opposite; (4) deleting all or part of the sequence of the E1B gene of the adenovirus vector or wild-type adenovirus so that the E1B gene cannot express the gene product of the active E1B region; All E1A genes are retained; among them: (i) If the adenovirus vector lacks the E1B gene and cannot express the gene product of the active E1B region, step (4) is not performed; (ii) if the adenoviral vector lacks the E1A gene, inserting the E1A gene downstream of the 3' side of the left ITR of the adenoviral vector and upstream of the IX gene, so that the adenoviral vector can express all gene products of the E1A gene after the insertion of the E1A gene; (5) deleting all or part of the sequence of the E3 region gene of the adenovirus vector or wild-type adenovirus so that the E3 gene cannot express the gene product of the active E3 region; wherein: If the adenovirus vector lacks all or part of the sequence of the E3 region gene and cannot express the gene product of the active E3 region, step (5) is not performed; The order of steps (2)-(5) can be arbitrarily changed, or steps (2)-(5) can be performed simultaneously to obtain the recombinant adenovirus vector with reduced replication-competent adenovirus contamination.

5. The method according to claim 3, further comprising: (6) performing synonymous mutation on the gene encoding the IVa2 protein of the adenovirus vector or wild-type adenovirus; wherein the order of steps (2) to (6) can be arbitrarily changed, or steps (2) to (6) can be performed simultaneously; Preferably, the method further comprises: (7) virus packaging: introducing the obtained recombinant adenovirus vector with reduced replication-competent adenovirus contamination into packaging cells (such as HEK293 cells) to obtain virus particles; Preferably, the adenoviral vector is pAd / PL-DEST, and the method comprises the following steps: (i) taking a pAd / PL-DEST plasmid and retaining the left and right ITRs of the pAd / PL-DEST plasmid; (ii) deleting the fragment containing the packaging signal (ψ) and the fragment containing the IX gene located 3′ downstream of the left ITR of the pAd / PL-DEST plasmid; (iii) inserting a fragment containing a packaging signal (ψ) and a fragment containing the IX gene into the region downstream of the gene in the E4 region of the pAd / PL-DEST plasmid and between the 5' side of the right ITR, wherein the inserted fragment containing the packaging signal (ψ) is located upstream of the 5' side of the right ITR, the inserted fragment containing the IX gene is located upstream of the inserted fragment containing the packaging signal (ψ), and the directions of the inserted fragment containing the packaging signal (ψ) and the inserted fragment containing the IX gene are opposite; (iv) inserting the E1A gene downstream of the 3' side of the left ITR of the pAd / PL-DEST plasmid and upstream of the IX gene, so that the pAd / PL-DEST plasmid can express all gene products of the E1A gene after the E1A gene is inserted; The order of steps (ii)-(iv) can be arbitrarily changed, or steps (ii)-(iv) can be performed simultaneously to obtain the recombinant adenovirus vector with reduced contamination by replicating adenovirus; Preferably, the adenoviral vector is pAd / PL-DEST, and the method comprises the following steps: ① Designing a first sgRNA near the insertion site of the E4 region of the pAd / PL-DEST plasmid, mixing the first sgRNA, Cas9 nuclease and the pAd / PL-DEST plasmid to obtain a pAd / PL-DEST plasmid treated with the first sgRNA and Cas9 nuclease; ② Using the pAd / PL-DEST plasmid as a template, PCR amplification was performed to obtain fragment A containing the IX gene and fragment B containing the packaging signal (ψ), respectively, wherein the fragment A and the 5' end of the cut of the pAd / PL-DEST plasmid treated with the first sgRNA and Cas9 nuclease have an overlapping sequence I of at least 10 bp, and the fragment B and the 3' end of the cut of the pAd / PL-DEST plasmid treated with the first sgRNA and Cas9 nuclease have an overlapping sequence II of at least 10 bp, and at the same time, there is an overlapping sequence III of at least 10 bp between the fragment A and the fragment B; wherein the fragment A maintains a forward direction, and the fragment B is in a reverse direction; ③ The pAd / PL-DEST plasmid obtained in step ① after being treated with the first sgRNA and Cas9 nuclease is mixed with fragment A and fragment B obtained in step ②, and recombined and connected to obtain an intermediate vector; ④ Design the second sgRNA and the third sgRNA, mix the second sgRNA and the third sgRNA, Cas9 nuclease and the intermediate vector to obtain the intermediate vector treated with the second sgRNA, the third sgRNA and the Cas9 nuclease: ⑤ Perform PCR amplification with the template synthesized by gene, and respectively amplify fragment C containing pAd / PL-DEST (downstream of the 3' side of the left ITR - at least 10 bp sequence upstream of the packaging signal (ψ)) - (downstream of the packaging signal (ψ) - at least 20 bp sequence upstream of attR1) - promoter - target gene - polyadenylation signal, and fragment D containing the E1A gene and the IVa2 C-terminal gene fragment with synonymous mutation, fragment C and the nick upstream of the intermediate vector treated with the second sgRNA, the third sgRNA and the Cas9 nuclease have at least 10 bp overlapping sequence IV, fragment D and the nick downstream of the intermediate vector treated with the second sgRNA, the third sgRNA and the Cas9 nuclease have at least 10 bp overlapping sequence V, and at the same time, fragment C and fragment D have at least 10 bp overlapping sequence VI; preferably, the promoter is CMV promoter, CAG promoter, PGK promoter or ACTB promoter; the polyadenylation signal is SV40 PA, BGH PA, hGH PA or rbGlob PA; ⑥ The intermediate vector obtained in step ④ after being treated with the second sgRNA and the third sgRNA and the Cas9 nuclease is mixed with the fragment C and the fragment D obtained in step ⑤, and recombined and connected to obtain a recombinant adenovirus vector with reduced contamination of replicative adenovirus; wherein, after the fragment C and the fragment D are connected, they replace the IVa2 C-terminal gene without synonymous mutation at the 3' side downstream of the left ITR of pAd / PL-DEST; More preferably, in step ①, the insertion site in the E4 region of the pAd / PL-DEST plasmid is a site corresponding to 35773 bp of wild-type Ad5; the first sgRNA sequence is as shown in SEQ ID NO: 11; More preferably, in step ②, fragment A contains a 3525-4080 bp fragment corresponding to the wild-type Ad5 IX gene; fragment B contains an Ad5 129-392 bp fragment of the packaging signal (ψ); the overlapping sequence I sequence is shown in SEQ ID NO: 31; the overlapping sequence II sequence is shown in SEQ ID NO: 32; and the overlapping sequence III sequence is shown in SEQ ID NO: 33; More preferably, in step ③, the intermediate vector sequence is as shown in SEQ ID NO: 13; More preferably, in step ⑤, the second sgRNA sequence is as shown in SEQ ID NO: 14; the third sgRNA sequence is as shown in SEQ ID NO: 15; the fragment containing pAd / PL-DEST (downstream of the 3' side of the left ITR-at least 10bp sequence upstream of the packaging signal (ψ))-(downstream of the packaging signal (ψ)-at least 20bp sequence upstream of attR1)-promoter-target gene-polyadenylation signal is a fragment containing pAd / PL-DEST (104-128bp)-(393-504bp)-CMV-hXGENE-SV40 PA, and its sequence corresponds to the sequence shown in nucleotides 104 to 2374 of SEQ ID NO: 8; the gene fragment containing the E1A gene and the IVa2 C-terminal gene fragment with synonymous mutations is a gene fragment containing pSurvivin-E1A-E1A terminal-IVa2 C-terminal gene fragment with synonymous mutations, and its sequence corresponds to the sequence shown in nucleotides 2375 to 4233 of SEQ ID NO: 8; the overlapping sequence IV is as shown in SEQ ID NO: ID NO:34; the overlapping sequence V sequence is shown in SEQ ID NO:35; the overlapping sequence VI sequence is shown in SEQ ID NO:36; the packaging signal (ψ) sequence is deleted in the pAd / PL-DEST (104-128 bp)-(393-504 bp)-CMV-hXGENE-SV40 PA fragment; the IX gene fragment is deleted in the IVa2 C-terminal gene fragment of pSurvivin-E1A-E1A terminal-synonymous mutation; preferably, the synonymous mutation has a total of 29 site mutations; More preferably, in step ⑥, fragment C and fragment D are connected to replace the nucleotide sequence of positions 104 to 3168 of pAd / PL-DEST shown in SEQ ID NO: 1; the sequence of the recombinant adenovirus vector for reducing contamination by replicating adenovirus is shown in SEQ ID NO: 8; More preferably, 24 mutations, a total of 29 mutations, are performed on the IVa2 C-terminus; the sequence of the IVa2 C-terminal gene fragment after synonymous mutations is shown in SEQ ID NO:

9. A host cell comprising the recombinant adenovirus vector with reduced replication-competent adenovirus contamination according to any one of claims 1 to 3.

7. A composition comprising the recombinant adenovirus vector with reduced replication-competent adenovirus contamination according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

8. A method for producing a recombinant adenovirus, comprising: (1) mixing the recombinant adenovirus vector with reduced replication-competent adenovirus contamination according to any one of claims 1 to 3 with a host cell, and introducing the recombinant adenovirus vector with reduced replication-competent adenovirus contamination into the host cell; (2) growing the host cell under conditions for producing the recombinant adenovirus; Preferably, the host cell is a HEK293 cell, preferably a HEK293A cell.

9. The method of claim 8, further comprising: (3) collecting the cell suspension, freezing and thawing it, and collecting the supernatant by centrifugation to obtain the recombinant virus suspension; Preferably, the method further comprises: (4) Purifying the recombinant virus suspension described in step (3).

10. Use of the recombinant adenovirus vector with reduced replication-competent adenovirus contamination according to any one of claims 1 to 3 and / or the composition according to claim 7 in the preparation of recombinant adenovirus.

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