Viral vectors for reducing replication-competent adenovirus contamination and construction methods thereof

Abstract

The present invention discloses a viral vector for reducing the contamination of replication-competent adenovirus and a construction method thereof. The viral vector of the present invention has a low probability of homologous recombination with the adenovirus E1 region gene integrated in the packaging cells, reduces the contamination of replication-competent adenovirus, and improves the safety and effectiveness of adenovirus gene therapy products.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to a viral vector for reducing the contamination of replication-competent adenovirus and a construction method thereof. Background Art

[0002] Adenovirus is an enveloped linear double-stranded DNA virus that is widely distributed in nature and there are at least more than 100 serotypes. The genome of mammalian adenovirus is about 36 kb long. Figure 1Schematic diagram of the genomic 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 its genome. Inside the ITR is the viral packaging signal (ψ), which is a cis-acting element necessary 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. Four early transcription units (E1, E2, E3, E4) undertake regulatory functions. Among them, the proteins expressed by early genes E1a and E1b are responsible for trans-activating the expression of other viral genes. The E2 protein is responsible for viral DNA replication. The E3 protein is responsible for the host cell immune response. The E4 protein inhibits host cell apoptosis. Late genes encode the structural genes of the virus and some gene regulatory proteins. The adenovirus E1 region gene is immediately activated after the virus enters the nucleus. Its encoded product is the main transcriptional regulator, regulating the functions of all early genes. The E1 region is an essential region for virus replication. Adenovirus lacking the E1 region is a kind of virus that must be replicated and proliferated in special engineering cells or with the assistance of non-defective adenovirus. Due to the deletion of the essential replication region, adenovirus can only replicate in helper cells expressing the E1 gene product. The most commonly used helper cell is the 293 cell line, which is transfected from human embryonic kidney cells with the left terminal fragment of Ad5 and integrates a fragment containing the adenovirus E1 region in its genome, and can continuously express the E1 region protein. The E2 region gene encodes DNA binding protein (DBP), precursor terminal protein (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 processes 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 adenovirus life cycle, so it is called an intermediate gene, and it is also called a delayed early gene because its expression follows immediately after the early genes. The E4 region is mainly related to the metabolism of viral messenger RNA, and also has the functions of promoting viral DNA replication and shutting down host protein synthesis. In addition, IVa2 is a transcriptional activator of the adenovirus major late promoter (MLP) and plays an important role in the activation of MLP.The expression of late proteins such as the major capsid proteins Hexon, Penton, and Fiber of adenovirus is regulated by MLP. Meanwhile, IVa2 plays an important role in the assembly of viral capsid proteins and the encapsidation of viral genomes. Therefore, in theory, recombinant adenoviruses lacking the IVa2 gene are blocked in the expression of structural proteins, the assembly of virus particles, and the encapsidation of genomes, and thus cannot produce progeny viruses.

[0003] Gene therapy products have become a hot topic in drug R & D at home and abroad in recent years. In the research, development, and large-scale production of gene therapy products, how to control the quality of products and ensure their safety and effectiveness is a key link for the successful application of such products. The biological functions of adenovirus have been well studied. It has low pathogenicity to humans, high transduction efficiency for both dividing and non-dividing cells, broad tissue tropism, and does not integrate into the host genome. Adenovirus can be modified into replication-defective virus vectors and oncolytic virus vectors with selective replication.

[0004] The first-generation adenovirus vector removes the E1 and / or E3 regions of the adenovirus genome. The deletion of E1 makes it unable to replicate on its own and can only rely on the trans-complementation provided by packaging cells such as HEK293A cells for replication and amplification, thus ensuring the safety of adenoviruses. The commonly used HEK293 cells for adenovirus packaging are immortalized cells formed by transfecting human embryonic kidney cells with sheared human adenovirus 5 (Ad5). This cell contains and expresses the transfected Ad5 gene, and the 1-4344 linear nucleotides of Ad5 are integrated into chromosome 19 (19q13.2) of HEK293 cells, with sequences identical to those of serotype 5 adenovirus, including the left terminal repeat sequence (ITR), packaging signal sequence (ψ), E1 gene, IX gene, and approximately 250 bp at the C-terminus of the IVa2 gene. Even if the E1 gene is deleted from the adenovirus vector, there is still a risk of homologous recombination between the packaging signal sequence upstream of the E1 gene and the 3' ends of the IX and IVa2 genes downstream of the E1 gene and the Ad5 1-4344 gene integrated into the genome of the packaging cells. 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 capable of self-replication and amplification - a replication-competent adenovirus, namely RCA virus (Replication Competent Adenoviruses, RCAs). Experiments have shown that the number of RCAs increases with the increase in the number of passages of the adenovirus vector on 293 cells. RCAs can self-replicate and amplify 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 use effect of recombinant adenovirus vectors and pose safety hazards. In gene therapy drugs using non-replicating adenoviruses as vectors, the control standard for RCAs is less than 1 RCA / 3×10 10 VP. How to reduce the contamination of replication-competent adenoviruses and improve the safety and effectiveness of adenovirus gene therapy products has become an urgent research topic. Summary of the Invention

[0005] In order to reduce the probability of homologous recombination between recombinant adenoviruses and host cells, the present invention provides an adenovirus vector for reducing the contamination of replication-competent adenoviruses and its construction method.

[0006] In one aspect, the present disclosure provides a recombinant adenovirus vector with reduced replication-competent adenovirus contamination. Compared with an adenovirus vector or wild-type adenovirus, the recombinant adenovirus vector with reduced replication-competent adenovirus contamination lacks all or part of the sequences of the E1B region gene and the E3 region gene. Among them, 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; a fragment containing the packaging signal (ψ) and a 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. Among them, 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. Among them, the inserted fragment containing the packaging signal (ψ) has at least the nucleotide sequence necessary for the viral genome to be incorporated into the viral capsid during virus assembly, and the inserted fragment containing the IX gene has at least the 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 replication-competent adenovirus contamination.

[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, which includes:

[0010] (1) Mixing the aforementioned recombinant adenovirus vector with reduced replication-competent adenovirus contamination with a host cell to introduce the recombinant adenovirus vector with reduced replication-competent adenovirus contamination into the host cell;

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

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

[0013] Gene recombination is a common phenomenon in the biological world. Homologous recombination is the exchange between the DNA sequences of any two genes with a homologous sequence segment. The condition for homologous recombination to occur is the existence of homologous sequences between two DNA molecules, regardless of 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, recombination is very difficult to occur. In the present disclosure, in order to reduce the probability of homologous recombination, the packaging signal and the IX gene homologous to the Ad5 gene integrated in the cell genome in the adenovirus vector are both moved to the E4 region; further, multiple-site synonymous mutations are performed on the 3'-terminal sequence of IVa2, and the sequences homologous to the Ad5 sequence transfected into the packaging cell in the E1 region are all transferred or subjected to synonymous mutations, and there are no large homologous sequence segments, thus reducing the probability of homologous recombination to the greatest extent; the IX gene is inserted forward in the E4 region, and the packaging signal sequence is inserted backward at the same position. The arrangement directions of the IX gene sequence and the packaging signal sequence are inconsistent with the Ad5 sequence transfected into the packaging cell, thereby also avoiding the possibility of recombination occurring in the E4 region. The experiments of the present disclosure prove that the above-mentioned modification of the adenovirus vector greatly reduces the probability of homologous recombination, thereby reducing the contamination of replication-competent adenovirus. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0018] Figure 5 It is an electrophoresis result diagram after digestion of the CR-rAd5 control virus plasmid, which is the result diagram of agarose gel electrophoresis detection of the digestion products after the virus plasmid is digested with the restriction enzyme XbaI.

[0019] Figure 6Normal HEK293A cells and HEK293A cells with cytopathic effect during the packaging process of CR-rAd5 control virus; In the figure: A are normal HEK293A cells; B are HEK293A cells with cytopathic effect; The magnification is 10×10 for both.

[0020] Figure 7 It is a result diagram of agarose gel electrophoresis detection of the digestion product after the genomic DNA of the low CR-rAd5 control virus is digested with the restriction enzyme BstZ17I-HF.

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

[0022] Figure 9 It is a comparison diagram of the C-terminal nucleotide sequence of synonymous mutant IVa2 and the wild-type nucleotide sequence.

[0023] Figure 10 It is an electrophoresis result diagram after digestion of the L-RCA-rAd5 virus plasmid, which is the result diagram of agarose gel electrophoresis detection of the digestion product after the virus plasmid is digested with the restriction enzyme XbaI.

[0024] Figure 11 Normal HEK293A cells and HEK293A cells with cytopathic effect during the packaging process of L-RCA-rAd5 virus; In the figure: A are normal HEK293A cells; B are HEK293A cells with cytopathic effect; The magnification is 10×10 for both.

[0025] Figure 12 It is a result diagram of agarose gel electrophoresis detection of the digestion product after the genomic DNA of the low RCA recombinant human adenovirus type 5 (L-RCA-rAd5) is digested with the restriction enzyme SpeI-HF.

[0026] Figure 13 It is an electrophoresis diagram of PCR identification detection using the low RCA recombinant human adenovirus type 5 (L-RCA-rAd5) as a template.

[0027] Figure 14 It is the change of the RCA level of the conditionally replicating recombinant human adenovirus type 5 (CR-rAd5) and the low RCA recombinant human adenovirus type 5 (L-RCA-rAd5) after continuous passage. Specific implementation mode

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. For those not specified in the embodiments, the operations are performed under conventional conditions or conditions recommended by the manufacturer.

[0029] For those reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchases.

[0030] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains. The terms used in the description 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" means one or more of that entity; for example, "a nucleotide sequence" should be 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" means a value or a composition within an acceptable error range of a specific value or composition as determined by those of ordinary skill in the art, and the acceptable error range will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, in accordance with the practice in the art, "about" or "consisting essentially of" can mean within one or more standard deviations. Alternatively, "about" or "consisting essentially of" can mean a range of up to 20%. In addition, especially with respect to biological systems or processes, the term can mean up to one order of magnitude or up to 5 times the value. When a specific value or composition is provided in the present application and the claims, unless otherwise stated, it should be assumed that the meaning of "about" or "consisting essentially of" is within the acceptable error range of that specific 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 value within the recited range and, where appropriate, fractions of such values (such as tenths and hundredths of an integer).

[0034] As used herein, the terms “adenovirus” and “adenovirus 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 “adenovirus particle” refer to the virus itself or its derivatives, and encompass all serotypes and subtypes as well as naturally occurring and recombinant forms. In one embodiment, such adenoviruses infect human cells.

[0035] As used herein, the term “adenovirus vector” refers to an adenovirus that has been genetically modified to behave differently from the native wild-type virus, wherein the adenovirus genome has been manipulated to accommodate a nucleic acid sequence that is non-native with respect to the adenovirus genome. Typically, an adenovirus vector is produced by introducing one or more mutations (e.g., deletions, insertions, or substitutions) into the adenovirus genome of an adenovirus to accommodate the insertion of a non-native nucleic acid sequence into the adenovirus. For example, an adenovirus vector can be modified such that it cannot replicate outside of a specific packaging cell line. In some aspects, the adenovirus vector is genetically modified to carry one or more genes encoding a protein of interest. The protein of interest can be a non-adenovirus protein. Examples of adenoviruses that can be used as viral vectors in the present invention include those adenoviruses 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, human, chimpanzee (e.g., ChAd1, ChAd3, ChAd7, ChAd8, ChAd21, ChAd22, ChAd23, ChAd24, ChAd25, ChAd26, ChAd27.1, 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 rhesus monkey adenoviruses (e.g., rhAd51, rhAd52, or rhAd53).

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

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

[0038] In one embodiment, the adenovirus packaging cell line of the present disclosure includes some of the coding sequences that make up the E1 region (e.g., E1A or E1B). As used herein, the term "E1A" refers to all the 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 by 46 amino acids, and they are spliced from the 12S mRNA. For the purposes of the present invention, the packaging cell line can express the 289 polypeptide, the 243 polypeptide, or both the 289 and 243 polypeptides. As used herein, the term "E1B" refers to all the gene products of the adenovirus E1B region, including three major polypeptides of 19kd and 55kd. The E1B 19kd and 55kd proteins are important in cell transformation. For the purposes of the present invention, the packaging cell line can express the 19Kd polypeptide, the 55Kd polypeptide, or both the 19Kd and 55Kd polypeptides.

[0039] As used herein, the term "E2" refers to a cis - acting element having at least 3 ORFs, all of which are involved in DNA replication, including the polymerase. The adenovirus E2 late promoter has been described, for example, by Swaminathan, S. and Thimmapaya, B. (1995) Curr. Top. Microbiol. 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 the genes E2A and E2B. In this case, the synthesis of E2 mRNA initially starts from the E2 early promoter. Approximately 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 that are present in the adenovirus genome and are expressed during 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 in vitro replication of the virus and may therefore be deleted in adenoviral vectors.

[0041] As used herein, the term "E4 region" refers to a group of genes that are present in the adenovirus genome near the right-hand terminal repeat (ITR) and are expressed during 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 are involved in lytic infection and tumorigenesis.

[0042] As used herein, the term "packaging signal" refers to the nucleotide sequence that is present in the viral genome and is 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 designated as "ψ".

[0043] The term "protein of interest" broadly refers to any protein that has a particular relevance in the industrial production of proteins. Proteins of interest include, but are not limited to, heterologous therapeutic proteins, marker proteins or proteins of host cells that have functions such as protein secretion, post-translational protein modification, translation, transcription, cell cycle regulation or nutrient metabolism. Such proteins of interest 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 the purpose of transformation (i.e., introducing heterologous DNA into a host cell). An exemplary type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another exemplary type of vector is a viral vector, into which additional DNA segments can be ligated into 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 that has the ability to incorporate a heterologous nucleic acid fragment (such as DNA) into a foreign cell and express it in the foreign cell. In other words, an expression vector includes nucleic acid sequences / fragments (such as DNA, mRNA) that are capable of being transcribed or expressed in a target cell. Many viral, prokaryotic and eukaryotic expression vectors are known and / or commercially available. Selection of an appropriate expression vector is within the knowledge of those skilled in the art. An expression vector may contain one or more regulatory sequences.

[0045] The terms "viral vector" and "virus" are used interchangeably herein and refer to any obligate intracellular parasite that lacks protein synthesis or energy - generating mechanisms. The viral genome can be RNA or DNA in a protein - coated structure that contains a lipid membrane. The terms virus and viral vector are used interchangeably herein. Viruses useful in the practice of the present invention include recombinant - modified enveloped or non - enveloped DNA and RNA viruses, preferably selected from the family Baculoviridiae, Parvoviridiae, Picornoviridiae, Herpesviridiae, Poxviridae, or Adenoviridiae. The virus is modified by DNA recombination techniques to contain an expression cassette for an exogenous transgene (e.g., a nucleic acid sequence encoding a gene of interest) and can be engineered to be replication - defective, conditionally - replicating, or replication - competent. A minimal vector system can also be employed, where the viral backbone contains only the sequences required for packaging of the viral vector and optionally contains a transgene expression cassette. The term "replication - defective" refers to a vector with highly attenuated replication in wild - type mammalian cells. To produce such vectors in large quantities, the missing functions are usually complemented by co - transfection with a helper virus or by genomic modification to establish 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 "conditionally - replicating viral vector" or "conditionally - replication - defective virus" as used herein refers to a replication - competent vector that is designed to achieve selective expression in a specific cell type. Such conditional replication can be achieved by operably linking a tissue - specific, tumor - specific, or cell - type - specific or other selectively - inducible regulatory control sequence to an early gene (such as the E1 gene of an adenovirus vector). Infecting a subject with a recombinant virus or non - viral vector can provide long - term expression of a gene of interest in the subject. In one embodiment, the nucleic acid sequence encoding the gene of interest in the 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 temporally - regulated promoter.

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

[0047] For autonomous replication, the vector can further contain an origin of replication that enables the vector to replicate autonomously in the 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. 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 required. The autonomous replication sequence can be any suitable sequence available to those skilled in the art that permits plasmid replication independent of chromosomal replication. The origin of replication can be any plasmid replicator that mediates autonomous replication functioning in the cell. Examples of origins of replication include, but are not limited to, the 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 high copy number (e.g., 150 to 200) of the vector per cell. When present in a vector, a medium copy origin of replication can result in a medium copy number (e.g., 25 to 50) of the vector per cell. When present in a vector, a low copy origin of replication can result in a low copy number (e.g., 1 to 3) of the vector per cell.

[0048] "Selectable marker gene" refers to a gene that allows for the easy selection of transformed cells, and the product thereof provides antibiotic resistance or virus resistance, heavy metal resistance, prototrophy of auxotrophs, etc. The selectable marker 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 proteins with 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, and so on. Auxotrophic markers such as URA3, ADE2, HIS3, LEU2, TRPl, or LYS2 allow for the screening of the expression of selectable marker genes.

[0049] As used herein, the term "pharmaceutically acceptable" means that the carrier or excipient does not cause any unnecessary or adverse effects in the subject to which they are administered at the dosages and concentrations employed. 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 can include a pharmaceutically acceptable, non-toxic carrier or diluent, which is defined as the medium commonly used to formulate pharmaceutical compositions for administration to animals or humans. The selection of the diluent should avoid affecting the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate buffered saline, Ringer’s solutions, dextrose solutions, 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 particular application.

[0050] In one aspect, the present disclosure provides a recombinant adenovirus vector with reduced replication-competent adenovirus contamination. Relative to an adenovirus vector or wild-type adenovirus, the recombinant adenovirus vector with reduced replication-competent adenovirus contamination lacks all or part of the sequences of the E1B region gene and the E3 region gene. Among them, 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; a fragment containing the packaging signal (ψ) and a 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. Among them, 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. Among them, the inserted fragment containing the packaging signal (ψ) has at least the nucleotide sequence necessary for the incorporation of the viral genome into the viral capsid during virus assembly, and the inserted fragment containing the IX gene has at least the nucleotide sequence capable of expressing the IX protein.

[0051] In some embodiments, there are synonymous mutations in the IVa2 protein-encoding gene of the recombinant adenovirus vector with reduced replication-competent adenovirus contamination.

[0052] In some embodiments, a gene of interest is inserted between the 3' side of the left ITR of the recombinant adenovirus vector with reduced replication-competent adenovirus contamination and 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 the hTERT promoter, the PEG-3 promoter, or the Survivin gene promoter. In some embodiments, a constitutive promoter or an inducible promoter is operably linked to the gene of interest. Preferably, the constitutive promoter is selected from the CMV promoter, the CAG promoter, the PGK promoter, or the ACTB promoter.

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

[0054] In some embodiments, the adenovirus vector is an adenovirus vector 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, 62, 63, 64, 65, 67, 69, 70, 71, 72, 73, 74, and 75, preferably an adenovirus vector of serotype 4, 5, 7, 26, 35, or 48, and preferably a human adenovirus vector.

[0055] In some embodiments, the adenovirus vector is an adenovirus vector of serotype 5 or 35.

[0056] In some embodiments, the adenovirus vector is a human adenovirus vector of serotype 5.

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

[0058] In some embodiments, the wild-type adenovirus is selected from wild-type adenoviruses 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, 62, 63, 64, 65, 67, 69, 70, 71, 72, 73, 74, and 75, preferably wild-type adenoviruses of serotype 4, 5, 7, 26, 35, or 48, and preferably human adenoviruses.

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

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

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

[0062] In some embodiments, the recombinant adenovirus vector for reducing 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 the group consisting of immunomodulatory molecules, antigen-binding molecules, antigens or ligands of the antigens, toxins, enzymes, or other active protein molecules.

[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 costimulatory 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-15, IL-15 receptor fusion protein, TGF-β, TGF-β trap, IL-10 trap, 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, functional fragments thereof, and derivatives thereof.

[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 functional fragments 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 target gene encodes a toxin. In some preferred embodiments, the toxin is diphtheria toxin, ricin, 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 adenovirus vector with reduced replication-competent adenovirus contamination further comprises an origin of replication and / or a selectable marker gene.

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

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

[0075] In some embodiments, the antibiotic or virus resistance gene is selected from one or more of ampicillin resistance gene, chloramphenicol resistance gene, neomycin resistance gene, G418 resistance gene, puromycin resistance gene, hygromycin resistance gene, bleomycin resistance gene, kanamycin resistance gene, methotrexate resistance gene, phleomycin resistance gene, and 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 following steps:

[0078] (1) Take an adenovirus vector or wild-type adenovirus, and retain the left and right ITRs of the adenovirus vector or wild-type adenovirus;

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

[0080] (3) Insert the fragment containing the packaging signal (ψ) and the fragment containing the IX gene 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 upstream of the 5' side of the right ITR, the inserted fragment containing the IX gene is 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) Delete 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; retain all genes of the E1A gene; wherein:

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

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

[0084] (5) Delete 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 all or part of the sequence of the E3 region gene of the adenovirus vector is deleted and the gene product of the active E3 region cannot be expressed, step (5) is not carried out;

[0086] Among them, the order of steps (2)-(5) can be arbitrarily adjusted, or steps (2)-(5) can be carried out simultaneously to obtain the recombinant adenovirus vector with reduced replication-competent adenovirus contamination.

[0087] In some embodiments, the method further includes: (6) carrying out synonymous mutation on the IVa2 protein coding gene of the adenovirus vector or wild-type adenovirus; wherein, the order of steps (2)-(6) can be arbitrarily adjusted, or steps (2)-(6) can be carried out simultaneously.

[0088] In some embodiments, the method further includes: (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 adenovirus vector is pAd / PL-DEST, and the method includes the following steps:

[0090] (i) Take the pAd / PL-DEST plasmid and retain the left and right ITRs of the pAd / PL-DEST plasmid;

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

[0092] (iii) Insert the fragment containing the packaging signal (ψ) and the fragment containing the IX gene between the downstream of the E4 region gene of the pAd / PL-DEST plasmid and the 5' side of the right ITR, wherein the inserted fragment containing the packaging signal (ψ) is upstream of the 5' side of the right ITR, the inserted fragment containing the IX gene is 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) Insert 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 after the E1A gene is inserted into the pAd / PL-DEST plasmid, all gene products of the E1A gene can be expressed;

[0094] Among them, the order of steps (ii)-(iv) can be arbitrarily adjusted, or steps (ii)-(iv) can be carried out simultaneously to obtain the recombinant adenovirus vector with reduced replication-competent adenovirus contamination.

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

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

[0097] ② Perform PCR amplification using the pAd / PL-DEST plasmid as a template to obtain fragment A containing the IX gene and fragment B containing the packaging signal (ψ) respectively. Fragment A has at least 10 bp overlapping sequence I with the 5' end of the incision of the pAd / PL-DEST plasmid treated with the first sgRNA and Cas9 nuclease, and fragment B has at least 10 bp overlapping sequence II with the 3' end of the incision of the pAd / PL-DEST plasmid treated with the first sgRNA and Cas9 nuclease. At the same time, there is at least 10 bp overlapping sequence III between fragment A and fragment B; wherein, fragment A remains forward and fragment B is reverse;

[0098] ③ Mix the pAd / PL-DEST plasmid treated with the first sgRNA and Cas9 nuclease obtained in step ① with fragment A and fragment B obtained in step ②, and perform recombinant ligation to obtain an intermediate vector;

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

[0100] ⑤Perform PCR amplification using the gene synthesis template to amplify fragments 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) - at least 20 bp sequence upstream of attR1 downstream of the packaging signal (ψ) - promoter - target gene - polyadenylation signal) and fragment D containing the E1A gene and the fragment of the C-terminal of IVa2 with synonymous mutations. There is at least 10 bp overlapping sequence IV between fragment C and the upstream of the incision of the intermediate vector treated with the second sgRNA, the third sgRNA and Cas9 nuclease. There is at least 10 bp overlapping sequence V between fragment D and the downstream of the incision of the intermediate vector treated with the second sgRNA, the third sgRNA and Cas9 nuclease. Meanwhile, there is at least 10 bp overlapping sequence VI between fragment C and fragment D. 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] ⑥Mix the intermediate vector treated with the second sgRNA, the third sgRNA and Cas9 nuclease obtained in step ④ with fragment C and fragment D obtained in step ⑤, and perform recombinant ligation to obtain a recombinant adenovirus vector with reduced replication-competent adenovirus contamination. Among them, after fragment C and fragment D are ligated, they replace the downstream of the 3' side of the left ITR of pAd / PL-DEST - the C-terminal gene of the unmutated IVa2.

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

[0103] In some preferred embodiments, in step ②, fragment A contains the 3525 - 4080 bp fragment corresponding to the wild-type Ad5 IX gene; fragment B contains the 129 - 392 bp fragment of Ad5 with the packaging signal (ψ); the overlapping sequence I sequence is as shown in SEQ ID NO:31; the overlapping sequence II sequence is as shown in SEQ ID NO:32; the overlapping sequence III sequence is as 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 upstream of the packaging signal (ψ)) - (downstream of the packaging signal (ψ) - at least 20 bp upstream of attR1) - promoter - target gene - polyadenylation signal is the fragment containing pAd / PL-DEST (104 - 128 bp) - (393 - 504 bp) - CMV-hXGENE-SV40 PA, and its sequence corresponds to the sequence shown by nucleotides at positions 104 - 2374 of SEQ ID NO:8; the fragment containing the E1A gene and the synonymous mutant IVa2 C-terminal gene is the fragment containing pSurvivin-E1A-E1A terminal - the synonymous mutant IVa2 C-terminal gene fragment, and its sequence corresponds to the sequence shown by nucleotides at positions 2375 - 4233 of SEQ ID NO:8; overlapping sequence IV is as shown in SEQ ID NO:34; overlapping sequence V is as shown in SEQ ID NO:35; overlapping sequence VI is as shown in SEQ ID NO:36.

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

[0107] The IX gene fragment is deleted in the pSurvivin-E1A-E1A terminal - the synonymous mutant IVa2 C-terminal gene fragment.

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

[0109] In some preferred embodiments, 29 mutations in total at 24 sites are made to the IVa2 C-terminus; the sequence of the synonymous mutant IVa2 C-terminal gene fragment is as shown in SEQ ID NO:9.

[0110] On the other hand, the present disclosure provides a host cell comprising the aforementioned recombinant adenovirus vector with reduced replication-competent adenovirus contamination.

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

[0112] On the other hand, the present disclosure provides a method for producing a recombinant adenovirus, which includes:

[0113] (1) Mixing the aforementioned recombinant adenovirus vector with reduced replication-competent adenovirus contamination with a host cell, and introducing the recombinant adenovirus vector with reduced replication-competent adenovirus contamination into the host cell;

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

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

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

[0117] (3) Collecting the cell suspension, subjecting it to freeze-thawing, and then centrifuging to collect the supernatant to obtain a recombinant virus suspension.

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

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

[0120] On the other hand, the present disclosure provides the use of the aforementioned recombinant adenovirus vector and / or composition with reduced replication-competent adenovirus contamination in the preparation of a recombinant adenovirus.

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

[0122] The features and properties of the present invention will be further described in detail below in conjunction with examples. Obviously, the present disclosure is not limited to the following examples and there can be many variations. Therefore, modifications or improvements made by those skilled in the art based on the disclosed content of the present disclosure shall fall within the scope of protection required by the present disclosure.

[0123] Example 1: Preparation of a Conditional Replication-competent Recombinant Human Adenovirus Type 5 Vector

[0124] In this example, a conditional replication-competent recombinant human adenovirus type 5 vector (CR-rAd5) was prepared, which was subsequently used as a control virus vector. It was based on the pAd / PL-DEST plasmid (purchased from Invitrogen, catalog number V49420, Figure 2, with the pAd / PL-DEST plasmid (https: / / www.snapgene.com / plasmids / viral_expression_and_packaging_vectors / pAd_PL-DEST) as the basic backbone, the pAd / PL-DEST plasmid lacks the E1 region and E3 region genes of Ad5. Among them, 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 all genes of E1A and E1B are deleted; the E3 region lacks partial genes of E3 CR1-alpha0 and E3 14.7K and all genes of E3 gp19K, ADP, E3RID-alpha, and E3 RID-beta. The full-length nucleotide sequence of the pAd / PL-DEST vector is as shown in SEQ ID NO:1.

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

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

[0127] 1.1 Preparation of the control virus vector

[0128] The pAd / PL-DEST plasmid was transformed into JM110 chemically competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd., product number DL1025S). Among them, JM110 is a strain lacking methyltransferases dam and dcm, and the extracted pAd / PL-DEST plasmid DNA can be cut by restriction enzymes sensitive to dam and dcm methylation. Single colonies obtained after transformation were picked to extract plasmids, and then the demethylated pAd / PL-DEST was digested with ClaI. Then, it was ligated in vitro with fragment 1 containing the CMV-XGENE-SV40 PA fragment (corresponding to 505-2632 bp of SEQ ID NO:2) synthesized by gene synthesis and fragment 2 containing the pSurvivin-E1A-E1Aterminal fragment (corresponding to 2633-4068 bp of SEQ ID NO:2). These two fragments (fragment 1 and fragment 2) were ligated to the pAd / PL-DEST vector digested with ClaI by in vitro recombination. The successfully constructed vector is a conditionally replicating recombinant human adenovirus type 5 vector (CR-rAd5).

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

[0130] (a) Transfer 100 ng of the pAd / PL-DEST plasmid into 100 μl of JM110 competent cells and transform by heat shock at 42 °C for 45 s. Single colonies obtained after transformation were 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 the ClaI restriction enzyme (NEB, R0197S) using the rCutsmart digestion buffer at 37 °C for 60 min. The large fragment (33151 bp) after digestion was recovered by gel extraction for standby. The reaction system is shown in Table 1:

[0132] Table 1 Reaction system

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

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

[0135] Table 2 Reaction System

[0136] Component 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 and the fragment 1 containing the CMV-XGENE-SV40 PA fragment and the fragment 2 containing the pSurvivin-E1A-E1A terminal fragment obtained by amplification are recombinantly ligated by the Gibson in vitro recombination method. The ligation reaction system is shown in Table 3; the reaction program is 50°C, 30 min.

[0138] Table 3 Reaction System

[0139] Component Volume (μl) pAd / PL-DEST (ClaI digested) 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 s. Screen positive clones by colony PCR method. After extracting the plasmid, use the restriction enzyme XbaI (NEB, product number R0145S) for enzyme digestion identification. Use rCutsmart enzyme digestion buffer and digest at 37°C for 30 min. Run the enzyme digestion products on a 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 plasmids with correct digestion verification were sent for sequencing. The plasmid verified correctly by sequencing was the CR-rAd5 control virus vector. The entire nucleotide sequence of the vector constructed by the above method is shown in SEQ ID NO:2. The reaction system of colony PCR is shown in Table 4; the reaction program is 98°C, 10 s; 56°C, 10 s; 72°C, 20 s; 30 cycles.

[0141] Table 4 Reaction System

[0142] Component Volume (μl) 1.1× GoldMix Ver.2 (TSINGKE, TSE102) 22 10μM Forward Primer 1 10μM Reverse Primer 1 Template DNA 1 Total 25

[0143] 1.2 Packaging of Control Virus

[0144] The successfully constructed control virus vector was subjected to large-scale plasmid extraction and endotoxin removal, linearized with PacI (NEB, catalog number R0547S) at 37°C for 1 h, then incubated at 65°C for 20 min to inactivate the endonuclease Pac I, and then purified by a DNA purification and recovery kit to recover the linearized viral plasmid DNA. When the confluence of HEK293A cells (Invitrogen, catalog number R70507) reached 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 viral plasmid DNA for transfection, and the cell status was observed daily. The steps are as follows:

[0145] (1) Prepare transfection solutions A and B: Add 4 μg of the recombinant plasmid linearized with PacI and recovered to 500 μl of Opti-MEM TM I serum-free medium (purchased from Thermo Fisher Scientific , catalog number 31985070), and pipette to mix evenly to obtain transfection solution A; Add 10 μl of Lipofectamine 2000 transfection reagent to 490 μl of Opti-MEM TM I serum-free medium, and pipette to mix evenly to obtain transfection solution B.

[0146] (2) Transfer all of transfection solution B to transfection solution A, and pipette to mix evenly to obtain transfection solution C, and let it stand at room temperature for 25 min.

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

[0148] (4) Add 10 ml of DMEM medium without antibiotics containing 10% FBS (purchased from Cytiva, catalog number SH30022.01) to the culture flask and culture for 10 - 14 days, observing the cell status every day. At 10 - 14 days after transfection, HEK293A cells will show a cytopathic effect (CPE). When more than 50% of the cells detach from the bottom of the culture flask, collect the cell suspension at this time. After freezing and thawing 3 times, centrifuge at 500×g for 10 min to collect the supernatant, and obtain the recombinant virus suspension. Normal HEK293A cells (A) and HEK293A cells showing cytopathic effect (B) are as Figure 6 shown.

[0149] 1.3 Restriction Enzyme Digestion Identification of Control Virus Genome

[0150] Extract the virus genomic DNA from the recombinant virus suspension, and use the restriction enzyme BstZ17I-HF (NEB, R3594S) to perform restriction enzyme digestion identification on the extracted virus genomic DNA. Use rCutsmart enzyme digestion buffer and digest at 37°C for 0.5 h. Observe the bands of the BstZ17I-HF enzyme digestion products by 1% agarose gel electrophoresis. The results are as Figure 7 shown. Two bands of 28375-bp and 6313-bp were observed, which were consistent with the expected results.

[0151] 1.4 PCR Identification of Control Virus

[0152] Using the virus genomic DNA as a template, use the 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 program is shown in Table 6.

[0156] Table 5 Identification Reaction System

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

[0158] Table 6 Identification Reaction PCR Program

[0159]

[0160] Analyze the PCR products by 1% agarose gel electrophoresis, and the results are as Figure 8 shown. A 579-bp band was observed, which was consistent with the expected results.

[0161] Subsequent sequencing verification of the control virus genome was performed to ensure the integrity of the virus sequence. By sending the sample to a sequencing company and sequencing the virus genome by NGS sequencing, the results were completely consistent with the designed sequence, indicating that the virus construction was successful. The virus with correct sequencing verification is the conditionally replicating recombinant human adenovirus type 5 (CR-rAd5).

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

[0163] In this example, a virus vector with reduced contamination of replicating adenovirus (L-RCA-rAd5) was prepared. The construction of the L-RCA-rAd5 virus vector was carried out in two steps: (1) Using the 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 inserted in reverse into the E4 region. At the same time, fragment 3 containing the IX gene Ad5 3525-4080 fragment (SEQ ID NO:6) was amplified and inserted forward upstream of the reverse complementary fragment of the Ad5 129-392bp fragment containing the packaging signal sequence inserted in reverse. The pAd / PL-DEST plasmid treated with sgRNA was mixed with fragments 3 and 4, and recombinant ligation was carried out by the Gibson in vitro recombination method to obtain the intermediate vector IX-phi-E4-DEST plasmid. (2) PCR amplification was carried out using the template of gene synthesis to amplify fragment 5 containing the pAd / PL-DEST (104-128bp)-(393-504bp)-CMV-hXGENE-SV40 PA fragment (corresponding to 104-2374bp of SEQ ID NO:8) and fragment 6 containing the pSurvivin-E1A-E1A terminal-synonymous mutated IVa2 C-terminal gene fragment (corresponding to 2375-4233bp of SEQ ID NO:8). After these two fragments were ligated, they replaced pAd / PL-DEST 104-3168bp. The IX-phi-E4-DEST plasmid treated with Cas9 nuclease and sgRNA was mixed with fragments 5 and 6 for recombinant ligation to obtain the L-RCA-rAd5 virus vector. The schematic diagram of its structure is shown in Figure 4 . The packaging signal (ψ) sequence was deleted from fragment 5 containing the pAd / PL-DEST (104-128bp)-(393-504bp)-CMV-hXGENE-SV40 PA fragment. The IX gene fragment was deleted from fragment 6 containing the pSurvivin-E1A-E1A terminal-synonymous mutated IVa2 C-terminal gene fragment.

[0164] Among them, the Ad5 129-392 bp fragment (SEQ ID NO: 5) containing the packaging signal (ψ) sequence (SEQ ID NO: 26) on the 3' side of the left ITR (5' ITR, SEQ ID NO: 24) and the Ad5 3525-4080 bp fragment (SEQ ID NO: 6, containing the IX gene promoter, the IX gene coding sequence (SEQ ID NO: 27) and the transcription termination sequence) containing the IX gene were deleted, and the two deleted fragments were inserted between the 5' side of the right ITR (3' ITR, SEQ ID NO: 25) downstream of the E4 gene. Among them, the deleted Ad5 129-392 bp fragment containing the packaging signal sequence was inserted in reverse at the 35773 bp of wild-type Ad5 (the nucleotide sequence of the wild-type Ad5 genome is as shown in SEQ ID NO: 7 (GenBank: AC_000008)), and the deleted Ad5 3525-4080 bp fragment (SEQ ID NO: 6) containing the IX gene was inserted in the forward direction upstream of the Ad5 129-392 bp fragment containing the packaging signal sequence inserted in reverse. At the same time, one synonymous mutation was made every 3-5 amino acids in the 333-450 aa interval of the IVa2 protein, and a total of 29 mutations at 24 sites were made to the base sequence corresponding to the amino acid sequence of this region. The full-length nucleotide sequence of the vector constructed by the above method is as shown in SEQ ID NO: 8; the comparison between the nucleotide sequence of the C-terminal of the IVa2 (SEQ ID NO: 9) after synonymous mutation and the wild-type nucleotide sequence (SEQ ID NO: 10) is shown in Figure 9 , and the nucleotides of the synonymous mutations are marked with underlines.

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

[0166] 2.1 Preparation of the virus vector (L-RCA-rAd5) reducing the contamination of replication-competent adenovirus:

[0167] (a) Design 0718-sgRNA near the 35773bp of wild-type Ad5, and the sequence is as shown in SEQ ID NO:11; synthesize the forward primer 0718-sgT7F for in vitro transcription of sgRNA, and the sequence is as shown in SEQ ID NO:12. Transcribe 0718-sgRNA using a one-step sgRNA in vitro transcription kit (Novoprotein, product number E369), and then cut the pAd / PL-DEST plasmid together with the NLS-Cas9 Nuclease protein (Novoprotein, product number E365-01A) to obtain the pAd / PL-DEST plasmid treated with sgRNA and Cas9 nuclease. The reaction system is shown in Table 7:

[0168] Table 7 Reaction System

[0169] Component 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) Perform PCR amplification using the pAd / PL-DEST plasmid as a template to obtain Fragment 3 containing the Ad5 3525-4080bp fragment of the IX gene and Fragment 4 containing the Ad5 129-392bp fragment of the packaging signal (ψ). There is a 28bp overlapping sequence 4 (SEQ ID NO:31: 5’-AATCGTCACTTCCGTTTTCCCACGTTAC-3’) at the 5’ end of the incision after sgRNA cleavage of Fragment 3, and a 26bp overlapping sequence 5 (SEQ ID NO:32: 5’-GTCACTTCCCATTTTAAGAAAACTAC-3’) at the 3’ end of the incision after sgRNA cleavage of Fragment 4. At the same time, there is a 20bp overlapping sequence 6 (SEQ ID NO:33: 5’-AAGCAAGTGTCTGGGCGAGT-3’) between these two fragments (Fragment 3 and Fragment 4); in addition, Fragment 3 containing the Ad5 3525-4080bp fragment of the IX gene is in the forward direction, while Fragment 4 containing the Ad5 129-392bp fragment of the packaging signal (ψ) is in the reverse direction. The two amplified fragments (Fragment 3 and Fragment 4) are recovered by gel cutting and reserved for use. The PCR reaction system is shown in Table 2; the reaction program is 98℃, 10s; 58℃, 20s; 72℃, 30s; 30 cycles.

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

[0172] Table 8 Reaction system

[0173] Component Volume (μl) pAd / PL-DEST (after sgRNA and Cas9 treatment) 3 Fragment 3 of Ad5 3525 - 4080bp fragment containing IX gene 0.5 Fragment 4 of Ad5 129 - 392bp fragment containing 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) 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 s. 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 reaction system of colony PCR is shown in Table 4; the reaction program is 98 °C, 10 s; 56 °C, 10 s; 72 °C, 20 s; 30 cycles.

[0175] (e) Design 0816-5sg1 (SEQ ID NO: 14) and 0816-3sg1 (SEQ ID NO: 15), and synthesize the forward primers 0816-5sg1-T7F (SEQ ID NO: 16) and 0816-3sg1-T7F (SEQ ID NO: 17) for in vitro transcription of sgRNA. Transcribe 0816-5sg1 and 0816-3sg1 using a one-step sgRNA in vitro transcription kit, and then cut the intermediate vector IX-phi-E4-DEST plasmid together with the NLS-Cas9 Nuclease protein. After the reaction, recover the large fragment by gel cutting for standby; the reaction system is shown in Table 9:

[0176] Table 9 Reaction system

[0177] Component 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) Perform PCR amplification using the template of gene synthesis to amplify fragment 5 containing the pAd / PL-DEST (104 - 128bp)-(393 - 504bp)-CMV-hXGENE-SV40 PA fragment (corresponding to 104 - 2374bp of SEQ ID NO:8) and fragment 6 containing the pSurvivin-E1A-E1A terminal-synonymous mutated IVa2 C-terminal gene fragment (corresponding to 2375 - 4233bp of SEQ ID NO:8). There is a 25bp overlapping sequence 7 (SEQ ID NO:34: 5’-TAGTGTGGCGGAAGTGTGATGTTGC-3’) upstream of the 0816-5sg1 incision in fragment 5, and a 30bp overlapping sequence 8 (SEQ ID NO:35: 5’-CCATATCCCTCCGGGGATTCATGTTGTGCA-3’) downstream of the 0816-3sg1 incision in fragment 6. Meanwhile, there is also a 20bp overlapping sequence 9 (SEQ ID NO:36: 5’-TCAATGTATCTTACTCACTC-3’) between these two fragments (fragment 5 and fragment 6). The two amplified fragments (fragment 5 and fragment 6) are recovered by gel cutting and reserved for later use. The PCR reaction system is shown in Table 2; the reaction program is 98°C, 10s; 60°C, 20s; 72°C, 120s; 30 cycles.

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

[0180] Table 10 Reaction system

[0181] Component Volume (μl) IX-phi-E4-DEST plasmid (after Cas9 and sgRNA treatment) 3 Fragment 5 of pAd / PL-DEST (104 - 128bp)-(393 - 504bp)-CMV-hXGENE-SV40PA fragment 0.5 Fragment 6 of pSurvivin-E1A-E1A terminal-synonymous mutated IVa2 C-terminal gene fragment 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 s. Screen positive clones by colony PCR method. After extracting the plasmid, perform enzyme digestion identification with the restriction endonuclease XbaI (NEB, product number R0145S), use rCutsmart enzyme digestion buffer, and digest at 37 °C for 30 min. Run the enzyme digestion product on a 1% agarose gel electrophoresis to observe the bands. The enzyme digestion identification results are shown in Figure 10 , and four bands of 18003-bp, 8583-bp, 6895-bp, and 3268-bp were observed, which met the expectations. The plasmid with correct enzyme digestion verification 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 with correct sequencing is the final target viral vector L-RCA-rAd5, and its full-length nucleotide sequence is shown in SEQ ID NO:8. The colony PCR reaction system is shown in Table 4; the reaction program is 98 °C, 10 s; 56 °C, 10 s; 72 °C, 20 s; 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 1 h, then incubated at 65 °C for 20 min to inactivate the endonuclease Pac I, and then purified through a DNA purification and recovery kit to recover the linearized viral plasmid DNA. At the same time, HEK293A cells were cultured in DMEM medium (purchased from Cytiva, product number SH30022.01), 10% fetal bovine serum was added to the medium, and the cells were placed in an incubator at 37 °C and 5% CO2 concentration. When the confluence of HEK293A cells reached 80%-90%, the transfection reagent Lipofectamine 2000 (purchased from Thermo Fisher Scientific, product number 11668019) was used to form a Lipofectamine2000-DNA complex with the linearized viral plasmid DNA for transfection, and the cell status was observed daily. The steps are as follows:

[0184] (1) Prepare transfection solutions A and B: Add 4 μg of the recombinant plasmid linearized with PacI and recovered to 500 μl of Opti-MEM TMResuspend it by pipetting in Iscove's Modified Dulbecco's Medium (purchased from Thermo Fisher Scientific, catalog number 31985070) to obtain transfection solution A; add 10 μl of Lipofectamine 2000 transfection reagent to 490 μl of Opti-MEM TM Resuspend it by pipetting in Iscove's Modified Dulbecco's Medium to obtain transfection solution B.

[0185] (2) Transfer all of transfection solution B to transfection solution A, and resuspend it by pipetting to obtain transfection solution C. Incubate at room temperature for 25 min.

[0186] (3) Add all of transfection solution C to a T25 flask seeded with HEK293A cells. Mix well by cross-shaking, then place it in a 37 °C, 5% CO₂ incubator for 6 h, and 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. Observe the cell status every day. At 10 - 14 days after transfection, HEK293A cells will show cytopathic effect. When more than 50% of the cells have detached from the bottom of the culture flask, collect the cell suspension at this time. Freeze-thaw 3 times and then centrifuge at 500 × g for 10 min to collect the supernatant to obtain the recombinant virus suspension. Normal HEK293A cells (A) and HEK293A cells showing cytopathic effect (B) are as Figure 11 shown.

[0188] 2.2 Restriction Enzyme Digestion Identification of Viral Genome

[0189] Extract viral genomic DNA from the recombinant virus suspension. Use the restriction enzyme SpeI-HF (NEB, R3133V) to perform restriction enzyme digestion identification on the extracted viral genomic DNA. Use rCutsmart enzyme digestion buffer and digest at 37 °C for 0.5 h. Observe the bands of the SpeI-HF enzyme digestion products by 1% agarose gel electrophoresis. The results are as Figure 12 shown. Three bands of 26607-bp, 7804-bp, and 264-bp are observed, which are consistent with the expectations.

[0190] 2.3 PCR Identification of Virus

[0191] Using the viral genomic DNA as a template, use the 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 program is shown in Table 6.

[0195] The PCR products were analyzed by 1% agarose gel electrophoresis, and the results were as Figure 13 shown. A 318-bp band was observed, which was consistent with the expectation.

[0196] Subsequently, the virus genome was sequenced and verified to ensure the integrity of the virus sequence. The virus genome was sequenced by sending the sample to a sequencing company and using NGS sequencing. The results were completely consistent with the designed sequence, indicating that the virus construction was successful. The virus with correct sequencing verification was the low RCA recombinant adenovirus type 5 (L-RCA-rAd5).

[0197] 2.4 Virus passage

[0198] The harvested virus suspension was inoculated into HEK293A cells with a confluence of 80%-90%. After 48-72 h, cytopathic effect appeared in HEK293 cells, and more than 50% of the cells detached from the bottom of the culture flask. The cell suspension was harvested. The cell suspension was centrifuged at 500×g for 10 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in 0.5 ml of DMEM medium. After three freeze-thaw cycles, the harvested virus suspension was then inoculated into HEK293A cells with a confluence of 80%-90%, and one virus passage was completed.

[0199] And so on, the harvested low RCA recombinant adenovirus type 5 (L-RCA-rAd5) and the control virus were each passaged 14 times in HEK293A cells. The control virus was the conditional replication recombinant human adenovirus type 5 (CR-rAd5) with the E1B region and E3 region deleted as shown in Figure 3 .

[0200] 2.5 Virus purification

[0201] Since adenovirus is packaged and amplified in HEK293A, genomic DNA of HEK239A cells may be contaminated during the extraction of virus genomic DNA, and it contains partial E1 region genes, which will affect the subsequent quantitative detection of back mutations. Therefore, SuperNuclease (purchased from Sino Biological, product number GMP-SSNP01) was first added to the harvested virus solution and incubated at 37°C for 60 min to remove some residual nucleic acids. Subsequently, the harvested virus suspension was purified using the VivaPure AdenoPACK 20RT adenovirus purification kit (purchased from Sartorius, product number VS-AVPQ022) to remove residual host proteins and nucleic acids and obtain virus particles.

[0202] 2.6 Quantitative analysis of back mutations in the virus

[0203] Quantify the virus and the copy number of the reverse mutants by fluorescence quantitative method with dyes. Take the viruses of the 2nd, 4th, 6th, 8th, 10th, 12th, and 14th passages of continuous passage to quantitatively analyze the virus particles and the number of reverse mutants. Respectively take 100 μl of the harvested virus suspension to extract the viral genomic DNA, and then use fluorescence quantitative PCR to quantify the number of virus particles and the number of reverse mutants. When quantifying the number of virus particles, dilute the viral genomic DNA 100 times, and do not dilute when quantifying the reverse mutants. 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] The primers for quantifying the number of virus particles are:

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

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

[0211] The primers for quantifying the number of reverse mutants 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 criterion is the number of reverse mutants in every 1×10 9 the number of virus particles. The quantitative results are as Figure 14 shown. The results show that: when continuously passaging to the 14th generation of the virus, the number of reverse mutants of low RCA recombinant adenovirus type 5 (L-RCA-rAd5) always remains at a low level during continuous passage; while the number of reverse mutants of the control conditional replication recombinant human adenovirus type 5 (CR-rAd5) always remains at a high level during continuous passage, and the number of RCA increases with the increase of the passage times of the adenovirus vector on 293 cells.

[0215] Not more than 1 copy of the revertant mutation in 3×10 10 adenovirus particles according to the requirements of the US Food and Drug Administration (FDA). The result determination criterion of the present invention is the number of revertant mutations in every 1×10 9 virus particles. The experimental results show that the low-RCA recombinant adenovirus type 5 constructed by the present invention will not produce more revertant mutations during continuous passage, and the effect of reducing the contamination of replication-competent adenovirus can be achieved.

Claims

1. A recombinant adenovirus vector with reduced contamination by a replication-competent adenovirus, wherein the sequence of the recombinant adenovirus vector with reduced contamination by a replication-competent adenovirus is shown in SEQ ID NO:

8.

2. The method for preparing the recombinant adenovirus vector with reduced contamination of replicative adenovirus according to claim 1, comprising the following steps: ① Designing a first sgRNA near the insertion site in 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; the insertion site in the E4 region of the pAd / PL-DEST plasmid is a site corresponding to 35773 bp of the wild-type Ad5; the first sgRNA sequence is shown in SEQ ID NO: 11; the full-length nucleotide sequence of the pAd / PL-DEST vector is shown in SEQ ID NO: 1; the wild-type Ad5 genome nucleotide sequence is shown in SEQ ID NO: 7; ② Using pAd / PL-DEST plasmid as template, PCR amplification was performed to obtain fragment A containing the IX gene and fragment B containing the packaging signal (ψ), respectively. The fragment A was kept in the forward direction, and the fragment B was in the reverse direction. Fragment A was a fragment containing the IX gene and corresponding to 3525-4080 bp nucleotides of wild-type Ad5; fragment B was a fragment containing the packaging signal (ψ) and corresponding to 129-392 bp nucleotides of wild-type Ad5. ③ The treated pAd / PL-DEST plasmid obtained in step ① is mixed with fragment A and fragment B obtained in step ②, and recombined to obtain an intermediate vector; the sequence of the intermediate vector is shown in SEQ ID NO: 13; ④ Designing a second sgRNA and a third sgRNA, mixing the second sgRNA and the third sgRNA, Cas9 nuclease and the intermediate vector obtained in step ③ to obtain an intermediate vector treated with the second sgRNA, the third sgRNA and Cas9 nuclease; the sequence of the second sgRNA is shown in SEQ ID NO: 14; the sequence of the third sgRNA is shown in SEQ ID NO: 15; ⑤ Performing PCR amplification with the gene synthesis template to amplify fragment C and fragment D respectively, wherein fragment C is the sequence shown by nucleotides 104 to 2374 of SEQ ID NO: 8; and fragment D is the sequence shown by nucleotides 2375 to 4233 of SEQ ID NO: 8; ⑥ Mix the treated intermediate vector obtained in step ④ with the fragment C and the fragment D obtained in step ⑤, and perform recombination and ligation to obtain a recombinant adenovirus vector with reduced replication-competent adenovirus contamination; the sequence of the recombinant adenovirus vector with reduced replication-competent adenovirus contamination is shown in SEQ ID NO:

8.

3. A host cell comprising the recombinant adenovirus vector with reduced replication-competent adenovirus contamination according to claim 1. 4 . A composition comprising the recombinant adenovirus vector with reduced replication-competent adenovirus contamination according to claim 1 , and a pharmaceutically acceptable carrier.

5. A method for producing a recombinant adenovirus, comprising: (1) mixing the recombinant adenovirus vector with reduced replication-competent adenovirus contamination as claimed in claim 1 with host cells, so that the recombinant adenovirus vector with reduced replication-competent adenovirus contamination is introduced into the host cells; (2) Growing the host cell under conditions for producing the recombinant adenovirus. The method according to claim 5 , wherein the host cell is a HEK293 cell.

7. The method according to claim 6, wherein the host cell is a HEK293A cell.

8. The method of claim 7, further comprising: (3) Collect the cell suspension, freeze-thaw it, and collect the supernatant by centrifugation to obtain the recombinant virus suspension.

9. The method of claim 8, further comprising: (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 claim 1 and / or the composition according to claim 4 in preparing recombinant adenovirus.

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