JPH10510987A - Improved adenovirus vectors and producer cells - Google Patents

Abstract

  (57) [Summary] One adenovirus vector whose adenovirus genome has been modified to reduce the host's immune and inflammatory response to the adenovirus vector. In one embodiment, the adenovirus vector comprises an adenovirus 5'ITR; an adenovirus 3'ITR; an adenovirus envelope signal; at least one DNA sequence encoding a protein or polypeptide of interest; It contains a promoter that controls the DNA sequence encoding the polypeptide of interest. The adenovirus vector lacks all or part of each of the adenovirus E1 and E4 DNA sequences, or lacks all or part of each of the adenovirus E1 and E2 DNA sequences, or each of the E1, E2 and E4 DNA sequences. Lacks all or part of the Also disclosed and claimed is one producer cell for producing adenovirus vector particles, wherein the producer cell comprises an adenovirus E1 DNA sequence and an adenovirus E4 DNA sequence, or comprises an adenovirus E1 DNA sequence. It contains DNA sequences and adenovirus E2a DNA sequences or contains adenovirus, E1, E2a and E4 DNA sequences.

Description

DETAILED DESCRIPTION OF THE INVENTION                Improved adenovirus vectors and producer cells   This application is a pending application Ser. No. 08/355, filed on Dec. 12, 1994. , 087 are partially inherited.   The present invention relates to adenovirus vectors, wherein the adenovirus genome is a vector. Has been modified to reduce the host's immune inflammatory response to the host. More details In particular, the invention relates to all or one of the adenovirus E1 and E4 DNA sequences. Part or all of the adenovirus E1 and E2 DNA sequences Is partly absent or all of each of the E1, E2 and E4 regions or Eliminate some or generate adenoviral particles from such vectors A functional promoter, or some operably linked to a producer cell Adenovirus vector with or without E3 region.                                Background of the Invention   The adenovirus genome is a linear double-stranded DNA molecule of about 36 kilobases versus length . Each end of the viral genome is an inverted terminal repeat (i.e., It has a short sequence known as ITR). A well-characterized adenowi The molecular genetic characteristics of Lus give it a vector that is advantageous for gene transfer. Adenow Knowledge of the genetic organization of the ills is based on placing large fragments of viral DNA in external sequences. Can be replaced. In addition Recombinant adenovirus is structurally stable; extensive post-amplification translocated virus observed It has not been.   Thus, adenovirus is a delivery vector that introduces the desired gene into eukaryotic cells Adenovirus binds to cell receptors and binds Internalized through pores, disrupts endosomes, and releases particles into the cytoplasm, Nuclear translocation and molecular expression of the denovirus genetic program will allow such genes to be Deliver to eukaryotic cells.   In general, the adenovirus genome consists of the E1, E2, E3, E4, And major late regions as well as several other subregions. The E1 region is transformed, Provides functions related to transcriptional deregulation and replication control. The E2 region is a DNA And are essential for other viral functions, including transcriptional regulation. E3 area Provides adenovirus defenses against the host immune system. E3 region is an external resistance A protein that inhibits the host's ability to provide viral proteins as well as the original Code. The E4 region is responsible for late gene expression and viral shutoff of host cell function. It is essential for several key viral functions, including chain and viral replication. The major late region encodes a major adenovirus structural protein.   Adenovirus vectors have at least the majority of the E1 and E3 sequences deleted Was built like so. The E1 deletion makes the vector replication incomplete and the E3 region deletion Will adenoize desired genes without exceeding the maximum allowable size of the packaged genome. Provides room for insertion into a viral vector. What the applicant has discovered When such a vector is placed in vivo in a target cell or organ, However, a sharp decrease in the expression of the desired gene occurs approximately 1-3 weeks after infection. It was. In addition, the inflammatory response evolves in the target organ and the adenovirus vector T cells directed to vector-transduced host cells It has also been found to guide the answer. This cytotoxic response is induced by the host It has the effect of removing invading cells. Also, the host is Develop a neutralizing antibody response to it.   Accordingly, it is an object of this invention to minimize host response to To provide adenovirus vectors that increase sex and expression; To provide a producer cell line that produces adenovirus particles from the vector.BRIEF DESCRIPTION OF THE FIGURES   The present invention will now be described with reference to the drawings. here   FIG. 1 shows the construction of plasmid pSE280-E1.   FIG. 2 is a map of the plasmid pMAMneo-Luc.   FIG. 3 is a construction diagram of the plasmid pMAMneo-E1.   FIG. 4 shows plasmid p from pSE280-E1 and pGRE5-2 / EBV. It is a construction diagram of GRE5-E1.   FIG. 5 is a map of the plasmid pGRE5-E1.   FIG. 6 is a construction diagram of the plasmid pMAMneo-E2A.   FIG. 7 is a map of plasmid pSE380.   FIG. 8 shows the construction of plasmids pSE380-E4 and pSE380-ITR / E4. It is a construction drawing.   FIG. 9 is a construction diagram of the plasmid pSE380GRE5 / E4.   FIG. 10 is a map of the plasmid pTZ18R.   FIG. 11 is a construction diagram of plasmids pTZE2A and pTZd1E2A.   FIG. 12 shows plasmid pSE380-E3.⁺FIG.   FIG. 13 shows plasmid pSE380-E3.⁺E4⁺And pTZE3⁺E4⁺Building FIG.   FIG. 14 shows the plasmid pTZE3.^-E4⁺, PSE280E3^-E4⁺, And pS PORT-1E3^-E4⁺FIG.   FIG. 15 is a map of the plasmid pSPORT-1.   FIG. 16 shows the plasmid pSPORT-1E2A.^-E3^-E4⁺FIG.   FIG. 17 is a map of plasmid pBR322.   FIG. 18 shows the plasmid pBR322-E2A.^-E3^-E4⁺And pBRad5- E2A^-E3^-E4⁺FIG.   FIG. 19 shows plasmid pSE380-E4.^-And pSE380E3⁺E4^-Structure It is a construction drawing.   FIG. 20 shows the plasmid pSE380E3.⁺E4^-It is a construction diagram of (2).   FIG. 21 shows plasmid pSE380E3.^-E4^-It is a construction diagram of (2).   FIG. 22 shows the plasmid pSPORT1 / E2A.^-E3^-E4^-It is a construction diagram of (2). You.   FIG. 23 shows genotype E1.⁺, E2A^-, E3^-, E4⁺Adenovirus vector with It is a production drawing of.   FIG. 24 is a map of pAvS6.   FIG. 25 shows genotype E1 by homologous recombination.^-, E2A^-, E3^-, E4⁺A with It is a construction diagram of a denovirus vector.   FIG.In test tubeGenotype E1 by ligation and transfection^-, E2A^-, E3^- , E4⁺FIG. 3 is a production diagram of an adenovirus vector having   FIG. 27 shows genotype E1 obtained by homologous recombination.^-, E2A^-, E3^-, E4⁺A with It is a production drawing of a denovirus vector.   FIG. 28 is a diagram of the vector Av3nLacZ.   FIG. 29 shows genotype E1.⁺, E4^-Is a production diagram of an adenovirus vector having .   FIG. 30 shows genotype E1 obtained by homologous recombination.^-, E2⁺, E3⁺, E4^-Ade with FIG. 3 is a production diagram of a novirus vector.   FIG.In test tubeGenotype E1 by ligation and transfection^-, E2⁺, E3⁺, E4^-FIG. 3 is a production diagram of an adenovirus vector having   FIG. 32 is a diagram showing generation of Av4nLacZ by homologous recombination.   FIG.In test tubeFIG. 4 is a diagram showing generation of Av4nLacZ by ligation.   FIG. 34 shows the production of an Av4 (4th generation adenovirus) type vector containing a transgene. FIG.   FIG. 35 is a diagram showing the construction of plasmid pAvS6-CFTR.   FIG. 36 shows an Av1-type vector (having the whole or a part of the E1 region deleted). ), Av2 vector (deleting the whole or part of the E1 region and Or an Av3 vector or Ad13 Immunoprecipitation of Adenovirus Hexon Expression in A549 Cells Infected at 27 Lot.   FIG. 37 shows infection with the Av1, Av2, or Av3 vector. E1⁺E2⁺Cells or E1⁺Immunization of adenovirus hexon expression in cells It is an epidemic sedimentation blot.   FIG. 38 shows an Av1 vector, an Av2 vector, or an Av3 vector. Infected E1⁺E2⁺Or E1⁺Adenovirus vector DNA replication in cells It is a blot showing the product.Summary of the Invention   The present invention provides a method for reducing the immune and inflammatory response of a host to a vector. The present invention provides an improved adenovirus vector having a modified nonovirus genome. This Une vector is reduced adenovirus compared to existing adenovirus vectors Has the ability to replicate and reduce the expression of adenovirus genes (eg, hexons) ing. Such modifications of the adenovirus genome are part of the adenovirus genome This is done through deletion and / or mutation of the minute.Detailed description of the invention   The adenovirus vectors described here include common types or generations And each of them is described below. This These vectors are adenovirus 5'ITR, adenovirus 3'ITR, Encodes a virus-envelope signal, protein or polypeptide of interest At least one DNA sequence to be converted to a protein or polypeptide of interest. Adenovirus containing promoter controlling DNA sequence encoding peptide Includes the Lus DNA genome. One generation of adenovirus vector (Av3 vector) Deletes all or part of the E1 region, and further deletes all or part of the E2 region (desired). Preferably, the whole or part of the E2a region is deleted. Adenovirus vector The second generation (Av4 vector) lacks all or part of the E1 region and Deleting all or part of the region (preferably all or part of the E2a region) In addition, the whole or part of the E4 region is deleted. Adenovirus vector The third generation (Av5 vector) lacks all or part of the E1 region, All or part of the region is deleted. Each of these vectors has its expression Contains a portion of the E3 region operably linked to a promoter that controls Not included.   These vectors encode proteins or polypeptides of interest The DNA sequence operably linked to the promoter that regulates its expression Below are sometimes referred to as transgenes. Addition under the control of an inducible promoter (i) Cell lines containing the inovirus structural gene include iE1 (E1 gene), iE2a (E2 a gene), iE1 / E2 (E1 and E2 genes), (i) E1 / E4 (E1 And E4 gene), or It is referred to as iE1 / E2a / E4 (E1, E2, and E4 genes).   In one embodiment, the adenovirus 5'ITR, adenovirus 3'IT R, adenovirus envelope signal, protein or polypeptide of interest At least one DNA sequence encoding a protein or protein of interest. One containing a promoter that controls the DNA sequence encoding the polypeptide An adenovirus vector is provided. This adenovirus vector is The whole or a part of the ills E1 (preferably at least the whole or a part of the E1a) ) And E4 DNA sequences. In a preferred embodiment, adenowi The Lus vector further excludes all or part of the adenovirus E1b DNA sequence. Have been. Such vectors are sometimes adenovirus E1^-E4^-As a vector Quoted.   As used herein, the term “part” refers to a region of the adenovirus genome (eg, For example, a part of the DNA sequence of E1, E2, E3, and / or E4) That the functions of such areas have been removed or impaired means. For example, a portion of the DNA sequence of a region of the adenovirus genome Expresses one or more proteins normally encoded by such regions Structures that are not removed or that eliminate or impair the function of such proteins Is removed to be expressed as   The determination of the amount of DNA sequence in the region to be removed is given in the lesson included here. By a person who is familiar with Is determined. Preferably all or substantially the entire coding region is removed. You.   In one preferred embodiment, at least reading frame 6 of the E4 DNA sequence is used. (ORF6) is removed. In one embodiment, at least the E4 DNA sequence is read. The trimming frames 3 and 6 are removed.   In another embodiment, the adenovirus 5'ITR, adenovirus 3'I TR, adenovirus envelope signal, protein or target polypeptide At least one DNA sequence encoding a protein and a protein or subject Comprising a promoter that controls a DNA sequence encoding the polypeptide A non-viral vector is provided. This adenovirus vector is adenovirus All or part of the E1 DNA sequence and all or part of the E2 DNA sequence (Preferably all or part of E2a). In the preferred embodiment In addition, the adenovirus vector further contains the entire adenovirus E1b DNA sequence. Or partly escaped. Such vectors are sometimes adenovirus E1^-E2^- As a vector.   In another embodiment, all or part of the E2b DNA sequence is deleted. I have. Further, in one embodiment, the entire or entire E2a and E2b DNA sequences are Is partially deleted.   In another preferred embodiment, the adenovirus 5'ITR, adenovirus 3'ITR, adenovirus envelope signal, protein or target Encodes a repeptide At least one DNA sequence and a protein or polypeptide of interest Adenovirus vector containing a promoter that controls the DNA sequence encoding the code Is provided. This adenovirus vector contains the adenovirus E1 (E1a And E1b), E2 and E4 DNA sequences in whole or in part. Have been. Such vectors are sometimes adenovirus E1^-E2^-E4^-vector Quoted as Desirably, this vector is capable of reading at least the E4 DNA sequence. The frame 6 has been spared.   In one preferred embodiment, the vector comprises the entire E2a DNA sequence. Has escaped some. Such vectors are sometimes adenovirus E1^-E2a^-E 4^-Quoted as a vector. In another embodiment, the vector is E2b. All or part of the DNA sequence is spared.   In one embodiment, the adenoviral vector described above comprises a transduced host. Allows cells to express the E3 region gene and allows adenoviruses to Operationally to a promoter that allows to retain part or all of the stem Contains at least a portion of the ligated adenovirus E3 DNA sequence. Adenowi The amount of the E3 DNA sequence contained in the virus vector is generally determined by the amount of the adenovirus vector. E3 DNA sequence that allows the host immune response to escape or disable Is the amount of Desirably, all genes in the E3 region are 11.6 kilograms causing cell lysis. Except for the gene encoding the dalton protein, it is included in the vector.   In another embodiment, the adenovirus vector comprises a gene in the E3 region. Not included.   The vector of the invention in a preferred embodiment is an adenovirus particle, wherein Genetic material contained in the virus is identified as deleted Lacking.   Thus, in a preferred embodiment of the present invention, the adenovirus vector Are located in regions identified as being deleted from the adenovirus genetic material Impairs the function of the protein encoded by it, in whole or in part The initial regions (E1, E2, E3 and E4) and the main Includes late-stage areas. Thus, if the adenovirus vector contains all of E1 and E2 If defined as excluding the body or part, the vector is thereby Does not disrupt or substantially impair the function of the encoded protein To include the E3 and E4 regions to the extent necessary. Major late-stage areas and And all other regions, including the small transcription unit, Will exist within the required range without disrupting or substantially impairing the function of the .   Adenovirus vectors are not necessarily limited to adenovirus 2 , Adenovirus 3, and any known adenovirus, including adenovirus 5 Derived from the ills serotype.   Generally, adenoviral vectors encode at least one additional therapeutic agent Contains DNA. The term "therapeutic agent" is commonly used Is used in a generic sense and includes therapeutic, prophylactic and replacement agents.   The DNA sequence coding for the therapeutic placed in the adenovirus vector must be Including but not limited to: CFTR gene; interfero Α, interferon β, and interferons such as interferon γ From IL-1, IL-1β, and interleukin 2 A gene encoding an interleukin, such as 14; encoding GM-CSF Gene encoding adenosine deaminase or ADA; Genes encoding cell growth factors, such as lymphokines, which are sphere growth factors; Genes encoding coagulation factors, such as offspring VIII and factor IX; T cell receptors LDL receptor, ApoE, ApoC, ApoAI and cholesterol Alpha-1 antitrypsin (α1AT) Pulmonary surfactant proteins such as SP-A, SP-B, and SP-C genes Protein gene; insulin gene, negative selectable marker or "suicide" gene, eg For example, herpes simplex virus thymidine kinase gene, cytomegalovirus Midine kinase gene and varicella-zoster virus thymidine kinase gene Virus thymidine kinase gene; Fc receptor in the antigen-binding region of an antibody And antisense inhibiting the replication of hepatitis B or non-A non-B hepatitis virus Antisense sequences that inhibit viral replication, such as virus sequences.   The DNA sequence (or transgene) encoding the therapeutic is genomic DNA Or a cDNA sequence. DNA sequence It is also a natural DNA sequence or an allelic variant thereof. The term "allelic variant" used here The term "body" refers to an allelic variant, which is an alternative form of the natural DNA sequence, The sequence of which replaces, deletes or adds one or more nucleotides The encoded protein or polypeptide or fragment or Does not substantially alter the function of the derivative of In one embodiment And the DNA sequence further comprises a leader sequence or a part thereof, a secretion signal or And / or further include a trailer arrangement or a part thereof.   The DNA sequence encoding at least one therapeutic agent is controlled by an appropriate promoter Below. Suitable promoters used are not necessarily limited to the following, Including: Adenovirus major late promoter and other adenovirus pro Motor; or heterologous, such as cytomegalovirus (CMV) promoter Promoter; Rous sarcoma virus (RSV) promoter; MMT promoter Inducible promoters, such as promoters and metallothionein promoters; heat shock promoters Albumin promoter; and ApoAI promoter. But this The scope of the invention should not be limited to specific external genes or promoters That must be understood.   One such vector is the adenovirus 5 genome (ATCC number VR-5). Or derived from viruses containing the mutations or deletions described here Modified or unmodified virus genome or plasmid such as adenovirus 5 Part of Combination of Plasmids Containing Fragments Derived Directly from the Linear Adenovirus Genome Directly fromIn test tubeConstructed by linking.   Alternatively, the vector may be an adenovirus gene with the desired modifications. Nome (adenovirus 5 genome or adenovirus 5E3-mutant Ad   d1327 (Zinmapaya, others,cell, Vol. 31, p. 543 (1983)) And the leftmost adenovirus ITR, E One region deletion, as well as a second plasmid containing the desired transgene (eg, FIG. 24) PAvS6) and homologous recombination in eukaryotic cells. Can be Alternatively, homologous recombination is performed with plasmid clones and linear Denovirus (Adenovirus 5, or Addl327 or described here) Adenovirus 5 derived from a virus containing the listed mutation or deletion And) fragments directly derived from the genome.   This vector is then transformed into a viral vector to produce infectious viral particles. Into a cell line that can complement the function of any essential genes deleted from Quality is imported. Generally, the cell line is infectious and adenovirus or adenovirus The presence of the glucocorticoid hormone can support the growth of the lus vector Supplies continuous virus production under (Ie, the cell line expresses the glucocorticoid hormone receptor). Cell line). Transfected with an essential adenovirus gene, Therefore, cell lines that can be used to purify infectious adenovirus particles are not necessarily Also include, but are not limited to, A549, KB, and HE-p2 cell lines.   Because the expression of certain viral genes is toxic to cells, the E1 region The E2a, E2b, and / or E4 regions are under the control of an inducible promoter. Such inducible promoters are not necessarily limited thereto, but include: Mu; mouse mammary tumor virus (MMTV) promoter (Archer, et al .;Rhinoceros Ence 255, 1573-1576 (March 20, 1992)) Adult minimal glucocorticoid response element promoter GRE5 (Mader, et al.,Nationwide Bulletin of the Academy of Sciences 90, 5603-5607 (June 1993)) Alternatively, tetracycline responsive promoters (Gossen, et al.,National Academy of Sciences -Bulletin 89, pp. 5547-5551 (June 1992)). One choice As an alternative, the E1 region is under the control of an inducible promoter, and E2a, E2b And / or the E4 region is under the control of their native promoter. like this In an alternative, the native promoter is transcriptionally driven by expression of the E1 region .   In one embodiment, the cell line is a whole adenovirus with a native promoter region. The ils E4 region is also ligated to the E1a region in the presence of a regulatable or inducible promoter. Region or the entire E1 region (including the E1a and E1b regions) Alternatively, the inducible promoter is, for example, mouse milk Atrial tumor virus (ie, MMTV) promoter, Or containing a glucocorticoid response element (GRE's) for transcriptional control Is such a promoter. Also in one embodiment, the E4 DNA sequence Is further expressed from a regulatable promoter such as the MMTV promoter. E1 And the E4 DNA sequence are contained in one expression vector or Included in vector. Desirably, the expression vector is integrated using the genome of the cell line. This is a plasmid vector.   In one embodiment, the producer cell line is an inducible vector (such as the MMTV promoter). A first plasmid containing E1a or the entire E1 DNA sequence under the control of And a second plasmid containing an E4 DNA sequence under the control of its native promoter. Including In such an example, the producer cell lines are E1 and E4 DNA Transduced with an adenovirus containing no sequence. The cells are then inducible (eg, Dexamethasone if the MMTV promoter is used) This inducer activates an inducible promoter such as the MMTV promoter, Provides more E4 DNA expression. The expression of E1 and E4 DNA is thus Enables purification of infectious adenovirus particles.   In another embodiment, the producer cell line comprises a (MMTV promoter or Regulation of inducible promoters (such as promoters containing lucocorticoid response elements) The first plasmid containing E1a or the entire E1 DNA sequence below, and (MMT V promoter or promoter containing glucocorticoid response element ) E4 DNA delivery under the control of an inducible promoter A second plasmid containing the sequence. Producer cell lines are then E1 and E4 DN Transduced with an adenovirus plasmid vector that does not contain the A sequence. Cells It is then exposed to an inducing agent (such as dexamethasone), where the inducing agent is 4 Promoter that controls DNA sequence (MMTV promoter or glucoside (E.g., a promoter that contains a corticoid response element) The expression of the array allows the production of infectious adenovirus particles. But E1 and E 4. The inducible or regulatable promoter controlling the DNA sequence is the same. It must be understood that there is no need to be.   Also in one embodiment, the E1a or E1 DNA sequence, and E2a An adenovirus lacking the DNA sequence or the E2a and E2b DNA sequences The ills vector contains all El and E2a to generate infectious viral particles. Alternatively, the E2b DNA sequence, and any other deleted from the viral vector It is transfected into a cell line that can supplement the function of any of these essential genes. In one embodiment In this case, the cell line contains E1a or the entire E1 region under the control of one promoter. For example, this promoter may be, for example, a mouse mammary tumor virus (ie, MMTV) promoter. Glucocorticoid response elements (GRE's) to motor or transcriptional regulation Including natural or regulatable promoters such as other promoters is there. The cell line may also contain its native promoter region or a preparation as described above. Under the control of a knotable promoter, the adenovirus E2a region or E2a and Includes E2b region. E1a or all E1, And E2a or E2a and E2b DNA sequences are combined into a single expression vector. Or in a different expression vector.   In one embodiment, the cell line comprises a promoter comprising a glucocorticoid response element. A first plasmid containing the E1 DNA sequence under the control of a MMTV promoter; A second plasmid containing the E2a DNA sequence under the control of Producer cells The system is an adenovirus plasmid vector without E1 and E2a DNA. Transduced. The cells then activate the E1 promoter and the MMTV promoter. Exposure to glucocorticoid hormones such as dexamethasone to activate To initiate expression of the E1 and E2a DNA sequences and Enables production of virus particles.   In another embodiment, the producer cell line is an inducible promoter (MMTV promoter). Or other promoters containing glucocorticoid response elements) A first plasmid containing the E1 DNA sequence, and its native promoter A second plasmid containing the E2a DNA sequence under control. E1 and E2a Expression of the DNA sequence is controlled by the control of an inducible promoter. Associated with a producer cell containing an E4 DNA sequence under the control of a native promoter This is achieved by the same approach.   In yet another embodiment, the producer cell line is under the control of the MMTV promoter. Of the first plasmid containing the E1 DNA sequence, and control of the MMTV promoter Below the E2a DNA sequence Containing a second plasmid. E1 leading to the production of infectious adenovirus particles and Expression of the E2a DNA sequence is achieved by exposing the cells to dexamethasone, This activates the MMTV promoter that controls the E1 and E2a DNA sequences. Sexualize.   In yet another embodiment, the E1, E2a, and E4 DNA sequences are deleted. The adenovirus vectors used are adenovirus E1, E2a, and E4. The cell line containing the region is transfected. If the vector has a deletion of the E2b DNA sequence , The cell line will further comprise the E2b region. E1, E2a, and Each of the E4 regions is under the control of its native promoter, or Any or all of the E2a and E4 regions may be derived as described above. Under the control of a conductive or regulatable promoter. E1, E2a, and E 4 The DNA sequence is contained in one expression vector, or E1, E2a, and The E4 DNA sequence may be contained in different expression vectors, or The E1, E2a, and E4 DNA sequences are contained in one expression vector and E1, E2a, and E4 DNA sequences are also contained in one expression vector. Expression of the E1, E2a and E4 DNA sequences was similar to that described above. Achieved using   The use of the adenovirus vector particles of this invention is part of a gene therapy procedure. OfIn vivoOrIn test tubeTransduction of eukaryotic cells and desired proteins Quality or therapeuticIn test tubeFor productionIn vivoIncluding cell transduction.   In one embodiment, the adenoviral vector particles provide a therapeutic effect to the host In an effective amount to doIn vivoIs administered.   In one embodiment, the vector comprises one plaque forming unit to about 10¹⁴Plaque Forming unit, preferably about 10⁶About 10 from plaque forming units¹³Plaque forming unit Is administered in an amount of The host is a mammalian host, including a human or non-human primate host. You.   Infectious adenovirus vector particles can be used to treat lung diseases or disorders (eg, cystic pancreatic). Is administered to the lungs when fibrosis is to be treated. Such administration, for example, Aerosol inhalation or bronchial instillation, or intranasal or intratracheal instillation It is due to entry.   In another embodiment, the infectious adenovirus vector particles are administered systemically, e.g. Intravenous administration (eg portal vein injection or peripheral vein injection) intramuscular administration, intraperitoneal It is administered by intravenous administration, intratracheal administration, or intranasal administration.   Adenovirus vector particles used in combination with a pharmaceutically acceptable carrier suitable for administration to patients Is administered. The carrier may be a liquid carrier (for example, a saline solution) or a solid carrier, for example, Microcarrier beads and the like.   Cells infected by infectious adenovirus particles are not necessarily limited But including: primordial nucleated blood cells, such as leukocytes, granulocytes, mononuclear cells, macro Phages, lymphocytes (including T and B lymphocytes), totipotent stem cells, and Progenitor cells such as tumor and tumor infiltrating lymphocytes (TIL cells); bone marrow cells; endothelial cells; Endothelial cells; epithelial cells; lung cells; keratinocytes; stem Hepatocytes including hepatocyte precursor cells; fibroblasts; other fibroblasts; mesenchymal cells; Mesothelial cells; parenchymal cells; vascular smooth muscle cells; brain cells and other nerve cells; Vesicles; gut stem cells; and myoblasts.   Treatment of various diseases including, but not limited to, infected cells Effective for: adenosine deaminase deficiency, sickle cell anemia, Mediterranean anemia, blood Brain diseases such as hemophilia, diabetes, α-antitrypsin deficiency, Alzheimer's disease, Oketoneuria, and other disorders such as growth disorders and heart disease, such as cholesterol Changes that cause the roll to be metabolized, or caused by a defect in the immune system.   In one embodiment, the adenovirus vector particles are capable of infecting lung cells. Actuated, the adenoviral vector particles provide CF useful in the treatment of cystic fibrosis. Includes TR gene. In another embodiment, the adenovirus vector comprises a lung interface. A gene encoding an active protein, such as SP-A, SP-B, or SP-C Gene, which makes the adenovirus vector defective in pulmonary surfactant protein Used to treat conditions.   In another embodiment, the adenoviral vector particles infect hepatocytes. Such adenoviral vector particles are useful factors in the treatment of hemophilia. Includes genes encoding coagulation factors such as progeny VIII and factor IX.   In another embodiment, the adenoviral vector particles infect hepatocytes. Used in such adenovirus vectors Tar particles are used for the prevention and treatment of acquired or congenital defects in hepatocyte (liver) function. Includes genes encoding useful polypeptides or proteins. For example, They are used to treat birth defects in low density lipoprotein (LDL) receptors. Can be used.   In another embodiment, the adenovirus particles are used to infect hepatocytes. To treat acquired infectious diseases, such as those arising from viral infections. Including genes encoding therapeutic agents used in Eg infectious adenovirus Particles are used to treat viral hepatitis, especially hepatitis B or non-A non-B hepatitis used. For example, an infectious disease containing a gene encoding an antisense gene Virus particles are used to infect hepatocytes to inhibit viral replication. I was able to. In this case, the vector containing the structural hepatitis gene in reverse or reverse orientation Infectious adenovirus particles containing the virus are introduced into hepatocytes and hepatitis Antisense genes that can inactivate irus or its RNA transcript To produce. Optionally, hepatocytes are infected with infectious adenovirus particles, This particle contains, for example, alpha interferon, which confers resistance to hepatitis virus. Includes genes encoding proteins.   In yet another embodiment, the adenovirus vector is negatively charged according to the invention. Selectable marker, or herpes simplex virus thymidine kinase (TK) Includes "suicide" genes such as genes. Such a vector is an adenovirus vector. The adenovirus vector is administered to the tumor, Used to treat tumors, including cancerous and non-malignant tumors, such as by direct injection into the tumor The adenovirus vector then transduces the tumor cells. Cells After transduction with an adenovirus vector, the mutual An agonist is administered to the patient, which kills the transduced tumor cells.   In another embodiment, at least one DNA sequence encoding a therapeutic agent is Virions containing animals can be used to study diseases or disorders and their treatment. Administered to the animal as a model. For example, a DNA sequence encoding a therapeutic agent Adenovirus vectors containing You. Following administration of such a vector containing a DNA sequence encoding a therapeutic agent, Animals are evaluated for expression of the therapeutic. From the results of such studies, the adeno Which for the treatment of diseases or disorders viral vector is associated with loss of the therapeutic agent To be administered.   In another embodiment, the adenovirus vector particles are capable of eukaryotic cells.In vivoso Used to infect. The eukaryotic cell is as described above. This eukaryotic cell The host then resides as part of the gene therapy procedure in an amount effective to produce a therapeutic result in the host. Mainly administered.   Optionally, a vector containing the desired gene or the gene encoding the therapeutic agent Particle is the desired cell lineIn test tubeUsed to infect Infected cells may have the desired protein or therapeuticIn test tubeProduce in.                                  Example   The invention will now be described in connection with the following examples. However, the scope of this invention The box is not intended to be limited thereby.                                 Example 1               E1 ^- / E2a ^- or E1 ^- / E4 ^- vector of               Developing cell lines for complementation Construction of plasmid having adenovirus 5 E1 sequence   Adenovirus 5 genome is digested with ScaI enzyme and separated on agarose gel A 6,095 bp fragment containing the left end of the viral genome was isolated. Perfect Adenovirus 5 genome registered as Genbank, accession number M73260 And incorporated herein by reference, this virus has accession number VR-5. American type culture in Rockville, United States, Maryland ・ Can be quoted from the collection. The ScaI 6,095 bp fragment was further 917 salts. It was digested with ClaI at base pairs and BglII at 3,328 base pairs. Generate A 2,411 base pair C1aI-Bg1II fragment was purified from an agarose gel, Superlinker shuttle plasmid pSE280 (Sandy, CA) (Ego Invitrogen), which forms pSE280-E Digested with C1aI and Bg1II (FIG. 1).   Adenovirus 5 DNA, XhoI flanking 552 to 924 base pairs and To synthesize DNA encoding the SalI restriction site, a polymerase chain reaction is used. (PCR) was performed. Use The primers used were:   5 'end, Ad5, 552-585 base pairs:   3 'end, Ad5, 922-891 base pairs:   This amplified DNA fragment (hereinafter sometimes referred to as fragment A) was then And C1aI, which cut at the native C1aI site (base pair 917), It is also ligated to the XhoI and C1aI sites of pSE280-E, thus Reconstruct the 5 'end of the E1 region starting 8 base pairs upstream of the codon.   Then from 3,323 to 4,090 base pairs adjacent to the EcoRI restriction site. PCR was performed to amplify adenovirus 5 DNA. Ply used Ma was as follows.   5 'end, Ad5, 3323-3360 base pairs:   3 'end-Ad5, 4090-4060 base pairs:   This amplified DNA fragment (hereinafter sometimes referred to as fragment B) is BglII Digested with the adenovirus 5Bg1II site (3,382 base pairs). And adenovirus 5, bp 552 to 4,090. PSE to reconstruct more complete E1a and E1b regions Ligated into the BglII and EcoRI sites of 280-AE. Generate Plus The mid is referred to as pSE280-E1 (FIG. 1).   The complete E1a / E1b region was cut from pSE280-E1 by EcoRI digestion. And blunted with Klenow and cut with Sail. The 3,567 base pair fragment Purified from agarose gel and expressed plasmid pMAMneo (California) , Palo Alto, Clontech), which is likewise pMAMneo Prepared by cleavage of XhoI at Luc (FIG. 2) at base pair 3,405, Klenow And cleaved with SalI at base pair 1,465. 8,399 base pairs The strip is gel purified and phosphatase added to form pMAMneo-E1 To the E1a / E1b fragment described above (FIG. 3).   Bacterial transformants containing the final pMAMneo-E1 construct were identified. plus Mid DNA was prepared and purified by binding to CsTFA. Circular plastic DNA at the XmnI site in the ampicillin resistance gene of pMAMneo-E1 Phenol / chloroform extraction and linearization prior to use for cell transfection Further purification by ethanol precipitation.   Alternative construction containing the complete E1a / b region under the control of the synthetic promoter GRE5 Was prepared as follows. Complete E1a / b region excised from pSE280-E1 Which was digested with XhoI and BamHI, resulting in a BamHI site 3 '. Was previously modified to be included in the E1 region. Contains E1a / b fragment The fragment from XhoI to BamHI forms pGRES-E1 (FIG. 5). To pGRE5-2 / EBV (Figure 4, Ohio, Cleveland, USB (Iochemicals) at the unique XhoI and BamHI sites.Construction of plasmid containing adenovirus 5 E2a sequence   The adenovirus 5 genome was digested with BamHI and SpeI, which Cleavage at base pairs 21,562 and 27,080, respectively. Fragment is agarose sedge And a 5,518 base pair BamHI-SpeI fragment was isolated. 5 The 518 base pair BamHI-SpeI fragment was further digested with SmaI The pair was cut at 23,912. The resulting 2,350 base pair BamHI-SmaI fragment The pieces were purified from an agarose gel and the superlinker shuttle plasmid pSE 280 to form pSE280-E2 BamHI-SmaI Was digested with BamHI and SmaI (FIG. 6).   If the PCR is 23,912 base pairs adjacent to the NheI and EcoRI restriction sites 24,730 in order to amplify adenovirus 5 DNA from the SmaI site. Was done. The primers used were as follows.   5 'end, Ad5 base pairs 24,732-24,708:   3 'end, Ad5 base pair 23,912-23,934:   This amplified DNA fragment was digested with SmaI and EcoRI to give Ad5 base pairs 2 Complete E2a from 4,730-21,562 SmaI and E of pSE280-E2Bam-Sma to reconstruct the region Ligation to the coRI site. The resulting plasmid is pSE280-E2a (FIG. 6).   To convert the BamHI site at the 3 'end of E2a to a SalI site, The 2a region was cut with BamHI and NheI to give pSE280-E2 a) and recombine at the unique BamHI and NheI sites of pSE280. Loaned (Figure 6). The E2a region is then p5 in the 5 'to 3' orientation, respectively. Clone into the NheI and SalII sites of the MAMneo multiple cloning site. Therefore, it was excised from this construction with NheI and Sail. The resulting construct is pMAMneo E2a (FIG. 6).   A bacterial transformant containing the final pMAMneo-E2a was identified. Plasmid DNA was prepared and purified by binding to CsTFA. Circular plasmid D NA is lined at the XmnI site in the ampicillin resistance gene of pMAMneo-E2a. Phenol / chloroform extraction and shaping prior to use for cell transfection Further purification by ethanol precipitation. As an option, the plasmid is linear with Bc1II And the latter is neo^RCleavage within the open reading frame of the gene, Was inactivated. This form of the plasmid can be used for co-transfection with an alternative selectable marker. Used when entry was required.Construction of Plasmid Containing Adenovirus 5 ITR / E4 Region   The adenovirus 5 genome is digested with SpeI, SpeI at base 27,082. And cut the two fragments on an agarose gel. Isolated. An 8,853 base pair fragment containing the left end of adenovirus 5 was isolated. Was.   The 8,853 base pair right terminal fragment further comprises StuI and salt at base pair 31,956. Digested with Eco47-III at base pair 35,503. Fragment is agarose gel And the Eco47-III fragment was separated from 3,547 base pairs StuI. Was. This fragment was used as the superlinker shuttle plasmid pSE380 (Invitro). Trogen) (FIG. 7) and StuI to construct pSE380-E4. And digested with Eco47-III (FIG. 8).   PCR was performed from base pair 35,499 adjacent to the BamHI restriction site to base pair 35, Performed to amplify adenovirus 5 DNA to the right ITR ending at 935. The PCR primers used were as follows:   5 'end, Ad5 base pairs 35,935-35,908:   3 'end, Ad5 base pairs 35,503-35,536:   This amplified DNA was digested with BamHI and Eco47-III, which Cleavage at Denovirus 5 Eco47-III site base pair 35,503, pSE3 Unique BamHI and pSE380-E4 to form 80-ITR / E4 And Eco47-III sites (FIG. 8).Construction of plasmid containing E4 region under control of introduced promoter   Under the control of the glucocorticoid-inducible GRE5 promoter, Expresses all of the denovirus 5 E4 open reading frame and regulates the SV40 promoter. Under the control, a plasmid containing the puromycin resistance gene was constructed as follows.   Construct pSE380-E4 (Figure 8) was cut with Eco47-3 and BamHI. And ligated to a 33 base pair synthetic oligonucleotide, which is adenovirus 5 From the Eco47-3 site at base pair 35,503 to the E4 at base pair 35,522 Includes ORF1 ATG. The resulting construct pSE380E4-ATG (FIG. 9) ) Contains all E4 open reading frames without promoter. E4 ORF is Xho I was excised from pSE380E4 / ATG as a BamHI fragment and pGRE 5 / Expression shuttle plasmid pSE38 to generate E4Puro (FIG. 9) 0GRE5 / SV40Puro^RXhoI and B in the multiple cloning site of Linked to the g1II site. Expression shuttle plasmid pSE380GRE5 / S V40Puro^RFrom pSE380 (FIG. 7) and pGRE5 / EBV (FIG. 4) Constructed from excised GRE5 promoter and puromycin resistance gene Was done.Transfection and selection of cells   Generally, this method involves calcium phosphate precipitation or other DNA delivery means. Single tissue culture of two plasmid constructs, each with a different viral gene It involved continuous introduction into cells. Cells are transfected with the first construct; The expression of the relevant drug resistance gene was selected to determine a stable component. Individual Cell clones have been identified and Function was verified. The appropriate candidate clone is then the second viral gene and the second Transfected with a second construct containing the two selectable markers. The transfected cells are then Selected to determine the stable components of the construct, cell clones were determined. Cell clones were assayed for functional expression of both viral genes.   To determine the most suitable cell line for the transfection, use continuous transfection and And selection was performed on the following parental cell types.   A549 (ATCC access number CCL-185);   Hep-2 (ATCC access number CCL-23); or   KB (ATCC access number CCL-17).   Appropriate selection conditions are for G418 and hygromycin B for the three cell lines. And confirmed by standard kill curve determination.Transfection of Cell Lines Using Plasmids Containing El and E2a Regions   neo^RTo linearize the plasmid in the reading frame and inactivate the gene, pMAMneo-E2a was digested with BclII. The cell line described above has a linear p MAMneo^REukaryotic expression cassette comprising E2a and SV40 promoter , Hygromycin resistance gene, and multiple clonins of pTZ18R (FIG. 10) Marker plasmid consisting of a polyadenylation signal cloned into the At a molar ratio of 10: 1. 48 hours after transfection, cells are Placed under glomycin selection and remain selected until drug-resistant colonies develop Was held. Clones were isolated and E2 using polyclonal antisera. E2a expression was selected by a protein staining and visualized by immunofluorescence. Was. E2a function is a temperature-sensitive mutation with a temperature-sensitive mutation in the E2a gene. Selected by complementation of the variant Ad5ts125 virus (Van der Brie G, others,Virology Journal15, Volume 348-354 (1975) ). Positive clones were identified and linearized with XmnI within the ampicillin resistance gene. Transfected with pMAMneoEl. Cells were selected for G418 resistance. G418 resistant colonies were stained using monoclonal antibodies for E1 protein. E1 expression (New York, Uniondale, Oncogene Yenises). The E1 function is such as pAvS6 (FIG. 20) as described below. Assayed by the ability to complement the E1 deleted vector.   In a preferred option, pMAMneo-E2a is Amp^RX with gene mnI was linearized, introduced into cells by transfection, and cells were G418-selected. Selected stable integration of this plasmid. Expresses the E2a gene Clones were identified and GRE5 enhanced E1a / b using 7 kilobase EcoRV. Domain plus hygromycin^RXmn from pGRE-E1 containing the gene (FIG. 5) Used for transfection of the I fragment. Cells were selected for hygromycin resistance and E1a / B and E2a expression were assayed.Transfection of Cell Lines Using Plasmids Containing El and E4 Regions   Circular plasmid pSE380-E4 had a 10: 1 molar ratio A cell line described with the hygromycin resistance plasmid described 48 hours after transfection, cells are placed under hygromycin selection and drug resistant colonies are established. Selection was kept until it occurred. Clones were then isolated and screened for E4 function . Specifically, the clones were 5 adenovirus deletions that deleted the entire E4 open reading frame. Natural mutant d1 1011 (bridge, others,Virology, 193, 794-80 1 (1993)) and produce an infectious virus of the E4 genotype. Clones were tested for their ability. Positive clone identified and ampicillin resistant Transfected with pMAMneoE1 linearized with XmnI in the gene. Cells Then G418 resistance was selected. G418 resistant colonies then selected E1 function. I was separated.Includes induced E1 and E2a plasmids using induced E4 expression plasmids Transfection of cells   Prior to generation of cells containing pMAMneo / E2a and pGRE5-E1 In the preferred option described above, a clone expressing both viral genes inducibly. Was identified. These cell lines are further defined as pGRE5 / E4 / SV40Puro^R Transfection with plasmid (FIG. 9) and puromycin-resistant colonies It was further modified by screening. Cell clones were selected, expanded and E4 deleted By assaying for dexamethasone-dependent complementation of the virus Ad5d11011 Inducible E4 function was analyzed (Bridge, et al.,Virology, 193, 794 -801 page (1993)). In one alternative, the E4 gene is Lacyclin-inducible promoter and manipulation Will be combined.                                 Example 2               E1 / E2a for vector construction of E2A deletion               Alternatively, development of a vector lacking the E1 / E4 intermediate Subcloning of the adenovirus 5 E2 region   Adenovirus 5 genomic DNA consists of BamHI (base pairs 21,562) and S cleaved with peI (base pair 27,082) restriction enzyme, 5,520 base pairs BamH The I-SpeI fragment was separated from the agarose gel. 5,520 base pairs BamH The I-SpeI fragment was previously used to generate plasmid pTZE2A (FIG. 11). PTZ18R plasmid modified to include a SpeI site at base pair 186 (New Jersey, Piscataway, Pharmacia) (Figure 10) Was.Deletion of E2A open reading frame   pTZE2A is located at base pairs 21,562 and 23,912 of adenovirus 5. Corresponding BamHI (base pairs 8,391) and SmaI (base pairs 2,350) Cleavage with restriction enzymes. 6,041 base pairs and 2,350 base pairs BamHI / The SmaI fragment was separated from the agarose gel. 2,350 base pairs BamHI / The SmaI fragment was digested with DraI restriction enzyme (adenovirus 5 base pairs 22,442). Digested and 880 bp DraI-BamHI was separated on an agarose gel. Was. The 880 base pair DraI / BamHI fragment is composed of the attached BamHI-BamHI and 6,041 bp BamHI / S via blunt-end SmaI-DraI ligation ligated to the maI fragment, which The plasmid pTZd1E2A was generated (FIG. 11). Such plasmids are Includes the virus 5 E2A region, where the DraI site (base pairs 22,442) From the E2A region corresponding to the SmaI site (base pairs 23,912) The base pair had been deleted.Subcloning of the adenovirus 5 E3 region   Adenovirus 5 genome was digested with SpeI (at 27,082 base pairs) . An 8,853 bp fragment containing the right end of adenovirus 5 was isolated. Adeno The 8,853 base pair right terminal fragment of virus 5 is Stul (base pair 31,956). Digested. This fragment was ligated into plasmid pSE380 (Invitrogen). Which is used to generate pSE380-E3 + (FIG. 12) with StuI and S digested with peI.Connection of E3 and E4 regions   Plasmid SE380-E3 + was digested with SpeI and StuI, resulting in 4,8 The 74 base pair fragment was Sp Sp to generate pSE380-E3 + E4 + (FIG. 13). The pSE380-ITR / E4 plasmid digested with eI and StuI (FIG. 8) ).   The E3 + / E4 + fragment was then 8,943 bases from pSE380-E3 + E4 +. Excised as a pair SpeI-BamHI fragment, pTZE3 + E4 + (FIG. 13) PTZ18R previously modified to remove an XbaI site to generate At the SpeI and BamHI sites.Deletion in the E3 region   pTZE3^-To generate E4 + (FIG. 14), the plasmid pTZE3 + E 4+ is at base pairs 28,592 and 30,470 of the adenovirus 5 genome Deleted 1,878 base pairs of the E3 region between the XbaI sites and digested with XbaI. Was.   Plasmid pTZE3^-E4 + was digested with SpeI and BamHI and pS E280-E3^-The SpeI and pSE280 plasmids were used to generate E4 +. And BamHI sites. pSE280-E3^-E4 + plasmid is B AmHI digested, blunted and recycled to remove BamHI sites Was. E3^-E4 + containing fragments were removed by restriction digestion with SpeI and Sail. And pSPORT1E3^-SpeI and S to generate E4 + (FIG. 14) pSPORT-1 digested with a1I (FIG. 15) (Gaysersba, Maryland) , Jibco / BRL) plasmid.Linkage between E2A region and E3 ⁻ / E4 ⁺ region   Plasmid pSPORT-1E3^-E4 + digested with SpeI and BamHI And pSPORT-1E2A^-E3^-Plus to generate E4 + (FIG. 16) It was ligated with the BamHI / SpeI fragment from the mid pTZd1E2A.Constructions involving E2A region deletion and modified E3 regions   In one alternative, pSPORT1 / E2A-E3 + E4 + (FIG. 16) Is pSPORT / E2A-E3 +^*E3 reading frame to generate E4 + A heterologous promoter, such as SV40, operatively linked to all or part of Xb including It is modified by inserting the aI fragment into a unique XbaI site.                                   21. Incorporation of the E2A ^- E3 ^- E4 + region into circular non-infectious adenovirus 5 plasmid Only   Plasmid pSPORT-1E2A^-E3^-E4 + is BamHI and Sa1I Digested. E2A^-E3^-The fragment containing the E4 + sequence is pBR322E2A^-E3^- BamHI and SalI digested plasmids to produce E4 + (FIG. 18) Sumid pBR322 (Figure 17) (Lifetime, Gaithersburg, Maryland) Knology).   The adenovirus 5 genome contains C1aI (base pair 917) and BamHI (base pair). 21,562) and the 20,645 bp fragment was pBRad5E2 A^-E3^-To generate E4 + (FIG. 18) (also C1aI and BamHI Plasmid pBR322E2A)^-E3^-Connected to E4 +.Intermediates for construction of vectors with E4 deletion   Plasmid pSE380-ITR / E4 is pSE380-E4^-(FIG. 19) BpU1102 (base pair 33) which removes all E4 open reading frames to generate , 129) and Eco47-3.   E4 region of plasmid pSE380-E3 + E4 + (StuI-BamHI) Shows that the pSE380-E3 + E4^-(FIG. 19) The pSE380 is removed -E4^-Was replaced with a StuI-BamHI fragment from   In a preferred option, pSE380E4^-Starting with E4 ORF1 ATG Larger E4 region deleted than reducing the entire 60 amino acid open reading frame to 7 amino acids Loss is made. This pSE380E4^-Preferred embodiment cited as (2) Is constructed as shown in FIG. pSE380E4^-Is Bpu1102 ( Adenovirus 25 base pairs 33,129) and AfII (Adenovirus 5 Cleaved at base pairs 33, 104) and bound in a 5 'suspension using Klenow Blunted pSE380E4^-Rejoined to form (2). this The alternative E4 region deletion was then excised as a StuI-BamHI fragment and FIG. PSE380E3 as shown by⁺E4^-PSE380 to generate (2) E3⁺E4⁺(FIG. 13) was replaced with the StuI-BamHI fragment.   Yet another modification is the adenovirus 5 bp 28,592 and 30,47. The XbaI region between 0 and pSE380E3 as shown in FIG.^-E4^-(2 PSE380E3 to produce⁺E4^-Deleted from.   As another option, Ad5 base pair 28 of pSE380E3 + E4 (2), XbaI region between 592 and 30,470, FIG. 21 shows pSE380E3 +^* To create E4- (2), where or all of the E3 area reading frame Xbal fragment containing a heterologous promoter, such as SV40, operably linked Will be replaced.Ligation of deletions in the E2A, E3, and E4 regions into a single plasmid   Adenovirus 5 DNA fragment containing both E3 and E4 region deletions in tandem The pieces were SpeI (adenovirus 5 bp 27,082) and Sail (Figure 22). ) By digestion of pSE380E3^-E4^-It is resected from (2). This fragment is shown in FIG. PSPORT1 / E2A as indicated by 2^-E3^-E4^-To form (2) To pSPORT1 / E2A^-E3^-E4⁺Within the unique SpeI-Sa1I fragment Will be replaced.   In one alternative, pSPORT / E2A-E3-E4 + (FIG. 16) The unique SpeI-Sa1I fragment was transformed into pSPORT1 / E2A-E3 +^*E4- PSE380E3 + to create (2)^*Sp removed from E4- (2) Replaced with the eI-Sa1I fragment.                         Virus vector production Production of vectors having genotypes E1 + E2A ^- E3 ^- E4 +   Plasmid pSPORT-1E2A^-E3^-E4 + is BamHI and Sa1I Digested with E2A^-E3^-The fragment containing the E4 + sequence was purified from an agarose gel. You. The unique BamHI site is Bam at 21,562 base pairs of adenovirus 5. HI. The purified fragment was digested with the left BamHI fragment of the adenovirus 5 genome. Pieces (21,562 base pairs) are ligated in vitro. The ligation mixture is then E1 + E2^-E3^-Vector for Production of E4 + Adenovirus 5 Vector (FIG. 23) Transfected into a suitable cell line that can complement the E2A deletion.Production of vectors with genotype E1 + E2A-E3 + ^* E4 +   In a manner similar to that described above and shown in FIG. 23, BamHI-Sa1I The fragment is pSPORT1 / E2A-E3 +^*Genotype E1 + isolated from E4 + E2A-E3 +^*Ad5, 21,5 to generate Ad5 virus with E4 Used for ligation with the 62 base pair left terminal BamHI fragment.Production of vectors having genotypes E1 ^- E2A ^- E3 ^- E4 +   E1 + E2^-E3^-The DNA of the E4 + adenovirus 5 vector was purified and C1 digested with aI. This restriction enzyme cuts the adenovirus 5 genome at 917 base pairs. Refuse. The right-hand fragment is purified (including the reporter gene or transgene) Double complement can provide both E1 and E2A functions in trans with AvS6 The cell line is co-transfected. pAvS6 (FIG. 24) is a shuttle plasmid. And the adenovirus 5'ITR, and the adenovirus envelope signal, E 1a enhancer sequence; promoter; tripartite leader sequence, insertion of foreign gene Cloning site for; poly A signal; and genomic base pair 3329 DNA segment corresponding to 5 genomes of adenovirus not exceeding from base pair 6246 including. pAvS6 is a published PCT application further published October 27, 1994 It is described by the number WO94 / 23582. Viral DNA fragment and pAvS6 Homologous recombination between the genotypes E1^-(Transgene) E2a^-E3^-E4⁺Line with Resulting in a shaped viral genome (FIG. 25). This vector also contains the double complementing cell line E 1⁺/ E2a⁺Is plaque purified using   In one alternative, genotype E1^-E2A^-E3^-Vector with E4 + E1 replicates when transfected into an appropriate complementing cell line^-(Reporter or Transgene) E2A^-E3^-Pre-existing for direct production of E4 + virus The left ITR-BamHI fragment of the E1 vector into pSPORT-1E2A^-E3^-E 4^-It is generated by ligation to the BamHI / SalI fragment of (Figure 26). However, this approach requires that the reporter or transgene contains a BamHI site. Need not be.   In yet another option (FIG. 27), plasmid pBRAd5-E2A^- E3^-E4 + is transformed into E1 by homologous recombination.^-(Reporter or transgene) E 2A^-E3^-To generate an E4 + vector, select (reporter gene or transfection (Comprising the gene) with pAvS6 into a double complementing cell line. Baek The plaques are plaque purified using a dual complementation cell line.   As an example of performing this method, genotype E1^-E2a^-E3^-Baek with E4 + Contains nuclear localized beta-galactosidase cDNA (PCT Application No. WO Adenovirus plasmid vector pAvS6 (described in US Pat. No. 95/09654). Produced using nLacZ. The resulting vector, Av3nLacZ (FIG. 28) ) Was generated by homologous recombination outlined in FIG.   In one alternative, the vector Av3nLacZ is used for the production of other vectors. Can be used as starting material for Av3n LacZ contains two C1aI sites, one containinglacZIn the coding area, One is in the polyadenylation signallacZAt the 3 'end (FIG. 28). Av3nLacZ was digested with C1aI and pAvS6 / introduced as described in FIG. By recombination with the gene, another Av3-type vector is created. This approach Chi is E1⁺The vector backbone is not present in the recombination and a new recombinant vector -, X-gal is B-gal negative contained in Agar overcoat during plaque purification. Offers the advantage of being able to choose.Production of vectors with genotypes E1-E2A-E3 + ^* E4 +   In a manner similar to that described previously and shown in FIG. 25, the virus E1 + E2A-E 3+^*The DNA from E4 + was digested with C1aI and the right fragment was Genotype E1-E2a-E3 +^*PA to generate a vector with E4 + Used to co-transfect E1 / E2A-expressing cells with vS6 / transgene. Used.   In one alternative, and in a similar way to that shown in FIG. The left terminal BamHI fragment from the existing E1-vector was pSPORT1 / E2A -E3 +^*Directly ligated to the BamHI / Sa1I fragment from E4 +, the genotype E 1-, transgene, E2A-E3 +^*E to generate a vector with E4 + Transfected into 1 / E2A expressing cells.Genotype E1 + E4 ^- production of the vector with the   Base pair 1 to SpeI site containing base protein at base 5 '(base pair 27,0 82) is a fragment of E4 Ligated to the SpeI / BamHI fragment purified from the plasmid containing the region deletion Was. In vitro ligation mixture was adenovirus 5E1 + E4^-Vector (Fig. 29) E4 + cell lines were transfected for production.   As an option, pSE380E3⁺E4^-(2), as shown in FIG. E1 with E4 deletion in the same manner as outlined in FIG.⁺E3⁺E4^-(2) C Used to generate irs. Another option is pSE380E3^- E4^-(2) corresponds to E1 as described in FIG.⁺E3^-E4^-(2) Adenovirus Used in the same way to generate theGenotype E1 ^- E4 ^- production of the vector with the   Adenovirus 5E1 + E4^-Genomic DNA is purified and C1aI restriction enzyme Digested. The right fragment is purified (including the reporter or transgene) Co-transfected dual complementing cell lines expressing E1 and E4 with pAvS6 It is. E4^-Homologous recombination between the viral fragment and pAvS6 is due to genotype E1^- (Reporter or transgene) E2A⁺E3 + E4^-Spawn vector with You. The vector (FIG. 30) is plaque purified using a dual complementation cell line.   Another option is SE380-E3 + E4^-SpeI / Bam from The HI fragment is E1^-(Reporter or transgene) E4^-Vector (Fig. 31) To generate the pre-existing E1 digested with SpeI.^-Vector left Directly ligated to the terminal fragment. But this approach is a reporter Alternatively, it requires that the transgene does not contain a SpeI site.   In one alternative, the homologous set described above (FIGS. 30 and 31) Change orIn test tubeAny of the linkages and E1 as described above.⁺E3⁺E4^-(2 ) Or E1⁺E3^-E4^-(2) Using any of the viruses, type E1^- (Transgene) E3⁺E4^-(2) or E1^-(Transgene) E3^-E4^-(2 ) Is produced.                                 Example 3 Production of Adenovirus Vectors with Genotypes E1 ⁻ , E2A ⁻ , E3 ⁻ , E4 ⁻   3 essential genes referred to as triple deletion vector or AV4 production vector An adenovirus vector lacking (E1, E2A, E4) is constructed as follows. Is done. Av3nLacZ was used as starting material and AV4nLacZ was homologous. Produced by either recombination or in vitro ligation. Av4nLacZ Use as starting material to construct any other transgene-containing Av4-type vector can do.   In one option, pSPORT1 / E2A^-E3^-E4^-Yu from (2) The neak BamHI-SalI fragment was isolated and a unique SrfI restriction site (AV Av3n cleaved at 3nLacZ base pair 27,098 (FIGS. 28, 32) Generates E1, E2A, and E4 viral genes with the left terminal fragment of LacZ. The emerging triple complementing cell line (as described in Example 1 above) ) Are co-transfected. Overlap between BamHI and SrfI Homologous recombination between these two DNA fragments in the coding region is due to genotype E1^- , NLacZ, E2A^-, E3^-, E4^-(Fig. 32).   In one alternative, pSPORT1 / E2A^-E3^-E4^-From (2) A unique SrfI-Sa1I fragment was isolated and genotype E1^-, NLacZ, E 2A^-, E3^-, E4^-At base pair 27,098 to produce a vector with Directly ligated to the left terminal fragment of AV3nLacZ cut with rfI. This concatenation The mixture was then purified to produce AV4nLacZ (FIG. 33) in a triple complementing cell line ( E1⁺, E2A⁺, E4⁺).   In one alternative, an AV4 vector containing a transgene other than nLacZ Is constructed. Purified DNA from Av4nLacZ was C1aI (see FIG. 34). Digest and purify the right end fragment. This fragment is the desired pAVS6 / transgene The triple complementation cells (E1⁺, E2A⁺, E4⁺Transfected) Genotype E1 by homologous recombination^-, Transgene, E2A^-, E3^-, E4^-A with The V4 type vector is produced (FIG. 34).Production of Adenovirus Vectors with Genotype E1-E2A-E3 + ^* E4-   In one embodiment, and in a manner similar to that shown in FIG. ORT1 / E2A-E3 +^*Unique BamHI / Sa1I from E4- (2) Fragments are separated and homologous The genotypes E1-, nLacZ, E2A-E3 +^*E4 (2) or Is AV4nLacZE3 +^*To generate a vector with a unique Srf E1, E2A, with the left terminal fragment of AV3nLacZ cut at the I restriction site, And E4 expressing cells.   In one embodiment, and in a manner similar to that shown in FIG. PORT1 / E2A-E3 +^*Unique SrfI-Sa1I from E4- (2) The fragment was isolated and ligated directly to the left terminal fragment of AV3nLacZ cut with SrfI. Used to tie. The ligated product is then genotype E1-, nLacZ, E2 A + E3 +^*A vector having E4- (2) or AV4nLacZE3 +^*To Cells that express E1, E2A, and E4 are transfected to produce.   Also in one embodiment, the modified E3 + comprises a transgene other than nLacZ and^* An AV4 vector containing the region is generated in a manner similar to that shown in FIG. be able to. AV4nLacZE3 +^*Is cleaved with C1aI and genotype E1 −, Transgene, E2A-E3 +^*To generate a vector with E4- (2) In addition, cells expressing E1, E2A, and E4 together with pAV6 / transgene When transfected.                                 Example 4 E1 / E2a or E1 / E4 expressing normal human CFTR cDNA also Of a recombinant adenovirus vector lacking E1 / E2a / E4   pAvS6 (FIG. 24) was linearized with EcoRV. normal The human CFTR cDNA sequence (nucleotides 75 to 4,725; No. M2868), plasmid pBQ4-7. (FIG. 35) was removed by PstI digestion followed by CFT with T4 polymerase. R cDNA ends were blunted (pBQ4-7 was purchased from The Ho Spital for Thick Children, L. C. Offered by Twee ). This CFTR cDNA is then used to prepare the RSV promoter and CFTR cD Linear p to create an operational linkage between the 5 'ends of the coding sequence of NA It was inserted into the plasmid of AvS6. The resulting plasmid pAvS6 CFTR (FIG. 35) were linearized by digestion with KpnI and E1 respectively as described above.^-/ E2^-Av3LacZ, E1^-/ E2^-/ E4^-Has a large C1 of Av5LacZ E1 / E together with the aI fragment or the large C1aI fragment from Av4nLacZ 2a, E1 / E4 or E1 / E2a / E4 expressing cells were co-transfected.   The iE1 / E2a cells co-transfected as described above were then overlaid with agonist. And cultured in a humidified atmosphere containing 5% carbon dioxide at 37 ° C until virus plaque formation. Was. Plaques are collected and recombinant adenovirus is further plaque purified and amplified And titrated as above (Rosenfeld, et al.,cell, 68 volumes, 143-1 55 pages (1992)). Adenovirus vector is Ad gene as described above. E1 / E2a, E1 / E4 or E1 / E2 / E4 deletion and normal human C FTR cDNA was evaluated for partial or total content (Mittele Dar, others,Human gene therapy5, Vol. 717-729 (1994)). C F1 epithelial cell line C1^-Such CFTR-expressing adenovirus for treating secretion deficiency Svector's ability was tested as previously described (Mitterderer, Et al., 1994).                                 Example 5 E1 / E2a deletion vector reduced expression of adenovirus late gene product Illustrating things   This example demonstrates the deletion of the E3 region sequence or the entire E1 / E2a without deletion. Or third-generation vectors with partial deletions reduce the ability to elicit a host immune response Prove that you have reduced. Hexon gene expression was measured by metabolic labeling. In addition, this expression is based on first generation vectors (eg, E1 / E3 deletion), second generation vectors (Eg, E1 deletion containing a temperature sensitive mutation in the E2a gene (Englehart ,other,Bulletin of the National Academy of Sciences91, 6196-6200 (1994) Year))) and the third generation vector described above.   Sex of these vectors in relation to a decrease in late gene expression due to E2a deletion In order to evaluate the performance, A549 cells were cultured with Av1LacZ4 (50-500 iu / Cells), a temperature sensitive mutation in the E2a gene, and a luciferase gene. Av2Luc1 (50-500 iu / cell), which is an E1 deletion vector containing Or infected with Av3LacZ1 (500-3000 iu / cell) at 37 ° C. The cells were cultured in a humidified atmosphere of 5% acid gas. As controls (induced Ad1 of Ad5 and And E2a area Of either iE1 / E2a cells or 293 cells expressing Av1LacZ4, Av with approximate multiplicity of infection (MOI) of staining unit (iu) They were infected with 2Luc1 or Av3LacZ1. The medium is then³⁵SM Changed to methionine-free DMEM (mitterader, et al., 1994) containing et The cells were further cultured for 24 hours. Metabolically labeled cells are then washed with phosphate buffered saline. Lysed, then lysed, then the antiproteolytic enzymes (PMSF, Lipase buffer (Ripa buffer) containing 800 μl It was scrapped. After mixing to homogenize cells, label the protein of³⁵S-Met uptake was quantified by routine trichloroacetate precipitation . 3 × 10⁶Aliquots of each incubation culture containing cpm were anti-adenovirus 5 hex Immunoprecipitated using the antibody and using SDS-page as described previously. Rated (Mitterder, others,Human gene therapyVolume 5, 717-729 (1994)). As a result, at the standard body temperature of 37 ° C., Av3LacZ1 A549 cells even at higher multiplicity of infection than LacZ4 or Av2Luc1 (FIG. 36) and 293 cells (FIG. 37, lanes 6-8) markedly with the hexon gene. It was shown to have reduced expression. As a control, all three vectors are vector Hexon in E1 / E2a expressing cells complementing various adenovirus gene deletions within (FIG. 36, lanes 1-3). This is the third generation vector Having improved (decreased) expression of the denovirus backbone gene Is shown.                                 Example 6 Example of E1 / E2a deletion vector reducing adenovirus genome replication Testimony   Third generation (E1 region with or without deletion of E3 region sequence)^-/ E2a^-)vector To demonstrate that has improved attributes regarding residual viral DNA replication³²P Metabolic labeling of vector-infected cells was performed. These experiments were performed in Example 5 above. This was done in a manner similar to that described. Evaluate replication deficiencies of these vectors Therefore, 293 and iE1 / E2 cells were Av1La at about 10 infectious units per cell. Infected with cZ4, Av2Luc1 or Av3LacZ1, as described above³²P -No PO containing orthophosphate_FourThe cells were cultured in the medium, and further cultured for an additional 8 hours. The cells were then washed with phosphate buffered saline followed by Hart sol. union) was dissolved in 0.8 ml. The viral DNA is purified and XbaI Digested with restriction enzymes, agarose gel electrophoresis and autoradiography as previously described He received a graphic (Mitterder, et al., (1994)).   This result indicates that Av3LacZ1 is more than Av2Luc1 or Av1Luc1. Also receive significantly less replication (FIG. 38). Thus the third generation Adenou Ils vector has improved function for adenovirus vector DNA replication have.   All patents and published information (including published patent applications) cited in this specification: And database access numbers and trustee access numbers are as if Individual patents, published information and And database access numbers and accession numbers are specific and individual Quotes the entire range as if directed to be incorporated as a citation Taken here as an example.   However, the scope of the present invention should not be limited to the specific examples described above. Must be understood. The invention may be practiced other than as specifically described. And within the scope of the following claims.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 38/21 AAM A61K 37/02 ADP 38/28 ACS 37/66 AAM 38/43 ADU 37/64 ADD 38/55 ADD 37 / 26 ACS 48/00 ACC 37/465 ADU C12N 5/10 9/14 L 7/00 C12N 5/00 B // (C12N 15/09 C12R 1:92) (C12N 5/10 C12R 1:91) (81 ) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), AU, CA, JP, NZ (72) Invention Godzilla, Mario United States, 20879 Maryland, Gaithersburg, Lions Crestway 8027 (72) Inventor Trappnell, Bruce United States, 20895 Mary Land, Kensington, Bird Road 4023

Claims (1)

[Claims] 1. An adenovirus vector, wherein the adenovirus genome is modified to reduce a host's immune and inflammatory response to said vector. 2. 2. The adenovirus vector of claim 1, wherein said vector encodes an adenovirus 5'ITR; an adenovirus 3'ITR; an adenovirus envelope signal; a protein or a polypeptide encoding a polypeptide of interest. The adenovirus vector lacks all or part of the adenovirus E1 DNA sequence and contains a promoter that controls the DNA sequence encoding the protein or polypeptide of interest. An adenovirus vector lacking all or part of a DNA sequence. 3. 3. The vector according to claim 2, wherein said vector further lacks adenovirus E1a and E1b DNA sequences. 4. 3. The vector according to claim 2, wherein said vector lacks at least the open reading frame 6 of the E4 DNA sequence. 5. 3. The vector of claim 2, wherein said vector further comprises at least a portion of an adenovirus E3 DNA sequence. 6. A producer cell for producing an adenovirus vector particle, wherein the producer cell contains an adenovirus E1 DNA sequence and an adenovirus E4 DNA sequence. 7. A method for producing infectious adenovirus particles, comprising transfecting a producer cell according to claim 6 with an adenovirus vector according to claim 3. Method. 8. An infectious adenovirus particle produced by the method of claim 7. 9. 9. A method of performing a gene therapy treatment, comprising administering to a host an infectious adenovirus particle according to claim 8 in an amount effective to produce a therapeutic effect in the host. how to. 10. A eukaryotic cell transfected with the adenovirus particles of claim 8. 11. 2. The adenovirus vector of claim 1, wherein said vector encodes an adenovirus 5'ITR; an adenovirus 3'ITR; an adenovirus envelope signal; a protein or a polypeptide encoding a polypeptide of interest. An adenovirus vector that lacks all or part of the adenovirus E1 DNA sequence and contains an E2 DNA sequence that contains a promoter that controls the DNA sequence encoding the protein or polypeptide of interest. An adenovirus vector, which lacks all or a part of the vector. 12. 12. The vector of claim 11, wherein said vector lacks adenovirus E1a and E1b DNA sequences. 13. 13. The vector according to claim 12, wherein said vector lacks all or part of the adenovirus E2a DNA sequence. 14． 12. The vector according to claim 11, wherein said vector lacks all or part of the adenovirus E2b DNA sequence. 15. 12. The vector according to claim 11, wherein said vector further comprises all or part of an adenovirus E3 DNA sequence. 16. A producer cell for producing an adenovirus vector particle, said producer cell comprising an adenovirus E1 DNA sequence and an adenovirus E2a DNA sequence. 17． A method for producing infectious adenovirus particles, comprising transfecting a producer cell according to claim 16 with an adenovirus vector according to claim 13. Method. 18. An infectious adenovirus particle produced by the method of claim 17. 19. 19. A method of performing gene therapy treatment, comprising administering to a host an infectious adenovirus particle according to claim 18 in an amount effective to produce a therapeutic effect in the host. how to. 20. A eukaryotic cell transfected with the adenovirus particles of claim 18. 21. The adenovirus vector of claim 1, wherein said vector encodes an adenovirus 5'ITR; and an adenovirus 3'ITR; an adenovirus envelope signal; a protein or a polypeptide of interest. An adenovirus vector comprising at least one DNA sequence; and a promoter controlling a DNA sequence encoding a protein or polypeptide of interest, said adenovirus vector comprising all or part of each of the adenovirus E1, E2 and E4 DNA sequences. An adenovirus vector characterized in that it lacks. 22. 22. The vector of claim 21, wherein said vector lacks all or part of an adenovirus E2a DNA sequence. 23. 22. The vector according to claim 21, wherein said vector lacks all or part of an adenovirus E2b DNA sequence. 24. 22. The vector of claim 21, wherein said vector further comprises all or part of an adenovirus E3 DNA sequence. 25. A producer cell for producing an adenovirus vector particle, said producer cell comprising an adenovirus E1, DNA E2a DNA sequence and an adenovirus E4 DNA sequence. cell. 26. A method for producing infectious adenovirus particles, comprising transfecting a producer cell according to claim 25 with an adenovirus vector according to claim 22. Method. 27. An infectious adenovirus particle produced by the method of claim 26. 28. 28. A method of performing a gene therapy treatment comprising administering to a host an infectious adenovirus particle according to claim 27 in an amount effective to produce a therapeutic effect in the host. how to. 29. A eukaryotic cell transfected with the adenovirus vector of claim 27.