HK1041499A - Defective adenoviruses and corresponding transcomplementant cell lines - Google Patents

Description

The invention concerns new defective adenoviral vectors which allow the transfer and expression of genes of interest in a host cell or eukaryotic organism and new complementation lines which trans-complement the essential viral functions which have been deleted from the genome of these recombinant adenoviruses.

Adenoviruses are DNA viruses that have a wide host spectrum. They have been identified in many animal species and many cell types. There are several serotypes that differ, notably in their genome sequence. Most human adenoviruses are low pathogenic and usually produce only mild symptoms.

The adenosine virus enters the permissive host cell via a specific receptor, then it is internalized and passes into endosomes. Their acidification contributes to a change in the conformation of the virus and its exit into the cytoplasm. Then, the viral DNA associated with certain viral proteins needed in the early stages of the replication cycle, enters the nucleus of the infected cells where its transcription is initiated by cellular enzymes. Replication of adenosine DNA takes place in the nucleus of the infected cells and does not require replication. The assembly of the virions also takes place in the nucleus.

The infectious cycle of adenovirus occurs in two stages: The early phase preceding the initiation of the replication of the adenoviral genome, which allows the production of regulatory proteins involved in viral DNA replication and transcription, and the late phase leading to the synthesis of structural proteins.

Generally speaking, the adenoviral genome consists of a linear, bi-chain DNA molecule approximately 36kb long that contains the coding sequences for over 30 proteins. At each end of the genome, there is a short sequence of 100-150 nucleotides depending on the serotype, reversed and designated ITR (Inverted Terminal Repeat). ITRs are involved in the replication of the adenoviral genome. The encapsulation region, approximately 300 nucleotides, is located at the 5' end of the genome just after ITR 5'.

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The early E1 region is located at the 5' end of the adenoviral genome and contains 2 viral transcription units, E1A and E1B respectively. This region codes for proteins that are involved very early in the viral cycle and are essential for the expression of almost all other adenovirus genes. In particular, the E1A transcription unit codes for a trans-activating protein of the transcription of other viral genes, which induces transcription from promoters of E1B, E2A, E2B, and E4 regions.

The products of the E2 region, which also includes two transcription units E2A and E2B, are directly involved in viral DNA replication. This region notably controls the synthesis of a 72kDa protein, which has a high affinity for single strand DNA and a DNA polymerase.

The E3 region is not essential for virus replication. It encodes at least six proteins that are thought to inhibit the host immune response to adenovirus infection. In particular, the glycoprotein gp19kDa is thought to inhibit the CTL response, which is responsible for the cytolysis of cells infected by host cytotoxic T cells.

The E4 region is located at the 3' end of the adenoviral genome and encodes for many polypeptides that are involved in late gene expression, late messenger stability (mRNA), early to late phase transition, and inhibition of cell protein synthesis.

Once viral DNA replication is initiated, transcription of the late genes begins. These take up the majority of the adenoviral genome and partially cover the transcription units of the early genes. But they are transcribed from different promoters and in an alternative splicing mode, so that the same sequences are used for different purposes. Most of the late genes are transcribed from the late major promoter MLP (Mor Promoter Late). This promoter allows the synthesis of a long transcriptional primary which is then matured into about twenty messenger RNAs (AKNm) from which the virion capsid terminal proteins are produced.

A number of adenoviruses are now well characterised genetically and biochemically, including human adenovirus type 5 (Ad5) whose sequence is disclosed in the Genebank database under reference M73260. The different genes were precisely located on the adenoviral genome which comprises 5' to 3' ITR 5' of 103 bp followed by the encapsulation region (Hearing et al., 1987, J. Virol., 61, 2555-2558) of about 300 bp, then early and late regions whose location is schematically shown in Figure 1, and finally ITR 3'.

It is clear from the above that adenoviruses have interesting characteristics that make them the vectors of choice for gene transfer of interest. Many recombinant adenoviruses are described in the literature (Rosenfeld et al., 1991, Science, 252, 431-434; Rosenfeld et al., 1992, Cell, 68, 143-155). Generally, they are derived from Ad5 and are defective in E1 function to prevent their spread to the environment and host organism. In addition, the non-essential E3 region may also be deleterious. Exogenous sequences are integrated instead of the E1 or E3 region.

Thus, these defective adenoviruses can only be propagated in a cell line that trans-complements the E1 function essential for viral replication. At present, the only complementation line that can be used is the embryonic kidney line 293 (Graham et al., 1977, J. Gen. Virol., 36, 59-72), which results from the integration into its chromosomes of a fragment of the Ad5 genome including the 5' end of the viral genome so that the 293 line complements the defective adenoviruses for E1. The 293 cells also contain sequences that occur in the recombinant adenovirus, such as ITR 5', the coding region and the 3B1 region for ECO1 proteins.

The feasibility of gene transfer using adenoviruses is now established. However, the question of their safety remains. Finally, they are able to transform certain cell lines into cultures, reflecting the potentially oncogenic power of some of the adenoviral genome expression products, mainly from the E1 region and probably E4, at least for some serotypes. Furthermore, the probability of genetic recombination between a previously defective adenovirus, including a recombinant adenovirus, and either a natural or recombinant adovirus (from accidental contamination or opportunistic infection of a host organism), or a fragment of adoviral protein that can be integrated into the complement line of two adoviruses is not likely to be negative. In addition, the possibility of a recombinant adovirus producing a naturally-occurring or active adovirus (from accidental contamination or opportunistic infection of a host organism), or a fragment of adoviral protein that can be integrated into the complement line of two adoviruses is not necessarily negative. In addition, the E1A1 virus is also capable of producing a partially defective adovirus that can be actively recombined in the same adovirus.

It is therefore desirable to have high-performance adenoviral vectors with the lowest risk for use in gene therapy to correct serious genetic defects in vivo and to treat certain diseases for which effective therapeutic approaches are not available.

In addition, there are questions about the derivation of the 293 line, which may compromise the acceptability of products for human use derived from it.It would be useful to have complementation lines whose origin and history are exactly known to produce recombinant adenovirus particles for human use.

(1) new defective adenoviral vectors have now been found which have been removed from specific regions of the adenoviral genome and are better suited to transferring an exogenous nucleotide sequence in vivo and (2) new characterised complementation lines which are pharmaceutically acceptable and thus have all the safety characteristics required for the production of products for human use.

The interest of these new vectors is that they have an increased cloning capacity allowing the insertion of one or more genes of interest of large size and maximum safety of employment. These deleterious mutations render these adenoviruses incapable of autonomous replication and cellular transformation without altering their ability to transfer and express a gene of interest.

Therefore, the present invention concerns a replication defective adenoviral vector capable of being encapsulated in a complement cell, derived from an adenovirus genome comprising 5' in 3', an ITR 5', an encapsulation region, an E1A region, an E1B region, an E2 region, an E3 region, an E4 region and an ITR 3', by deletion: (i) all or part of the E1A region, and all of the part of the E1B region coding for early proteins; or (ii) all or part of the E1A region, and all or part of at least one region selected from the E2 and E4 regions; or (iii) all or part of the E1A region, and part of the encapsulation region.

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An adenoviral vector according to the invention is defective for replication but capable of being replicated and encapsulated in a complement cell providing it with the trans product (s) for which it is defective in order to generate an adenoviral particle (also called defective adenovirus) unable to replicate autonomously in a host cell but nonetheless infectious because it has the ability to deliver the vector into a host cell.

A first variant is that an adenoviral vector according to the invention is derived from the genome of a natural or wild adenovirus by deletion of all or part of the E1A region and part of the E1B region including all the sequences coding for early proteins. A preferred mode is that it concerns the promoter and the sequences coding for the expression products of the E1B region i.e. early proteins and does not include all or part of the termination signal of the transcription that covers the trans sequences coding for late protein IX.This deletion shall include at least the sequences between nucleotides 1634 and 3509 of the adenoviral genome whose sequence is as disclosed in the Genebank database under reference M73260. The purpose of this deletion is to reduce or delete the common sequences between an adenoviral vector of the invention and the adenoviral genome fragment embedded in a complement line, e.g. line 293. Furthermore, it shall eliminate from an adenoviral vector of the invention sequences whose expression products are potentially oncogenic, at least in conjunction with the expression products of the E1A region.

In addition, an adenoviral vector of the invention is further derived from the genome of a natural or wild adenovirus by deletion of all or part of: The following information is provided for the purpose of the calculation of the amount of the aid:

It is clear that an adenoviral vector according to the invention may have one of the three deletions described above or two of them in any combination or all of the deletions.

In a particularly advantageous mode, an adenoviral vector of the invention is selected from only part of the E3 region and preferably from the part that does not contain the gp19kDa protein coding sequences. The presence of the gp19kDa protein coding sequence in an adenoviral vector of the invention will allow infected cells to escape host immunosurveillance; an important criterion when the therapeutic protocol requires multiple repeated doses. It is preferable to place the gp19kDa protein coding sequences under appropriate controls to allow their expression in the host cell, i.e. the elements necessary for transcription of RNA sequences and translation of RNA into the latter.These elements include in particular a promoter. Such promoters are well known to the public and are inserted upstream of the coding sequence by conventional genetic engineering techniques. The promoter selected will preferably be a constitutive promoter not activated by any of the E1A region expression products. Examples include the promoter of the HMG (Hydroxy-Methyl-Glutaryl coenzyme A reductase) gene, the early promoter of SV40 virus (Simian 40), the LTR (Long Terminal Repeat) of RSV (Rose Sarcoma Virus) or the promoter of a higher PG (Phosphosphocercer-glyucate kinase) gene of eukaryote.

In addition, an adenosiviral vector of the invention may optionally be deleted from the portion of the E3 region corresponding to the promoter region, which will be replaced by a heterologous promoter region, such as one of the above.

A second variant is that an adenoviral vector of the invention is derived from the genome of a natural or wild adenovirus by continuous or discontinuous deletion of all or part of the E1A region and all or part of at least the E2 and/or E4 regions. Such deletion increases the chances of cloning genes of interest. On the other hand, removing all or part of the E4 region also reduces or removes sequences coding for potentially oncogenic products.

As above, an adenoviral vector of the invention may also be deprived of all or part of the E1B and/or E3 regions and, in particular, by a mode of execution as mentioned above (such as deletion of the part of the E1B region comprising all sequences coding for early proteins and the part of the E3 region not coding for gp19kDa).

Finally, according to a third variant, an adenoviral vector according to the invention is derived from the genome of an adenovirus by deletion of all or part of the E1A region and part of the encapsulation region.

Partial deletion of the encapsulation region significantly reduces the likelihood of uncontrolled spread of an adenoviral vector of the invention when the adenoviral vector is in the presence of a wild adenoviral, and affects its encapsulation functions in such a way that even if the defective function of the adenoviral is trans-complemented by a wild adenoviral, it cannot be effectively encapsulated in relation to the genome of the competing wild adenoviral.

The deletions of the encapsulation region will be selected according to 2 criteria: a reduced capacity to be encapsulated but at the same time a residual efficiency compatible with industrial production. In other words, the encapsulation function of an adenoviral vector according to the invention is substantially maintained, although to a lesser degree. Attenuation can be determined by conventional techniques of titration by infection of an adequate lineage and evaluation of the number of lysis ranges. Such techniques are known to the art.

Of course, an attenuated adenoviral vector of the invention may also include at least one or some combination of the deletions mentioned above.

An adenoviral vector according to the present invention is derived from the genome of a natural or wild adenovirus, preferably from a canine, avian or human adenovirus, preferably from a human adenovirus type 2, 3, 4, 5 or 7, and preferably from a human adenovirus type 5 (Ad5). In the latter case, deletions of the adenoviral vector according to the invention are indicated by reference to the nucleotide position of the Ad5 genome specified in the Genebank data bank under reference M73260.

A particular preference is given to an adenoviral vector according to the invention derived from the genome of a human adenovirus type 5, by deletion: (i) the entire early protein coding portion of the E1B region extending from nucleotide 1634 to nucleotide 4047; and/or (ii) the E4 region extending from nucleotides 32800 to 35826; and/or (iii) the portion of the E3 region extending from nucleotides 27871 to 30748; and/or (iv) the portion of the encapsulation region: The number of nucleotides in the nucleotide family is very small, ranging from 270 to 346, from 184 to 273, from 287 to 358.

Preferably, an adenoviral vector of the invention is derived from the genome of a wild or natural adenovirus by deletion of at least 18%, at least 22%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% and in particular 98,5%.

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An exogenous nucleotide sequence may consist of one or more genes of interest and preferably of therapeutic interest. In the present invention, a gene of interest may code for either an anti-sense RNA or an mRNA that will then be translated into a protein of interest. A gene of interest may be of the genomic type, complementary DNA type (cDNA) or mixed type (minigene, in which at least one intron is delleted). It may code for a mature nucleotide, a precursor to a mature protein, including a precursor to be secreted and thus including a peptide, a chimeric protein from the fusion of a sequence of different origin or a protein that has a natural mutation or mutation to enhance biological properties.

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Among the genes of interest for use in the present invention are: genes coding for cytokines, such as interferon alpha, interferon gamma, interleukins; genes coding for membrane receptors, such as receptors recognised by pathogens (viruses, bacteria, or parasites), preferably by HIV (Human Immunodeficiency Virus); genes coding for clotting factors, such as factor VIII and factor IX; genes coding for dystrophinel; genes coding for insulin; genes coding for proteins directly or indirectly involved in cell ion channels, such as CFTR (Cystic Fibrosis Transmembrane Regulator) protein; genes coding for anti-sensitivity or inhibitory activity of proteins produced by a caprocarboxylic RNA; genes coding for proteins directly or indirectly involved in cell ion channels, such as CFTR (Cystic Fibrosis Transmembrane Regulator) protein; genes coding for anti-sensitivity or inhibitory activity of proteins produced by a caprocarboxylic RNA;in the genome of a pathogenic organism, or by a cell gene, the expression of which is deregulated, for example an oncogene;genes coding for a protein that inhibits enzymatic activity, such as α1-antitrypsin or a viral protease inhibitor;genes coding for pathogenic protein variants that have been mutated to alter their biological function, such as HIV virus TAT trans-dominant variants that are able to compete with the natural protein for binding to the target sequence, preventing HIV activation;genes coding for classes of antigenic agents to increase host cell immunity;genes coding for proteins of major histocompatibility complex II and I,The TK-HSV-1 virus gene has a significantly higher affinity for certain nucleoside analogues (such as acyclovir or gancyclovir) than the viral TK enzyme, converting them into monophosphate molecules, themselves convertible by cellular enzymes, into nucleotide precursors, which are toxic. These nucleotide analogues are incorporated into DNA molecules in the process of synthesis, and therefore mainly into DNA in the replication state of cells.This incorporation allows specifically to destroy dividing cells like cancer cells.

This list is not exhaustive and other genes of interest may be used in the context of the present invention.

In addition, according to another embodiment of the invention, an adenoviral vector of the invention may also include a non-therapeutic gene coding for a non-adenoviral transcription trans-activating protein. Of course, the gene (s) in the E1A region coding for a trans-activating protein will be avoided, the expression of which may render the adenovirus non-defective. The gene coding for the Saccharomyces cerevisiae protein Gal4 will be preferred. Its expression will allow the vector to propagate in a complement line as described below. Such expression is more sophisticated and may be of interest due to toxicity problems in the transcription of the complement gene; for example, those coding for the production of complement proteins that are necessary for the control of its continuous expression, such as the expression of a complement protein, may be of interest.

The invention also relates to an adenoviral particle and a host eukaryotic cell containing an adenoviral vector according to the invention. e cell is preferably a mammalian cell and preferably a human cell and may include the said vector in an integrated form in the genome or preferably in an unintegrated form (episome).

An adenoviral particle of the invention may be prepared by passing through any complement line providing trans the functions for which an adenoviral vector of the invention is defective, e.g. line 293 of the previous art. These preparation techniques are known to the art (Graham and Prevec, 1991, Methods in Molecular Biology, vol. 7, 109-128, Ed.: E.J. Murey, The Human Press Inc.).

Therefore, the present invention also concerns a complement line containing a complement element, including in particular a part of the E1 region of the genome of an adenovirus excluding ITR 5', such complement element being capable of trans-complementing a defective adenoviral vector and being integrated into the genome of said complement line or inserted into an expression vector.

Err1:Expecting ',' delimiter: line 1 column 92 (char 91)Thus, an adenoviral vector defective for function E1 will have to be propagated in a complement line for E1 (capable of providing trans the or all of the proteins encoded by the E1 region which the vector cannot produce), a vector defective for functions E1 and E4 will be propagated in a complement line for E1 and E4 (providing the essential proteins encoded by the E1 and E4 regions) and finally a vector defective for functions E1, E2 and E4 will be propagated in a complement line for all three functions.and does not require specific completion.

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According to a specific embodiment, a complementation line according to the invention is intended to trans-complement an adenoviral vector defective for function E1.

For the purposes of this invention, a complement line according to the invention may include all or part of the E1A region of an adenovirus genome and: (i) all or part of at least one region of the adenoviral genome selected from regions E1B, E2 and E4, or (ii) all or part of at least two regions E1B, E2 and E4 of that genome, or (iii) all or part of regions E1B, E2 and E4 of that genome.

In the present invention, these regions may be controlled, if necessary, by appropriate means of expression, but they are preferably controlled by their own promoter, inducible by the transcriptional trans-activating protein encoded by the E1A region.

As an indication, a complement line in variant (ii) comprising regions E1A, E1B and E4 is intended for the preparation of an adenovirus defective for functions E1 and E4 deleted from all or part of the corresponding regions.

In an advantageous mode, a complement line of the invention includes in particular all or part of the E1A region and all sequences coding for the early proteins of the E1B region.

In addition, according to a variant of this embodiment, a complement line according to the invention may also be devoid of the promoter region of the E1A region. In this case, the part of the adenoviral genome coding for the early proteins of the E1A region will be placed under the control of an appropriate and functional heterologous promoter in the said complement line. It may be isolated from any eukaryotic or viral gene. However, the use of an adoviral promoter from an early region will be avoided. It may be an emptive promoter. Examples include the SV40 virus promoters, the THSK-V gene and the PGK-1 murin gene.

Err1:Expecting ',' delimiter: line 1 column 653 (char 652)In the case of a hybrid promoter, the activation and expression of the genes encoded by the E1A region under its control will only be induced in the presence of the Gal4 protein. Then, the products of expression of the E1A region will be able to induce the expression of the other early regions E1B, E2/or E4 possibly included in a complementation line according to the invention.This particular embodiment of the invention avoids the production in a constitutive (possibly toxic) manner of the adenoviral proteins necessary for complementation. Thus, induction can be triggered in the presence of a defective adenoviral vector according to the invention expressing the Gal4 protein. However, such a line can also be used to prepare any defective adenoviral vector, provided, however, that the Gal4 protein is provided in trans.

Generally, a complement line includes a part of the genome of an adenovirus that is advantageously derived from an animal adenovirus, such as a canine or avian adenovirus or preferably a human adenovirus and, in particular, type 2 or 5.

A complement line according to the invention includes, inter alia, the part of the genome of a human adenovirus type 5 extending: (i) from nucleotide 100 to nucleotide 5297 of the sequence as disclosed in the Genebank database under reference M73260, or (ii) from nucleotide 100 to nucleotide 4034, or (iii) from nucleotide 505 to nucleotide 4034.

The advantage is that the part of the genome according to (ii) is inserted upstream of a transcription termination signal, such as the polyadenylation signal of the SV40 virus (Simian Virus 40) or the rabbit ß-globin gene, whereas the part according to (iii) that includes neither the E1A region promoter sequences nor the E1B region transcription termination signal is placed under the control of an appropriate promoter, such as a Gal4 protein inducible promoter, and a transcription termination signal, such as the rabbit ß-globin gene. ß Such a complementation lineage is considered safe because it lacks the majority of the sequences common to a particularly defective adenovirus.

On the other hand, a complement line according to the invention may contain the part of the E4 region of a human adenovirus type 5 starting from nucleotide 32800 and ending at nucleotide 35826 of the sequence as disclosed in the Genebank database under reference M73260.

In addition, a complement line of the invention may contain the entire genome of a natural adenovirus, except for the encapsulation region and 5' and 3' ITRs, and preferably the part of the genome of a human adenovirus type 5 from nucleotide 505 to nucleotide 35826 of the sequence disclosed in the Genebank reference M73260. For the purposes of this invention, the sequence is preferably controlled by an appropriate promoter. A promoter inducible by the protein vector Saccharomyces cerevisiae E41, will be used. Such a complement line of the protein is recommended to provide a minimum set of essential functions, including the transcription and the prevention of E24, and a complement vector E24.

In the context of the present invention, this may be any gene coding for a selection marker, these being generally known to the artisan, with the advantage of an antibiotic resistance gene and preferably a gene coding for the puromycin acetyltransferase (pacin gene) conferring puromycin resistance.

In the present invention, the gene coding for a selection marker may be placed under control of appropriate elements allowing its expression. It may be a constitutive promoter, such as the early SV40 virus promoter, but a promoter inducible by the trans-activator protein encoded by the E1A region, in particular the adenoviral promoter E2A, will be preferred. Such a combination will introduce selection pressure to maintain the expression of E1A region genes in a complement lineage as per the invention. However, for the purposes of the present invention, the selected promoter may be modified by deletion, mutation, substitution and/or addition of nucleotides.

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Alternatively, a complement lineage of the invention may be derived from primary cells, including retinal cells taken from a human embryo.

The invention also relates to a method of preparation of an adenoviral particle according to the invention, according to which: an adenoviral vector of the invention is introduced into a complement line capable of complementing in the said vector in such a way as to obtain a transfected complement line,the said complement line is cultured under appropriate conditions to allow the production of the said adenoviral particle, and the said particle is recovered in the cell culture.

Of course, the adenoviral particle can be recovered from the culture medium but also from cells according to conventional protocols.

In a preferred way, a process of the invention implements a line of complementation of the invention.

The invention also concerns the therapeutic or prophylactic use of an adenoviral vector, adenovirus particle, host eukaryotic cell or complement line according to the invention.

Finally, the present invention relates to a pharmaceutical composition comprising an adenoviral vector, adenovirus particle, eukaryotic cell or complement cell as therapeutic or prophylactic agent, as the invention requires, in combination with a pharmaceutically acceptable medium.

The composition according to the invention is intended in particular for the preventive or curative treatment of diseases such as:genetic diseases such as hemophilia, mucoviscidosis or myopathy, Duchene's and Becker's disease,cancers such as those induced by oncogenes or viruses,retroviral diseases such as AIDS (acquired immunodeficiency syndrome resulting from HIV infection) and recurrent viral diseases such as viral infections caused by the herpes virus.

A pharmaceutical composition according to the invention may be manufactured conventionally. In particular, a therapeutically effective amount of a therapeutic or prophylactic agent may be combined with a medium such as a diluent. A composition according to the invention may be administered by aerosol or by any conventional pharmaceutical route in use in the art, in particular by oral, subcutaneous, intramuscular, intravenous, intrapulmonary or intra-tracheal intravenous route. Administration may be performed in a single or repeated time interval or several times after a certain dose. The route of administration and the dose may vary according to various parameters, for example the individual or the pharmaceutical route or the treatment of the disease.

The invention also extends to a treatment method whereby a therapeutically effective amount of an adenoviral vector, adenoviral particle, eukaryotic cell or complement line is administered to a patient who requires such treatment.

The present invention is described more fully by reference to the following Figures and using the following examples.

Figure 1 is a schematic representation of the genome of human adenovirus type 5 (represented in arbitrary units from 0 to 100), showing the location of the individual genes.

Figure 2 is a schematic representation of the vector pTG6546.

Figure 3 is a schematic representation of the vector pTG6581.

Figure 4 is a schematic representation of the vector pTG6303.

Figure 5 is a schematic representation of the pTG1660 and pTG1661 vectors.

The test chemical is a test chemical that is used to test the presence of a substance in the test chemical.

Figure 7 is a schematic representation of the vector pTG5913.

Figure 8 is a schematic representation of the vector pTG8512.

Figure 9 is a schematic representation of the vector pTG8513.

Figure 10 is a schematic representation of the vector pTG8514.

Figure 11 is a schematic representation of the vector pTG8515.

Examples

The following examples illustrate only one way of doing the present invention.

The constructs described below are performed using general genetic engineering and molecular cloning techniques, detailed in Maniatis et al., (1989, Laboratory Manual, Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, NY). All cloning steps using bacterial plasmids are performed by passing through the strain Escherichia coli (E. coli) 5K or BJ, while those using vectors derived from the M13 phage are performed by passing through E. coli NM 522.

On the other hand, the cells are transfected using standard techniques well known to the professional, such as the calcium phosphate technique (Maniatis et al., above).

The fragments inserted into the different constructions described below are indicated precisely according to their position in the nucleotide sequence: the genome of Ad5 as disclosed in the Genebank database as reference M73260, the genome of adenovirus type 2 (Ad2) as disclosed in the Genebank database as reference J01949, the genome of SV40 virus as disclosed in the Genebank database as reference J02400.

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We're building a vector that includes the 5' ITR of the Ad5 genome (nucleotide 1 to nucleotide 103),the Ad5 encapsulation region between nucleotides 104 to 458 in which the portion from nucleotide 184 to nucleotide 273 is deleted and thymine (T) at position 176 is modified into a cytosine (C) to create a restriction site AatII,an expression cassette of a gene of interest comprising 5' 3' MLP of Ad2 (nucleotides 5779 to 6038),the KpnI-XIba-HindIII and SVHI restriction sites,the human DNA fragment for the CFTR protein (except for amino acids corresponding to the sequence of Riordan 2524 and 1035),the transcription site of the virus at position 2640 and the signal at position 2670 (except for the nucleotide P and P in the nucleotide 2640 and 2665),the human DNA fragment for the amino acids at position 2638 and the transcription site of the SID at position 2640 and the nucleotide P in the nucleotide 2670 (except for the nucleotide 3365 and P in the nucleotide 2670),the DNA fragment at position 2638 and the nucleotide P in the nucleotide 3365 and the nucleotide P in position 2665 (except for the nucleotide 3365 and P in the nucleotide 1065),the nucleotide P in position 2665 and the nucleotide 2665 and the nucleotide 1065 in position 2670;

The first step is to clone the pMLP11 isolated EcoRI SmaI fragment between the EcoRI and EcoRV sites of the M13TG131 vector (Kieny et al., 1983, Gene, 26, 91-99) This construction is derived from pMLP10 (Levrero et al., 1991, Gene, 101, 195-202) and differs from the parent vector by introducing a SmaI site at the HindIII site. The vector M13TG6501 is obtained. It is subjected to directed mutagenesis to detect sequences between nucleotides 184 to 273 of the encapsulation region. The directed mutagenesis is carried out using a commercial fragment kit (Amersham) as recommended by the supplier, and the vector is implemented in the pAMG4 vector designated by EcoRG17K. The M13TG4 is introduced into the region of the re-identification site, thus being referred to as the M13TQ2 (EcoRG11N1), and the sequence is processed by a free-flowing NO2 (EcoRG11N1), which is referred to as the EcoRG11K.

The resulting vector, pTG6500, is partially digested by PstI, processed with the DNA polymerase of phage T4 and then digested by PvuI. The PvuI-HpaI fragment isolated from pTG5955 (derived from pMLP11) is inserted into this vector. This fragment contains the SV40 virus transcription termination signal and the portion of the Ad5 genome extending from nucleotide 3329 to nucleotide 6241. The pTG6505 vector thus generated is partially digested by SphI, processed with the DNA polymerase of phage T4 and then digested by PvuI. This is used to bind the SphI site located in polymerase 525 to the PvuI fragment. The result is the polymerase A65T11 (AADTN), which is produced in 1991, after digestion by PTRADN, and is a form of the drug KTN, which is treated with the drug CFG525 and is a drug called KTN, and is a treatment for the human digestion of CFG525 (TradN, KTN, KTN, KTN, KTN, KTN, KTN, KTN, KTN, KTN, KTN, KTN, KTN, KTN, KTN, KTN, KTN, KTN, KTN, KT, KTN, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, KT, K

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The M13TG6501 vector is subjected to directed mutagenesis using the OTG4173 oligonucleotide (SEQ ID NO: 2) and then the mutated fragment is reintroduced into pMLP11, as previously reported, to generate the pTG6501 vector. The latter is digested by SphI, treated with T4 phage DNA polymerase, and then by PvuI. PTG6546 (Figure 2) is obtained by cloning the PvuI-KpnI fragment (the KpnI site has been cleared) isolated from pTG6525 and containing human CFTRc DNA.

Err1:Expecting ',' delimiter: line 1 column 72 (char 71)

The vector M13TG6501 is subjected to directed mutagenesis to delete the sequences between nucleotides 287 and 358 of the encapsulation region and to modify thymines at positions 275 and 276 into guanines to introduce a NcoI site. The mutagenesis is performed using the oligonucleotide OTG4191 (SEQ ID NO: 3) to give M13TG6507. The latter is cleaved by BglII, treated with Klenow DNA polymerase and then by IRI and the corresponding mutated fragment is digested as found in pMLP11 digested by EcoI and SmaI. PTG6504 is generated from the polymerase before the SHI fragment is isolated (TNNXP2 and PTG656 are treated by PATHNXP2 and PATHNXP4 to give the polymerase PATHNXP2 which is inserted into the polymerase PATHNXP6 and the polymerase PATHNXP6 is obtained by the treatment of PATHNXP65 (TG656 and PATHNXP65) which is obtained by the treatment of PATHNXP6 and PATHNXP65 (TG656 and PATHNXP6 and PATH).

4. Generation of a defective and attenuated recombinant adenovirus.

Recombinant defective adenoviruses are generated by co-transfection in cells 293 of either pTG6525, pTG6526 or pTG6546 linearized by ClaI and genomic DNA from Ad-dl324 (Thimmappaya et al., 1982, Cell, 31, 543-551) also digested by ClaI, to generate a recombinant virus by homologous recombination. After 8 to 10 days, individual ranges are isolated, amplified in cells 293 and analyzed by restriction mapping.

The virus AdTG6546 is placed in competition by co-infection with Ad-CFTR (Rosenfeld et al., 1992, Cell, 68, 143-155) which has a wild-type encapsulation region. 293 cells are infected with 5 ufp (range-forming unit) of Ad-CFTR and 5 ufp of AdTG6546 per cell. In parallel, the total viral DNA is isolated by the Hirt method (Gluzman and Van Doren, 1983, J. Virol., 45, 91-103) and the viral DNA then encapsulated after treatment of the cells with 0.2% deoxyhydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydrogenase (DNNNNase) I) with 10 μg/ml of deoxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydro

The expression level of the CFTR protein is measured in the cell extracts of 293 cells infected with AdTG6546 The analysis is performed by Western blot using the technique described in Dalemans et al. (1991, Nature, supra) using the monoclonal antibody MATG1031 However, any other antibody that recognizes antigenic epitopes of the CFTR protein can be used A product of an expected molecular weight of about 170 kDa is detected As an indication, the production level is approximately equivalent to that obtained in the unattenuated Ad-CFTR virus infected cell extracts.

EXAMPLE 2 Generation of a deletion of defective adenovirus from the E1A region and the complete sequence coding for the early proteins from the E1B region. 1. Obtaining a recombinant adenovirus for the expression of the CFTR protein (AdTG6581)

Such an adenovirus is generated from a plasmid vector pTG6581 comprising 5' to 3': The 5' ITR of Ad5 (nucleotide count 103),the Ad5 encapsulation region (nucleotide count 104 to 458),an exogenous nucleotide sequence containing an expression cassette, which includes the following: * the MLP of Ad2 (nucleotides 5779 to 6038), followed by the three tripartite leaders also of Ad2 (nucleotides 6039 to 6079; nucleotides 7101 to 7175; nucleotides 9637 to 9712); these leaders are included to increase the translation efficiency of the downstream inserted sequences,* a polylinker comprising 5' to 3' restriction sites XbaI, HindIII, BamHI EcoRV, HpaI and NotI usable for cloning a gene of interest*, a gene of interest, such as the gene coding for the CFTR protein,* the termination signal of isolated SV40 virus transcription (nucleotides 2543 to 2618), adenoid genome portion of the nucleotides 4024 to 6241,

The AD5 genome fragment extending from nucleotide 4047 to nucleotide 4614 is amplified by PCR from the genomic DNA of Ad5. The PCR reaction implements the sense-amorce OTG5021 (SEQ ID NO: 4), comprising at its 5' end a BamHI site to facilitate further cloning steps, and the anti-sense-amorce OTG5157 (SEQ ID NO: 5). The resulting fragment is then treated with the Klenol DNA polymerase, before being cloned into the SmaI site of M1318 (Gibco BRL), resulting in M13T65G17. The sequence generated by the PCR fragment is verified by the classical enzymatic method (Sanger et al., 1977, Nat., USA, Acad. 544, 73, 63).

In addition, the PvuI-SmaI fragment is isolated from pMLP11 and cloned between the PvuI and KpnI sites of pTG6511 (example 1.1) after the KpnI site was cleared by treatment with T4 phage DNA polymerase using standard methods.

The latter is digested by the enzymes SalI and BstXI and bound to two fragments, the purified BamHI-BstXI fragment of M13TG6517 and the XhoI-BglII fragment of pTG6185. This includes the SV40 virus end-transcription signal encoded by the XhoI and BglII restriction sites. However, any other plasmid with the same end-sequence and appropriate restriction sites could be used. Finally, the vector pTG6555 is obtained, in which an adapter containing two free-terminal restriction sites, EcoV and HpaI, is inserted into the single site. This adapter is derived from the reassociation of the oligon OTG64 and pTG65 (Oligon PQRN) in human cells.

The corresponding recombinant adenovirus AdTG6581 is generated by co-transfection of pTG6581 and Ad dl324 cleaved by ClaI into a complement line for function E1, such as line 293 or a line from example 6, according to the classical protocol.

2. Obtaining a recombinant adenovirus for the expression of IFNγ.

The pTG6303 vector (Figure 4) is obtained by cloning in the HpaI site of pTG6580 of the HpaI-SmaI fragment of M13TG2437, which is derived from the cloning in a vector M13TG130 (Kieny et al., 1983, supra) of the interferon gamma (IFNγ) gene sequence as specified in Gray et al. (1982, Nature, 295, 503-508). The recombinant adenovirus AdTG6303 is obtained by conventional techniques by homologous recombination resulting from the co-transfection of pTG6303 and dl324 Adaris linar by ClaI into a complement line for E1.

3. Construction of an adenovirus that has been removed from region E1 and in which region E3 is placed under the control of a constitutive promoter.

The pTG1670 vector is obtained by cloning between the AatII and BamHI sites of the p polyII vector (Lathe et al., 1987, Gene 57, 193-201) of a PCR fragment containing the RSV (Rous Sarcoma Virus) LTR3' (Long Terminal Repeat). The PCR reaction uses the pRSV/L vector (De Wet et al., 1987, Mol. Cell. Biol. 7, 725-737) as the matrix and the OTG5892 and OTG5893 (SEQ ID NO: 8 and 9) initiators.

The 5' part of the E3 region (nucleotides 27588 to 28607) is amplified by PCR from the pTG1659 vector and with the help of the OTG5920 and OTG5891 (SEQ ID NO: 10 and 11) primers. The latter is constructed in several steps. The BamHI-AvrII fragment (nucleotides 21562 to 28752) is obtained from the genomic DNA of Ad5 and then cloned between the same pTG7457 sites to generate pTG1649. Finally, the pTG7457 vector is a final p19 (Gibco BRL) polylinker modified to contain a site AvrII. The Eco-AvrII fragment (KRI) is then inserted into the pTG1616 (PATR) vector (PATR) to give an example of PATR.

The EcoRI fragment of the Ad5 corresponding to nucleotides 27331 to 30049 is isolated from a genomic DNA preparation and subcloned in the pBluescript-Sk+ (Stratagene) previously cleaved by EcoRI. pTG1669 is obtained. This is mutated (kit Amersham example) by inserting a BamHI site either at position 27867 (mutagen oligonucleotide OTG6079; SEQ ID NO: 12) or at position 28249 (mutagen oligonucleotide OTG5080; SEQ ID: 13). The resulting Eco-BHI polymer fragment containing the two vectors pTG1676 is obtained by inserting a BTR-V vector in the anterior vector of the BamHI site.

An adenovirus particle is generated by homologous recombination in a complement line for function E1, between the AatII fragment of pTG1679-E3+ and an adenoviral vector such as Ad dl324 or Ad-RSVβ-gal, which contains the β-galactosidase gene in place of the E1 region (Stratford-Perricaudet et al., 1992, J. Clin. Invest., 90, 626-630).

EXAMPLE 3 Construction of recombinant adenoviral vector with improved cloning capacity by partial deletion of E1 and E3 regions The structure of pTG6590△E3

The fragment containing the part of the Ad5 genome between nucleotides 27325 and 27871 is amplified by PCR from a genomic DNA preparation of Ad5 and with the aid of the primers OTG6064 and OTG6065 (SEQ ID NO: 14 and 15).

The amplified fragment is cloned into the SmaI site of M13mp18 to yield M13TG6523. The EcoRI-BsmI fragment is isolated from the latter to be introduced into the pTG6590 vector cleaved by the same enzymes. pTG6590△3, which contains the 3' part of the adenoviral genome (nucleotides 27082 to 35935) is obtained by deleting the E3 region between nucleotides 27872 to 30740, while pTG6590 is then cleaved from a smaller part of the E3 region (position 28592 to 30470). The pTG6590 vector is obtained by generating a fragment corresponding to the 35228 nucleotides in the pTG653 (nucleotide vector 2782 to 35935), which is then used to prepare the drug for digestion from the X-ray region, and then by the METTG1313 (TG651 to METTG1384) to generate another fragment, which is then used to digest the pTG651 (nucleotide vector 3522 to 35935), which is then generated by the PTG651 (TG651 to METG1388) vector.

As an indication, the pTG6584 vector is a pUC19 (Gibco BRL) vector containing the Ad5 sequences extending from the single site SpeI (position 27082) to the beginning of the promoter region of the E4 region (position 35826).

2. Construction of an adenoviral vector deleted from the E1 region and the non-expressing part of E3 gp19kDa

The part of the E3 region of Ad5 encoding for gp 19kDa (nucleotides 28731 to 29217) is obtained by PCR from a preparation of genomic DNA of Ad5 and by implementing the OTG5455 and OTG5456 (SEQ ID NO: 18 and 19) primers. The generated fragment is introduced into the SmaI site of M13mp18 to give M13TG6520. The EcoRI-XbaI fragment of the latter is isolated, cloned into the AatII site of pTG1670 (example 2.3), the sites being made free by treatment with Klenow polymase DNA.

3. Obtaining adenoviral particles.

Recombinant viral particles are obtained by ligation of isolated SpeI fragments of the genomic DNA of AdTG6303 or AdTG6581 and either of the vectors in examples 3.1 and 3.2 and then the ligation mixture is transfected into a complement line for function E1.

EXAMPLE 4 Construction of an adenovirus deleted from regions E1 and E4.

The parts of the adenoviral genome extending from nucleotides 31803 to 32799 and 35827 to 35935 are amplified from a genomic DNA preparation of Ad5 and the precursors OTG5728 and OTG5729 (SEQ ID NO: 20 and 21) and OTG5730 and OTG5481 (SEQ ID NO: 22 and 16) respectively. After about ten cycles of amplification, the reaction is continued from an aliquot of the two reaction mixtures by implementing the oligonucleotides OTG5728 and OTG5781. The amplified fragment extends from nucleotides 31803 to 35935 with deletion of the entire E4 region (Eco positions 32800 to 3582621). After digestion and cleavage, it is removed from the same sites to give M131365TRI and M131318TRI.

M13TG6521 is digested by EcoRI, processed by DNA polymerase klenow and then cleaved by BstXI. The 0.46 kb fragment containing ITR 3' is inserted between the BamHI site made free by DNA polymerase klenow treatment and the BstXI site of pTG6584 (example 3.1).

A synthetic DNA fragment from the reassociation of the oligonucleotides OTG6060, OTG6061, OTG6062 and OTG6063 (SEQ ID NO: 23 to 26) is introduced into the PacI site of pTG6588 resulting in pTG8500 in which the signals of termination of transcription of the late L5 genes are enhanced.

An adenoviral particle (Ad△E4) with a genome deletion from the entire E4 region (nucleotides 32800 to 35826) and the XbaI fragment from the E3 region (nucleotides 28592 to 30470), is generated by binding the isolated SpeI fragments of pTG8500 or pTG6588 and Ad5. The binding mixture is transfected into a complement cell line for the E4 function, e.g. the W162 line (Weinberg and Ketner, 1983, Proc. Natl. Sci. Acad. USA, 80, 5383-5386). An adovirus defect for the E1 and E4 functions (E1, E1, E4) is obtained by transfection into a complement line for the E1 and E4 function (e.g. the E4 line of the E4G658 or the DL8 line of the E4G658 gene).

On the other hand, the following procedure can also be followed: the isolated SpeI-ScaI fragment of pTG1659 (example 2.3) is cloned into the pTG6588 vector cleaved by these same enzymes, to obtain pTG6591, which contains the Ad5 sequences of the nucleotides 21062 to 35935 but, as before, deleted from the entire E4 region and the XbaI fragment of the E3 region. The synthetic DNA fragment described above is introduced into the PacI-digested pTG6591 vector and pTG6597 is generated.

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The assembly of these different elements is carried out using standard molecular biology techniques, and the production of infectious virions containing such a vector is carried out as described above in a complement line in Example 7.

EXAMPLE 6 - Construction of a complement cell capable of trans-complementing the E1 function 1. Construction of a complement cell comprising the E1 region of nucleotides 100 to 5297 (pTG6533)

It consists of: a cassette of expression of the pac gene, which is under the control of the early SV40 promoter (nucleotides 5171 to 5243) and contains in 3' the signal for termination of the SV40 transcription (nucleotides 2543 to 2618). The pac gene used corresponds to a fragment from nucleotide 252 to nucleotide 905 of the sequence disclosed by Lacalle et al. (1989, Gene, 79, 375-380) and contains 4 mutations compared to the published sequence (C in position 305 replaced by A; C in position 367 replaced by T; insertion of a G in position 804 deletion of a G in position 820), a protein from the ADOME containing 100 nucleotide fragments from the E.291 and E.291 regions, and appears to produce a signal for their termination of the transcription of E.291 and E.292 regions, as well as a fragment of the E.291 and E.293 regions, which does not appear to be a signal for the recombination of the E.291 and E.292 regions.

The construction is carried out in several steps, detailed below. The p polyIII-I* vector (Lathe et al., 1987, Gene, 57, 193-201) is subjected to digestion by the AccI and EcoRI enzymes. The pTG6164 isolated EcoRI-ClaI fragment is cloned into the treated vector.

The pTG6164 plasmid is derived from pLXSN (Miller D, 1989, Bio/Techniques, 7, 980) and includes the pac gene which is under the control of the early promoter of SV40 virus. Briefly, the HindIII-KpnI fragment of pLXSN is introduced into M13TG131 to produce M13TG4194. The NheI-KpnI fragment of pMPSV H2 K IL2R (Takeda et al., 1988, Growth Factors, 1, 59-66) is introduced into M13TG4196. This is digested by HindIII-KpnI and the fragment from a digested and partially purified HindIII-KpnI fragment is cloned and obtained from PNXSN. This is obtained from PNXSN.

The pTG6528 vector is digested by PstI and the PstI fragment isolated from pTG6185 (example 2.1) containing the SV40 termination signal is introduced at this site, resulting in pTG6529. The pTG6529 is digested by EcoRI-HpaI and bound to two fragments, a BspEI-BcgI fragment (positions 826 to 5297) purified from the genomic DNA of Ad5 and a PCR-generated fragment at the ends of EcoRI and BspEI, to give pTG6531. The PCR fragment is generated by genetic amplification from the genomic DNA of the genes NO564 and OTG45 and the AMORGEN (as shown in IDQ 27 and 28 in the previous section) and is amplified by digesting and binding the BSI.

The pTG6531 vector includes the 2 transcription units (the E1 region and the pac gene) in the same orientation. To avoid interference at the transcription level, they are placed in a spike orientation (reverse one relative to the other) by processing pTG5531 by BamHI and linking.

The pTG6533 vector is transfected into a mammalian cell line, e.g. the Vero (ATCC, CCL81) or A549 (ATCC, CCL185) line by the calcium phosphate technique. The transfected cells are cultured as recommended by the supplier and are placed 24 hours after transfection in a selective medium containing puromycin (6 μg/ml concentration). Resistant clones are selected on which the expression of E1 region genes are evaluated to determine the most productive clone, which can be used as a complementary line for the preparation of an adenovirus defective for E1, such as the one detailed in Example 2.

The expression of the sequences coding for early E1 region proteins is analysed by Northern blot using appropriate probes labeled with the isotope 32P. The production of E1A region coded proteins is detected by immunoprecipitation after cell labelling with the isotope 35S and using a commercial antibody (Oncogene Science Inc., DP11 reference).

Err1:Expecting ',' delimiter: line 1 column 335 (char 334)

Finally, these cells can be infected with Ad-RSV-βgal (Stratford-Perricaudet et al., 1992, supra) and titrated by agar as soon as a cytopath effect is observed. Generally, the following procedure is followed: cells are infected with a 10 m (multiple infection) level. Approximately 48 hours after infection, with the cytopath effect visible, the cells are lysed and β-galactosidase activity is dosed according to conventional protocol (see, for example, Maniatis et al., 1989). Positive clones are reinfected at a weaker level. 48 hours after infection, the surnant and viral cells are collected according to the classical factors. The agar is determined by the titration method using the 293.

2. Construction of a complement line comprising the E1 region of nucleotides 505 to 4034 (pTG6557, pTG6558, pTG6559, pTG6564 and pTG6565)

The vectors pTG6557, pTG6558 and pTG6559 include: (i) a cassette of expression of the pac gene (nucleotides 252 to 905 as before) under control: The E2A promoter of Ad2 (nucleotides 27341 to 27030) (in pTG6558);the E2A promoter of Ad2 deleted sequences from nucleotides 27163 to 27182 (for pTG6557).Such a mutation allows the baseline level of the E2A promoter to be decreased, without affecting inducibility by the trans-activator protein encoded by E1A, or early SV40 promoter for pTG6559. In all three cases, it also contains the SV40 virus transcription termination signal in 3' (nucleotides 2543 to 2618); and (ii) an expression cassette containing the part of the E1 region of Ad5 from nucleotides 505 to 4034.This portion of the adenoviral genome contains all sequences coding for the early proteins of the E1A region, the termination signal of the transcription of the E1A unit, the E1B promoter (induced by the trans-activator protein encoded by E1A) and all sequences coding for the E1B region. It also includes the sequences coding for the IX protein, which overlap with the E1B region. However, it lacks the E1A region promoter and the termination signal of the transcription of the E1B and IX transcriptional units. In order to allow the expression of the E1 region sequences, the 5' adenylid fragment is introduced,the mouse PGK promoter and in 3' the termination signal of the transcription of the rabbit ß-globin gene (nucleotides 1542 to 2064 of the sequence disclosed in the Genebank under reference K03256).

Optionally, any nucleotide sequences, e.g. isolated from pBR322 (Bolivar et al., 1977, Gene, 2, 95-113), may also be inserted between the expression cassettes of the pac gene and the E1 region to avoid possible transcriptional interference.

The construction of these vectors is carried out in several stages, which are outlined below.

First, the part of the Ad5 genome from nucleotide 505 to nucleotide 826 is amplified by PCR from a genomic preparation and OTG5013 precursors which includes a 5' site PstI useful for the subsequent cloning steps (SEQ ID NO:29) and OTG4565 overlapping the BspE1 site (SEQ ID NO:28) and the PCR-generated fragment is processed at the Klenow DNA polymerase and then introduced into the SmaI site of M13mp18 resulting in M13TG6512.

The vector pTG6533 (example 6.1) is digested by the EcoRI and BspE1 enzymes. The vector is bound to the PstI-BspE1 fragment isolated from M13TG6512 and the EcoRI-PstI fragment isolated from pKJ-1. The latter comprises the portion of the mouse PGK promoter between nucleotides -524 and -19, the sequence of which is reported in Adra et al. (1987, Gene, 60, 65-74). This step results in pTG6552 and allows the mouse PGK promoter to be inserted upstream from the E1 region of the Ad5 starting at nucleotide 505.

In addition, the XhoI-BamHI fragment, the end of which is cleared by Klenow DNA polymerase treatment, is purified from pBCMG Neo (Karasuyama et al., 1989, J. Exp. Med., 169, 13-25). This fragment, which includes the transcription termination signal of the rabbit ß-globin gene, is introduced between the SmaI and BamHI sites of the poly pII-Sfi/Not-14* vector (Lathe et al., 1987, Gene, 57, 193-201). The resulting pTG6551 vector is then digested by the SphI and EcoRV enzymes to insert a fragment of the genome of NO5 from the 3665Q of the nucleotide matrix 403.

The PCR fragment is processed by Klenow DNA polymerase before being cloned into the SmaI site of M13mp18, generating M13TG6516. After sequence verification, the PCR fragment is extracted by digestion by BglII, processing with Klenow DNA polymerase and digestion by SphI. It is inserted between the SphI and EcoRV sites of pTG6551 resulting in pTG6554.

On the other hand, the pTG6529 vector (example 6.1) is digested by the HpaI and HindIII enzymes. The 2.9 kb fragment containing the pac gene followed by the SV40 virus transcription termination signal is purified and bound to the SmaI-HindIII fragment isolated from pE2 Lac (Boeuf et al., 1990, Oncogene, 5, 691-699), which carries the E2A promoter of Ad2.

pTG6556 is digested by the EcoRI and BamHI enzymes. The pTG6552 isolated EcoRI-SacII fragment and pTG6554 isolated SacII-BamHI fragment are inserted between these sites to obtain the pTG6558 vector. The same step performed on pTG6550 and pTG1643 (example 7.1) generates pTG6557 and pTG6559 respectively.

pTG6557 and pTG6558 are digested by EcoRV, a single site located between the two expression cassettes (pac gene and region E1). In this site, a 1.88kb EcoRV-PvuII fragment isolated from pBR322 (Bolivar et al., supra) is cloned to remove the two promoters.

The pTG6557, pTG6558, pTG6559, pTG6564, and pTG6565 vectors are transfected into the A549 cell line. As before, puromycin-resistant clones are selected and E1 region expression is checked. E1-expressing clones are intended to amplify and spread adenoviruses that are defective for E1 function. The production of E1 expression products is accompanied by cytotoxic effect but the Southern analysis does not reveal rearrangements of vectors. After infection with Ad-RSV-β, several clones are capable of amplifying the virus by a factor greater than 100.

3. Construction of a complement cell inducible by the Gal4 protein of Saccharomyces cerevisiae.

These vectors include the E1 part of the Ad5 region from nucleotide 505 to 4034, but the expression of the E1A region sequences is controlled by an inducible promoter consisting of a portion of the Ad2 minimal MLP promoter (TATA box and transcription initiation signal; nucleotides -34 to +33) and an activation sequence of the Gal 10 gene that is activated by the Gal4 protein. The consensus activation sequence of 17 nucleotides (17MX), which corresponds to the Gal4 binding site, is specified in Webster et al. (1988, 52, 169).

A first DNA fragment containing a dimer of the 17MX sequence (SEQ ID NO: 32 and 33) followed by the minimal MLP promoter of Ad2 and containing a SalI site at its 5' end and a BamHI site at its 3' end is synthesized. The SalI site is made free by treatment with Klenow DNA polymerase.

Each of these fragments is introduced into the BglII site of p poly II to generate pTG1656 and pTG1657 respectively.Then the following two fragments are introduced into each of the previously digested PstI-BamHI vectors: the PstI-XbaI fragment isolated from pTG6552 (Example 6.2) and the XbaI-BamHI fragment isolated from pTG6559 (Example 6.2).

A549 cells are co-transfected with pTG1643 (the expression vector for the pac gene) and either pTG1660 or pTG1661. The clones are selected for their puromycin resistance and studied as previously reported. About 50% of A549-1660 and A549-1661 clones produce E1 region expression products. However, production is accompanied by a cytotoxic effect, changing the morphological appearance of the cells.

The integration and non-rearrangement of plasmids in the cell genome is verified by Southern. No substantial modification of the integrated plasmids (pTG1643, pTG1660, and pTG1661) can be detected in the analyzed product clones. The inducibility of expression of the E1A region-encoded sequences in the presence of Gal4 (by transformation by a plasmid allowing the constitutive expression of the Gal4 protein) can also be verified.

After infection of several producing clones with Ad-RSV-Bgal at a mean of about 2, two A549-1660 clones are able to amplify the viral stock by a factor greater than 100.

EXAMPLE 7 - Creation of a complement line for all functions essential to the replication of an adenovirus.

A vector comprising the entire adenoviral genome of Ad5 is constructed with the exception of ITR 5', ITR 3' and the encapsulation region.

The vector pTG6528 (example 6.1) is digested by the enzymes PstI and BglII between which a DNA fragment chemically synthesized according to standard protocol consisting of oligonucleotides of OTG5039 and OTG5040 (SEQ ID NO: 34 and 35) is inserted. The oligonucleotide sequence is designed so that the PstI cloning site is not reconstituted and an EcoRV site is introduced. pTG1639 is obtained, which is linearized by digestion by EcoRV and bound to an XbaI-BamHI fragment whose ends are crossed by treatment with Klenol DNA polymerases. This fragment is suitable for any SV40.ow virus carrier with a signal from the transcription sites of the plasmid used.

The pTG1640 vector thus generated is digested by BamHI and BglII and the carrier fragment of the pac gene expression cassette is introduced into the BglII site of the pPolyII-Sfi/Not-14 vector.* This results in pTG1641. This is linearized by NotI and processed at the Klenow DNA polymerase.

The pTG1643 is linearized by XhoI and a hybrid XhoI fragment containing a 17MX dimer is inserted into this site followed by the minimum promoter of the TK-HSV-1 gene (nucleotides 303 to 450 of the sequence disclosed in the Genebank database under reference V00467 and completed in 3' of an XhoI site).

This construction, pTG1647, serves as the base vector for introducing between the PstI and BamHI sites a fragment of the Ad5 genome ranging from nucleotide 505 to nucleotide 35826. First, pTG1647 is digested by PstI and BamHI and then bound to the PstI-ClaI fragment of pTG6552 (example 6.2) containing the Ad5 part of the nucleotides 505 to 918 and to the ClaI-BamHI fragment (positions 918 to 21562) prepared from the Ad5 DNA.

In addition, the 3' part of the Ad5 genome is assembled in the ppolyII-Sfi/Not-14* vector. This is linearized by BamHI and the BamHI-AvrII fragment (nucleotides 21562 to 28752) of the Ad5 genome is introduced and a PCR fragment corresponding to nucleotides 35463 to 35826 of the Ad5 is generated from the genomic DNA of Ad5 and the OTG5024 (SEQ ID NO: 36) and OTG5025 (SEQ ID NO: 37) precursors and has a BamHI site in 5'.

The BamHI fragment containing the adenoviral sequences is introduced into the BamHI site of the previous stage vector containing the 5' portion of the adenoviral genome lacking the 5' ITR and the encapsulation region.

A complement line capable of complementing all the functions of a defective adenovirus is generated by transfection into a cell line, such as A549, according to the protocol described in the previous examples.

This can also be done by constructing four vectors containing almost the entire adenoviral genome which will be reassembled into a single vector in the final step. pTG1665 is the cloning of the BspE1 fragment (nucleotides 826 to 7269) isolated from a genomic DNA preparation of Ad5 at the XmaI site of p poly II-Sfi/Not-14 *; pTG1664 is generated by inserting the NotI fragment (nucleotides 6503 to 1504) isolated from a genomic DNA preparation of Ad5 at the NotI site of the same vector. pTG1662 is obtained by inserting the AatII fragment (nucleotides 10754 to 23970) is of an Ad5 genomic DNA preparation into the AatII site of p poly II. pTG1659 contains part 3 of the Ad5 genome (Example 2.3).

Then a fragment containing an inducible expression system such as the Gal4 inducible promoter described in example 6.3 or 7 or a previously known promoter such as the metallothioneine or tetracycline promoter is introduced upstream of the 5' sequences of Ad5 (nucleotides 505 to 918) into the pTG1665 vector digested by AatII and ClaI. Finally, the NotI fragments of pTG1664, AatII of pTG1662 and BamHI of pTG1659 are cloned successively into the previous vector and the corresponding sites.

A complement line is generated by co-transfection of the previous vector and pTG1643 and puromycin-resistant clones are isolated. This line is specifically intended to amplify and encapsulate the example 5 adenoviral vectors defective for E1, E2, and E4 functions and late functions.

EXAMPLE 8 - Construction of a line of complementation for the functions E1 and E4.

The vector pTG1647 (example 7) is digested by the PstI-BamHI enzymes and 3 fragments are introduced into the vector: The PstI-XbaI fragment of pTG6552 (example 6.2) containing the Ad5 sequences from nucleotide 505 to nucleotide 1339, the XbaI-SphI fragment of pTG6552 containing the Ad5 sequences from nucleotide 1340 to nucleotide 3665, and the SphI-BamHI fragment of pTG6554 (example 6.2) containing the Ad5 sequences from nucleotide 3665 to 4034 and a termination signal for transcription.

The resulting vector is cut by BamHI and three fragments are introduced into the site: a fragment digested by BamHI-AflII generated by PCR corresponding to the Ad5 sequence located between positions 32800 to 33104.Ad5 genomic DNA is used as matrix and the primers OTG5078 (SEQ ID NO: 38) and OTG5079 (SEQ ID NO: 39),the fragment AflII-AvrII isolated from the genomic DNA of Ad5 (nucleotides 33105 to 35463),the fragment AvrII-BamHI generated by PCR using the primers OTG5024 and OTG5025 (see example 7).

The resulting vector is introduced into a cell line according to the protocol described above to form a complement line for functions E1 and E4.

In addition, such a lineage may also be obtained by the following protocol:

The E4 region of the Ad5 genome (nucleotides 32800 to 35826) is reconstituted in several steps. The portion from nucleotides 33116 to 32800 is synthesized by PCR from the genomic DNA of Ad5 with the pair of amores OTG5078 and OTG5079 (SEQ ID NO: 38 and 39), then inserted into the EcoRV site of M13TG130, to generate M13TG1645.

The BamHI-AflII fragment of the latter is engaged in a binding reaction with the AflII-AvrII fragment of Ad5 (nucleotides 33104 to 35463) and the pTG7457 vector digested by BamHI and AvrII.

The E4 region is then completed by extracting the fragment corresponding to nucleotides 35826 to 35457 by PCR from a genomic DNA preparation of Ad5 and the OTG5024 and OTG5025 precursors (SEQ ID NO: 36 and 37). This is inserted into the SmaI site of M13mp18 to give M13TG1646 The AvrII-EcoRI fragment is isolated from the latter and cloned between the AvrII and EcoRI sites of pTG1650 to obtain pTG1652.

The BamHI fragment containing the E4 region of Ad5 is isolated from pTG1652 and cloned in the BamHI site of pTG1643, pTG6559 (example 6.2) or in the SspI site of pTG6564 (example 6.2) after rendering the free sites, to generate pTG1653, pTG1654 and pTG1655 (Figure 6) respectively.

A complement cell is generated by conventional techniques capable of trans-complementing the E1 and E4 functions by: (1) transformation of pTG1653 in cell line 293, or (2) transformation of pTG1654 or pTG1655 in cell line A549.

Generally, the expression of products from E1 and E4 regions is accompanied by a cytotoxic effect. A number of clones 293-1653 are capable of complementing both E1-deleted adenoviruses and E4-deleted adenoviruses.

Another alternative is to do the following.

The mutated vector is designated M13TG6522. It is digested by PstI, processed at the DNA polymerase of the T4 phage and then by AvrII and bound to a purified pTG1652 EcoRI (Klenow) -AvrII fragment (example 8) to give pTG6595. The latter is cleaved by HpaI and the 0.8 kb fragment obtained from pTG5913 (indicative Figure 7) is introduced into the PstI and processed by the Klenow gene in the PstG4 phage and then by AvrII and bound to a purified pTG1652 EcoRI (Klenow) -AvrII fragment (example 8) to give pTG6595.

Simultaneously, the vectors pTG1643 and pTG6559 (example 6) are linearized by BamHI and a synthetic fragment from the reassociation of the oligonucleotides OTG6141 and OTG6142 (SEQ ID NO: 41 and 42) is inserted to obtain pTG8508 and pTG8507 respectively.

On the other hand, the introduction of the BamHI fragment of pTG1652 into the pTG8508 or pTG8507 vector linearized by the same enzyme results in pTG8514 and pTG8515 respectively (Figures 10 and 11).

Cell lines transfected by pTG8512 or pTG8515 will complement an adenovirus defective for function E4, while those resulting from pTG8513 or pTG8514 transfection are intended to amplify and propagate adenoviruses defective for function E1 and E4. Similarly, transfection of pTG8512 or pTG8515 into cells 293 will complement adenoviruses defective for function E1 and E4.

Claims (31)

1. A composition of adenovirus particles containing a defective adenoviral vector for replication derived from the genome of an adenovirus by deletion of at least all or part of the E1A region which can be obtained by a process that: - What?

   (i) the adenoviral vector is introduced into a complement cell line characterized by the presence of a complement element, including in particular a part of the E1 region of the genome of an adenovirus excluding the ITR 5'; the complement element is capable of trans-complementing a defective adenoviral vector and is either integrated into the genome of the complement cell line or inserted into an expression vector;

   (ii) the said transfected complement cell line is cultured under appropriate conditions to allow the production of the said adenovirus particle; and

   (iii) a composition containing the above adenovirus particles is obtained from the cell culture.
2. The composition according to claim 1, characterised by the fact that the replication defective adenoviral vector is a vector derived from the genome of an adenovirus comprising 5' by 3' an ITR 5', an encapsulation region, an E1A region, an E1B region, an E2 region, an E3 region, an E4 region and an ITR 3', by deletion in the E1A region and in at least one region of that genome selected from the E1B, E2, E3, E4 and encapsulation regions.
3. The composition according to claim 1 or 2, characterised by the fact that the replication defective adenoviral vector is a vector derived from the genome of an adenovirus comprising 5' by 3', a 5' ITR, an encapsulation region, an E1A region, an E1B region, an E2 region, an E3 region, an E4 region and a 3' ITR, by deletion - What?

   (a) in region E1A; or

   (b) in Region E1A and in Region E1B; or

   (c) in Region E1A, in Region E1B and in Region E2; or

   (d) in Region E1A and in Region E2; or

   (e) in Region E1A, in Region E3 and in Region E1B; or

   (f) in Region E1A and in Region E3; or

   (g) in Region E1A, in Region E1B, in Region E2 and in Region E3; or

   (h) in Region E1A, in Region E2 and in Region E3; or

   (i) in Region E1A, in Region E1B and in Region E4; or

   (j) in Region E1A and in Region E4; or

   (k) in Region E1A, in Region E1B, in Region E2 and in Region E4; or

   (i) in Region E1A, in Region E2 and in Region E4; or

   (m) in Region E1A, in Region E1B, in Region E3 and in Region E4; or

   (n) in Region E1A, Region E3 and Region E4; or

   (o) in Region E1A, in Region E1B, in Region E2, in Region E3 and in Region E4; or

   (p) in region E1A, in region E2, in region E3 and in region E4.
4. The composition according to claim 2 or 3, wherein the said deletions in the regions of the genome of an adenovirus are deletions that prevent the production of at least one expression product encoded by those regions.
5. The composition according to claim 1 in which the adenoviral vector is derived from the genome of an adenovirus by deletion as defined in claim 3 or 4 and by deletion of part of the encapsulation region.
6. The composition according to one of claims 2 to 5 in which deletion of the E1B region of the adenoviral genome is a partial deletion by maintaining the part of the E1B region coding for the pIX protein.
7. The composition according to one of claims 2 to 6, in which deletion of the E3 region of the adenoviral genome is a partial deletion by maintaining the part of the E3 region coding for the gp19kDa protein.
8. The composition according to claim 7, in which the part of the E3 region encoding gp19kDa protein is controlled by elements appropriate for the expression of that protein in the host cell.
9. The composition according to one of the claims 2 to 6, in which the E3 region of the adenovirus genome is completely deleted.
10. The composition according to one of the claims 1 to 9, characterised by the complement cell line used in the preparation process including: - What?

    (i) all or part of the E1A region of an adenovirus genome; and

    (ii) all or part of at least one region of the genome selected from regions E1B, E2 and E4.
11. The composition according to one of the claims 1 to 9, characterised by the complement cell line used in the preparation process including: - What?

    (i) all or part of the E1A region of an adenovirus genome; and

    (ii) all or part of at least two of the E1B, E2 and E4 regions of the genome.
12. The composition according to one of the claims 1 to 9, characterised by the complement cell line used in the preparation process including: - What?

    (i) all or part of the E1A region of an adenovirus genome; and

    (ii) all or part of the regions E1B, E2 and E4 of the genome.
13. The composition according to one of claims 10 to 12, characterised by the complement cell line used in the preparation process including all or part of the E1A region and all of the E1B region of the genome of an adenovirus coding for early proteins.
14. The composition according to one of the claims 1 to 13, characterised by the fact that the complement cell line used in the preparation process includes in particular part of the genome of an adenovirus selected from canine, avian and human adenoviruses.
15. The composition according to claim 14, characterised by the fact that the complement cell line used in the preparation process includes in particular part of the genome of a human adenovirus type 5.
16. The composition according to claim 15, characterised by the fact that the complement cell line used in the preparation process includes in particular the part of the genome of a human adenovirus type 5 - What?

    (i) extending from nucleotide 100 to nucleotide 5297;

    (ii) extending from nucleotide 100 to nucleotide 4034; or

    (iii) extending from nucleotide 505 to nucleotide 4034.
17. The composition according to claim 15 or 16, characterised by the complement cell line used in the preparation process including the part of the E4 region of the genome of a human adenovirus type 5 extending from nucleotide 32800 to nucleotide 35826.
18. The composition according to claim 15, characterised by the fact that the complement cell line used in the preparation process includes, inter alia, the part of the genome of a human adenovirus type 5 extending from nucleotide 505 to nucleotide 35826.
19. The composition according to one of claims 1 to 18, characterised by the fact that the complement cell line used in the preparation process includes a part of the E1A region of the genome of an adenovirus which is devoid of its natural promoter and is under the control of an appropriate promoter.
20. The composition according to claim 19, characterised by the fact that in the complement cell line used in the preparation process, the part of the E1A region is controlled by an inducible promoter by a non-adenoviral transcriptional trans-activating protein.
21. The composition according to claim 20, characterised by the fact that in the complement cell line used in the preparation process, the non-adenoviral transcription activating trans-protein is encoded by a recombinant adenoviral vector, which is controlled by elements necessary for its expression in a host cell and contains a gene that may encode in particular for a transcription activating trans-protein Gal4 of Saccharomyces cerevisiae.
22. The composition according to claim 20 or 21, characterised by the fact that in the complement cell line used in the preparation process, the said part of the E1A region is controlled by a promoter inducible by a trans-activating Gal4 transcription protein of Saccharomyces cerevisiae.
23. The composition according to one of claims 1 to 22, characterised by the fact that the complement cell line used in the preparation process also includes a gene coding for a selection marker.
24. The composition according to claim 23 characterised by the presence in the cell line of the complement used in the preparation process of the selection gene code for puromycin acetyl transferase.
25. The composition according to claim 23 or 24, characterised by the selection gene in the complement cell line used in the preparation process being controlled by an inducible promoter by a transcriptional trans-activating protein encoded by the E1A region of the genome of a wild adenovirus, including the E2 region promoter of that genome.
26. The composition according to one of claims 1 to 25, characterised by the fact that the complement cell line used in the preparation process is derived from a pharmaceutically acceptable cell line.
27. The composition according to claim 26, characterised by the fact that the complement cell line used in the preparation process is derived from a cell line selected from the Vero, BHK, A549, MRC5, W138 and CHO lines.
28. The composition according to one of claims 1 to 25, characterised by the fact that the complement cell line used in the preparation process is derived from a retinal cell of a human embryo.
29. The composition according to one of claims 1 to 28, characterised by the fact that the complement cell line used in the preparation process contains a complement element capable of trans-complementing the E1 function of a defective adenoviral vector, such complement element being in particular devoid of ITR 5', the encapsulation region, the promoter of the E1A region and the termination signal of the transcription of the transcriptional units E1B and pIX.
30. The composition according to claim 29, characterised by the fact that in the complement cell line used in the preparation process, the said complement element of function E1 is controlled by a heterologous promoter, in particular the promoter of the mouse PGK gene.
31. The composition according to claim 29 or 30, characterised by the fact that in the complement cell line used in the preparation process, the said complement element of the E1 function is controlled by a signal terminating the heterologous transcription, in particular that of the rabbit beta globulin gene.