The present invention relates to new viral vectors permitting the transfer and expression of genes of interest in a host cell or body, the expression of the viral genes being regulated so as to be functional in a complementation cell and nonfunctional in the host cell or body. It also relates to the cells containing these now vectors, as well as to a method for preparing infectious viral particles intended for therapeutic use. The invention in of very special interest in relation to prospects for gene therapy, in particular in man.
The possibility of treating human diseases by gene therapy has changed in a few years from the stage of theoretical considerations to that of clinical applications. The first protocol applied to man was initiated in the US in September 1990 on a patient who was genetically immunodeficient as a result of a mutation affecting the gene coding for adenine deaminase (ADA). The relative success of this first experiment encouraged the development of now gene therapy protocols for various genetic or acquired diseases (infectious diseases, and viral diseases in particular, such as AIDS, or cancers). The large majority of the protocols described hitherto employ viral vectors to transfer the therapeutic gene to the cells to be treated and to express it therein.
To date, retroviral vectors are among the ones most widely used on account of the simplicity of their gene. . or, &part from their restricted capacity for cloning, they present two major drawbacks which limit their systematic use: on the one hand they chiefly infect dividing cells, and on the other hand, an a result of their integration at random in the genome of the. host cell, the risk of insertional mutagenesis is not insignificant. For this reason, many scientific teams have endeavored to develop other types of vector, among which those originating from adenoviruses, adeno-associated viruses (AAV), cytomegaviruses, poxviruses and herpesviruses may be mentioned. Generally speaking, their organization and their infection cycle are amply described in the literature available to a person skilled in the art.
In this connection, the use of adenoviral vectors has8 been seen to be a prosing alternative. Adenoviruses have been demonstrated in many animal species, have a broad host range, have little pathogenicity and do not present the drawbacks associated with retroviruses since they are nonintegrative and replicate also in resting cells. As a guide, their genome consists of a linear, double-stranded DNA molecule of approximately 36 kb carrying more than about thirty genes, both early genes necessary for viral replication and late structural genes (see FIG. 1).
The early genes are divided into 4 regions dispersed in the adenoviral genome (B1 to E4; E standing for early). They contain 6 transcription units which possess their own promoters. The late genes (L1 to L5; L standing for late) partially overlap the early transcription units and are, for the most part, transcribed from the major late promoter (MLP).
At the present time, all the adenoviral vectors used in gone therapy protocols lack most of the E1 region essential for replication, in order to avoid their dissemination in the environment and the host body. Some of then contain additional deletions, in particular in the nonessential E3 region, enabling their cloning capacity to be increased. The genes of interest are introduced into the viral DNA in place of one or other deleted region. Deletion of the E1 region renders the viral genome deficient for replication. However, E131 viruses my be propagated in a complementation call line, which supplies in trans the deleted viral functions to generate an infectious viral particle. Line 293, established from human embryonic kidney cells, which complements the E1 function effectively (Graham et al., 1977, J. Gen. Virol. 36, 59-72), is commonly used. The E3 region is nonessential and does not need to be complemented.
While the feasibility of gone transfer using these first generation vectors is now well established, the question of their safety remains unresolved. Apart from the safety aspects (risk of generating RCAs, that is to say replication competent particles), the problem of their toxicity arises. in effect, the first clinical trials have revealed the induction of inflammatory responses associated with the expression of the viral genes in the host.
Second generation adenoviral vectors have recently been proposed in the literature. They retain the In cis regions necessary for replication of the virus in the infected cell (ITRs and encapsidation sequences) and contain substantial internal deletions aimed at abolishing the bulk of the viral genes whose expression in vivo is not desirable. However, these vectors of the prior art have some drawbacks which limit their exploitation at an industrial level. It in, in effect, necessary to have at one's disposal now lines complementing the collective deleted functions and enabling viral particles to be produced at a high titer. In point of fact, such a line, in order to be optimal in term of capacity for growth and yield of viral particles, in specially difficult to generate on account of the cytotoxicity of the adenoviral genes.
The present invention enables these drawbacks to be remedied. On the one hand, a new line derived from line 293 complementing the E1 and E2 or E4 adenoviral functions, for the amplification of conventional second generation adenoviral vectors, has now been constructed, in which line the expression of the E2 or E4 regions is directed by a promoter equipped at its 5′ and with so-called “operator” sequences of the bacterial tetracycline operon, these sequences hereinafter being designated tet O. The synthesis of the corresponding expression products will be activated only in the presence of an inducer which can be produced by the adenoviral vector or by the line itself. Similarly, a repressor may be added to the culture medium when complementation is no longer desired.
On the other hand, new adenoviral vectors from which the majority of the E1 and E3 regions have been deleted have now been generated, in which vectors the transcription units of the remaining viral regions (E2, E4 and/or L1-L5) are regulable with the object of permitting their expression when infectious viral particles are to be generated and of inhibiting it in the host cell. in the examples which follow, the regulation is effected by the tet O sequences. Their insertion On the 5′ side of the TATA box generates a promoter from which the baseline level of transcription in minimal but may be strongly stimulated in the presence of the inducer mentioned above. Thus, the production of viral proteins is activated in a 293 line expressing the inducer, which will enable infectious viral particles to be formed. In contrast, it in considerably reduced in the infected host cell which does not naturally produce the inducer of bacterial origin. The regulation of the viral genes has no effect on the expression of the exogenous nucleotide sequence placed under the control of a promoter that does not respond to tetracycline.
The adenoviral vectors of the present invention provide an advantageous approach to the drawbacks inherent. in the use of the vectors of the prior art, since they combine safety of use and ease of production. On the one hand they may be propagated in a conventional complementation line with a high titer compatible with industrial requirements, and on the other hand they enable an exogenous nucleotide sequence to be transferred in vivo, and to be expressed stably while limiting the adverse effects (inflammatory responses in the host). They are most especially suitable for human gene therapy.
Accordingly, the subject of the present invention in a viral vector, characterized in that it comprises an expression unit containing one or more viral genes; said expression unit being functional in a complementation cell and nonfunctional in a host cell.
For the purposes of the present invention, a “viral vector” is obtained from a parent virus whose genome has been modified. These modifications may be diverse (deletion, mutation and/or addition of one or more nucleotides) and localized in the coding regions of the viral genome or outside these regions, for example in the promoter regions. As a guide, some viral sequences may be deleted, rendered nonfunctional or replaced by other sequences, and In particular an exogenous nucleotide sequence whose expression is sought in a host cell.
A viral vector according to the invention may be derived from a wide variety of viruses, such as herpesviruses, cytomegaloviruses, AAV (adeno-associated virus) and poxviruses, and in particular vaccinia, fowlpox or canarypox virus. Such vectors, as well as the techniques for preparing them, are known to a person skilled in the art.
However, a vector which is especially suitable for the present invention is an adenoviral vector. It may be derived from an adenovirus of human, canine, avian, bovine, =urine, ovine, porcine or simian origin, or alternatively from a hybrid comprising adenoviral genome fragments of different origins. The adenoviruses CAV-1 or CAV-2 of canine origin, DAV of avian origin or alternatively Bad type 3 of bovine origin (Zakharchuk et al., 1993, Arch. Virol., 128, 171-176; Spibey and Cavanagh, 1989, J. Gen. Virol., 70, 165-172; Jouvenne et al., 1987, Gene, 60, 21-28; Mittal et al., 1995, J. Gen. Virol., 76, 93-102) may be mentioned more especially. However, an adenoviral vector derived from a human adenovirus, preferably of serotype C and, as an absolute preference, of type 2 or 5 (Graham and Prevect, 1991, Methods in Molecular Biology, vol. 7, p 109-128; Ed: E. J. Murey, The Human Press Inc.), will be preferred.
An advantageous embodiment of the present invention consists of a vector which is defective for replication, in which one or more viral genes necessary for replication are deleted or rendered nonfunctional. Such a vector, incapable of autonomous replication, will be propagated in a complementation cell. The term “complementation cell” denotes a cell capable of supplying in trans the function(s) for which a vector according to the invention is defective. In other words, it is capable of producing the proteins(s) necessary for the replication and encapsidation of said vector, early and/or late proteins, which it cannot itself produce and which are necessary for the formation of an infectious viral particle. By way of illustration, since a preferred adenoviral vector according to the invention lacks most of the E1 region, use will be made of a complementation cell such as line 293. capable of supplying in trans the collective proteins encoded by the E1 region which the vector cannot produce. “Infectious viral particle” is understood to mean a viral particle having the capacity to infect a host cell and to cause the viral genome to enter the latter.
According to a preferential embodiment, a viral vector according to the invention in recombinant. Thus, it will comprise an exogenous nucleotide sequence placed under the control of the elements necessary for its expression in a host cell. “Exogenous nucleotide sequence” refers to a nucleic acid which can be of any origin and which is not normally present in the genome of a parent virus employed in the present invention or, if it is present, in a different genomic context. In the context of the invention, the exogenous nucleotide sequence perhaps [sic] made up of one or more genes, and especially gene(s) of therapeutic interest.
Generally speaking, the exogenous nucleotide sequence can code for an antisense RNA and/or an mRNA which will then be translated into a protein of interest. It may be of the genomic type, of the complementary DNA (cDNA) type or of mixed type (minigene, in which at least one intron is deleted). It may code for a mature protein or a precursor of a mature protein, in particular a precursor intended to be secreted and comprising a signal peptide. Moreover, the encoded product may be all or part of a protein as found in nature (native or truncated protein), or alternatively a chimeric protein originating from the fusion of sequences of diverse origin or else a mutated protein displaying improved and/or modified biological properties. Such proteins may be obtained by the conventional techniques of molecular biology.
In the context of the present invention, it can be advantageous to use the genes coding for the following polypeptides:
cytokines or lymphokines (interferons α, β and γ, interlukins and in particular IL-2, IL-6, IL-10 or IL-12, tumor necrosis factors (TNF), colony stimulating factors (GM-CSF, C-CSF, M-CSF, etc.);
cell or nuclear receptors (receptors recognized by pathogenic organisms (viruses, bacteria or parasites), and preferably by the HIV virus (human immunodeficiency virus) [lacuna] or their ligands;
proteins involved in a genetic disorder (factor VII, factor VIII, factor IX, dystrophin or mini-dystrophin, insulin, CFTR (cystic fibrosis trans-membrane conductance regulator) protein, growth hormones (HGF) [lacuna];
anzymes (urease, renin, thrombin, etc..)
enzyme inhibitors (α1-antitrypsin, antithrombin III, inhibitors of viral proteases, etc.);
polypeptides having an antitumor effect capable of at least partially inhibiting the initiation or progression of tumors or cancers (antisense RNAs, antibodies, inhibitors acting on cell division or on transduction signals, expression products of tumor suppressing genes, for example p53 or Rb, proteins that stimulate the immune system, etc.);
proteins of the major histocompatibility complex classes I or II, or regulatory proteins that act on the expression of the corresponding genes;
polypeptides capable of inhibiting a viral, bacterial or parasitic infection and/or its development (antigenic polypeptides having immunogenic properties, antigenic epitopes, antibodies, trans-dominant variants capable of inhibiting the action of a native protein by competition, etc.);
toxins (herpes simplex virus 1 thymidine kinase (HSV-1 TK), ricin, cholera or diphtheria toxin, etc.) or immunotoxins; and
markers (β-galactosidase, luciferase, etc.).
It should be pointed out that this list is not limiting and that other genes may also be employed.
Moreover, an exogenous nucleotide sequence employed in the present invention may comprise, in addition, a selectable gone enabling the transfected cells to be selected or identified. There may be mentioned the neo gene (coding for neomycin phosphotransferase) conferring resistance to the antibiotic G418, the dhfr (dihydrofolate reductase) gene, the CAT (chloramphenicol acetyltransferase) gene, the pac (purosycin acetyltransferase) gene or alternatively the gpt (xanthine guanine phosphoribosyltransferase) gene. Generally speaking, the selectable genes are known to a person skilled in the art.
Elements necessary for the expression of an exogenous nucleotide sequence in a host cell are understood to mean the collective elements permitting its transcription into RNA (antisense RNA or mRNA) and the translation of an mRNA into protein. Among these, the promoter assumes special importance. It may be isolated from any gone of eucaryotic or even viral origin, and may be constitutive or regulable. Alternatively, it can be the natural promoter of the gene in question. It will be preferable to employ a promoter different from the one included in the unit for the expression of the viral genes (defined below). Moreover, it may be modified so as to improve the promoter activity, to abolish a region that inhibits transcription, to render a constitutive promoter regulable or vice versa, to introduce a restriction site, etc. There may be mentioned, by way of examples, the promoters of the HSV-1 TK, murine or human PGK (phosphoglycerate kinase), α1-antitrypsin (liver-specific) and immunoglobulin (lymphocyte-specific) genes, the SV40 virus (simian virus 40) early promoter, a retroviral LTR or alternatively the adenoviral MLP. promoter, in particular of hum adenovirus type 2.
Naturally, an exogenous nucleotide sequence employ d in the present invention can, in addition, comprise further elements necessary for expression (intron sequence, signal sequence, nuclear localization sequence, transcription termination sequence, translation initiation site of the IRES or an other type, etc.) or alternatively for its maintenance in the host cell. Such elements are known to a person skilled in the art.
As stated above, a viral vector according to the present invention comprises an expression unit containing one or more viral genes and having the advantageous feature of being functional in a complementation cell and nonfunctional in a host cell. “Functional” is understood to mean an expression of the viral genes in a sufficient amount and for a sufficiently long time to permit the formation of an infectious viral particle. “Nonfunctional” is understood to moan an expression of the viral genes which is reduced (preferably by a factor of at least 10, or even a zero expression) relative to their level of expression in the parent virus. The functional character manifests itself in the production of the products of the viral genes included in the expression unit, it being possible for these products to be demonstrated by the standard techniques of molecular biology, immunology, biochemistry or enzymology. The nonfunctional character manifests itself in the absence of production or alternatively in the production of the viral products at a reduced level.
Advantageously, said expression unit comprises one or more heterologous regulatory sequence(s) not present in the parent viral DNA. Use may be made of sequences that respond to a regulator of the repressor type acting negatively on the expression, or preferably to a regulator of the inducer type exerting a positive action. A regulatory sequence employed in the context of the present invention may be of any origin, viral, prokaryotic or alternatively eukaryotic. Generally speaking, regulatory sequences are described in the literature available to a person skilled in the art. It is also possible to employ a homolog whose sequence is modified relative to the native sequence but which exerts a similar or improved regulatory function. These modifications may result from the addition, deletion and/or replacement of one or more nucleotides.
In accordance with the objectives pursued by the present invention, a regulatory sequence is capable of modulating the expression of the viral genes at different levels: transcription, elongation, transport, stability of the mRNAs or alternatively translation. It may be present at various places in said expression unit, for example in the promoter, especially when its effect lies at transcriptional level (preferably upstream of the TATA box, up to a few hundred base pairs from the latter), or in the transcribed (and if possible noncoding) sequences when its action in exerted at a subsequent step of the transcription. It is possible to employ from 1 to 25 regulatory sequences, advantageously from 1 to 20, preferably from 1 to 10 and, as an absolute preference, from 1 to 7.
For the purposes of the present invention, the term “inducer” denotes a molecule which has the capacity of initiating or activating the expression of the viral genes placed under the control of a regulatory sequence, either directly by binding to said regulatory sequence, or indirectly via other cellular or viral factors. It can also prevent the action of a repressor. In contrast, a “repressor” has the capacity to inhibit or block the expression of the viral genes placed under the control of a regulatory sequence on which it acts, this taking place either directly or indirectly.
These definitions may be illustrated by the example of the lactose (lac) operon. The lac gone codes for a repressor which binds to a short regulatory sequence termed “operator”, thereby preventing the transcription of the structural genes coding for the enzymes of the metabolic pathway of lactose. In the presence of the inducer, the latter binds to the repressor and converts it to an inactive form which can no longer bind to the operator, thereby enabling transcription to take place.
It is also possible to nvisage using portions or analogs of these regulators in order to improve their efficacy or modify. their specificity (for example anhydrotetracycline, ten times as effective as tetracycline for inhibiting transcription from a promoter comprising the regulatory sequences derived from the tetracycline operon, or the reverse transactivator described recently by Gossen at al., 1995, Science, 268, 1766-1769). Moreover, a regulator employed in the present invention can be a hybrid protein originating from the fusion of polypeptides of different origins. A preferred combination consists of a polypeptide capable of recognizing or binding a regulatory sequence employed in the present invention (for example derived from the tetracycline repressor (tet R) or estrogen repressor ER) and a polypeptide capable of activating expression (for example the activation domain of the Ga14 or VP16 proteins, capable of interacting with transcription factors).
Table 1 below lists some of the regulatory sequences and regulators which can be used in the context of the present invention:
| ||Inducer (+)/ || || |
|Origin of the regulatory sequences ||Repressor (−) ||Insertion site ||Bibliographic reference |
|MRE (metal-responsive element) ||metal ions (+) ||5′ TATA ||Makarov et al., 1994, |
|metallothionein gene || || ||Nucleic Acids Res. 22, |
| || || ||1504-1505 |
|tryptophan operon ||tryptophan (−) ||5′ TATA ||Yanofsky et al., 1981, |
| || || ||Nucleic Acids Res. 9, |
| || || ||6647-6668 |
|lac operator ||product of the lacI gene (−) ||5′ TATA ||Miller and Reznikoff |
| || || ||(Eds), The operons (Cold |
| || || ||Spring Harbor Laboratory, |
| || || ||New York (lacuna) |
|tet operator ||VP16-TetR protein (+) ||5′ TATA ||Gossen and Bujard, 1992, |
| || || ||Proc. Natl. Acad. Sci. USA |
| || || ||89, 5547-5551 |
|tet operator ||TetR (−) ||3′ TATA ||Kim, 1995, J. Virol. 69, |
| || || ||2565-2573 |
|TAR (transactivation responsive ||TAT (+) ||transcribed ||Steffy and Wong-Staal, |
|region) || ||3′ sequence ||1991, Microbiological |
| || || ||Reviews 55, 193-205 |
|RRE (REV responsive element) ||REV (+) ||transcribed ||Steffy and Wong-Staal, |
| || ||3′ sequence ||1991, Microbiological |
| || || ||Reviews 55, 193-205 |
|GRE (glucocorticoid responsive ||glucocorticoid (+) ||5′ TATA ||Israel and Kaufman, 1989, |
|element) || || ||Nucleic Acids Res. 17, |
| || || ||4589-4604 |
|PRE (progesterone responsive ||progesterone (+) ||5′ TATA ||Gronemeyer et al., 1987, |
|element) || || ||EMBO J. 6, 3985-3994 |
|Ga14 UAS (Ga1 4 upstream ||Ga14 (+) ||5′ TATA ||Webster et al., 1988, Cell |
|activating sequence) || || ||52, 169-178 |
|ERE (estrogen responsive element) ||Estrogen (+) ||5′ TATA ||Klein-Hitpaβ et al., 1986, |
| || || ||Cell 46, 1053-1061 |
According to a particular embodiment of the present invention, regulatory sequences derived from the bacterial tetracycline operon, designated in the literature “operator” (tet O), are used. Generally speaking, the tetracycline resistance operon in encoded by the transposon TN10 (Hillen et al., 1984, J. Mol. Biol. 172, 185-201). Regulation is effcted by a short nucleotide sequence termed “operator” (tet O) which constitutes a binding site for various regulators. Thus, the binding of the tetacycline [sic] repressor (tet R) or of the antibiotic tetracycline considerably decrease the level of transcription. On the contrary, an activation effect is obtained by employing a protein, designated in the literature “tetracycline transactivator (tTA)”, which results from the fusion between tat R and the. 130 C-terminal a acids of the activation domain of the VP16 protein of the herpes simplex virus. Goosen and Boujard (1992, Proc. Natl. Acad. Sci. USA 89, 5547-5551) have recently shown that this regulatory system is functional in eukaryotic cells. The expression of a reporter gone placed under the control of several copies of tet O upstream of basic transcription sequences (TATA box, transcription startsite, etc.) in detectable by coexpression of tTA and inhibited by the addition of tetracycline. Rim's group (1995, J. Virol. 69, 2565-2573), for its part, utilizes the tet O sequences positioned downstream of the TATA box of a promoter, and in this case transcription is inhibited by the action of tetR.
In the context of the present invention, the combination “tet O-minimal promoter” (in the 5′ to 3′ direction), giving rise to a promoter whose baseline transcription level in naturally very low but activable by the inducer tTA and repressible by tetracycline, is most especially preferred. However, it in also possible to use a promoter in which the tet O sequences are placed on the 3′ side of the TATA box, or alternatively on each side of the latter, and which is repressible by a repressor comprising the sequences of tet R that recognize tet O.
As regards the preferred variant, an adenaoviral vector according to the invention preferably consists of the genome of an adenovirus lacking all or part of the E1 region and, alternatively, all or part of the E3 region. Advantageously, it in preferable to retain a portion of the E3 region, and in particular the portion corresponding to the gene coding for the gp19k (Gooding and Wold, 1990, Critical Reviews of Immunology 10, 53-71), said portion not being included in an expression unit as defined above but placed under the control of a conventional homologous (E3) or heterologous promoter. It is self-evident that it is possible to carry out other modifications of the viral genome, in particular in the E4 or E2 region. It may be advantageous to introduce mutations or additional deletions. To illustrate this point, the temperature-sensitive mutation affecting the DBP (standing for DNA binding protein) gene of the E2A region (Ensinger and Ginsberg, 1972, J. Virol. 10, 328-339).
According to a preferential embodiment, an adenoviral vector according to the invention comprises an expression unit containing one or more viral genes of the E2, E4 or L1-L5 regions. An advantageous variant consists in retaining from the E4 region only the sequences coding for ORFs 3, 6 and/or 7 (these limited deletions of the E4 region not necessitating complementation of the E4 function; Ketner et al., 1989, Nucleic Acids Res. 17, 3037-3048).
It can also be advantageous to have at one's disposal an adenoviral vector comprising several expression units, it being possible to envisage all the combinations (E2 and E4, E2 and L1-L5, E4 and L1-L5 or E2, E4 and L1-L5). Regulatory sequences acting in the same manner, and preferably positively (for example TAR, RRE, tet O, etc.), will then be chosen. For reasons of simplicity of implementation, preference is given to the case where the units carry identical regulatory sequences enabling the adenoviral vector to be propagated in a complementation line comprising a single inducer.
The invention also relates to an infectious viral particle, as well as to a eukaryotic host cell comprising a viral vector according to the invention. Said host cell is advantageously a mammalian cell, and preferably a human cell, and can comprise said vector in integrated form in the genome or in nonintegrated form (episome). It can be a primary or tumor cell of hematopoietic (totipotent stem cell, leukocyte, lymphocyte, monocyte or macrophage, etc.), muscular, pulmonary, hepatic, epithelial or fibroblast origin.
The subject of the present invention is also a complementation cell, characterized in that it comprises an inducer and/or a repressor (regulator). Depending on the requirements, the latter may be added to the culture medium, or produced stably or transiently by the cell itself. Preferably, a complementation cell according to the invention is modified by the introduction of a DNA fragment coding for said regulator. All standard means for introducing a nucleic acid (synthetic, viral or plasmid vector, naked DNA, etc.) into a cell may be used in the context of the present invention, such as, for example, transfection, electroporation, microinjection, lipofection, adsorption and protoplast fusion. In the context of the invention, it can be advantageous to generate a complementation cell which produces only a single regulator, and in particular an inducer. However, it can also be advantageous to have at one's disposal cells producing several inducers.
According to an advantageous embodiment, a complementation cell according to the invention is capable of complementing In trans a viral vector according to the invention, and especially an adenoviral vector. In this connection, a complementation cell for the E1 and/or E4 function will advantageously be chosen. Accordingly, the invention also relates to a cell intended for the complementation of a second generation adenoviral vector which is defective for the E1 function and another adenoviral function (late or early). Such a cell comprises all or part of the 3E region of an adenovirus whose expression is controlled by any promoter, and all or part of a region of an adenovirus other than the E1 region, placed under the control of a promoter equipped with regulatory sequences, for example at its 5′ end with at least one and preferably one to 20 tet O sequence(s) which is/are activable by the transactivator tTA. An advantageous variant consists of a minimal promoter derived from the CMV virus (cytomegalovirus), upstream of which lie 7 tat O sequences in a head-to-tail orientation. The inducer may be introduced into the complementation cell according to the invention prior to, concomitantly with or subsequently to the adenoviral sequences placed under the control of the tet O sequences or, as mentioned above, it may be added to the culture medium. It in also possible to envisage expressing the inducer (for example tTA) in the complementation cell, and adding the repressor (for example tetracycline) to the culture medium when the expression of the adenoviral genes is no longer desired.
By way of preferred examples, the adenoviral region other the E1 region consists of:
(i) all or part of the E4 region, and in particular the sequences coding for open reading frames 6 and 7 (ORFs 6/7) of the latter, or alternatively
(ii) all or part of the E2 region, and in particular the sequences coding for the DBP protein (DNA binding protein) or a temperature-sensitive mutant of the latter.
Naturally, a complementation cell according to the invention can, in addition, comprise a third adeno-viral region whose expression is placed under the control of the appropriate elements. In this connection, a preferred cell is intended for the complementation of an adenoviral vector which is defective for the collective early functions which are essential for replication, and comprises all or part of. the E1, E2 and E4 regions, one or other or both of the latter two regions being placed under the control of a promoter equipped with regulatory sequence(s) as defined above.
One of the advantages of a complementation cell according to the invention is that it permits the production at high titer of infectious viral particles from a conventional viral vector or viral vector according to the invention. The viral titer of the final preparation (after purification) is advantageously greater than 5×109 pfu/ml, preferably greater than 1×109 pfu/ml, as an absolute preference greater than 5×109 pfu/ml and, as yet a further preference, greater than 5×1010 pfu/ml. The term pfu is understood to refer to a particle capable of forming a plaque by infection of permissive cells. The techniques for evaluating the number of pfu are conventional and known to a person skilled in the art. The agar technique (Graham and Prevec, 1991, supra) may be mentioned. Generally, the viral titer in pfu/ml is equal to or less than the actual number of infectious viral particles capable of carrying out their gene transfer function. In effect, a certain percentage is incapable of propagating effectively and hence of generating lytic plaques on permissive cells. The titer of infectious viral particles produced by a complementation cell according to the invention is advantageously greater than 5×109 ifu/ml, preferably greater than 1×1010 ifu/ml, as an absolute preference greater than 5×1010 ifu/m [sic] and, as yet a further preference, greater than 5×1011 ifu/ml. The term ifu is understood to refer to an infectious particle capable of infecting a nonpermissive target cell and of transferring its genome and permitting the expression of the genes carried by the latter. The number of ifu in estimated by the number of target cells expressing the gone of interest or a viral gone. A person skilled in the art is aware of the techniques to be employed for detecting their expression: by immunofluorescence, western blotting or alternatively staining, etc. For example, when the gene of interest consists of the LacZ gone, the protein β-galactosidase may be visualized by staining with X-Ga1 (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside) and the number of blue cells is counted. When the CFTR therapeutic gone is employed, the expression product may be visualized by western blotting. It in also possible to look for cells expressing the adenoviral DBP, penton or fiber proteins using specific antibodies.
A complementation-cell according to the invention may be generated from various cell lines by transfection of suitable portions of the adenoviral genome and a DNA fragment coding for a regulator. Among the lines which can be envisaged, there may be mentioned the Vero kidney (monkey), BHK (hamster), MDCK (dog) and MBDK (bovine) lines, the CHO line (hamster) or alternatively the human lines (HeLa, A549, MRC5, W138, etc.) available in collections such as the ATCC (Rockville, USA). However, an especially suitable cell is line 293. The use of primary lines such as primary human retina cells may also be envisaged.
An infectious viral particle according to the invention may be prepared according to any conventional technique in the field of the art (Graham and Prevect, 1991, supra), for example by cotransfection of a vector and an adenoviral fragment into a suitable cell, or alternatively by means of a helper virus supplying in trans the nonfunctional viral functions. It is also possible to envisage generating the viral vector in vitro in Escherichia coli (E. coli) by ligation or alternatively homologous recombination (see, for example, French Application 94/14470).
The invention also relates to a method of preparation of an infectious viral particle comprising a viral vector according to the invention, according. to which:
(i) said viral vector in introduced into a complementation cell capable of complementing in trans said vector, so as to obtain a transfected complementation cell,
(ii) said transfected complementation cell in cultured under suitable conditions to permit the expression of th viral genes and the production of said infectious viral particle, and
(iii) said infectious viral particle is recovered in the cell culture.
Naturally, the infectious viral particle may be recovered from the culture supernatant, but also from the cells. According to an advantageous embodiment, an adenoviral vector and a complementation cell according to the invention are employed. According to another variant, use may be made of a conventional complementation cell. It may be necessary to add an inducer to the culture medium in the case where the expression unit comprises activable regulatory sequences. The amount to be used depends on the actual nature of the inducer. A person skilled in the art will quite obviously be able to Adapt the optimal concentration in accordance with the specific data.
The invention also relates to a method of preparation of an infectious viral particle comprising a conventional viral vector, employing a complementation cell according to the invention. By way of example, the introduction of a viral vector which is defective for the E1 and E4 functions (E1- E4-) into a 293 cell expressing (i) ORFs 6/7 of the E4 region under the control of a minimal promoter equipped at its 5′ and with 7 tet O sequences, and (ii) the. transactivator tTA directed by the same promoter, will ale viral particles to be generated which are deficient for replication but infectious with respect to a host cell.
The subject of the invention in also a pharmaceutical composition comprising as therapeutic or prophylactic agent a viral vector, an infectious viral particle, a complementation cell or a eukaryotic host call according to the invention, in combination with a vehicle which is acceptable from a pharmaceutical stand- point. The composition according to the invention in intended especially for the preventive or curative treatment of disorders such an:
genetic disorders (hemophilia, cystic fibrosis, diabetes or myopathy, Duchęne's [sic] myopathy and Becker's myopathy, etc.),
cancers, such as those induced by oncogenes or viruses,
viral diseases such as hepatitis B or C and AIDS (acquired immunodeficiency syndrome resulting from infection with HIV), and
recurrent viral diseases such as the viral infections caused by herpesvirus.
A pharmaceutical composition according to the invention may be manufactured in a conventional manner. In particular, a therapeutically effective amount of a therapeutic or prophylactic agent in combined with a vehicle such as a diluent. A composition according to the invention may be administered locally, systemically or by aerosol, especially via the intragastric, subcutaneous, intracardiac, intramuscular, intravenous, intraperitoneal, intratumoral, intrapulmonary, intranasal or endotracheal route. The administration may take place in a single dose or a dose repeated one or several times after a certain time interval. The appropriate administration route and dosage vary in accordance with various parameters, for example with the individual or the disorder to be treated or alternatively with the gene(s) of interest to be transferred. In particular, the viral particles according to the invention may be formulated in the form of doses of between 104 and 1014 pfu (plaque forming units), advantageously 105 and 1013 pfu and preferably 106 and 1011 pfu. The formulation can also include an adjuvant or an excipient which is acceptable from a pharmaceutical standpoint.
Lastly, the present invention relates to the therapeutic or prophylactic use of a viral vector, of an infectious viral particle, of a complementation cell or of a eukaryotic host cell according to the invention, for the preparation of a medicinal product intended for the treatment of the human or animal body, and preferably by gene therapy. According to a first possibility, the medicinal product may be administered directly in vivo (for example by intravenous injection, in an accessible tumor, in the lungs by aerosol, etc.) . It is also possible to adopt the ex vivo approach, which consists in removing cells from the patient (bone marrow stem cells, peripheral blood lymphocytes, muscle calls, etc.), transfecting or infecting them in vitro according to the techniques of the art and readministering then to the patient. In the case where the expression unit comprises regulator sequences that respond to a repressor, it is possible to envisage administering the repressor in order to block or limit the expression of the viral genes (prior, concomitant or subsequent administration of the repressor or coexpression via conventional vectors).
The invention also extends to a method of treatment, according to which a therapeutically effective amount of a viral vector, of a viral particle, of a eukaryotic host cell or of a complementation cell according to the invention is administered to a patient requiring such treatment.