You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

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### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


The present invention relates to novel helper vectors allowing defective recombinant viral vectors, which have the characteristic of being provided with recombination sequences recognized by a recombinase, to be complemented. It likewise relates to a complementation cell expressing the recombinase as well as a method of preparation of recombinant viral vectors in the form of infectious viral particles allowing the transfer and the expression of genes of interest in a cell or a host organism. The invention is of very particular interest for gene therapy prospects, especially in man.
The possibility of treating human diseases by gene therapy has passed in the course of a few years from the stage of theoretical considerations to that of clinical applications. The first protocol applied to man was initiated in the United States in September 1990 on a patient who was genetically immunodeficient because of a mutation affecting the gene coding for adenine deaminase (ADA). The relative success of this first experiment encouraged the development of new gene therapy protocols for various genetic or acquired diseases (infectious diseases and especially viral diseases such as AIDS or cancers). The great majority of the protocols described until now employ viral vectors to transfer and express the therapeutic gene in the cells to be treated.
The interest in adenoviruses as gene therapy vectors has already been touched on in numerous documents of the prior art. In fact, the adenoviruses have a wide spectrum of hosts, are not very pathogenic and do not have the disadvantages connected with the retroviruses since they are nonintegrative and replicate equally in quiescent cells. By way of information, their genome is formed of a linear and double-stranded DNA molecule of approximately 36 kb carrying regions acting in cis (ITR 5xe2x80x2 and 3xe2x80x2 encapsidation region of the viral genome and ITR 3xe2x80x2) and additionally about thirty genes, at the same time early genes necessary for viral replication and late structure genes (see FIG. 1).
The early genes are divided into 4 regions dispersed in the adenoviral genome (E1 to E4; E for early). They comprise 6 transcriptional units which have their own promoters. The late genes (L1 to L5; L for late) partly cover the early transcription units and are, for the majority, transcribed starting from the major late promoter MLP.
At the present time, all the adenoviral vectors used in gene therapy protocols are devoid of the major part of the E1 region essential for replication, in order to avoid their distribution in the environment and the host organism (first generation vectors). This deletion makes the viral genome deficient for replication. However, the E1 viruses can be propagated in a cell line which complements the E1 function to generate an infectious viral particle. The 293 line, established starting from human embryonic kidney cells, is currently used, in the genome of which is integrated the left 5xe2x80x2 end of the type 5 adenovirus (Graham et al., 1977, J. Gen. Virol. 36, 59-72).
The majority of adenoviral vectors of the prior art comprise supplementary deletiors. Certain of these have been introduced in the E3 region with the aim of increasing the cloning capacities but do not need to be complemented to the extent where the E3 region is nonessential for replication. More recently, second generation vectors have been proposed in the literature. They conserve the in cis regions (ITRs and encapsidation sequences) and comprise important internal deletions aimed at suppressing the main part of the viral genes whose expression can be responsible for inflammatory responses in the host. In this respect, a minimal vector which is deficient for all of the coding viral regions represents a choice alternative.
The techniques of preparation of adenoviral vectors are widely described in the literature. Firstly, the complete genome is formed by homologous recombination in the 293 line (see especially Graham et Prevect, 1991, Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols; Ed E. J. Murray, The Human Press Inc. Clinton, N.J.) or in Escherichia coli (technique described in the French Application No. 94 14470).
It is then necessary to propagate the vector in order to form a stock of viral particles containing it. This production step is critical and must allow high titers of infectious particles to be attained to be able to consider development on a large scale with a view to the preparation of clinical batches. If the first generation adenoviral vectors can be propagated relatively easily in the 293 cell line, the only complementation line described to date and capable of efficiently expressing E1, such is not the case for second generation vectors. In fact, according to the same basic principle, such a vector must be complemented for the essential functions which it cannot express.
The complementation can be provided xe2x80x9cin transxe2x80x9d by the cell line employed (designated complementation cell line). It is then necessary to have new lines complementing several essential viral functions (E1 and E2, E1 and E4 or E1, E2 and E4). However, the various attempts carried out until now give the impression that the coexpression of several adenoviral regions is potentially toxic, such that the line risks not being optimal in terms of growth capacity and yield of viral particles, these two criteria being indispensable for industrial exploitation.
Another alternative is based on the use of a supplementary viral element, called xe2x80x9chelper virusxe2x80x9d introduced into the line at the same time as the adenoviral vector (two-component system) At the present time, an adenovirus from which the E1 region has been deleted and which is capable of synthesizing the expression products of other adenoviral regions is currently used. The co-transfection of such a helper virus and of an adenoviral vector in the 293 line allows the formation of viral particles.
However, a major disadvantage of this method is that the cells produce a mixed population of viral particles, one type comprising the recombinant vector and the other type the helper virus. In practice, the preparations mainly contain viral particles of helper virus, the contamination being able to reach and even exceed 90%. The presence of the helper virus is not desirable in the context of a therapy applied to man and therefore necessitates the employment of cumbersome and costly physical separation techniques, such as ultracentrifugation. In addition, this technology is not very well adapted to the production of vectors of complex structure, such as the second generation vectors, to the extent where, very often, the helper virus has a selective advantage (more rapid replication).
The unresolved problem to this day of the production of recombinant adenoviral vector particles with a high titer is an obstacle to the development of gene therapy.
A novel helper virus has now been constructed by insertion of direct repetitions on both sides of the encapsidation region. The action of a recombinase recognizing them involves the excision of the genetic material situated between them. This deletion does not have any notable consequence on the expression of the viral genes but limits the encapsidation of the helper virus in a viral particle. Thus, the employment of the two-component procedure described above in a cell line expressing the recombinase will allow preparations enriched in adenoviral vector particles of interest to be produced.
The present invention follows from the perfection of a genetic technique based on the use of recombination sequences and of a recombinase to produce mainly the recombinant viral vector and to limit the contamination by the helper virus. The aim of the present invention is to put at the disposal of the public a novel helper virus able to express the genes which it carries (that is to say capable of exercising its function of trans-complementation) but unable to be propagated in the presence of a recombinase. The solution provided by the present invention combines safety cf use (preparation enriched in recombinant viral vector), simplicity (production in a conventional cell line in the presence of recombinase) and efficiency (high titer compatible with industrial needs). It is very particularly adapted to the production of second generation adenoviral vectors.
For this reason the present invention relates to a helper virus for the production of a recombinant viral vector defective for replication, characterized in that it comprises a first recombination sequence at 5xe2x80x2 and a second recombination sequence at 3xe2x80x2 of a region essential for the propagation of said helper virus; said recombination sequences being recognized by a recombinase.
The term xe2x80x9chelper virusxe2x80x9d designates a vector able to trans-complement in full or in part a recombinant viral vector defective for replication. It is thus able to produce at least one polypeptide, early and/or late, which the recombinant vector cannot produce itself and which is necessary for the formation of a viral particle. xe2x80x9cIn fullxe2x80x9d signifies that the helper virus is capable of complementing the whole of the viral genome essential for replication of which the recombinant viral vector is devoid and xe2x80x9cin partxe2x80x9d signifies that the complementation is limited to a part of the defective functions.
In the context of the present invention, a helper virus derives from a natural virus such as found in nature as well as from a virus whose genome comprises modifications with respect to that of the parent virus from which it is descended. These modifications can have been introduced in vitro by genetic engineering techniques. They can be different (deletion, mutation and/or addition of one or more nucleotides) and localized in the encoding regions of the viral genome or outside of these. The modification can, for example, allow one or more gene(s) essential for viral replication to be inactivated with the aim of likewise rendering it defective that is to say incapable of autonomous replication.
The human adenoviruses of serotype C and, more particularly, of type 2, 5 or 7 represent particularly preferred viruses in the context of the invention. However, it is likewise possible to resort to other adenoviruses, especially of animal origin (canine, bovine, murine, avian, ovine, porcine or simian). It is more particularly possible to mention the canine adenoviruses CAV-1 or CAV-2, avian viruses DAV or even bovine viruses Bad of type 3 (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). However, it may also be of interest to have a helper virus derived from a poxvirus (vaccinia virus, fowlpox, canarypox . . . ), retrovirus, herpesvirus, cytomegalovirus, adenovirus-associated virus (AAV) or even at a hybrid virus comprising fragments of different origin.
The characteristic of the helper virus according to the invention is that it comprises at least two recombination sequences inserted at 5xe2x80x2 and at 3xe2x80x2 of a sequence essential for its propagation. An essential sequence designates all or part of a viral gene, elements necessary for the expression such as a promoter or, in a preferred manner, elements acting in cis (ITR, LTR, encapsidation sequence . . . ). By way of information, the first and/or second recombination sequences can be positioned in the interior or immediately at 5xe2x80x2 and 3xe2x80x2 of the essential region up to about several hundreds of bp.
In the sense of the present invention, a xe2x80x9crecombination sequencexe2x80x9d is formed by a nucleic acid sequence (DNA or RNA) recognized by a recombinase able to induce a recombination event. Usually, a recombination sequence has at least 10 base pairs (bp), advantageously 15 to 80 bp, preferably 20 to 60 bp and, in a very preferred manner, 30 to 50 bp. According to an advantageous embodiment, a helper virus according to the invention comprises two copies of an identical or closely similar recombination sequence (at least 70% sequence identity and, in a preferred manner at least 90%). For this reason we shall talk of sequence repetitions. In this respect, the repetition can be reversed (the two recombination sequences present in the helper virus have a reverse orientation with respect to one another, one being in the direction 5xe2x80x2 to 3xe2x80x2 and the other 3xe2x80x2 to 5xe2x80x2) or direct (same orientation 5xe2x80x2 to 3xe2x80x2 or 3xe2x80x2 to 5xe2x80x2). This second form will be preferred.
The recombination is accompanied by a pairing of the recombination sequences, by a cleavage of a target sequence at their level and by a ligation of the cleaved ends. The enzyme able to promote the recombination is designated xe2x80x9crecombinasexe2x80x9d. The recombination between direct repetitions leads to the excision of sequences between them. On the other hand, recombination between reversed repetitions involves the reversal of the genetic material located between them.
Generally speaking, the recombination sequences and the recombinases are described in the literature accessible to the person skilled in the art. They can be of any viral, phagic, prokaryotic or eukaryotic origin (yeast, fungus or even higher eukaryote). In addition, they can be obtained by the conventional molecular biology techniques (cloning, amplification by chain reaction (PCR for Polymerase Chain Reaction) or by chemical synthesis.
As preferred examples, mention will be made of loxP recombination sequences (described in the identifier of sequence SEQ ID NO.: 1), FRT (SEQ ID NO.: 2) and R (SEQ ID NO.: 3) recognized by the recombinases CRE, FLP and R, respectively (see, for example, the review article Kilby et al., 1993, TIG 9, 413-420).
A helper virus particularly adapted to the present invention is derived from the genome of an adenovirus and comprises the ITRs 5xe2x80x2 and 3xe2x80x2, an encapsidation region and at least one viral gene selected from the genes of the E1, E2, E4 and L1-L5 regions and defective in the recombinant adenoviral vector as well as a first recombination sequence at 5xe2x80x2 and a second recombination sequence at 3xe2x80x2 of the encapsidation region. Preferably, they are formed by loxP sequences positioned in the same orientation with respect to one another. According to a first variant of interest, the encapsidation region can be attenuated (reduced capacity for encapsidation) to favor the encapsidation of the recombinant viral vector. The attenuation can be obtained by deletion of a part of the encapsidation region. The means of attenuating an encapsidation region are indicated in the Application WO 94/28152.
According to a second variant of interest, the helper virus can include an expression cassette of a recombinase, and allowing especially an inducible expression or the production of an inactive recombinase activatable according to needs (defined below). The insertion takes place in an appropriate region of the helper virus and, preferably, outside of the localized region between the recombination sequences.
According to a particular embodiment intended to increase the safety aspect, a helper virus according to the invention can comprise a first recombination sequence and a second recombination sequence at 5xe2x80x2 and at 3xe2x80x2 of several regions essential to its propagation. For reasons of simplicity of employment, the case will be preferred where the recombination sequences are identical or related so as to be recognized by an identical recombinase.
The present invention likewise relates to a complementation cell line comprising a DNA fragment coding for a recombinase. It can be generated from various cell lines by introduction of appropriate portions of the viral genome and of the fragment in question. A line able to complement the E1 and/or E4 function of an adenovirus is more particularly preferred. Mention will be made of the lines 293 (Graham, 1997 supra) and 1653 (described in the Application WO 94/28152) modified by the introduction of the DNA fragment coding for a recombinase.
All the standard means for introducing a nucleic acid into a cell can be employed in the context of the present invention (synthetic, viral, plasmid vector, naked DNA . . . ). Of course, said DNA fragment can be integrated into the cell genome or remain in the episome state. For the aims of the present invention, it can comprise the elements necessary for its expression. These will be, preferably, elements conferring an inducible expression in response to an inducer. Such elements are known to the person skilled in the art. It is possible to mention, by way of information, promoters inducible by metals (promoter of the metallothnioneine gene), by hormones (promoter comprising elements responding to glucocorticoids GRE, to progesterones PRE, to estrogens ERE . . . ), by viral inducers (promoter comprising the TRA or RRE sequence responding respectively to the TAT or REV protein of the human immunodeficiency virus HIV) or by various cellular inducers (promoter comprising the UAS-Gal4 sequence (for Upstream Activating Sequence Ga14) responding to Gal4 or the operators of the tetracycline bacterial operon responding positively to the transtetracycline activator tTA.
In addition, a DNA fragment in use in the present invention codes for a recombinase able to recognize the recombination sequences present in a helper virus according to the invention. It is preferred to employ a recombinase selected from the group formed by CRE, FLP and R. However, it is equally possible to resort to a DNA fragment coding for a homolog of a recombinase whose sequence is modified with respect to the native sequence but exercising a similar or improved function. These modifications can result from the deletion, addition or substitution of one or more nucleotide(s). They can likewise be a hybrid protein resulting from the fusion of polypeptides from various origins, especially a polypeptide having a recombinase activity and the other a linking region. A recombinase particularly adapted to the present invention is formed by a hybrid protein, designated CRE-ER, resulting from the fusion of the recombinase CRE and of the linking region to the ligand of the human estrogen receptor; (Metzger et al., 1995, Proc. Natl. Acad. Sci. USA 92). The latter insofar as such is inactive and its biological activity is activated in the presence of a hormonal ligand such as estradiol.
The invention likewise relates to a process for preparing a viral particle comprising a recombinant viral vector, which comprises the following steps:
(a) Preparing a recombinant viral vector deficient for replication;
(b) Preparing a helper virus according to the invention;
(c) Introducing the recombinant viral vector and the helper virus into an appropriate cell line;
(d) Culturing said cell line under appropriate conditions to allow the production of the viral particle in the presence of a functional recombinase able to recognize said first and second recombination sequences; and
(e) Recovering the viral particle in the cell culture.
In the sense of the present invention, a defective recombinant viral vector derives from a virus in the genome of which certain sequences have been deleted, rendered nonfunctional, mutated or even substituted by other sequences and, more particularly, a heterologous DNA fragment (normally not present in the parent virus). The insertion takes place in an appropriate region of the viral genome, so as to allow its expression in a host cell. A host cell is formed by any eukaryotic cell infectable by a viral particle containing said recombinant viral vector.
The heterologous DNA fragment in use in the present invention can be descended from a eukaryotic organism, from a prokaryote or from a virus other than that in which it is inserted. It can be isolated by any technique conventional in the field of the art, for example by cloning, PCR or chemical synthesis. It can be a fragment of genome type (comprising all or part of the whole of the introns), of complementary DNA type (cDNA, devoid of intron) or of mixed type (comprising all or part of at least one intron). In addition, it can code for an antisense RNA and/or a messenger RNA (mRNA) which will then be translated into a polypeptide of interest, the latter being able to be (i) intracellular, (ii) membranous present at the surface of the host cell or (iii) secreted into the external medium. In addition, it can be a polypeptide as found in nature (native) or a portion of the latter (truncated) or equally a chimeric polypeptide arising from the fusion of sequences of various origins or even mutated and having improved or modified biological properties.
In the context of the present invention, it can be advantageous to use a DNA fragment coding for a cytokine (interleukin including IL-2, interferon, colony-stimulating factor . . . ), a cell or nuclear receptor, a ligand, a clotting factor (factor VII, factor VIII, factor IX . . . ), CFTR protein (Cystic Fibrosis Trans-membrane Conductance Regulator), insulin, dystrophin, a growth hormone, an enzyme (renin urease, thrombin . . . ), an enzyme inhibitor (inhibitor of a viral protease, xcex11-antitrypsin . . . ), a polypeptide with antitumor effect (product of suppressor genes of tumors, polypeptide stimulating the immune system . . . ), a polypeptide able to inhibit or slow down the development of a bacterial, viral or parasitic infection (antigenic polypeptide, trans-dominant variant . . . ) an antibody, a toxin, an immunotoxin and finally a label (luciferase, xcex2-galactosidase, product conferring resistance to an antibiotic . . . ). Of course, this list is not limiting and other genes can likewise be employed.
Advantageously, the heterologous DNA fragment is placed under the control of elements necessary for its expression in the host cell. xe2x80x9cNecessary elementsxe2x80x9d designates all of the elements allowing the transcription of said DNA fragment to RNA (antisense RNA or mRNA) and the translation of the mRNA to polypeptide. These elements comprise a regulatable or constitutive promoter, which can be heterologous or on the contrary homologous to the parent virus. It is possible to mention, as examples, the promoter of the human or murine PGK gene (Phospho Glycerate Kinase), the early promoter of the SV40 virus (Simian Virus), the LTR of RSV (Rous Sarcoma Virus), the TK promoter (Thymidine Kinase) of the HSV-1 virus (Herpes Simplex Virus) and the adenoviral promoters E1A and MLP. The necessary elements can, in addition, include additional elements (intron sequence, secretion signal sequence, nuclear localization sequence, translation initiation site, transcription termination poly A signal . . . ). Although it is not a preferred variant, it is indicated that the viral vector can likewise comprise a DNA fragment coding for the recombinase.
It is within reach of the person skilled in the art to generate a recombinant viral vector in use in the present invention. He will quite certainly know how to adapt the technology as a function of the specific data (type of vector, heterologous DNA fragment . . . ). According to a preferred variant, the vectors capable of being employed in the context of the present invention are recombinant adenoviral vectors defective for all of the viral functions or even all of the functions with the exception of E4. Such vectors are described in the International Application WO 94/28152.
A helper virus according to the invention is obtained by insertion in a viral genome of a first and a second recombination sequence on both sides of a region essential for replication and, preferably, of the encapsidation region, it being possible for the latter to be attenuated or nonattenuated. The person skilled in the art knows the regions essential for the replication of a virus and is able to carry out such a construction by applying the classical techniques of molecular biology. According to the variant mentioned above, it can likewise comprise a DNA fragment coding for a recombinase and, especially, the CRE-ER hybrid. According to a preferential embodiment, a helper virus according to the invention and the recombinant viral vector which it allows production of derive from the same parent virus and, in a very preferred manner, from an adenovirus.
After the actual construction step, the helper virus and the recombinant viral vector are introduced into an appropriate cell line. All the standard means for introducing a nucleic acid into a cell can be used in the context of the present invention, for example transfection, electroporation, microinjection, lipofection, adsorption and fusion of protoplasts. It is indicated that they can be co-introduced (concomitant fashion) or introduced separately (the helper virus according to the invention previously or subsequently to the recombinant viral vector).
Although any cell line can be employed in the context of the present invention, a complementation line is especially preferred. Recourse will be had to a line of the prior art (293, 1653 . . . ) when a recombinant viral vector or a helper virus comprising the DNA fragment coding for the recombinase is employed. On the other hand, when this is not the case, use will be made of a complementation cell line according to the invention.
After transfection, the cell line is cultured under appropriate conditions to allow the production of viral particles. A procedure according to the invention can, in addition, comprise an amplification step previous to the culture step in the presence of the functional recombinase. The aim of this step is to increase the quantities of helper virus and of recombinant viral vector in order to improve the yields. It can be carried out by culture in any permissive line or in the appropriate line in use in the present invention before the addition, expression or activation of the recombinase.
This first culture step is followed by a second step carried out in the presence of a functional recombinase able to recognize said first and second recombination sequences. In the context of the procedure according to the invention, this recombinase can be added to the cell culture, for example in substantially pure form. However, according to another very preferred and already mentioned variant, the recombinase is produced by one of the constituents of the procedure according to the invention, namely the recombinant viral vector or, in a preferred manner, the helper virus or the cell line. Once produced in functional form, the recombinase will cause the excision of the essential region of the helper virus localized between the recombination sequences, with the aim of preventing or reducing its propagation.
In addition, when a recombinase is employed whose expression is inducible by an inducer or the CRE-ER hybrid protein whose biological activity is dependent on a hormonal ligand, the culture step in the presence of the functional recombinase is carried out by the addition to the culture medium of the inducer or the ligand.
According to an advantageous embodiment intended to increase the safety of a procedure according to the invention, the helper virus and the recombinant viral vector are defective and can be conversely complemented, in total or in part. A variant of interest consists in employing (i) a helper adenovirus according to the invention defective for the functions E1 and E4 and comprising the loxP sequences at 5xe2x80x2 and at 3xe2x80x2 of the encapsidation region, (attenuated or nonattenuated) (ii) a recombinant vector defective for all the functions with the exception of E4 and (iii) a 293 cell line producing the CRE-ER hybrid recombinase. According to another advantageous alternative, a procedure according to the invention employs (i) a helper adenovirus according to the invention defective for the functions E1 and E4, comprising the loxP sequences at 5xe2x80x2 and at 3xe2x80x2 of the encapsidation region (attenuated or nonattenuated) and producing the CRE-ER hybrid recombinase, (ii) a recombinant vector defective for all of the functions with the exception of E4 and (iii) a conventional cell line 293.
The viral particles are recovered from the cell culture, from the medium or after lysis of the cells. Advantageously, a procedure according to the invention comprises an additional step of purification of the recombinant viral vector particles. Although the choice of the technique is wide and within the reach of the person skilled in the art, it is possible to mention more particularly ultracentrifugation on a cesium chloride or sucrose gradient.
Finally, the invention likewise relates to a procedure for preparing a viral particle comprising a recombinant viral vector by means of a helper virus, according to which the ratio viral particles of recombinant adenoviral vector to those of helper virus is greater than 50%, advantageously greater than 60%, preferably greater than 70% and, in a very preferred manner, greater than 80%.
The invention likewise relates to a recombinant viral vector particle obtained by a procedure according to the invention as well as to a eukaryotic host cell 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 . . . ), muscular, pulmonary, tracheal, hepatic, epithelial or fibroblast origin.
The invention likewise relates to a pharmaceutical composition comprising as therapeutic or prophylactic agent a recombinant viral vector particle obtained by a procedure according to the invention or a eukaryotic host cell according to the invention, in combination with a carrier acceptable from a pharmaceutical point of view. The composition according to the invention is intended in particular for the preventive or curative treatment of diseases such as:
genetic diseases (hemophilia, mucoviscidosis, diabetes or myopathy, that of Duchxc3xaane and Becker . . . ),
cancers, such as those induced by oncogenes or viruses,
viral diseases, such as hepatitis B or C or AIDS (acquired immunodeficiency syndrome resulting from infection by HIV), and
recurrent viral diseases, such as viral infections caused by the herpesvirus.
A pharmaceutical composition according to the invention can be produced in a conventional manner. In particular, a therapeutically efficacious quantity of a therapeutic or prophylactic agent is combined with a carrier such as a diluent. A composition according to the invention can be administered by aerosol, locally or even systemically. The routes of administration envisagable within the context of the present invention can be intragastric, subcutaneous, intracardiac, intramuscular, intravenous, intraperitoneal, intratumor, intrapulmonary, nasal or intratracheal. The administration can take place in a single or repeated dose one or more times after a certain delay interval. The appropriate route of administration and the appropriate dose vary as a function of various parameters, for example of the individual or of the disease to be treated or even of the recombinant gene(s) to be transferred. In particular, the viral particles according to the invention can 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 likewise include an adjuvant which is acceptable from a pharmaceutical point of view.
Finally, the present invention relates to the therapeutic or prophylactic use of a recombinant viral vector particle obtained by a procedure according to the invention or of a eukaryotic host cell according to the invention for the preparation of a medicament intended for the treatment of the human or animal body and, preferentially, by gene therapy. According to a first possibility, the medicament can be administered directly in vivo (for example in an accessible tumor, in the lungs by aerosol . . . ). It is likewise possible to adopt the ex vivo approach which consists in taking cells from the patient (stem cells of the bone marrow, peripheral blood lymphocytes, muscle cells . . . ), in transfecting or infecting them in vitro according to the techniques of the art and in readministering them to the patient.
The invention likewise extends to a method of treatment according to which a therapeutically efficacious quantity of a recombinant viral vector particle obtained by a procedure according to the invention or of a eukaryotic host cell according to the invention is administered to a patient having need of such a treatment.
The present invention is described more completely with reference to the following figures and with the aid of the following examples.