HK1034283B - Use of triplex structure dna sequences for transferring nucleotide sequences - Google Patents

Description

Err1:Expecting ',' delimiter: line 1 column 188 (char 187)

The purpose of the invention is therefore to define and provide new means which may be used, for example, in gene therapy or transgenesis protocols for the creation of transgenic animals or plants or recombinant cells or cell lines. e means include the development of new vectors capable of transferring a nucleotide sequence, and in particular a sequence of therapeutic interest, into target cells in the human or animal body.

A major limitation of known gene therapy approaches to date is the vectorization of the gene of therapeutic interest. Oncovirus-derived retroviral vectors, mainly MoMLV, have been widely used for gene transfer. Their application is greatly limited by the fact that oncoviruses only integrate into actively dividing target cells. On the contrary, lentiviruses have the unique ability among retroviruses to infect differentiated, non-mitotic cells, and are interesting viral candidates for the development of new vectors. While retaining the advantages of an oncoviral vector (lack of immunity, stable integration), non-mitotic vectors could allow in vivo differentiation of tissue and find a significant therapeutic application (muscle, lung, and liver), as well as finding a field of gene expression.

Several attempts to construct retroviral vectors from lentivirus have been reported, including work by Poznansky M. et al. (J. Virol 1991, 65, 532-6), Naldini et al. (Science, 1996, 272, p. 263-7) from HIV retrovirus and by Poeschla EM et al. (Nature Medicine, 1998, 4, p. 354-7) from IVF retrovirus.

In particular, Zufferey et al. (Nature Biotechnology (1997) 15: 871-875) describe an HIV-1 derived vector whose viral genes are deleted and examine the influence of different vector constructs providing the particle envelope-coding sequence on the level of transduction. Zufferey et al. do not discuss the construction of the transfer vector, nor do they disclose vectors comprising a polynucleotide comprising cPPT and CTS regions.

WO97/12622 (Verma) proposes a transfer vector (likely to carry a transgene) which is devoid of retroviral genes and which also does not contain the polynucleotide comprising the cPPT and CTS regions. WO97/12622 discusses the existence of nuclear import determinants located in the GAG protein (NLS signal) and in the VPR and MA gag proteins

The inventors looked for determinants involved in the mechanism of entry of the retroviral genome into the nucleus of infected cells (nuclear import mechanism).

Err1:Expecting ',' delimiter: line 1 column 326 (char 325)

The mechanisms of entry of retroviral DNA into the nucleus are markedly different from one retroviral family to another. The lentivirus genome is capable of crossing the nuclear membrane of the interphasic nucleus via addressing and then translocating its pre-integration complex (linear DNA and associated proteins) through the nuclear pore. Thus, these viruses are able to replicate in the absence of target cell division. They specifically infect differentiated mitotic tissue macrophages and nucleotide disorders, cells at the center of HIV transmission, dissemination, and physiopathology.

The virus determinants responsible for the nuclear import of HIV1 virus DNA have been investigated by the inventors. The identification and functional understanding of the molecular mechanisms of HIV pre-integration complex nuclear import is of important fundamental interest. The inventors identified an original HIV-1 genome nuclear import mechanism whereby this import would be governed by a DNA structure, a triplex at the center of linear DNA molecules, generated by steps specific to lentiviral retroscription.

The triplex DNA structure at the centre of linear DNA molecules generated by lentiviral retrotranscription, particularly in the HIV retrovirus, has been described by the inventors in various previous publications (Charneau P. et al., J. Mol. Biol. 1994, 241, 651-662; Chameau P. et al., Journal of Virology, May 1991, pp. 2415-2421; Chameau P. et al., Journal of Virology, 1992, vol. 66, pp. 2814-2820).

The triplex DNA structure for viral retroscription is a polynucleotide with a central initiation cis-active region, or polypurine tract (cPPT), and a cis-termination active region (CTS), which initiates the transcription of a single strand+ whose synthesis is initiated by the PPT region at the centre of the HIV or other lentivirus genome and interrupts the synthesis of a second strand+, whose synthesis is initiated at a 3' PPT site upstream of the retroviral LTR (Figure 1).

The formation of the triplex DNA structure is the result of a discrete strand displacement event within the retrovirus genome, blocked by the CTS sequence (Charneau et al, J. Mol. Biol., 1994).

Err1:Expecting ',' delimiter: line 1 column 71 (char 70)

The triplex DNA structure thus formed during retroscription allows, or at least contributes to, the entry of the retroviral genome into the cell nucleus, thereby allowing non-mitotic cells to become infected.

Based on the identification of this mechanism required for retrovirus entry into the nucleus of target cells, the inventors developed a new generation of lentiviral vector, including the triplex DNA region. The introduction of a DNA fragment from the HIV-1 genome, including cis-active cPPT and CTS sequences, into an HIV vector system increases gene transduction in cells by stimulating the nuclear import rate of the vector DNA. This generation of triplex lentiviral vectors significantly improves gene transduction in cells, mitotic and non-mitotic.

a nucleotide sequence of retroviral or retroviral-like origin, which can be synthesised, comprising cPPT and CTS regions active in cis in general retroscription, and in particular 2 associated polynucleotides when placed in the normal retroviral genome, each containing at least 10 nucleotides.

Err1:Expecting ',' delimiter: line 1 column 296 (char 295)

The application also describes a nucleotide sequence comprising three DNA strands consisting of, on the one hand, the CTS region (or an equivalent region in the case of a genome origin used other than HIV1 but having the same properties as the CTS region published by Charneau et al, J. Mol. Biol., 1994) and, on the other hand, upstream of CTS, a region with approximately 90 to 110 nucleotides, preferably 99 nucleotides in the case of HIV1.

Err1:Expecting ',' delimiter: line 1 column 155 (char 154)

Err1:Expecting ',' delimiter: line 1 column 89 (char 88)

For example, the triplex sequence is that shown in Figure 11F for HIV1 or Figure 11G. The triplex, when present in vivo in a vector that can be used to penetrate the nuclear membranes of eukaryotic cells, stimulates the import of DNA into the nucleus of the cell to be modified or transduced.

The application discloses the use of this triplex sequence alone or in a vector to introduce nucleotide sequences to which the triplex sequence is bound into the nucleus of the recipient eukaryotic cell.

The application thus describes a recombinant vector characterised by a polynucleotide with a central initiation cis-active region (cPPT) and a termination cis-active region (CTS), both of retroviral or retroviral-like origin, which also includes a specific nucleotide sequence (transgene or sequence of interest) and signals for regulating retroscription, expression and encapsulation of retroviral or retroviral origin.

In particular, the invention relates to a recombinant lentiviral vector characterised by its intended to be transcribed for GAG, POL and ENV protein sequences or a portion of these polypeptides sufficient to allow the formation of lentiviral vector particles, and by its inclusion of: (b) a polynucleotide derived from the lentiviral genome in which it is capable of adopting a triplex DNA conformation during retranscription, such polynucleotide being a DNA structure comprising a central cis-active initiation region (cPPT) and a cis-active termination region (CTS), etc.

The invention also concerns a recombinant vector characterised by its intended to be transcribed for GAG, POL and ENV protein sequences or a portion of these polypeptides sufficient to allow the formation of lentiviral vector particles, and by its inclusion: (b) a polynucleotide containing a central initiation cis-active region (cPPT) and a termination cis-active region (CTS) originating from a retrotransposon, such a polynucleotide consisting of a DNA structure, forming a triplex DNA during retranscription, etc.) a nucleotide sequence of interest.

The term polynucleotide used refers to any sequence of nucleic acid, whether single stranded or double stranded or triple stranded, whether it is DNA e.g. cDNA, or RNA.

For example, the invention relates to the transfer of transgenes for therapeutic purposes, in particular as part of somatic gene therapy protocols, to insert a modulating or repair nucleotide sequence of deficient activity into the somatic cells of an organism to correct malfunction of an endogenous gene, or to enable the expression of an additional function, in particular a suppressive function of gene expression or activity, for a therapeutic purpose.

Err1:Expecting ',' delimiter: line 1 column 60 (char 59)

For the purposes of the present invention, and as an example, the nucleotide sequences of interest may be genes or parts of genes or sequences derived from genes, e.g. cDNA or RNA. They may also be antisense sequences, negative mutant sequences of a given gene, or sequences involved in the functions of transcription, expression, gene activation, or sequences suitable for the activation of prodrugs or cytotoxic substances.

The transgene sequences of the invention may also have an activity of stimulation or induction of the immune, cellular or humoral response, for example when used to transform antigen presenting cells.

The application describes the preparation of vectors for use in gene therapy in various fields, such as in hereditary diseases involving alterations in a gene, such as Duchenne myopathy, mucovicidosis, neurodegenerative diseases and acquired diseases such as malignancies that naturally lead to a weak immune response. e vectors also allow for the consideration of immunotherapy treatments to stimulate the response to pathogens, for example the production of CTL, for example in the case of diseases such as cancer or pathologies such as AIDS, or to decrease the response to antigen in the case of autoimmune diseases.

The invention also relates to the provision of means for the development of immunogenic compositions or vaccines, prophylactic or therapeutic compositions or immunogenic compositions.

Lentiviral vectors containing triplex DNA as described above may also be used for the construction of transgenic animals by transduction of genes into embryonic lineages or cells.

The vector of the invention contains a transgene inserted under the control of regulatory sequences of transcription or expression, viral or otherwise.

The transgene can be included in an expression cassette containing the appropriate sequences for regulating its expression in a cell.

A particularly interesting first embodiment of the invention is one in which the recombinant vector is characterised by the fact that the retroviral sequences of origin it contains are derived from the genome of a lentivirus.

Err1:Expecting ',' delimiter: line 1 column 90 (char 89)

Such a sequence may be obtained by any known means of identifying and isolating nucleotide sequences from their origin, in particular including cloning and/or amplification steps, or by synthesis using any available technique.

Alternatively, the vector of the invention is characterised by the sequence of retroviral-like origin being derived from a retrotransposon, for example the yeast retrotransposon TY1 (Heyman T et al).

The recombinant vector thus described may be, for example, a plasmid recombined by a retroviral or retroviral-like construction and a transgene, if any, contained in an expression cassette.

The recombinant vector may also be a retrotransposon, a phage, such as a λ-phage or a filamentous phage that can be introduced into bacteria, or a vector capable of transforming yeasts, such as a YAC.

Such a vector can be used for cell transduction, and in particular for encapsulation cells and/or target cells, by any known method, including transfection or infection or transduction, for example by an adenovirus or AAV-type vector containing the triplex lentiviral vector.

A vector of the invention is thus defined as one or more additional vectors that provide the sequences coding for the structural polypeptides of the genome of a selected retrovirus, in particular a lentivirus, or the structural polypeptides of a retrotransposon.

In this respect, the vector of the invention is transcribed by the addition of sequences coding for the GAG, POL and ENV polypeptides or for a portion of these polypeptides sufficient to allow the formation of retroviral particles intended to vector the recombinant vector lacking the viral genes and containing the transgene for which expression is sought.

A vector according to the invention may be characterized by the fact that the transgene or sequence of interest is contained in an expression cassette comprising regulatory signals for transcription and expression.

In general, vectors used for transcompletion into retroviral or retroviral-like proteins are devoid of encapsulation signals.

In this connection, vectors prepared according to the techniques of Goldman et al. (1997) which can be used for the transcompletion of a recombinant vector according to the invention will be mentioned.

This application describes recombinant retroviral vector particles including: (a) a gag polypeptide corresponding to lentivirus nucleoproteins or functional derived polypeptides (GAG polypeptides), (b) a polyppeptide consisting of lentivirus RT, PRO, IN proteins or functional derived polypeptide (POL polypeptide), (c) an envelope polypeptide or functional derived polypeptides (ENV polypeptides), (d) a recombinant nucleotide sequence comprising a specific nucleotide sequence (transgene or sequence of interest) will be placed under the control of transcription and expression regulators, a sequence containing these transcription and expression regulators will be located in a central or a cis- or cis-like region of the retrovirus, containing a central or a retrovirus-like region of reception and expression (TTS or cis- or cis-like regions) or a region of the retrovirus-like region of the retrovirus.

The application also discloses recombinant retroviral vector particles including: (a) a nucleotide sequence called gag sequence coding for lentivirus nucleoproteins or for functional derived polypeptides (GAG polypeptides); (b) a nucleotide sequence called pol sequence coding for RT, PRO, IN and RN proteins of a lentivirus or for a functional derived polypeptide (POL polypeptide); (c) signals regulating transcription and expression of gag and pol sequences; (d) a nucleotide sequence called gag sequence coding for envelope polypeptides or for functional derived polypeptides (GAG polypeptides); (c) a nucleotide sequence called pol sequence coding for envelope polypeptides or for functional sub-derived polypeptides (ENV polypeptides), the sequence being placed in the controlled regions of the signal origin or expression of the signal, or in a region containing a central or a transcription or expression controlled region (CIS-like region), with or without the controlled signal origin or expression of the signal; or (c) a signal-like region containing a central or a transcription or expression controlled region (CIS-like region) containing a signal-like region or a region of transcription or expression of the signal-like region, or a region containing a signal-like region of expression of the signal-like region or expression of the signal-like region of the signal-like region of the signal-like region of the signal-like region of the signal-like region of the signal-like region of the signal-like region of the signal-like region of the signal-like region of the signal-like region of the signal-like region of the signal-like-transcription or of the signal-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription-transcription

In particular, the invention relates to particles selected from (1) recombinant lentiviral particles whose genome is a recombinant nucleotide sequence comprising a determined nucleotide sequence (transgene) under the control of regulatory signals for transcription and expression, and including signals for retranscription, expression and encapsulation of lentiviral origin and a polynucleotide derived from a lentiviral genome, in which it is capable of adopting a triplex DNA conformation during retranscription, the said polynucleotide being a DNA structure comprising a central initiation cis-trans region (cPPT) and a cis-terminal region (CTS) of which the recombinant nucleotide sequence is a recombinant gene derivative (CTS2);controlled by transcription and expression regulatory signals, and comprising retroscription, expression and encapsulation regulatory signals of lentiviral origin with a partially deleted LTR sequence in the U3 region and a polynucleotide derived from a lentiviral genome, in which it is capable of adopting a triplex DNA conformation during retroscription, the polynucleotide being a DNA structure comprising a central initiation active region (cPPT) and a cis-termination cis-active regions (CTS) :3) of recombinant lentiviral vector particles including: (a) a gag polypeptide corresponding to lentivirus nucleoproteins or functional derived polypeptides (GAG polypeptides),(b) a polypeptide consisting of RT, PRO, IN proteins from a lentivirus or a functional derived polypeptide (polypeptide POL), (c) an envelope polypeptide or functional derived polypeptide (ENV polypeptide), and (d) a recombinant nucleotide sequence comprising a specific nucleotide sequence (transgene) controlled by regulatory signals for transcription and expression, a sequence containing regulatory signals for retranscription, expression and encapsulation of viral origin whose LTR sequence is partially deletion in the U3 region, and a polynucleotide which is a central cis-initiating region-active DNA structure (PPT) a cis-active termination region (CTS), these regions being of lentiviral origin and inserted into a functional orientation with these signals,the polynucleotide is capable of adopting a triplex DNA conformation during lentiviral retroscription; or (4) recombinant lentiviral vector particles including: (a) a gag polypeptide corresponding to lentivirus nucleoproteins or functional derived polypeptides (GAG polypeptides), (b) a polyppeptide consisting of lentivirus RT, PRO, IN proteins or a functional derived polypeptide (POL polypeptide), (c) an envelope polypeptide or functional derived polypeptides (ENV polypeptides), and (d) a recombinant nucleotide sequence lacking lentiviral genes and comprising a determined nucleotide sequence (transgene) determined under the control of transcription and expression regulators, a sequence containing transcription regulators,The test chemical is a polynucleotide that is a DNA structure with a central initiation cis-active region (cPPT) and a termination cis-active region (CTS), these regions being of lentiviral origin and functionally oriented with these signals, and that polynucleotide is capable of adopting a triplex DNA conformation during lentiviral retroscription.

The application also describes a nucleotide sequence comprising a polynucleotide with a central initiation cis-active region (cPPT) and a termination cis-active region (CTS), of retroviral or retroviral-like origin, each of which encodes an internal nucleotide sequence, the cPPT and CTS cis-active regions being inserted within the said nucleotide sequence, in a functional orientation with regulatory signals of retroviral or retroviral-like origin.

The polypeptides GAG and POL are nucleoprotein polypeptides derived from precursors cleaved by viral protease. POL polypeptides include retrovirus reverse transcriptase (RT), protease (PRO), integrase (IN), and Rnase H (RN). Other proteins from the retrovirus are also used for constructing vector particles where appropriate.

The gag,pol and env sequences used to construct the retroviral vector particles can, where appropriate, be modified by mutation, e.g. by point mutation or by deletion or insertion of one or more nucleotides, or by recombinant chimeras from different retroviruses e.g. between HIV1 and HIV2 or HIV1 and CAEV (Caprine Arthritis Encephalitis Virus), provided that they allow the production of functional polypeptides for the production of viral particles capable of vectorising the transgene to be expressed.

The advantage is that the recombinant vector or recombinant vector particles of the invention are such that the transgene is under control of regulatory signals of transcription and expression of non-retroviral origin. A promoter that may be used to control transgene expression is, for example, the CMV promoter, the PGK or EF1α promoter described in Tripathy, SK et al. (PNAS 1994, 91, p. 11557-11561).

According to a variant embodiment of the invention, however, the transgene can be placed under control of regulatory signals previously identified as being of retroviral or retroviral-like origin, in particular under control of the LTR sequence.

A lentivirus used to derive the retroviral construction according to the invention may be selected from among HIV retroviruses, e.g. HIV-1, HIV-2 or any different isolate of these two types, or for example from among CAEV (Caprine Arthritis Encephalitis Virus), EIAV (Equine Infectious Anemia Virus), VISNA, SIV (Simian Immunodeficiency Virus), or FIV (Feline Immunodeficiency Virus).

A particularly advantageous vector according to the invention is the vector characterized by the polynucleotide being a DNA sequence comprising the cis-active region of central initiation (cPPT) and the termination region (CTS) of the genome of an HIV-1 retrovirus or any other lentivirus.

The central PPT sequence or cPPT is a relatively conserved sequence in lentiviruses and is identified by the presence of many purine residues, some of which are shown in Figure 11H. Mutations, even single-point mutations, in one of these regions can abolish their functional character related to the formation of triplex DNA structures.

The identification of cPPT sequences is facilitated by the fact that in lentiviruses, the central termination sequence is a repetition in the genome center of a polypurine sequence located at the upper (5') edge of the 3' LTR in all retroviruses. This cPPT sequence may be an exact repetition, as in HIV-1, or slightly modified in other lentiviruses (Figure 11H). The central termination sequence of CTS is characteristic in HIV-1 (Charneau et al, 1994). It is located a few hundred nucleotides downstream of the cPPT sequence. CTS sequences are found in other lentiviruses at a further 100 nucleotides (80 to 120 nucleotides) downstream of the CPPT sequence. The central termination sequence is likely to be in the 11A of the CTSE.

The CTS sequence of the EIAV lentivirus has recently been characterized (Scott R. Stetor et al., Biochemistry 1999, 38, P 3656-67). According to these authors, in EIAV the cPPT and CTS sequences are 5' AAC AAA GGG AGG GA 3' and 5' AAA AAA TTT TGT TTT TAC AAA ATC 3', respectively.

The preferred polynucleotides for use in the present invention include, for example, the sequences shown in Figure 11, and more specifically the sequences between the two cPPT and CTS regions, including the sequences of these regions.

If necessary, the nucleotide sequence including cPPT, the internal polynucleotide (i.e. binding the cPPT to the CTS sequence) and the CTS sequence may be mutated point-wise, or mutated by deletion or insertion of nucleotides.

The invention covers any mutated sequence for cPPT or CTS having at least 60% identity with the naturally homologous cis-active nucleotide sequence from which it originated.

Modifications of the nucleotide sequence of the PPT or cPPT or CTS regions can be introduced to construct the triplex DNA according to the invention.

The identity of the variants of the nucleotide sequence with respect to the so-called natural sequences is calculated strictly in relation to the individual cPPT or CTS and not to the nucleotide sequence of the complete triplex DNA.

The region between the cPPT and the CTS is a polynucleotide that can be either the one found in the original retroviral genome between the CTS and the PPT or be different from it provided that the triplex DNA retains its properties in the nuclear import of the polynucleotide to bring the nucleotide sequence of interest into the nucleus.

The polynucleotide described in the application may be introduced into a replicating or non-replicating vector, which in the case of a retroviral vector is a non-replicating type vector.

For the preparation of large quantities of retroviral vector particles, adenoviral-type vectors may be used in which the polynucleotide corresponding to the retroviral genome containing the triplex DNA sequences and those of the gag, pol, and env genes has been introduced.

These adenoviral vectors may eventually be made replicative by introducing a replication origin sequence.

In Figure 11G, the cPPT and CTS sequences of HIV-1 are framed.

In any case, mutated sequences will be used that retain the ability to form a DNA triplex when the genome is retranscribed into the target cell.

A recombinant vector according to a particular embodiment of the invention may therefore include all or part of the retroviral or retrotransposon LTR sequences, the PBS retroviral sites, and PPT 3'-terminal, the retroviral sequence necessary for encapsulation of the vector genome in the vector particle.

A particular vector according to the invention is the plasmid pTRIP.EGFP, registered at the CNCM (National Collection of Microorganism Cultures of the Institut Pasteur, France) on 15 April 1998 under number I-2005.

Another vector of the invention is the plasmid pTRIP.MEL-IRES-GFP, filed with the CNCM on 20 April 1999 under No. I-2185. This vector is the plasmid pTRIP.MEL-IRES-GFP, shown in Figure 14.

A particular recombinant vector of the invention is characterized in that gag, pol and env sequences are also derived from sequences of a lentivirus, particularly an HIV retrovirus, particularly HIV-1 or HIV-2.

According to another embodiment of the invention, the gag and pol sequences are derived from an HIV retrovirus and the env sequence is derived from a retrovirus distinct from HIV or a virus, e.g. vesicular somatitis virus (VSV).

In general and depending on the expression of the transgene being sought, one will choose to use an env coding sequence for env polypeptides that are amphotropic relative to the host in which the transgene is to be expressed, or on the contrary one will choose env coding sequences for env polypeptides that are ecotropic.

The application describes recombinant vector particles comprising a recombinant nucleotide sequence comprising a specific nucleotide sequence (transgene or sequence of interest), controlled by transcription and expression regulatory signals, retroscription, expression and encapsulation regulatory signals and a polynucleotide with a central cis-active initiation region (cPPT) and a cis-active termination region (CTS).

It also discloses recombinant vector particles comprising a recombinant nucleotide sequence containing a determined nucleotide sequence (transgene) under the control of regulatory signals for transcription and expression, regulatory signals for retrotranscription, expression and encapsulation of retrotransposon and a polynucleotide with a central cis-active initiation region (cPPT) and a cis-active termination region (CTS), these regions being derived from a retrotransposon and placed in a functional orientation with the regulatory signals for retrotransposon.

In addition, recombinant vector particles are also described including: (a) a GAG polypeptide corresponding to the nucleoproteins of a retrotransposon or functional derived polypeptides, (b) a POL polypeptide corresponding to the RT, PRO, IN proteins of a retrotransposon or functional derived polypeptide, (c) transcription and expression regulatory signals of gag and pol sequences, (d) a recombinant nucleotide sequence comprising a determined nucleotide sequence (transgene), controlled by transcription and expression regulatory signals, a sequence containing transcription, expression and receptor transducer regulatory signals and a polynotide containing a central receptor-activated region (CTPR) and a receptor-activated region (CTSP), with these receptors located in a central receptor-activated region (CTPR) and a receptor-activated region (CTSP), with these receptors located in the receptors.

In addition, the application reports recombinant vector particles resulting from the expression: (a) a nucleotide sequence called gag sequence coding for the nucleoproteins of a retrotransposon or for functional derived polypeptides (GAG polypeptides), (b) a nucleotide sequence called pol sequence coding for the RT, PRO and IN protein of a retransposon or for a functional derived polypeptide (POL polypeptide), (c) transcription and expression regulatory signals of gag and pol sequences, these particles comprising a recombinant nucleotide sequence comprising a specific nucleotide sequence (transgene), placed under the control of transcription and expression regulatory signals, a signal regulatory sequence containing a central receptor and receptor-receptor region (CTPR) with a cis- and cis-transcription and expression-controlled region (CTS) and a cyclic receptor-receptor region (CTS) containing a central receptor and receptor-receptor.

The application also describes recombinant retroviral-like particles including: (a) a polynucleotide containing a central initiation cis-active region (cPPT) and a termination cis-active region (CTS), these regions being derived from a retrotransposon and placed in a functional orientation with the regulatory signals of the retransposon, (b) a polypeptide corresponding to the nucleoproteins of a retransposon or to functional derived polypeptides (GAG polypeptides), (c) a polynucleotide corresponding to proteins RT, PRO, IN of a retrosposon or a derived polypeptide (polypeptide), (d) a polypeptide of a viral envelope, (e) a recombinant nucleotide sequence containing a sequence of regulatory or transcriptional signals (including transcriptional, regulatory, regulatory and regulatory sequence), and (d) a sequence of nucleotides containing a sequence of transcriptional, regulatory and regulatory functions (including transcriptional, regulatory and regulatory, regulatory and regulatory of transcriptional, regulatory and regulatory and regulatory of expression of signals).

The invention relates, for example, to a recombinant vector as defined above, in which the regulatory signals for retranscription, expression and encapsulation and the polynucleotide comprising the cPPT and CTS regions are derived from a retrotransposon, e.g. a yeast retrotransposon.

In general, regulatory signals for transcription and expression of the transgene or sequences coding for polypeptides of vector particle structure, when not of retroviral or retroviral-like origin, are advantageously inducible or conditional signals, which may lead to tissue-specific expression.

The invention also includes recombinant cells characterized by recombination with a vector according to one of the above definitions.

The cells can be transfected in a transient manner or, on the contrary, in a stable manner, either as encapsulation cells or as target cells, especially cells in which a therapeutic effect is sought by transgene expression.

Interestingly enough, recombinant cells capable of expressing the transgene through transduction using a vector of the invention are differentiated non-mitotic eukaryotic cells.

The invention, however, also allows the preparation of recombinant non-mitotic primary eukaryotic cells, or mitotic cells.

Examples include lung cells, brain cells, epithelial cells, astrocytes, microglia, oligodendrocytes, neurons, muscle cells, liver cells, dendritic cells, neurons, bone marrow cells, macrophages, fibroblasts, lymphocytes and blood cells.

The invention therefore concerns therapeutic compositions, characterised by the inclusion of a vector as described above, or a recombinant cell as defined above.

The invention also relates to an immunogenic composition including a vector as described above or recombinant cells as defined above, which is likely to lead to an immune, cellular or humoral response in a particular host.

The application describes a polynucleotide as defined above, comprising retroviral or retroviral-like cPPT and CTS regions, and gives access to its use for the nuclear import, in particular ex vivo into specific cells, of a nucleotide (transgene) sequence.

In addition, the polynucleotide as defined above is associated with a nucleotide sequence of interest or a transgene.

Finally, the application discloses the use of a polynucleotide with a central initiation cis-active region (cPPT) and a termination cis-active region (CTS), these regions being of retroviral or retroviral-like origin, for the transfection or transduction of eukaryotic cells with a transgene or polynucleotide of interest.

It shall also report on the use of a recombinant vector or polynucleotide as defined in the application for in vivo transduction.

Other features and advantages of the invention are shown in the following examples and figures.

The legend of the figures The following table shows the results of the analysis:

Lentiviral genome retroscription differs from oncogene retroviruses in that the strand+ is synthesized in two separate halves. An upstream segment is initiated at a central copy of the polypurine tract (cPPT), characteristic of lentiviral genomes. Upstream strand+ synthesis ends after a discrete strand movement to the center of the genome. Blocking strand movement by reverse transcriptase is governed by a cis-active sequence to be separated from the HIV genome: the Central Termination Sequence (CTS). The final product of lentivirus retroscription is linear DNA with a central three-strand (central triplex) DNA structure about a hundred nucleotides long.

FIG. 2: Plasmids used for the production of HIV vector particles

The vector particles are produced by cotransfection of three plasmids: the vector plasmid comprising (pTRIP) or non-vector plasmid (pHR) cis-active sequences responsible for triplex formation, an encapsulation plasmid providing trans structural proteins and enzymes of the particle (pCMVΔR8.2 or pCMVΔR8.91 , Naldini et al, 1996 and Zufferey et al, 1997), and a VSV virus envelope expression plasmid (VSV-G).

Only the relevant parts of the co-transfected plasmids in Hela cells are presented (Naldini et al PNAS oct 1996, Zufferey et al Nature Biotech. 1997).

The pCMVΔR8.2 or pCMVΔ8.91 encapsulation plasmids allow expression of the proteins from gag and pol.

The pHR-TRIP vector plasmids are derived from the pHR'CMVlacZ plasmid (Naldini et al): a wild or mutated triplex sequence has been inserted and the reporting lacZ gene changed or not to EGFP.

Fig. 3 : Impact of the triplex on the transduction of EGFP in Hela cells

Hela cells, grown in 8-chamber Labtek, are transduced by different vectors expressing the autofluorescent EGFP protein. Infections are normalized by the amount of capsid protein (Dupont ELISA P24 kit) at 2 ng of P24 per inoculum. 48 hours post-infection, the cells are attached to the 1% PFA PBS, mounted in mowiol, and then viewed under a fluorescence microscope. Three independent fields are shown for the original vector without triplex (HR.EGFP, top), for the vector with triplex (TRIP.FPEG, bottom), or for a vector containing a mutated, non-triplex sequence (TRIP D.FPEG, right). A. The different transcripts are shown in the presence of transcriptapine, a transcription factor that inhibits the function of HIV-1.

Fig. 4 Quantification of the rate of transduction of the EGFP gene by HIV vectors with or without triplex

Hela cells transduced by 2 ng P24 of EGFP vectors with or without triplex are trypsinated 48 hours post-infection. Percentages of EGFP-expressing positive cells are calculated by flow cytometry (FITC channel). In all cases, transduction is inhibited in the presence of nevirapine, an HIV-1 retrotranscriptase inhibitor. In Figure 4C, the presence of triplex DNA is observed in the vector stimulated by GFP transduction (or other gene of interest) in mitosis or non-mitotic cells. This transduction is multiplied by a factor of 20 compared to results obtained with non-triplex sequence vectors (e.g., Nald et al., 1996).

Fig. 5 Quantification of the rate of transduction of the LacZ gene by HIV vectors with or without triplex

The impact of triplex on transduction is calculated by infection of Hela cells, cultured in 96-well plates, by different vectors expressing the LacZ reporter gene. 48 hours post-infection, the culture plates are lysed and beta-Galactosidase activity is measured using a luminescent reaction kit (Boehringer). Each transduction is performed in triplicate with a normalised innoculum at 2 ng of P24.

Upper panel: Hela cells are proliferating.

Bottom panel: Hela cells blocked in the cycle by aphidicoline.

The transduction of the LacZ gene is multiplied by a factor of 6 with a vector containing a triplex sequence versus a vector without a triplex sequence.

Fig. 6a and b: Impact of triplex on ex vivo transduction of the EGFP gene in rat primary spinal cells

Primary explant cells of rat spinal cord are infected with 300 ng of P24 for each vector with and without triplex, and observed under fluorescence microscopy as before.

Fig. 7: Impact of triplex on the in vivo transduction of the EGFP and luciferase genes in rat brains FIG 7-a-1: Transduction at the injection site

The EGFP gene transfer is performed by direct injection into the striatum of the rat brain of 2 microlitres of vector corresponding to 50 ng of P24.

Observation of the cuts in fluorescence microscopy shows a high transduction of EGFP in the presence of the triplex (left panel), and very low without (right panel). Figure 7-a-2: another representative section of the experiment described above.Figure 7-b: Quantification of the impact of the triplex on in vivo transduction in the brain.Figure 7-b-1: Impact of the triplex on the transduction of the luciferase-coding gene in Hela cells in vitro.The graph shows the production of luciferase quantified by luminescence measurement (Promega kit ®).The presence of the triplex in the vector results in an increase in the transduction of the luciferase gene, by a rat factor 8.Figure 7-b-2: In vivo quantification of luciferase activity in the brains of vectors important for luciferase, with or without triplex.The presence of a luciferase-coding stimulator in mice or StratFIGNAD: 7-FIGNAD-3: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: 8-FIGNAD: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In vivo: In the number of the number of the number of the number of the number of the number of the number of the number of the number of the number of the

A quantitative test to monitor retroscription kinetics, nuclear import and the integration or circulation of vector DNA into transcribed cells has been developed, which is an advantageous substitute for PCR amplification detection of two-LTR rings, markers of the nuclear import of viral DNA into the nucleus of the infected cell (Bukrinsky et al., Nature 1993, 365, pp. 666-669).This probe reacts with different fragments: the 0.77 Kb internal fragment, common to all vector DNA forms, and whose quantification in the phosphorimager indicates the total amount of retranscribed vector DNA, a 1.16 Kb distal fragment specifically indicating the amount of unintegrated linear DNA. After further digestion by the Xhol enzyme, the circles with one and two LTRs appear at 1.4 Kb and 2 Kb respectively.In the case of a nuclear import defect, the expected profile of vector DNA in transduced cells is an accumulation of unintegrated linear DNA. Conversely, if the linear vector DNA enters the nuclear compartment of the cell, most of the linear DNA is integrated into the cell chromatin and circulates.

Fig. 9a: Analysis of the nuclear import rate of the vector DNA.

The southern blot analysis, 48 hours post-transduction in Hela cells, shows a nuclear import defect typical in the case of the triplex-free vector (HR GFP) or containing the triplex sequence in the inverse, non-functional orientation (TRIP.GFP). In the case of these vectors, the signal corresponding to the unintegrated linear DNA is equivalent to the total DNA signal, indicating that the bulk of the vector DNA remains in linear form instead of integrating. In the case of the TRIP.GFP vector, the signal intensity corresponding to the triplex-free DNA is much lower than the total signal, indicating that a significant fraction of the vector DNA has been imported into the vector and is not integrated into the DNA.

Quantification with the Southern blot phosphorimager shown in Figure 9b shows that 48 hours post-transduction, the majority of the DNA of triplex-free vectors is as unintegrated linear DNA, few vector DNAs integrate or circulate. The triplex-free vectors (HR-GFP and TRIPinv-GFP) show a typical nuclear import defect. Conversely, in the case of the TRIP-GFP vector, more than 60% of the DNA is integrated into the transome of the generated cell and little vector DNA remains as unintegrated linear DNA. The introduction of the triplex sequence into the vector has completely completed the nuclear import vector HR-GFP to a level of nuclear import defect.The vector DNA profile obtained in the TRIP-GFP vector is comparable to that of a wild HIV-1 virus. This result demonstrates that the triplex sequence is the only nuclear import determinant missing from the HR-GFP construct. Only the integrated form of the vector DNA is active. 11A - 11F: Polynucleotide sequence including the cPPT and CTS regions of CAEV, EIAV, VISNA, SIVAGM, HIV-2ROD, HIV-1LAI viruses.Figure 11G represents the triplex DNA sequence of HIV1. The cis, cPPT and CTS active regions are delineated and printed in bold.Figure 11H shows the alignment of the cPPT and PPT3' sequences in some lentiviruses. The upper line corresponds to the PPT 3' sequence present in all retroviruses upstream of LTR 3. The lower line corresponds to the internal repetition of the so-called cPPT sequence in lentiviruses.

The following table shows the data:

Figure 12 shows the production of CTL in vitro from human dendritic cells transduced by the triplex vector with a melanoma CTL polypeptide of interest as the gene, which consists of epitopes with sequences described in Figure 15.

These dendritic cells are brought into contact with mononuclear cells (PBLo). CTL activity is measured after restimulation by the corresponding antigenic peptides.

On the axis of the abscissa, the ratio of effector cells to target cells is shown. Figure 13: Cytotoxic response after immunisation of mice with the TRIP.MEL-IRES-GFP vector

The strain of E. coli containing the pTRIP-MEL-IRES-GFP vector was registered with the CNCM on 20 April 1999 and has the number of accession I-2185. FIGURE 15: Sequences of melanoma specific HLA A2.1 CTLs epitopes included in the polyepitope construction of the TRIP.MEL-IRES-GFP vector. The purpose of highlighting the polyepitope sequences is to individualize each epitope.FIGURE 16: Very high efficiency transduction of CD34+ stem cells by triplex HIV vectors.

FACS flow cytometry analysis of transduction of the GFP gene in CD34+ hematopoietic stem cells by the TRIP-GFP vector. The percentage of CD34+ cells transduced by the TRIP-GFP vector is greater than 85%. This efficiency is significantly higher than the transduction rates previously obtained with a triplex-free HIV vector (HR-GFP) in CD34+ cells (Miyoshi H et al., Science 1999, 283, p 682-6).

The Commission has already adopted a proposal for a directive on the approximation of the laws of the Member States relating to the labelling of foodstuffs. Construction of the vector plasmids:

The pTRIP-LacZ and pTRIP-EGFP plasmids are derived from the pHR'CMVlacZ construction (Naldini et al, 1996).The LacZ reporter gene of pHR'CMVlacZ was replaced by the ORF of the autofluorescent EGFP protein.The EGFP gene was amplified by PCR from the pEGFP-N1 plasmid (Clontech) using the faithful thermostable polymerase Pfu (Stratagene).

The sequences of PCR initials used are as follows: The Commission shall be assisted by the European Parliament and the Council in the preparation of the annual report.

The PCR amplification was carried out in 30 cycles under the following conditions: The test chemical is used to determine the concentration of the test chemical in the test medium.

The BamHI and XhoI restriction sites were added at 5' and 3' respectively of the EGFP PCR fragment to insert it in an oriented manner into the pHR'CMV vector fragment, which was itself digested by BamHI and Xhol.

A 184 bp fragment from the central region of the HIV-1 genome, including the cis-active cPPT and CTS boxes, which are responsible for the formation of the triplex during HIV retranscription, was inserted into the ClaI site of the pHR-EGFP and pHR'CMVlacZ plasmids, upstream of the CMV promoter. The central triplex region was amplified by PCR from full proviral plasmids of the HIV-1 LAI genome including the wild triplex sequence (pBRU3; Chameau et al, 1991), mutated in the CTS termination cis-active sequence (pCTS; Chameau et al, 1994) or mutated in the central initiation cis-active sequence (cPPT225p; Chameau et al, 1992).

The sequences of PCR initials used are as follows: The Commission shall be assisted by the Member States in the preparation of the annual report on the implementation of the programme.

The PCR reaction conditions were identical to those described above.

The Triplex PCR fragments, digested by Narl, were inserted into the ClaI site of the pHR GFP and pHR'CMV LacZ plasmids by competitive ligation/digestion T4 DNA ligase/ClaI to eliminate the recirculating vector on itself during the ligation.

The resulting plasmids are called pTRIP.EGFP in the correct orientation of the triplex and pTRIPinv.EGFP in the reverse, non-functional orientation. Vectors containing a mutated version of the triplex are called pTRIP X EGFP, X corresponding to the code of the original mutant virus (AG, D, CTS or 225) (Charneau et al. J. Mol. Biol. 1994, Charneau et al. J. Virol 1992). From the different plasmids pTRIP.EGFP or pTRIP X EGFP, the EGFP gene was replaced by Lac by XhoI/BamHI oriented exchange. The resulting plasmids are called pTRIP.Z, pTRIP.Z, pTRIP.Z, pTRIP.Z, pTRZZZ, respectively.

Construction of HR luc and TRIP luc vectors

The BamHI-EGFP-XhoI fragment of the RH GFP and TRIP GFP vectors was replaced by the BamHI-Luc-XhoI fragment of the pGEM-luc plasmid (Promega) coding for luciferase.

Production of non-infectious vector particles

The production of HIV vectors was carried out according to a modification of the protocol described in Naldini et al. 1996 The vector particles were produced by transient calcium phosphate co-transfection of human 293T cells (ATCC), cultured in DMEM (ICN), SVF 10%, penicillin, streptomycin. The test chemical is a test chemical that is used to test the presence of a specific type of virus, such as a virus that causes a disease, such as vesicular stomatitis virus (VSV), 15μg of vesicular stomatitis virus (VSV) envelope coding plasmid, pMD.G (Naldini et al, 1996) 30μg of encapsulation plasmid, pCMVΔR8.2 (Naldini et al, 1996) or pCMVΔR8.91 (Zufferey et al, 1997).and 30μg of various pHR or pTRIP vector plasmids.

The co-precipitates calcium-phosphate/DNA were left in contact with the cells for 24 hours, the medium was then collected every 24 hours until day 3 post-transfection. Cell debris from the vector surfactants was removed by low-speed centrifugation. The vector surfactants were preserved by freezing at -80°C.

Particulate vector concentration

The use of the highly stable VSV-G envelope to pseudotyping the vector particles allows their concentration by ultracentrifugation. The vector supernatants, collected by the method described above, were ultracentrifuged in conical 30 ml (Beckman) tube bottoms, 90 min at 17000 rpm at +4°C in a SW 28 (Beckman) rotor. The coils were then taken up in 190†μl of PBS, centrifuged for 5 min at 2000 rpm to remove the non-resuspensible debris, aliquoted and frozen at -80°C.

Transduction of cells into culture

HeLa cells (ATCC) were transduced by addition of vector surfactants, ultracentrifuged or not, in the presence of 10 μg/ml Dextran DEAE. Hela cells were grown in DMEM (ICN) medium supplemented with 10% fetal calf serum (FCS). HeLa cells were spread to 20,000 cells/well in 96 well plate the day before infection and then transduced to a final volume of 200μl. Vector inoculum were normalized according to capsid protein concentration (P24), calculated using a commercial ELISA test (DuPont). Gene transfer efficiency was measured, based on 24 to 48 hours post-infection experiments.The transduction rate of vectors expressing the LacZ reporter gene was detected either by XGaI staining in situ (Charneau et al, 1992) or by a light-metric reaction according to a commercial kit (Boehringer) following the instructions of the supplier. In the case of vectors expressing the EGFP reporter gene, the transduction rate was qualitatively evaluated by direct observation of living cells under a fluorescence microscope on the FITC channel. The quantification of the number of cells expressing the EGFP marker was achieved by flow cytometry (FITC channel).The 96-well culture plates were rinsed twice with PBS and then lysed with 100μl of PBS 1% NP40. The EGFP fluorescence was read using a plate fluorimeter (Victor, Wallac) with an excitation filter at 475 nm and an emission filter at 510 nm.

Hela cells that had been stopped in their G1/S transition cell cycle were prepared under the same conditions as before with 24 hours prior treatment with 4 μM aphidicoline (Sigma) transduction, under which conditions more than 95% of the incorporation of tritiated thymidine was inhibited.

Ex vivo transduction of primary cells

Primary cultures of rat spinal cord cells were prepared as follows: marrow from 13-14 day old rat embryos were dissected under binoculars. Tissues were maintained in L15 medium (Gibco) supplemented with 3.6 mg/ml glucose during all stages. Nerve cells were dissociated by incubation in trypsin (0.05% w/v) for 15 min at 37°C. Trypsic digestion was inhibited by the addition of 10% foetal veal serum (SVF) and low-speed centrifugation. Cell cultures were collected from L15 medium (Gibco) containing 3.6 mg/ml of glucose at 100 μg/ml (Nasehel) by mechanical agitation.

The spinal cells were seeded in 24-well plates containing glass lamellae 12 mn in diameter and coated with poly-DL-ornithine (6 μg/ml) and laminin (3 μg/ml). The cell cultures were maintained in neurobasal medium (Gibco) containing supplement B27, 2% SVF, 0.5 mM L-glutamine, 25 μM beta-mercaptoethanol and 25 μM L-glutamate. The cultures were treated after 24 hours with 10 μg/ml 5' fluoride oxide to prevent colonisation of the culture by non-neuronal cells.

In vivo transduction of EGFP in rat brain

Vectors expressing the EGFP marker protein were used in in vivo experiments.

Injections into the rat brain with 2μl of HR.EGFP or TRIP.EGFP vector were performed on 5-week-old OFA spage dawley rats. Initially, the rats were put to sleep by intraperiotonal injection of Image 500 (Rhône Merieux). The injections were performed into the striatum of each hemisphere using a stereotactic guide, with a 5μl Hamilton needle, at the rate of 2μl/5min. The rats were sacrificed a week or more after injection, by infusion with PBS and then with 2% paraformaldehyde (PFA). The brains were then removed and kept to contain only the part of the injection site, which was removed by visible lesion. A post-exposure scan was performed in the PFA microscope.

In vitro and in vivo comparison of HR luc and TRIP luc vectors

HeLa cells were spread out one day before transduction to 20000 cells per well in 96 well plates. Transductions were performed with the same amount of vector particles, standardized on the p24 content of the preparations: 1 ng of p24 per well, in triplicate, in the presence of 10μg/ml of DEAE-dextran. Two days after transduction, luciferase activity was measured using a Promega kit (following the manufacturer's instructions) and a Wallac microplate measuring apparatus (Vic).

Vector injections into the striatum of OFA spage dawley rats and C57B6 mice were performed. 2 μl of a preparation of HR luc or TRIP luc containing 25 ng of p24 was injected (n=4). The animals were slaughtered 4 days later, striata were collected and luciferase activity measured by the same technique as before, measuring in parallel the total protein (Pierce kit).

Examples 1 Basic aspects: nuclear import of the pre-integration complex HIV-1: the role of the central triplex

The mechanism of HIV retroscription differs from that of oncogene retroviruses in that the synthesis of the plus strand (brin +) in two distinct halves (Figure 1). An upstream segment is initiated at a central copy of the polypurine tract (cPPT), characteristic of lentiviral genomes. Upstream strand synthesis ends after a discrete strand displacement to the center of the genome. Blocking strand displacement by reverse transcriptase is governed by a new active cis-sequence of the HIV genome: the CTS (Central Termination Sequence).

Analysis of the replication defect of the initiation and central termination mutants showed that the replication cycle of the initiation or central termination mutants of retroscription aborted at a stage after viral DNA synthesis and after the routing of the retroscription complex to the nuclear envelope. When the structure of the viral DNA present in infected cells is analysed, it is found that the phenotypes of the initiation and termination mutants are similar. In both cases, the overall amount of retroscript DNA is not affected by mutations in the cPPT or CTS.Nucleus/cytoplasm splitting and nucleus permeabilisation experiments subsequently showed that these linear DNA molecules are associated with the nucleus, but that their integration and/or circularisation can only occur after the nuclear envelope has dissolved, which indicates that the viral DNA of mutants is retained outside this envelope. Furthermore, nuclease attack experiments of nucleases purified from cells infected by DNasel immobilized on gold bullet deposition still show the accumulation of mutant linseed against the cytoplasmic DNA of the nuclear membrane.Finally, precise quantification of the integrative capacity of linear DNA molecules with or without a native central triplex has recently shown that the central triplex does not influence the integration of linear DNA into a heterologous DNA target in vitro.

The replicative failure of mutant viruses to initiate or terminate central retroscription is therefore related to the nuclear import of their pre-integration complex, and more specifically the translocation step through the nuclear pores. Lentiviruses, and HIV in particular, have developed an original retroscription strategy that aims to create the triplex at the center of the unintegrated DNA molecules, which is essential for the entry of the viral genome into the nucleus of an interphase cell. This mechanism distinguishes lentiviruses from all other retroviruses whose access to the DNA to the integration site depends on the disorganization of the nuclear membrane during mitosis.

2. Generation of lentiviral vectors containing the cis-active sequences responsible for the formation of the triplex 2.1 Principle and interest of lentiviral vectors

The generation of effective lentiviral vectors requires knowledge of the determinants responsible for active nuclear import and therefore for infection of non-mitotic cells.

The discovery of the involvement of the triplex in the nuclear import of the HIV-1 genome has important implications for the construction of efficient lentiviral vectors. It involves the conservation, within the vector construction, of cis-active sequences responsible for the formation of the triplex DNA during lentiviral retroscription. The application in vectorology of this fundamental work is to join the central cPPT-CTS region in the lentiviral vector constructions so as to create the triplex DNA structure during the retroscription of the vector genome.These vectors are replacement vectors, i.e. the entire retroviral genome is deleted and then replaced, between the two LTRs and the encapsulation sequence, by the reporter gene or the gene of therapeutic interest (Miller et al, 89). According to the inventors, this type of construction is not optimal for lentiviral vectors because of the need for the central cPPT-CTS region for the nuclear import of viral DNA. However, HIV vectors constructed on the same principle as retroviral vectors derived from one but pseudotyped by the highly concentrated stomatogenic envelope of the vesicle virus (SGV) and ultrasound,Err1:Expecting ',' delimiter: line 1 column 566 (char 565)

Err1:Expecting ',' delimiter: line 1 column 92 (char 91)

In this system (Figure 2), the HIV vector particles are produced by transient co-transfection of three plasmids: an encapsulation plasmid expressing all viral proteins except the HIV envelope, a plasmid expressing the VSV-G envelope and a vector plasmid, pHR-CMV LacZ, comprising the first HIV PCTRs, the bi-vital signal of HIV encapsulation and a lacFPZ expression cassette. Over time, the triple-gen was replaced by a fluorescent gene coding for a highly reversible version of the vector (EGPTPE), responsible for the correct insertion of the protein into the central region of the cell, and the C-GREPTPE gene was amplified in vivo.

2.3. Rapid and sensitive test for helper virus in lentiviral vector preparations: Err1:Expecting ',' delimiter: line 1 column 87 (char 86)

The production of vector particles from three independent plasmids with a minimum of homologous sequences minimizes the likelihood of generating a helper virus capable of replication. In addition, the encapsulation construction was deleted for the HIV envelope and for all so-called replication-auxiliary genes (Vif, Vpr, Vpu, Nef). The encapsulated vector genome contained only 2 LTRs, the sequences needed for encapsulation and the triplex sequence. However, each vector stock was tested for the presence of infectious helper viruses. MT4 cells were infected in triplicate,The test was performed with a micro-plaque overnight, then washed extensively and re-cultured for 5 days to amplify the innoculum. P4 indicator cells (HeLa CD4 LTR-LacZ) were then infected with MT4 cells and supernatated for 3 days to detect the infectious particles produced. Finally, X-gal in situ staining was performed. In this way, any infectious particle produced is detectable as a blue syncitia. This protocol allows the detection of an innoculum of HIV of 0.25 pg of P24, about 3200 physical particles.If the virus is infectious, or even in 10,000, the protocol should probably detect a single infectious particle.

The vector supernatants are consistently free of HIV-infecting particles.

Effect of triplex on the efficiency of transduction by vectors in vitro

In a first step (Figure 3), the effect of the insertion of the central triplex on HeLa cell transduction was measured. HeLa cells were infected with a transfection surgeon of the wild central triplex vector (WTPTP GFP), a vector without this sequence (HR GFP), or with a mutant triplex sequence (WTP GFP D). Mutant D is a mutant of the cPPT that prevents central initiation of the + strand and thus the formation of the central triplex (Figure 3).

GFP transduction in Hela cells was increased in the presence of a wild triplex and was reduced to baseline in the presence of a non-functional triplex.

This title gain can be quantified by using the reporting gene LacZ (Figure 4). These cells were transduced into triplicate by normalizing by reference to the amount of p24 protein. The transductions were performed on cells blocked or not dividing with aphidicoline, which blocks cells in G1/S. The vectors used were HRZ (without triplex), TRIP Z (with triplex), TRIP Z (the triplex sequence is inverted, it is non-functional and does not lead to the formation of a central triplex).Figure 4A: The transduction gain of β by vectors containing triplex is 6 to 10 times. It is lost when triplex is not formed (TRIPFZ).

In addition, the same results are obtained in HeLa cells, whether or not the encapsulation plasmid used in the production of the vector particles is deleted in the accessory genes Vif, Vpr, Vpu, Nef.

Effect of triplex on the efficiency of transduction by ex vivo vectors

The impact of triplex on EGFP transduction in non-mitotic primary cells was subsequently measured. Primary rat spinal cord explants enriched with neurons were transduced with ultracentrifugal vector supernatants. Transductions were performed with less than 10μl of ultracentrifugal vector containing the same number of particles, normalised on the number of ng of p24 capsid protein.

Figure 6: The vector with a triplex sequence translates a much larger number of rat spinal cord primary explant cells than the vector without a triplex.

2.6. Impact of triplex on in vivo transduction in the brain

The effect of triplex on EGFP transduction in vivo was then measured by direct injection into the rat brain. The same volume (2μl) of triplex- or triplex-free vector surgeon with the same amount of p24 protein was injected into the striatum. While a large number of transduced cells were detected in triplex-injected rats (Figure 7a), few EGFP-expressing cells were detected in the brains of triplex-free rats at the exact injection site, visible from the needle lesion.

In Figure 7b, the construction of HIV vectors (with or without triplex DNA sequence) expressing the luciferase reporter gene (HR Luc and TRIP.Luc) allowed precise quantification of the impact on gene transduction in the brain. In vitro a factor 8 increase was observed in HeLa cells (Figure 7-b1). A similar benefit was obtained after direct in vivo injection into the striatum of rat (Figure 7-b2) or mouse (Figure 7-b3).

2.7.Impact of trip/ex on nuclear import of the vector genome

A test to track over time all the vector DNA forms in the transduced cell has been developed by the inventors: linear DNA, circles with 1 or 2 LTRs but also integrated provirus. This test is based on Southern's detection of viral DNA using a cutting and selection strategy to differentiate between the different forms of retroviral DNA (see Figure 8).The internal fragment will allow the total vector DNA present in cells to be calculated after quantification by the Phosphorimager. A 1.16 Kb fragment corresponds to the distal fragment of the unintegrated linear DNA, another 3.3 Kb corresponds to the unintegrated circles. After quantification of the signals with a phosphorimager, the nuclear import rate is indicated by the percentage of viral DNA in integrated and circular form (viral nuclear DNA) relative to cyclastic DNA.The first preliminary blots showed an intracellular DNA profile characteristic of a nuclear import defect in the case of triplex-free vectors or those whose central region of the HIV-1 genome was inserted upside down. Indeed, the signal intensity corresponding to the linear DNA was equivalent to that of the total DNA signal, 48 hours after infection. In other words, the processing of the vector DNA is mostly blocked at the non-integrated linear stage, very few molecules integrate (Figure 9).indicating that most of them were imported into the transduced cell nucleus and then integrated.

2.8. Study of the effect of the position of the DNA triplex on vector construction

In all lentiviruses, the cis-active sequences cPPT and CTS are found, which are responsible for the formation of the triplex during retroscription. In all cases, this triplex is located a few nucleotides away from the center of the linear DNA genome. This central position of the triplex could be important for the optimal functioning of this determinant of translocation through the nuclear pore.Err1:Expecting ',' delimiter: line 1 column 354 (char 353)In order to test this hypothesis, the inventors set out to clone, instead of the reporting genes, a bank of randomly sized fragments (partial digestion Sau3A), the size distribution of the cloned fragments being analysed before and after transduction of target cells. In the case where the central position of the triplex would be important for its function, the constraint to build a vector that is symmetrical with respect to the triplex would be significant.This is the same as the triplex.

2.9. Transfer in vivo to different differentiated tissues

Err1:Expecting ',' delimiter: line 1 column 80 (char 79)

2.10. High efficiency gene transfer in haematopoietic stem cells by triplex HIV vectors.

Hematopoietic stem cells are major targets for the treatment of a large number of blood, muscle or neurological genetic disorders and infectious diseases. The major difficulty for gene transfer by oncovirus-derived retroviral vectors such as MoMLV into these cells is that they divide very little and that induction of mitosis by cytokine therapy usually results in a loss of their purifying totipotency. In Figure 16, the results of transduction of the GFP gene into CD34 stem cells by TRIP-GFP vector show GFP expression in more than 85% of cells. The efficiency of transduction of CD34 stem cells by CD34-GFP vector is very low, as their ability to purify and clot is intact (Miyashi, 1998, p. 682-63, HRG, H. 283).

2.11 Use of triplex sequence lentiviral vectors for embryonic cell transduction; Application to the construction of transgenic animals or modified cell lines.

Retroviral vectors are potentially interesting tools for the construction of transgenic animals via egg transduction (Rubenstein et al., 1986, PNAS, 83, p 366-368) or ES cells (Friedrich and Soriano, 1991, Genes Dev. 5, p 1513-1523). The use of lentiviral vectors is likely to increase the transduction efficiency of these totipotent cells. Our preliminary results of transduction of mouse embryonic cells by the TRIP-GFP vector show a high transfer efficiency of the GFP gene but also a complete extinction of transcription of the GFP transgene. Certain viral sequences in particular the primary binding site (PBS) are specifically constructed to interfere with this extinction. These vectors are suspected to be based on the AOL, AOL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, COL, C

2.12.Immunogenic composition for prophylactic and/or therapeutic uses:

A new immunization strategy is triplex lentivirus vectors.

The first part of the book

Err1:Expecting ',' delimiter: line 1 column 947 (char 946)

Initial study: comparison of different vaccine strategies

Using HHD mice, different vaccine strategies were previously compared. (iv) By selecting 5 tumour epitopes randomly, the inventors compared five clinically applicable immunization strategies: (i) synthetic peptides with Freund's incomplete adjuvant, (ii) lipopeptides, (iii) recombinant yeast Ty particles in the mice, independently the epitopes were fused in C-terminal to the P1 protein. (iv) intramuscular administration of recombinant DNA coding for hepatitis B virus glycoprotein to the epitopes in its pre-S2 portion, (v) simultaneous injection of polycytic cell-laden cytoproteins into the cells after in vitro differentiation and differentiation of the cell structures from the S2 or S3 epitopes. (Type 10A) A response of more than 25 million cytoproteins was observed in all the cells, with a potential for diffusion of cytoproteins from the S2 or S3 epitopes (Type 10A) or from the S4 or S5 epitopes (Type 95/11), which were detected in the test paper, showing a response of more than 10 million cytoproteins per unit of epitopes (Typ 10A) or 10 million copies per unit of epitopes per unit of epitopes (Typ 10A) (Typ B) (Typ 95/11307/95) (Typ 95/115)).

Ty or bare DNA particulate antigens have been shown to be effective strategies for inducing cytotoxic responses. However, large-scale production of Ty particles is difficult. In addition, there is concern that the introduction of hydrophobic and multiple epitopes into the pre-S2 segment of the hepatitis B virus glycoprotein does not result in a sharp reduction in the production of particles by CHO cells (the method of preparation of the current hepatitis vaccine). Recombinant lentiviral (HIV-1) vectors produced as pseudotypes have been extensively deleterious, but retain the triplex DNA sequence, the ability to cross the nuclear membrane of cells that are not dividing and potentially represent a new and more effective strategy compared to the above vaccine strategies.

Materials, methods and results Transgenic mice

HHD mice, express a monocatenary construction in which the peptide presentation domains (a1, a2) of the HLA-A2.1 molecule are covalently associated at the N-terminus with human β2-microglobulin. The a3 domain and intracytoplasmic portion of the HLA-A2.1 molecule are replaced by their H-2Db molecule equivalent (Pascolo S. et al. 1997). These mice allow the study of the immunogenicity of epitopial peptides and different vaccine strategies in a comparative manner.

Construction of the TRIP-MEL IRES GFP vector

The ECRI site of the ECRI site was filled with the DNA polymerase T4 creating the ECRI site, and a fragment containing a polyp and a melanocytes CTP was generated by a consensus polymer vector, the ECRI site was then cloned with the PCI site and the PCI site was acquired by the ECRI site and acquired by the ECRI site, and the ECRI site was acquired by the ECRI site, and the ECRI site was acquired by the ECRI site: 5G, 3G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 5G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6G, 6

Efficacy of in vitro transduction of dendritic cells (CD) by GFP lentiviral vectors with or without triplex

Murine CDs were obtained from the marrow of transgenic HHD mice in the presence of IL4 and GM-CSF. Human CDs were obtained from healthy donors of haplotype HLA-A2.1 (see below). These cells were transduced by LV vectors with or without triplex using different concentrations (75, 150, and 300ng-p24 lentiviral vector for 5,105 cells).

GFP expression in CDs was measured by FACS on days 2, 5 and 10. The mean fluorescence intensity values corresponding to cell transduction efficiency showed that triplex lentiviral vectors have a transduction capacity 5 to 7 times higher than human CDs compared to non-triplex lentiviral vectors.

Induction of primary CTL responses using human dendritic cells transduced by the TRIP-MEL-IRES-GFP vector

Immature human CDs were obtained from healthy donors with HLA-A2.1 haplotype in the presence of GM-CSF and IL13 (IDM, Paris, France).Immunophenotyping of these cells by monoclonal antibodies against CD1, CD4, HLA-ABC, HLA-DR, CD80, and CD86 showed their immaturity with a CD purity of over 91%.

The CDs obtained were transduced by the TRIP-MEL-IRES-GFP vector at a concentration of 100 ng p24/vector for 1,106 cells. The effectiveness of TRIP-MEL-IRES-GFP CD transduction was investigated by measuring GFP expression by FACS. The mononuclear cells (CMN) from the same donor were stimulated by the previously transduced CDs. After three stimulations, the cytotoxic activity of these cells was tested on T2 cells individually loaded with 4 epitope peptides using a 4-hour CTL test. The epitope peptides M-3, gp100.154, GnTV/NA17A, and Tyrosinase-36-D were selected from their previous immunity experiments.

Specific cytotoxic responses were observed against all the epitopes tested.

Direct immunisation of HD mice with the TRIP-MEL-IRES-GFP vector

HHD mice were immunized with 2.5 μ/p24 of the TRIP-MEL-IRES-GFP vector by subcutaneous (SC), intravenous (IV) and intraperitoneal (IP) mice. On day 11 of immunization, the splenic cells of each mouse were individually stimulated by melanoma epitope peptides for 6 days, including 2 days in the presence of 10% TCGF. The lytic activity of these cells was then tested on RMAS cells loaded with the corresponding peptides or on HeLa-HHD cells translated by TRIP-MEL-IRES-GFP vector.

The results obtained for each mouse are represented in terms of specific lysis on RMAS cells (Table 1) and on transduced Hela-HHD cells (Table 2). The best results were obtained after administration of the vector via both SC and IP routes in terms of both lysis and number of simultaneously induced responses in a given mouse.

Lyses spécifiques obtenues après immunisation de souris HHD par LV contenant un polyépitope de mélanome Stimulation in vitro pour chaque souris individuelle SC au jour 8 en présence de TCGF et de peptide
S.C.	1	25	4	13	8	54	17	17	4	4	10
2	44	1	5	11	89	10	11	4	3	6
3	39	0	19	21	81	29	26	6	4	0
I.V.
1	7	7	1	8	25	5	8	3	4	3
2	10	8	0	13	70	13	16	5	9	14
3	24	6	5	5	65	15	16	3	11	10
4	5	3	13	12	14	10	5	0	3	0
I.P.
1	30	10	2	3	57	9	6	4	3	2
2	63	8	7	17	72	11	19	9	7	7
3	21	7	8	16	72	14	32	0	7	7

S.C.	2	18	15	24	62	15	20	12	20	18
I.V.	8	10	15	23	50	14	29	10	10	18
I.P.	24	18	15	25	62	15	32	14	18	20

The Commission

The results demonstrate the ability of triplex lentiviral vectors to induce highly effective immune responses. Their immunogenic power has been shown not only in vitro on human dendritic cells but also evaluated in the HLA-A2.1 transgenic mouse model by different routes of administration. Notably, specific CTL responses were obtained for the ten CTL epitopes in the melanoma poly-epitope. Lysase percentages against various melanoma antigens are also higher than those obtained in the same HHD mice with other vaccine strategies such as lipopeptides, recombinant triplex or HBV pseudo-particle vaccination. As a result, vaccine strategies based on DNA or tumor vectors are applicable to a variety of pathologies.

The Bible is a book of wisdom.

Blomer U, Naldini L, Kafri T, Trono D, Verma IM, Gage FH. Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J Virol. 1997 Sep; 71(9): 6641-6649.Charneau P, F. Clavel. 1991. A single-stranded gap in human immunodeficiency virus unintegrated linear DNA defined by a central copy of the polypurine tract. J. Virol., vol. 65, N° 5, 2415-2421.Chameau P., Alizon M. and Clavel F. (1992). A second origin of plus strand synthesis is required for optimal HIV replication. J. Virol. 66: 2814-2820.Chameau P. G. Mirambeau, P. Paulo, S. Roux, S.Bucus, H.B. F. 1994.The following is a list of the most commonly used genes for the treatment of cystic fibrosis: Hum Gene Ther. 1997 Dec 10; 8 ((18): 2261-2268.Heyman T., Agoutin B., Friant S., Wilhelm F.X., Wilhelm M.L., 1995, Plus-Strand DNA Synthesis of the Yeast Retrotransposon Ty1 is Initiated at Two Sites, PPT1 Next to the 3' LTR and PPT2 Within the pol. PPT1 is sufficient for Ty1 Transposition.Naldini L, Blomer U, Gage FH, Trono D, Verma IM. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad U S A. 1996 Oct 15; 93(21): 11382-11388.Naldini L. Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma Trono, Trono D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. Apr. 1996 12; 2722559): 263-267.Miller, Rosman GJ. Improved retroviral vectors for gene transfer and expression.Tech (1989), Vol. 7, 980-990. Pascolo, S. N., Verma F. M., Berno, U. F. G., SmithHLA-A2.1-restricted education and cytolytic activity of CD8+ T lymphocytes from ß2 microglobulin (ß2m) HLA-A2.1 monochain transgenic, H-2Db, ß2m double knockout mice. J. Exp. Med. 185, 2043-2051.Poeschla E.M., Wong Staal F., Looney D.J. Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vector - Nature Medicine, vol. 4, no. 3: 354-357.Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Biotechnology; Sep. 1997; Nat: 1587-875.

Claims (36)

1. A recombinant lentiviral vector, characterized in that it is intended to be transcomplemented for sequences coding for the GAG, POL and ENV polypeptides or a portion of these polypeptides that is sufficient to allow lentiviral vector particles to be formed, and in that it comprises:

   a) regulatory signals, for reverse transcription, expression and packaging, of lentiviral origin;

   b) a polynucleotide derived from a lentiviral genome within which it is capable of adopting a triplex DNA conformation during reverse transcription, said polynucleotide being a DNA structure comprising a cis-acting central initiation region (cPPT) and a cis-acting termination region (CTS); and

   c) a nucleotide sequence of interest placed under the control of regulatory signals for transcription and expression of non-retroviral origin.
2. The recombinant vector according to claim 1, characterized in that it comprises:

   a) regulatory signals, for reverse transcription, expression and packaging, of lentiviral origin;

   b) a polynucleotide derived from a lentiviral genome and obtained by cloning, amplification or synthesis and comprising a cis-acting central initiation region (cPPT) and a cis-acting termination region (CTS), said regions being of lentiviral origin; and

   c) a sequence of nucleotides of interest placed under the control of regulatory signals for transcription and expression of non-retroviral origin.
3. The recombinant lentiviral vector according to claim 1 or 2, characterized in that it is free of lentiviral genes.
4. The recombinant vector according to any one of claims 1 to 3, characterized in that the polynucleotide forming a DNA triplex during viral reverse transcription comprises a cis-acting cPPT region and a cis-acting CTS region each containing at least 10 nucleotides.
5. The recombinant vector according to any one of claims 1 to 4, characterized in that the sequences of lentiviral origin are derived from the viruses HIV, CAEV, EIAV, VISNA, SIV or FIV.
6. The recombinant vector according to claim 4, characterized in that the cPPT and CTS sequences are derived from the genome of a HIV-1 or HIV-2 lentivirus.
7. The recombinant vector according to any one of claims 1 to 6, characterized in that the nucleotide sequence of interest is contained in an expression cassette comprising regulatory signals for transcription and expression.
8. The recombinant vector according to any one of claims 1 to 7, characterized in that the polynucleotide is a DNA sequence comprising the cis-acting central initiation region (cPPT) and the cis-acting termination region (CTS) of the genome of a HIV-1 lentivirus.
9. The recombinant vector according to any one of claims 1 to 7, characterized in that the polynucleotide comprises the cPPT and CTS regions of a sequence selected from SEQ ID NO: 9 to SEQ ID NO: 14 or in that it is one of said sequences, mutated in particular by deletion or insertion of one or more nucleotides provided that the polynucleotide allows a triplex to be formed during reverse transcription.
10. The recombinant vector according to any one of claims 1 to 9, characterized in that it is the plasmid pTRIP.EGFP deposited with the CNCM on 15th April 1998 with accession number I-2005.
11. The recombinant vector according to any one of claims 1 to 9, characterized in that it is the plasmid pTRIP.MEL-IRES-GFP deposited with the CNCM on 20th April 1999 with accession number I-2185.
12. A recombinant vector, characterized in that it is intended to be transcomplemented for sequences coding for the GAG, POL and ENV polypeptides or a portion of these polypeptides that is sufficient to allow lentiviral vector particles to be formed, and in that it comprises:

    a) regulatory signals for reverse transcription, expression and packaging originating from a retrotransposon;

    b) a polynucleotide comprising a cis-acting central initiation region (cPPT) and a cis-acting termination region (CTS) originating from a retrotransposon, said polynucleotide consisting of a DNA structure forming a DNA triplex during reverse transcription; and

    c) a nucleotide sequence of interest.
13. The recombinant vector according to claim 12, characterized in that the retrotransposon is a yeast retrotransposon.
14. The recombinant vector according to claims 1 to 3, characterized in that the regulatory signals for reverse transcription and packaging are constituted by:

    • all or a portion of the retroviral LTR sequences;

    • the retroviral sites PBS and PPT 3'; and

    • the retroviral sequence necessary for packaging of a genome vector in a vector particle.
15. The recombinant vector according to claim 14, characterized in that its LTR sequence is partially deleted in the U3 region.
16. A system of recombinant vectors for the production of retroviral particles, comprising:

    a) a recombinant vector according to any one of claims 1 to 12; and

    b) one or more vectors for transcomplementation of the recombinant vector by providing sequences coding for the GAG, POL and ENV polypeptides or for a portion of said polypeptides sufficient to allow retroviral particles to be formed.
17. Recombinant lentiviral particles the genome of which is a recombinant nucleotide sequence comprising a predetermined sequence of nucleotides (transgene) placed under the control of regulatory signals for transcription and expression, and comprising regulatory signals for reverse transcription, expression and packaging of lentiviral origin and a polynucleotide derived from a lentiviral genome within which it is capable of adopting a triplex DNA conformation during reverse transcription, said polynucleotide being a DNA structure comprising a cis-acting central initiation region (cPPT) and a cis-acting termination region (CTS), said genome of particles being deprived of lentiviral genes.
18. Recombinant lentiviral particles the genome of which is a recombinant nucleotide sequence comprising a predetermined sequence of nucleotides (transgene) placed under the control of regulatory signals for transcription and expression, and comprising regulatory signals for reverse transcription, expression and packaging of lentiviral origin, the LTR sequence of which is partially deleted in the U3 region and a polynucleotide derived from a lentiviral genome within which it is capable of adopting a triplex DNA conformation during reverse transcription, said polynucleotide being a DNA structure comprising a cis-acting central initiation region (cPPT) and a cis-acting termination region (CTS).
19. Recombinant lentiviral vector particles comprising:

    a) a gag polypeptide corresponding to nucleoproteins of a lentivirus or to functional polypeptide derivatives (GAG polypeptides);

    b) a pol polypeptide constituted by the RT, PRO, IN proteins of a lentivirus or a functional polypeptide derivative (POL polypeptide);

    c) an envelope polypeptide or functional polypeptide derivatives (ENV polypeptides); and

    d) a recombinant nucleotide sequence comprising a predetermined nucleotide sequence (transgene) placed under the control of regulatory signals for transcription and expression, a sequence containing regulatory signals for reverse transcription, expression and packaging of lentiviral origin, the LTR sequence of which is partially deleted in the U3 region, and a polynucleotide that is a DNA structure comprising a cis-acting central initiation region (cPPT) and a cis-acting termination region (CTS), said regions being of lentiviral origin and being inserted in a functional orientation with said signals, said polynucleotide being capable of adopting a triplex DNA conformation during lentiviral reverse transcription.
20. Recombinant lentiviral vector particles comprising:

    a) a gag polypeptide corresponding to nucleoproteins of a lentivirus or to functional polypeptide derivatives (GAG polypeptides);

    b) a pol polypeptide constituted by the RT, PRO, IN proteins of a lentivirus or a functional polypeptide derivative (POL polypeptide);

    c) an envelope polypeptide or functional polypeptide derivatives (ENV polypeptides); and

    d) a recombinant nucleotide sequence that is free of lentiviral genes and comprising a predetermined nucleotide sequence (transgene) placed under the control of regulatory signals for transcription and expression, a sequence containing regulatory signals for reverse transcription, expression and packaging of lentiviral origin and a polynucleotide which is a DNA structure comprising a cis-acting central initiation region (cPPT) and a cis-acting termination region (CTS), said regions being of lentiviral origin and being inserted in a functional orientation with said signals, said polynucleotide being capable of adopting a triplex DNA conformation during lentiviral reverse transcription.
21. Recombinant particles according to claim 19 or 20, characterized in that the gag, pol and env polypeptides are derived from sequences of a HIV retrovirus, in particular HIV-1 or HIV-2.
22. Recombinant particles according to claim 19 or 20, characterized in that the gag and pol polypeptides are derived from sequences of a HIV retrovirus and the env polypeptide is derived from a virus that is different from HIV.
23. Recombinant particles according to claim 19 or 20, characterized in that the env_polypeptide is an amphotropic ENV polypeptide.
24. Recombinant particles according to claim 19 or 20, characterized in that the env_polypeptide is an ecotropic ENV polypeptide.
25. Recombinant particles according to claim 19 or 20, characterized in that the env_polypeptide is derived from the vesicular somatitis virus (VSV).
26. Recombinant cells, characterized in that they are recombined with a recombinant vector according to any one of claims 1 to 15 or with recombinant vector particles according to any one of claims 17 to 25.
27. Recombinant cells according to claim 26, characterized in that they are non-mitotic differentiated eukaryotic cells.
28. Recombinant cells according to claim 26, characterized in that they are mitotic cells.
29. Recombinant cells according to claim 26, characterized in that they are primary eukaryotic cells or immortalized cell lines.
30. Cells according to claim 26, characterized in that they are lung cells, brain cells, epithelial cells, astrocytes, microglia, oligodendrocytes, neurons, muscle, hepatic, dendritic, neuronal cells, bone marrow stem cells, macrophages, fibroblasts, hematopoietic cells or lymphocytes.
31. A composition for therapeutic use, characterized in that it comprises a recombinant vector according to any one of claims 1 to 15 or recombinant vector particles according to any one of claims 17 to 25 or recombinant cells according to any one of claims 26 to 30.
32. An immunogenic composition, characterized in that it comprises a recombinant vector according to any one of claims 1 to 15 or recombinant vector particles according to any one of claims 17 to 25 or recombinant cells according to any one of claims 26 to 30.
33. Use of a recombinant vector according to any one of claims 1 to 15 or of recombinant vector particles according to any one of claims 17 to 25, for ex vivo transfection or transduction of non-mitotic differentiated cells.
34. Use of a recombinant vector according to any one of claims 1 to 15, or of recombinant vector particles according to any one of claims 17 to 25, for ex vivo transfection or transduction of primary cells or immortalized cell lines.
35. The recombinant vector according to any one of claims 1 to 15 or recombinant vectors according to any one of claims 17 to 25, for in vivo transduction of non-mitotic differentiated tissue for the purposes of gene therapy.
36. The recombinant vector according to any one of claims 1 to 15 or vector particles according to any one of claims 17 to 25, for the preparation of immunogenic, vaccination, prophylactic or therapeutic compositions.