WO2005061721A1

WO2005061721A1 - Novel high-titre retroviral vector and transduction methods

Info

Publication number: WO2005061721A1
Application number: PCT/ES2004/000558
Authority: WO
Inventors: Manuel Angel GONZALEZ DE LA PEÑA; José Luis ABAD MINGUEZ
Original assignee: Cellerix, S.L.
Priority date: 2003-12-17
Filing date: 2004-12-16
Publication date: 2005-07-07
Also published as: ES2235638B1; ES2235638A1

Abstract

The invention relates to a novel high-titre retroviral vector and transduction methods. More specifically, the invention relates to novel retroviral vectors that can produce high-titre, non-replicative, infectious retroviral particles and to methods of using said particles for the transfer of gene constructions of interest. In particular, the invention relates to retroviral vectors which are derived from MoMLV and which comprise a nucleotide sequence that is characterised by general formula: 5'-[CMV-R-U5]-[PBS]-[SD]-Ψ+-[MCS2]-[SA]-[MCS]-[PPT]-[LTR]-3', as well as the uses of said vectors.

Description

New high-title retroviral vector and transduction methods.

The present invention relates to new retroviral vectors capable of producing high-grade non-replicative infective retroviral particles and methods for the use of said particles in the transfer of gene constructs of interest.

Background of the invention

Retroviruses are a group of viruses characterized by having as single-stranded RNA + genetic material. All retroviruses contain three main coding regions in their genome: Gag, which directs the synthesis of the structural proteins of the viral particle (matrix, capsid and nucleocapsid), Pro-Pol, which determines the synthesis of proteins with enzymatic activity (protease , reverse transcriptase and integrase), and Env, which encodes envelope glycoproteins (surface and transmembrane) (Duesberg et al., 1970). The retrovirus cycle begins with the interaction of virion envelope glycoproteins with specific cell receptors. This interaction leads to the entry of the viral particle into the cell cytoplasm. Upon entry into the cell cytoplasm, this viral genomic RNA is converted to double stranded DNA through the first of the fundamental processes of its biological cycle: reverse transcription. Thanks to this process, the viral RNA, which at its 3 'end contains the region called U3 (unique region 3), results in a DNA in which U3 is duplicated and is represented on both sides of the chain, which allows Transcription of the integrated provirus, since U3 acts as a promoter. An analogous phenomenon occurs with the U5 sequence (single region 5), which is located at the 5 'end of the retroviral genomic RNA and contains the transcription terminator sequence thereof. These duplications of U5 and U3 during reverse transcription are due to the existence of sequences such as the polyurethane tract (PPT, immediately prior to 5 'end of U3), the first binding site (PBS, immediately after the 3' end of U5) and repeated regions (called R) located on both sides of the genome, which direct the synthesis of proviral DNA chains ( Baltimore, 1970; Temin et al., 1970; Coffin et al., 1997). Once the reverse transcription is finished, the newly formed copy DNA is the object of the second of the fundamental processes of the retroviral cycle: integration, thanks to which the viral DNA is inserted in non-predetermined places of the cell chromosomes, constituting the so-called provirus. This viral DNA behaves like a cellular locus in the course of cell divisions and is the one that encodes the materials necessary for the formation of new viral particles, which constitute the progeny of the viral particle that originated the process.

At the ends of the LTR regions (English acronym for Long Term Repeats, formed by the succession of U3-R-U5 sequences) there are key sites for the integration of proviral DNA into the target cell. Adjacent to the LTR regions, as described above, sequences necessary for the reverse transcription of the viral genome, PBS and PPT respectively are located, in addition to a sequence necessary for the efficient encapsidation of viral RNA in retroviral particles (the sequence Ψ). The 800 nucleotide region of the Moloney virus genome of murine leukemia (MoLV) that begins just after the SD (splicing donor site) and extends within the region encoding the gag polyprotein is sufficient to drive the efficient packaging of a heterologous transcript (Adam et al., 1988; Dornburg et al., 1988).

The processing of the viral mRNA (messenger RNA) is carried out from the SD, located next to the PBS, to the splicing acceptor site (SA), the latter, included within the coding region of the polyprotein pol. Retroviral vectors and their various applications have been described by numerous authors, (Mann et al., 1983; Cone et al., 1984; Miller, 1990), and US Patent Nos. 4,405,712; 4,861,719; 4,980,289 and PCT applications Nos. WO 89 / 02,468; WO 89 / 05,349 and WO 90 / 02,806. From these publications it is inferred that the usual thing regarding this type of vectors is to replace a portion of the retroviral RNA with a heterologous gene of interest, keeping the sequences of LTR, Ψ, PBS and PPT intact, such that when the retroviral particle Recombinant introduces its RNA into the target cell during the infective process, the heterologous gene of interest is also introduced into the cell, and thus is incorporated into the cellular genome in the form of DNA as if it were part of the cellular genome. . Thus, the presence of the gene of interest within the genome of the host cell leads to the expression of the heterologous protein in said cell.

Retroviral vectors as a method to introduce heterologous genes into target cells have been widely used for the following reasons: (1) Because of their broad cell tropism and their high efficiency in incorporating genetic material into the nucleus of replicative target cells; (2) For the relatively high levels of gene expression; (3) Because the type of gene expression they produce is stable due to the integration of the viral genome into the cell genome; (4) Because retroviral infection per se does not produce toxic effects on infected cells; (5) Because retroviral particles do not usually induce significant immune responses; (6) Because of the potential capacity that these vectors possess to regulate their expression in a tissue-dependent manner and / or cell target, as well as to regulate their expression over time; (7) Due to the knowledge that is already available of these types of vectors and their various applications (Romano et al., 2000). Even so, a disadvantage presented by retroviral vectors is that they only infect replicative cells, there being serious difficulties in infecting those cells characterized as non-replicative or quiescent (Roe et al., 1993). In addition, there are several cell types that have traditionally been difficult to transduce by retroviral vectors even though they have been stimulated to enter the replicative cycle, some examples include T lymphocytes, B lymphocytes, monocytic cells and dendritic cells. This is particularly frequent for primary cells, including stem cells. For these types of cells listed above, those skilled in the art have suggested other methods of gene transfer, including the direct administration of purified DNA as a method of incorporating genetic material into the target cells.

To try to improve the efficiency of retroviral vectors, methods have been suggested that focus on the induction of replication of the target cells or towards increasing the efficiency of replication of said cells, to allow retroviral infection of the target cells. In the case of T cells, there are various non-specific stimulation methods in vivo that are efficient enough to induce their replication (such as IL-2, mitogens, and anti-CD3 or anti-CD28 antibodies). However, there are difficulties in obtaining an efficient transduction of T lymphocytes using stimulation methods that are compatible with clinical and commercial uses, since they frequently alter the relative proportion of the different cell subpopulations present in the blood supply (Movassagh et al. ., 2000). The retroviral vectors of the present invention allow efficient transduction of primary cells stimulated with methods compatible with clinical uses of said cells, although said methods do not produce stimulation as potent as others more nonspecific and incompatible with the clinical one.

As we mentioned earlier, the realization of an efficient retroviral gene transfer in primary cells such as T lymphocytes or stem cells is not trivial. The methods currently used for retroviral transduction of these cells have a number of practical limitations, among which one of the most important lies in the low retroviral titres obtained with most of the available retroviral vectors, titers that are normally in the range from lxl O ⁴ to lxlO ⁶ infective units per milliliter (Ul / ml).

The methods of producing retroviral particles by stable transfection they yield low titers, and co-cultivation of stable producing lines of retroviral particles with the target cell is usually required. In addition, it has often been necessary to add large volumes of the preparation containing retroviral vectors to the cells that are to be transduced to achieve a useful transduction frequency. The method of producing infective retroviral particles by transient transfection of the packaging cell is much more efficient than stable packaging lines, but only some retroviral vectors such as those of the present invention are designed to be used by transient transfection.

Additionally, many of the most frequently used retroviral vectors encode genes that confer resistance to certain antibiotics to the cells that express them. This selection helps to compensate for low retroviral transduction efficiency, but at the same time it may have certain undesirable associated effects. Previous work indicates that prolonged in vitro culture of human lymphocytes during the more than two weeks required by antibiotic selection induces impoverishment of the immune repertoire of these cells. This effect is independent of the retroviral infection process (Ferrand et al., 2000). Selective pressure induced by antibiotic selection can induce structural reorganizations in the integrated vector and / or silencing of the gene of interest (Bandyopadhyay et al., 1984; Breuer et al., 1993; Schott et al, 1996). In addition, some of the products derived from antibiotic resistance genes show an immunogenic capacity in clinical studies that affects the survival of cells used as a therapeutic agent (Riddell et al., 1996). The high-titre retroviral vectors of the present invention allow to infect primary cells with an efficiency that makes subsequent selection with antibiotics unnecessary. In addition, its design allows to house other types of marker genes that involve selection processes of infected cells faster than antibiotic resistance selection.

It is an object of the present invention to provide new vectors retroviral derived from the Moloney virus of MoMLV murine leukemia with the capacity to produce high-titre retroviral supernatant consisting of infective and replication-deficient retroviral particles and methods for the use of said retroviral supernatants in gene transfer to cells resistant to standard transduction techniques, such as T lymphocytes, stem cells or any other type of cell lines or primary cells. These transduced cells can be used in research or administered to patients by techniques known to those skilled in the art.

Brief Description of the Invention

A first aspect of the invention is to provide new replication-derived MoMLV retroviral vectors comprising a specific set of sequences with an optimized arrangement for the production of an infective but replication-deficient retroviral particle, which confers on said vectors the ability to produce high-titre retroviral supernatants for retroviral transduction of cells and cell lines difficult to transduce so far. A second aspect of the invention is to provide host cells transfected, transformed and / or infected with the retroviral vectors of the present invention.

A third aspect of the present invention is to provide methods for the stable or transient production of infective retroviral particles and for the infection of target cells with said retroviral particles.

A fourth aspect of the present invention consists in the application of the retroviral vectors of the present invention for the production of transgenic animals by infection of embryonic stem cells with said retroviral particles, for gene therapy, production of retroviral libraries and Expression and / or overexpression of proteins or RNA.

Other aspects of the present invention will be apparent to one skilled in the art in view of the description of the invention.

Brief description of the figures

Figure 1 shows the structure of the pRV proviral genome. MCS and MCS2, polycloning sites; SD, splicing donor site; SA, splicing acceptor site; Ψ *, MoMLV extended packaging signal; CMV, early cytomegalovirus promoter; U3, R and U5, elements of the LTR of MoMLV and PBS, the first binding site. The restriction sites indicated are unique within the plasmid pRV. Figure 2 shows the structure of the 3 'LTR of the retroviral vector pRV (SEQ

ID NO 1). Dotted lines indicate deletions that can be made in the U3 region of the 3 'LTR to construct a self-activating pRV vector. The corresponding restriction targets are shown. MCS, polycloning site; RD, direct repetition; CAAT, CAAT box; TATA, TATA box.

Detailed description of the invention

In a first aspect, the invention provides new retroviral vectors derived from MoMLV characterized in that they consist of a specific set of sequences with an optimized arrangement for the production of an infective but replication-deficient retroviral particle, which confers on said vectors the ability to produce supernatants. High titre retrovirals for retroviral transduction of cells and cell lines difficult to transduce so far. These vectors comprise the retroviral plasmid pRV (SEQ ID NO. 1), whose construction is illustrated in Example 1 of the present invention, which includes sequences, derived from MoMLV or heterologous, optimized for transcription, encapsidation and retrotranscription of the retroviral genomic RNA, for the correct processing (splicing) of the mRNA, for the integration of the proviral DNA in the genome of the target cell and for the correct translation of the mRNA and expression of heterologous genes of interest in the target cells of the particles retrovirals produced from the pRV vector and other derivatives thereof. Figure 1 shows the structure of the pRV proviral DNA. The present invention also includes the complementary strand of SEQ ID NO. 1 and DNA sequences that hybridize under high astringency conditions with SEQ ID NO. 1 or its complementary strand. Nucleic acids with sequence identity or a high degree of homology can be detected by hybridization under high astringency conditions, for example, at 50 ° C or more in SSC solution 0, lx (15 mM sodium chloride / 1.5 sodium citrate mM). Nucleic acids having a region of high homology or identity with the sequences described in the present invention, eg allelic variants, or versions engineered, etc., bind to the sequences described in the invention under high conditions. astringency. Other conditions of high astringency are known in the state of the art that could also be used to identify nucleic acids that retain a high degree of homology with the described sequences.

The MoMLV regions encoding env and pol have been completely removed from the replication-deficient MoMLV retroviral vector of the present invention, and the gag coding region has been partially removed. The gag region that has not been removed is part of the extended packaging signal, which comprises positions 215-1038 of the MoMLV virus. The elimination of all these sequences is aimed at the maximum reduction of viral sequences that by homologous recombination can cause retroviral particles competent for replication. In addition, the elimination of these sequences reduces the size of the proviral genome of the vector of the present invention up to 2.5 kb, which allows cloning inserts of up to 7.0 kb. Retroviral vectors that only retain the minimum packaging signal, (positions 215-563 of MoMLV), produce retroviral titers 10 to 100 times lower than those that possess the extended packaging signal as in the vector of the present invention. , which includes 455 bp from the gag coding region. (Naviaux et al., 1992)

The replication-deficient MoMLV-derived retroviral vector of the present invention has the U3 region of MoMLV 5 'LTR replaced by the cytomegalovirus (CMV) early promoter. The production of the retroviral genomic RNA, being under the control of the CMV promoter, allows the number of copies of the retroviral genome to be significantly increased inside the packaging line, so that the retroviral titers of this vector are around an order of magnitude higher than those that retain the 5 'LTR native to MoMLV. In another preferred embodiment of the invention, other heterologous promoters are provided in the same position as that of the CMV promoter, including but not limited to the Rous sarcoma virus (RSV) promoter.

The replication-deficient MoMLV-derived retroviral vector of the present invention includes donor and splicing acceptor sites that favor export to the cytoplasm of subgenomic RNA, which enhances expression in the target cell of the heterologous genes included in the vector.

The replication-deficient MoMLV retroviral vector of the present invention includes a polycloning site (MCS2) that precedes the splicing acceptor site that includes, but is not limited to, the following restriction sites: Bgl II,

Hpa I, Sph I. This MCS 2 polycloning site allows the inclusion of heterologous sequences (eg cis regulatory sequences or internal promoters).

The replication-deficient MoMLV retroviral vector of the present invention includes as a splicing acceptor site (SA), a synthetic sequence based on a partially degenerated consensus sequence of the focus-forming Spleen virus (SFFV), which does not include open reading phases (ORF) or aberrant translation starts (ATG), coming from the 3 'end of the MoMLV Pol gene. This synthetic sequence only retains 26 base pairs homologous to the MoMLV Pol gene. Many of the retroviral vectors containing SA sites include open reading frames (ORF) that can interfere with the correct expression of the heterologous genes encoded by the retroviral vector. The synthetic sequence or oligonucleotide included in the retroviral vector of the present invention to act as an SA site, as it does not contain ORFs, eliminates the subsequent possibility of expression of abnormally rounded aberrant peptides that may interfere with the correct expression of the heterologous genes included in the proviral DNA. Additionally, the size of the vector is reduced by approximately 350 base pairs and viral sequences (from the Pol gene) that can cause recombination events capable of generating viruses competent for replication are eliminated. Additionally, the retroviral vector of the present invention has a polycloning site called MCS located next to the SA site, which includes, but is not limited to, the following restriction sites: Bam HI-Xho I-Eco RI-Swa I - Sac II - Cía I - Not I - Sal I. The presence of this MCS allows the inclusion in the retroviral vector of all types of heterologous sequences (eg genes applied in gene therapy or research, selection markers such as gfp, ΔNGFR, ΔGHR, Neo, LacZ and / or β-Geo [most of these markers will be described in more detail in the examples], promoters or other expression regulatory sequences or internal ribosome entry sites, IRES). The MCS site allows you to enter a single sequence (Example 2) or several simultaneously in the same construction (Examples 3 and 4). The inclusion of one or two IRES in the pRV MCS allows bi- or tricistronic constructs to be made for the simultaneous expression of two or three genes in the cell where said constructs are introduced, either by transfection or by retroviral transduction (Example 4). Heterologous sequences can be introduced interchangeably at the MCS, MCS 2 polycloning site or both simultaneously. In another embodiment of the invention the method for the production of self-activating retroviral vectors from the retroviral vector described in the present invention is included, as well as said self-activating retroviral vectors. These vectors have a truncated LTR3 'in its plasmid DNA form, which, by reverse transcriptase action is duplicated in the integrated provirus so that the latter has the two truncated LTRs (Anson 2004). Truncated LTRs are constructed by partial deletion of the U3 region of the viral LTR3 '. This U3 region constitutes the viral promoter and has in its sequence two direct repeats (RD) that act as enhancers for the transcription of the viral RNA. In addition, the U3 region possesses the transcriptional elements called CAAT and TATA boxes respectively, and which, together, constitute the minimum viral promoter. The deletion of one or more of these elements affects, to a greater or lesser extent, the levels of expression obtained from the U3 viral promoter. Self-activating vectors have advantages over those that preserve the U3 region in its entirety, since they lack viral sequences that could cause viruses competent for replication, by homologous recombination with exogenous or endogenous viruses. In addition, the elimination of viral sequences that act as transcriptional activators reduces the risk of insertional mutagenesis in gene therapy protocols, by preventing the activation of oncogenes.

In another embodiment of the invention, the method for obtaining infectious viral particles deficient in replication is included by transfection into packaging cell lines of the pRV vector (SEQ ID NO 1 or its complementary strand or DNA sequences that hybridize with them under conditions of high requirement) or retroviral constructions directly derived from said retroviral vector. These packaging lines may or may not stably express the necessary genes (gag, pol, env) for the formation of retroviral particles containing the retroviral vectors of the present invention. In the event that the packaging cell does not express said auxiliary genes, the retroviral vector is co-transfected with them in said cell (Example 5). The transfected packaging line as described above is grown under conditions and a culture medium that favors the production and release to said medium of infectious viral particles containing the retroviral vectors described in the present invention. The titres obtained from the retroviral vectors directly derived from pRV (described in the present invention) are very high, with values that do not fall in any case of lxl O ⁶ Ul / ml, and even reach lxlO ⁸ Ul / ml (Table I). The importance of these retroviral titres is evident when compared with those produced from other retroviral vectors (Table III). Only the titles of vectors that produce a greater number of copies of their proviral DNA in the packing cell are comparable with the vectors described in the present invention by including sequences that confer said retroviral plasmid an self-replicating ability in the packing cell. The high retroviral titres produced by the vectors described in the present invention facilitate retroviral transduction of cell lines and primary cells with an efficiency superior to that obtained by the most usual means of transfection (Table H).

Another embodiment of the invention includes the RNA sequence derived from the retroviral vector of the present invention, the DNA sequence of the retroviral provirus produced in the target cell during the reverse transcription process of the RNA derived from the retroviral vector and the mRNA produced from said retroviral provirus.

In yet another embodiment of the invention are included retroviral particles obtained by transfecting host cells (commonly referred to as packaging cells) with the retroviral vector of the present invention or RNA derived from said vector, host cells transformed or transfected with said retroviral vectors and cells. hosts infected or transduced with the retroviral particles obtained from the retroviral vectors described in the present invention.

Another aspect of the invention relates to a method of introducing heterologous or homologous nucleotide sequences in target cells comprising the infection of the cells with the retroviral particles obtained from the retroviral vectors described in the present invention. Other preferred uses of these retroviral vectors consist of the production of transgenic animals by transduction of embryonic stem cells with retroviral particles produced from the retroviral vectors described in the present invention, the production and screening of retroviral libraries constructed in these retroviral vectors, for the preparation of a pharmaceutical composition applicable for use in gene therapy and those pharmaceutically compatible vehicles with said composition, for cloning and gene selection and for the expression and / or overexpression of proteins or RNA.

Another aspect of the invention includes the use of the retroviral vectors described in the present invention for the production and / or purification of heterologous peptides or proteins whose genes are cloned at some position of the MCS polycloning site described in Figure 1 and in Example 1 of the present invention. Proteins are obtained from host cells transfected with the retroviral vector containing the heterologous protein gene (s), or transduced with the infective particles produced from said retroviral vectors. The host cells are cultured under conditions that favor the expression of the protein whose nucleotide sequence has been cloned into the MCS, so that the protein produced is collected from the culture medium and / or the host cells themselves.

Next, the Examples describe in greater detail certain embodiments of the present invention.

Examples

The following Examples are presented to illustrate, but do not limit this invention:

Example 1: Construction of the retroviral vector pRV 1.1.- Construction of pUCm. The pUC18 vector is digested with enzymes

Aat II and Afl III and in said fragment a synthetic polycloning site is cloned constituted by the hybridization of the oligonucleotides with sequences SEQ ID NO 2 and SEQ ID NO 3. The vector named pUCm originates. 1.2.- Construction of pUCRV-1. The CMV promoter is amplified by PCR from the pcDNA3 vector, oligonucleotides SEQ ID NO 4 and SEQ ID NO 5 are used. The PCR product is cloned into a vector destined for the reception of PCR products (pCR 2.1 or pCR -Blunt, from Invitrogen or pGEM-T from Promega, e.g.), the resulting construct is digested with Pme I and Sac I and the corresponding fragment is cloned into the Pme I and Sac I targets of pUCm. The resulting vector is called pUCRV-1.

1.3.- Construction of pUCRV-2. The retroviral vector pFB is digested with the Sac I and Bgl II enzymes, the fragment corresponding to the RU5 region of MoMLV and the extended packaging signal is cloned into the corresponding Sac I and Bgl II targets of pUCRV-1, the resulting vector is called pUCRV-2.

1.4.- Construction of pUCRV-3. The pUCRV-2 vector is digested with the enzymes Bgl II and Bam HI. In said targets the synthetic fragment constituted by the hybridization of oligonucleotides SEQ ID NO 6 and SEQ ID NO 7, corresponding to the MCS2 polycloning site and the region of the splicing acceptor site is introduced. The resulting vector is called pUCRV-3.

1.5.- Construction of pUCRV-4. The vector pUCRV-3 is digested with the enzymes Bam HI and Sal I. In said targets the synthetic fragment consisting of the hybridization of the oligonucleotides SEQ ID NO 8 and SEQ ID NO 9 is introduced, corresponding to the MCS polycloning site. The resulting vector is called pUCRV-4.

1.6.- Construction of pUCRV-5. The vector pUCRV-4 is digested with the enzymes Sal I and Nhe I. In said targets the synthetic fragment consisting of the hybridization of the oligonucleotides SEQ ID NO 10 and SEQ ID NO is introduced

1 1, corresponding to the polyurethane tract and a fragment of the 3 'LTR of

MoMLV The resulting vector is called pUCRV-5. 1.7.- DepRV construction. The pBabePuro vector (Morgenstern et al, 1990) is digested with the enzymes Nhe I and Sap I. The fragment corresponding to the 3 'LTR of MoMLV is cloned into the Nhe I and Sap I targets of pUCRV-5. The resulting construction is called pRV (Figure 1, SEQ ID NO 1). The location of the most relevant pRV sequences is as follows: a. Early-intermediate promoter of human cytomegal virus (CMV): positions 23-604 of SEQ ID NO 1 b. RU5 region of the 5 'LTR: positions 641-785 of SEQ ID NO 1 c. First junction site (PBS): positions 786-803 of SEQ ID NO 1 d. Splicing donor site: positions 846-847 of SEQ ID NO 1 e. Extended packing signal: positions 855-1700 of SEQ ID NO 1 f. Polycloning site 2 (MCS 2): positions 1703-1726 of SEQ ID NO 1 g. Region of the splicing acceptor site: positions 1727-1783 of SEQ ID NO 1 h. Site acceptor of splicing positions 1780-1781 of SEQ ID NO 1 i. Polycloning site (MCS): positions 1784-1861 of SEQ ID NO 1 j. Polyurethane tract: positions 1862-1903 of SEQ ID NO 1 k. 3 'LTR: positions 1904-2497 of SEQ ID NO 1

Example 2: Expression of a heterologous gene cloned at the polycloning site (MCS) of pRV. 2.1.- Construction of pRV-gfp. Digestion of the vector pEGFP-Nl with the enzymes Xho I and Not I, the resulting fragment, gfp, is cloned into pRV cut with Xho I and Not I. The originating construct is called pRV-gfp.

Example 3: Construction of retroviral vectors with a translation cassette consisting of an internal ribosome input sequence (IRES) and any heterologous gene. 3.1.- Construction of pRV-IRES-gfp. The vector pIRES-2-EGFP is cut with

Bam HI, is treated with the Klenow fragment of E. coli DNA polymerase and blunt ends are ligated to remove the Bam HI target from the vector. The vector thus treated is cut with Xho I and Not I, and the IRES-gfp fragment is cloned into pBacPAK8 digested with Xho I and Not I. The resulting construct is called pB-IRES-gfp. pB-IRES-gfp is cut with the Sac π and Not I enzymes and cloned into pRV cut with Sac II and Not I to obtain the retroviral vector pRV -IRES-gfp.

3.2.- Construction of pRV-IRES-ΔGHR (Truncated growth hormone receptor). Amplification of the truncated growth hormone receptor ΔGHR (Garcia-Ortiz et al., 2000) with oligonucleotides of the following characteristics:

Oligo sense: with a cohesive end at the 5 'end for the Ncol target, followed by the first 10 to 25 nucleotides corresponding to the ΔGHR sequence. The end corresponding to Neo I contains the initial methionine sequence and the first nucleotide of the first codon of ΔGHR (CATGG).

Oligo antisense: with a cohesive end at its 5 'end for the Not I target, followed by the first 10 to 25 nucleotides corresponding to the ΔGHR sequence. The PCR product is cloned into a vector for the reception of PCR products (pCR 2.1 or pCR-Blunt, from Invitrogen or pGEM-T from Promega, e.g.), the resulting construct is digested with Neo I and Not I and the fragment corresponding to ΔGHR is cloned into pB-IRES-gfp (Example 3.1) at the corresponding Neo I and Not I sites.

The pB-IRES-ΔGHR construct is cut with Sac II and Not I and the IRES-ΔGHR fragment is cloned into pRV cut with Sac π and Not I. The originating construct is called pRV-IRES-ΔGHR.

This method is applicable for the construction of analog vectors, pRV-IRES-Gen, provided that the first base of the first codon after the initial methionine of the gene is a G (guanine). 3.3.- Construction of pRV-IRES-Neo. Neo PCR amplification from the plasmid pcDNA3.1

Oligos used: Oligo sense: at the 5 'end it has a Bsa I target (GGTCTC) followed by the NCATG sequence (where N can be interchangeably A, T, C or G) that contains the cohesive end of the Ncol target, followed by the first 10 to 25 nucleotides corresponding to the sequence of Neo. The fragment corresponding to Neo I contains the initial methionine sequence and the first nucleotide of the first Neo codon (CATGA).

Oligo antisense: with a cohesive end at its 5 'end for the Not I target, followed by the first 10 to 25 nucleotides corresponding to the Neo sequence.

The PCR product is cloned into a vector for the reception of PCR products (pCR 2.1 or pCR-Blunt, from Invitrogen or pGEM-T from Promega, e.g.), the resulting construct is digested with Bsa I and Not I and the fragment corresponding to Neo is cloned into pB-IRES-gfp at the corresponding Neo I and Not I sites.

The pB-IRES-Neo construct is cut with Sac II and Not I and the IRES-Neo fragment is cloned into pRV cut with Sac II and Not I. The originated construct is called pRV-IRES-Neo.

This method is applicable for the construction of analog vectors, pRV-

IRES-Gen, regardless of whether the first base of the first codon after the initial methionine of the gene is a G (guanine), such as the truncated nerve growth factor receptor (ΔNGFR), the β-Galactosidase (LacZ) gene ) or the combined marker β-Galactosidase / Neo (β-Geo).

Example 4: Construction of bicistronic retroviral vectors for the simultaneous expression of two heterologous genes The construction of bicistronic retroviral vectors for the simultaneous expression of two heterologous genes was carried out, in which the different elements were arranged as follows: a heterologous gene any one precedes a translation cassette consisting of an internal ribosome input sequence (IRES), preferably derived from picornavirus, and more preferably from the encephalomyocarditis virus, and any second heterologous gene.

The assembly is cloned into the polycloning site (MCS) of pRV, using any possible combination of the restriction sites present in said polycloning site.

4.1.- Cloning of pRV-WASp-IRES-gfp. Amplify the coding sequence of WASp (Wiscott Aldrich Protein Syndrome) by PCR with oligos that leave cohesive ends for BamHI and Eco RI at the 5'and 3 'ends respectively of the WASp cDNA. The PCR product is cloned into a vector for the reception of PCR products (pCR 2.1 or pCR-Blunt, from Invitrogen or pGEM-T from Promise, p. ex.), the resulting construct is digested with Bam HI and Eco RI and the fragment corresponding to WASp is cloned into the Bam HI and Eco RI targets of pRV-IRES-gfp, resulting in the construction pRV-WASp-IRES-gfp .

Example 5: Generation and titration of retroviral particles The following example illustrates the generation of infective and defective retroviral particles in replication for use in research, gene therapy or others.

To generate recombinant particles, constructs such as those cited in the above examples 1 to 4 are co-transfected, in line 293, or derived from 293, such as 293T; with packaging constructions encoding the proteins of the gag-pol and env genes, expressed from a single vector or more preferably from two different constructs, preferably a construction to express gag-pol and another for e «v. The cotransfection is carried out by the calcium phosphate precipitation method. The transfection medium is renewed at 8 hours. 24 hours after transfection, the medium for collecting the retroviral supernatant is added to the packaging cells. 48 hours after transfection, the supernatant is collected with the retroviral particles. This supernatant is filtered with a low protein retention filter and a pore size of 0.45 μm. The supernatant can be used fresh for retroviral transduction or stored frozen at -80 ° C.

To titrate the retroviral supernatant, different procedures were used depending on the nature of the selection gene encoded by the retroviral vector.

5.1.- Retroviral vectors encoding selectable markers by cytometry (eg GFP, ΔNGFR, ΔGHR). 24 hours before infection, 1.5x10 ⁵ NIH 3T3 cells are plated in complete DMEM, per well of a 6-well plate (p6). At the time of titration we count the cells of a well, typically there are around 3x10 ⁵ cells per well. For titration, complete DMEM is prepared with 8 μg / ml of polybrene. 1 ml of well infection medium is put of p6 and an adequate volume of supernatant so as not to saturate the assay. Said volume, in the case of retroviral supernatants obtained in 293T by transient transfection of vectors such as those described in examples 1 to 4, is 1-20 μl, which implies a dilution of 1: 1000 to 1:50.

After 4 to 6 hours of incubation at 37 ° C, the medium is replaced with fresh complete DMEM. The cultures are maintained for an additional 2-3 days, until the cytometry quantification of the percentage of cells expressing the marker, so we avoid obtaining erroneous results due to the phenomenon of pseudoinfection, caused by non-integrated but transcriptionally active retroviral genomes. The titer is calculated as infective units per mi (Ul / ml), by the expression: (% of positive cells) x (number of cells at the time of infection / 100) x (dilution factor).

5.2.- Retroviral vectors encoding an antibiotic resistance gene (eg Neomycin). For titration, serial dilutions (10 ^{~ 3} to 10 ^"7 ) of the supernatant are made in complete DMEM with 8 μg / ml of polybrene. The diluted supernatants are added onto NIH 3T3 cells plated 24 hours before (lml diluted supernatant / lxlO ⁵ cells / well p6) and incubate for 4 to 6 hours at 37 ° C. The medium is then replaced with fresh complete DMEM and, 24 h after transduction, the selection is started with 0.5 mg / ml of G418 Ten to fifteen days later, antibiotic resistant colonies are stained with crystal violet (0.2% in 20% methanol), and finally counted in. A positive colony must contain at least 20 cells. The titer is calculated as antibiotic-resistant colony-forming units for me (CFU Antib. ^R / ml) by the expression: (No. of resistant colonies x supernatant dilution factor).

5.3.- Retroviral vectors encoding the β-galactosidase (LacZ) gene. The procedure is analogous to that of example 5.1, what varies is the method of detecting expression of the marker gene. The expression of this marker can evaluated quantitatively by flow cytometry, using the FluoReporter lacZ Flow Cytometry Kit from Molecular Probes (Cat. F-1930). The expression of the LacZ gene can also be detected by staining with X-Gal, a substrate of β-galactosidase that originates a blue product when processed by said enzyme, thus coloring the cells where it is expressed.

Example 6: Titles of retroviral vectors Table I shows representative examples of ranges of retroviral titers of retroviral particles produced with different envelopes from retroviral vectors derived from the retroviral vector pRV described in Examples 2 to 4.

Retroviral particles were obtained according to example 5 and titrated according to examples 5.1 and 5.2 depending on the nature of the selection marker.

Example 7: Retroviral transduction efficiencies Table II shows representative examples of the retroviral transduction efficiencies obtained for different cell lines and primary cells transduced with retroviral particles obtained from the retroviral vectors derived from pRV described in Examples 2 to 4. Retroviral (stable) transduction percentages correspond to the results of a characteristic experiment. In the case of non-retroviral methods, the values correspond to those obtained temporarily with the most efficient transfection method among the following three: electroporation, calcium phosphate and lipoplejos (commercial reagent). Examples include cell lines and primary cells, such as T lymphocytes, stem cells or undifferentiated progenitors. These transduction levels are compared, when available, with the most efficient usual transfection methods among those tested, such as electroporation, lipocomplexes (lipofectamine) or transfection with calcium phosphate.

Example 8: Comparison between the titles of the retroviral vector series pRV, pL, pCL and pLZR. In this example, the titers of retroviral supernatants obtained from retroviral vectors derived from pRV are compared according to the procedure described in example 5, and described in examples 2 to 4, with the titres obtained from the vectors pLXSΔN, pCLXSN and pLZR-CMV-gfp. The pLXSΔN vector (Ruggieri et al., 1997) has an LTR derived directly from MoMLV, which directs the expression of the heterologous gene X, and an internal promoter of SV40, which directs the expression of the ΔNGFR marker. This vector lacks the mixed CMV-LTR promoter, and has a MoMLV derived splicing acceptor site. The pCLXSN vector (Naviaux et al., 1996), unlike the previous one, does have a mixed LTR, with a CMV promoter, however, it shares with the leader sequence and, in particular, the area corresponding to the acceptor site of splicing The pCLXSN vector has a selection marker consisting of the neomycin resistance gene, its expression is directed by the SV40 promoter. The most modern vector is pLZR-CMV-gfp (Yang et al., 1999), with a mixed CMV promoter and a design for the production of high-titre retroviral particles based on presence, on the plasmid construction that supports the genome proviral, of the EBNA-1 and OriP sequences, origin of replication and nuclear antigen of the Epstein Barr virus, which give the plasmid an auto-replicative capacity in the packaging cell. The pLZR-CMV-gfp leader is, however, derived from MoMLV.

The comparison of titles is made between vectors that share the same selection marker and that are pseudotyped with the same envelope, whenever possible. Table III shows a comparison of typical retroviral titres obtained from different retroviral vectors directly derived from the retroviral vector pRV described in the present invention, with the titles of other retroviral vectors. Vectors derived from pRV have significantly higher titres than vectors with older designs, such as pLXSΔN and pCLXSN. The titers are comparable for pLZR-CMV-gfp and the vectors described in the present invention, although they lack the self-replicating sequences of the pLZ series vectors. Example 9: Construction of self-activating retroviral vectors

9.1 Subcloning of the viral LTR3 '. Cut the pRV vector (SEQ ID NO 1) with the Xhol and Pací enzymes to extract the retroviral LTR3 '. The resulting fragment is subcloned into a suitable intermediate vector, such as pBacPAKδ or pBluescript cut with Xhol and Pací.

9.2 Deletion of a fragment of U3. Remove a fragment from the U3 region by digestion with one or two restriction enzymes. If the Nhel enzyme is used together with any of the following enzymes, the following elements are removed from the U3 region (Figure 2): - RDs by digesting with Nhel and Xbal - RDs and CAAT box when digesting with Nhel and BssHLI o Sacl - The RD, the CAAT box and the TATA box when digesting with Nhel and Kasl.

9.3 Subcloning of the truncated LTR and construction of the self-activating retroviral vector.

Extract the truncated LTR from the intermediate vector by cutting with Xhol and Pací and bind with the pRV vector (SEQ ID NO 1) digested with Xhol and Pací. The resulting vector is pRV with an LTR3 'where the U3 region has been partially deleted, resulting in a self-activating retroviral pRV vector.

Table I

nd. does not determine or

Table m

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Claims

Claims 1. MoMLV-derived retroviral vector comprising a nucleotide sequence characterized by the general formula:

5 * - [CMV-R-U5] - [PBS] - [SD] -Ψ + - [MCS2] - [SA] - [MCS] - [PPT] - [LTR] -3 ', where: a. [CMV-R-U5] represents the 5 'LTR where CMV is defined as a nucleotide sequence belonging to the CMV promoter or any promoter b. [SA] corresponds to positions 24 to 80 of SEQ ID NO. 6c. [LTR] corresponds to positions 1904 to 2497 of SEQ ID NO. one

2. Retroviral vector according to claim 1, wherein the [LTR] sequence is truncated.

3. Retroviral vector according to claim 1, comprising a DNA sequence selected from the group consisting of: a. The sequence SEQ ID No. 1 or its complementary strand; and b. DNA sequences that hybridize under high astringency conditions with the DNA sequences defined in section (a)

4. Retroviral vector according to any one of claims 1 to 3, comprising at least one heterologous sequence cloned into any of the restriction targets of the MCS polycloning site.

5. Retroviral vector according to any one of claims 1 to 3, comprising at least one heterologous sequence cloned into any of the restriction targets of the MCS2 polycloning site.

6. Retroviral vector according to claims 4 to 5, wherein said sequences Heterologous comprise one or more copies of the sequence of an internal ribosome entry site (IRES)

7. Retroviral vector according to claims 4 to 5, wherein at least one of said heterologous sequences comprises a marker gene.

8. Retroviral vector according to the preceding claim, wherein said marker gene is selected from the group consisting of: gfp, ΔNGFR, ΔGHR, Neo, Lac Z, β-Geo.

9. Retroviral genomic RNA sequence of the retroviral vector according to claims 1 to 8.

10. DNA sequence of the retroviral provirus produced in the target cell during the reverse transcription process of the retroviral genomic RNA according to claim 9.

11. mRNA derived from the retroviral pro virus according to claim 10.

12. Infectious retroviral particle characterized in that said particle contains the

Genomic retroviral RNA according to claim 9.

13. Host cell characterized in that it has been transfected or transformed with a retroviral vector according to any one of claims 1 to 8 or with the retroviral genomic RNA of claim 9.

14. Host cell characterized in that it is infected with an infective retroviral particle according to claim 12.

15. Method for the production of infective retroviral particles characterized in that a packaging cell line is transfected with a retroviral vector according to any one of claims 1 to 8, and the transfected packaging line is cultured under conditions that allow the release of retroviral particles containing the retroviral genomic RNA according to claim 9.

16. Method for the production of infective retroviral particles characterized in that a cell line is co-transfected with a retroviral vector according to any of claims 1 to 8 and constructs that express auxiliary genes for the packaging of the retroviral genome and the transfected cell line is cultured under conditions that allow the release of retroviral particles containing the retroviral genomic RNA according to claim 9.

17. Method for introducing heterologous or homologous nucleotide sequences into target cells comprising the infection of said target cells with retroviral particles according to claim 12.

18. Use of the retroviral vector, according to any of claims 1 to 8, for the preparation of a pharmaceutical composition for use in gene therapy.

19. Use of the retroviral vector, according to any of claims 1 to 8, for cloning and gene selection and the construction of retroviral libraries.

20. Use of the retroviral vector, according to any of claims 1 to 8, for the expression and / or overexpression of proteins or RNA, and the production and / or purification of peptides or proteins.

21. Pharmaceutical composition comprising a retroviral vector according to any one of claims 1 to 8, further comprising a pharmaceutically compatible carrier.