WO2008125720A1

WO2008125720A1 - Expression vectors which include the human pklr gene promoter and use thereof for preparing pharmaceutical compositions for somatic genetic therapy with specific expression in erythroid cells

Info

Publication number: WO2008125720A1
Application number: PCT/ES2008/070069
Authority: WO
Inventors: José Carlos SEGOVIA SANZ; Nestor W. Meza; Juan Antonio Bueren; Elena Almarza Novoa; Guillermo GÜENECHEA AMURRIO; María Eugenia ALONSO FERRERO
Original assignee: Centro De Investigaciones Energéticas, Medioambientales Y Tecnológicas (Ciemat)
Priority date: 2007-04-17
Filing date: 2008-04-15
Publication date: 2008-10-23
Also published as: ES2325711B1; ES2325711A1

Abstract

Expression vectors which include the human PKLR promoter gene and use thereof for preparing pharmaceutical compositions for somatic genetic therapy with specific expression in erythroid cells. The present invention concerns the use of regulatory sequences of the promoter of the PKLR gene in the production of vectors, with the exception of vectors comprising adeno-associated viruses, for use in the preparation of pharmaceutical compositions intended for somatic genetic therapy in erythroid tissue.

Description

EXPRESSION VECTORS UNDERSTANDING THE PROMOTER OF THE HUMAN PKLR GENE AND ITS USE FOR THE ELABORATION OF PHARMACEUTICAL COMPOSITIONS INTENDED FOR SOMATIC GENE THERAPY WITH SPECIFIC EXPRESSION IN ERYTHROID CELLS.

FIELD OF THE INVENTION

The present invention falls within the field of genetic engineering. Specifically, the invention relates to the use of the human gene promoter that regulates the expression of the R-form of pyruvate kinase (hRPK) in the production of vectors, to be used in the preparation of pharmaceutical compositions intended for somatic gene therapy. Through the use of the PKLR gene promoter, the generation of vectors in which the transgene is specifically and moderately expressed in cells of the erythroid differentiation line has been achieved and more specifically, in the final stages of erythrocyte differentiation.

BACKGROUND OF THE INVENTION

The development of gene therapy as a generalized procedure for the treatment of diseases will depend on the positive balance between the clinical benefit and the risk associated with this new therapeutic intervention. Although the efficacy of gene therapy to produce clinical improvement in patients with inherited disorders (1-6) is already evident, reservations have recently emerged regarding the safety of such therapy. Experimental studies have demonstrated the generation of leukemia in mice associated with the activation of endogenous oncogenes by integrated provirus (7,8). In addition, similar results have been observed in human patients, whose hematopoietic stem cells (CMHs) were transduced with gamma-retroviral vectors containing viral promoters (9,10).

Human erythrocyte pyruvate kinase (RPK) is a glycolytic enzyme that is expressed only in erythroid lineage cells. During erythroid differentiation, the RPK expression gradually increases through a mechanism dependent on the activation of its promoter by specific transcriptional factors of erythroid tissue (11). The expression of RPK is greater in basal and polychromatophilic erythroblasts (11-13).

Human RPK is encoded by the PKLR gene, with an approximate extension of 12 kb. It has a total of 12 exons and 11 introns that encode the RPK iso form, and the LPK iso form produced in the hepatic parenchyma. The two initial exons are specific for each iso form, l (R) for RPK and l (L) for LPK, for specific liver expression. Transcriptional control depends on the specific activity of the promoters flanking each exon 1 (R and L), allowing differential expression in erythroid (RPK) and liver tissue (LPK) respectively. The 5 'region flanking the first exon (IR) has two CAC boxes, four GATA motifs and a new transcription regulatory element called PKR-REl with the motif (CTGTC) located between the 250 base pairs upstream of the start of transcription , which together produce a specific promoter activity in erythroid cells (14-30). Therefore, the expression of the RPK isoenzyme occurs with a high temporal specificity and absolute tissue specificity, only in erythroid tissue, as a consequence of the characteristics described for its promoter and regulatory regions.

The use of specific tissue promoters to express the protein of interest in cells committed to the differentiation of a particular lineage would avoid adverse effects associated with the ectopic expression of the protein in unwanted cells, which has been one of the objectives since the beginning of gene therapy. In addition, in the case of ubiquitous promoters, such as virals, their ability to transactivate endogenous genes, including oncogenes, has been proven after their stable insertion into the genome of the stem cells. The interest of limiting the influence of the promoter present in the vector on the expression of genes close to its integration site has been reinforced by recent observations that indicate that both gamma-retroviral vectors and lentiviral vectors have a high probability of insertion in nearby DNA regions or within genes (31-34). The expression Moderate and specific trangenes with promoters that are not expressed in stem cells would limit the chances of generating leukemia as a result of transactivation of oncogenes in stem cells. One of the main difficulties in generating vectors with expression specificity has been the size of the regulatory sequences, in terms of the number of base pairs necessary to have all the regulatory regions necessary to confer the required specificity. The larger the promoter size, the lower specificity it will have.

Some documentation pertaining to the state of the art that relates to the present invention should be mentioned. First, cite the website http://egp.gs.washington.edu (see selection Finished Genes Table) where information regarding the human PKLR gene located in the q21 band of chromosome 1 is located.

Among this information are several scientific articles of interest. Thus the article Biochemical and Biophysical Research Communications 188 (2): 516-523 (1992), is related to a structural analysis of the PKLR gene and the identification of specific promoter activity in erythroid cells.

Likewise, in the article with bibliographic reference Nucleic Acids Research 20 (21): 5669-5676 (1992), the result of the investigation on the mechanisms responsible for the specificity in erythroid cells of said promoter is offered.

Finally, in articles with reference Blood 101 (4): 1596-1602 (2003) and Blood Cells, Molecules and Diseases 34: 186-190 (2005), information concerning a regulatory element of the specific erythroid promoter of the PKLR gene is provided .

On the other hand, within the patent documentation, two documents must be mentioned. WO 02/29103, refers to a system for the diagnosis and evaluation of the progression of liver cancer, hepatocellular carcinoma or metastasis, of a liver tumor, in a patient by detecting the level of expression of two or more genes (one of them would be the human PKLR gene) in a liver tissue sample. Similarly, WO 02/28999 relates to a method of detecting granulocyte activation by detecting the differential expression of the associated genes (one of them would be the specific erythroid promoter of the human PKLR gene) with said activation in a DNA microarray (biochip). This method allows the monitoring of the various states of different diseases, as well as evaluating pharmacotoxicity.

On the other hand, the European patent document EP 1 193 272, corresponding to a vector of an adeno-associated virus useful in gene therapy, should be cited. Said vector contains an inducible promoter that is the promoter of the pyruvate kinase gene. However, said promoter is the specific liver promoter of the PKLR gene, whose sequence is different from the specific erythroid tissue promoter proposed herein. The liver-specific promoter would not regulate the expression of the PKLR gene in erythroid cell lines.

Mention should also be made of WO 89/02469, concerning an enhancer element of the transcription, specific to human erythroids. Said element provides an improvement in genetic constructs for transfection of erythroid lineage cells. Such constructs will be useful in gene therapy of disorders in erythroid cells, such as thalassemia or hemoglobinopathies. However, in this patent the sequence used is of a larger size (800 bp) than that of the present invention, therefore, it is less specific. In addition, the sequence described in said patent regulates the expression of a gene other than the PKLR gene. It is the β-globin gene, whose homology with the PKLR gene is nil.

The present invention is directed to the use of the specific erythrocyte promoter (RPK) in the construction of vectors, with the exception of vectors comprising adeno-associated viruses, to be used in the preparation of pharmaceutical compositions intended for somatic gene therapy in erythroid tissue . Therefore, the present invention differs from the state of the art because it is not focused on the "per se" promoter, nor is it associated with adeno-associated vectors and its use is specifically directed to the erythroid tissue. DESCRIPTION OF THE INVENTION

Brief Description of the Invention

In the present invention integrative vectors have been developed comprising the promoter and the regulatory regions of the PKLR gene by directing the expression of the gene of interest. More specifically, the sequences of the human genome have been incorporated between the 507 base pairs (bp) upstream of the start of transcription of the PKLR gene and the 94 bp of the open reading frame of the PKLR gene, which had been described as responsible for the deficiency in pyruvate kinase in a patient with hemolytic anemia (8). Together, the promoter region (SEQ ID NO: 1) comprises 601 bp, an optimal size for the construction of specific integrative vectors that do not activate other genes (oncogenes) close to the PKLR gene. The results obtained demonstrate a moderate, stable and specific expression of erythroid lineage cells, both in stable lines of this lineage and in differentiated human primary cells in vitro towards said lineage. Thus, the use of these promoter sequences of moderate and tissue-specific expression limits the problem of most of the vectors present in the state of the art which, when comprising promoters that direct a very high expression of the transgenes ubiquitously in all cells, they facilitate the transactivation of other genes, including oncogenes, in stem cells with the ability to cause leukemia. Therefore, the generation of vectors with promoters that direct the expression of the transgene specifically in differentiated cells constitutes a safer tool for gene therapy.

Description of the figures

Figure 1

Scheme of the generated lentiviral vectors: 1. LSinRPKCG: carries the specific sequences of the PKR promoter by directing the expression of the CopGreen fluorescent protein.

2. LSinPGKEG: carries the regulatory sequences of the promoter of the phosphoglycerate kinase enzyme (PGK) of ubiquitous tissue expression. It is used as a control vector.

Figure 2

to. Representative figure of the expression analysis of the fluorescent protein in three cell lines. In the axis of ordinates (Y) the cell lines used are represented: HL60, non-erythroid differentiation, and cell lines K562 and HEL, both with erythroid differentiation. The abscissa axis (X) represents: CN (non-transfected cells), PKLR (cells transfected with the LSinRPKCG vector) and PGK (cells transfected with the LSinPGKEG vector).

The cells were transduced in vitro with the vectors represented in Figure 1 (LSinPGKEG, LSinRPKCG), grown in vitro for 60 days and their expression analyzed by flow cytometry. Each dot-plot (represented in each of the nine graphs included in this figure) represents, on the abscissa axis (X), the intensity of expression of the fluorescent protein and, on the ordinate axis (Y ), the measured cell size of the cells analyzed. The positive cell window was chosen based on an expression in uninfected cells (CN) of less than 1%. The percentage values included in each of the nine graphs indicate the percentage of positive cells in each dot-plot.

b. Study of the activity and specificity of expression of PKR promoters in immortalized cell lines, transduced with the LSinPGKEG and LSinRPKCG lentiviruses.

On the abscissa axis (X) they are represented, in descending order: • The VCN: average number of viral DNA integrations per cell evaluated by quantitative real-time PCR.

• Type of vector used for transduction: LSinPGKEG lentiviral vector (black bars) and LSinRPKCG lentiviral vector (white bars). • Cell lines used:

I. K562: line of human origin of myelomonocytic and erythroid differentiation. II. HEL: line of human origin of erythroid differentiation.

III. HL60: leukemic line of human origin of myelomonocytic differentiation.

IV. HELA: line of human origin from a cervical carcinoma of epithelial differentiation.

V. WEHI: murine origin line of myelomonocytic differentiation. SAW. 3T3: Murine fibroblast differentiation line of origin. • Cell origin: H (human) and M (murine).

The ordinate axis (Y) represents:

• In the graph above: quantification of the percentages of transduction measured as expression of the fluorescent protein.

• In the graph of the medium: the intensity of expression measured as the average intensity of fluorescence (MFI) evaluated by flow cytometry.

• In the graph below: the activity of the promoter through the evaluation of the transcription index (TI, see detail in description of the invention) by quantitative PCR in real time.

Figure 3

Analysis of the activity of the RPK promoter in human primary cells transduced with lentiviral vectors carrying the green fluorescent protein directed by the RPK promoter in liquid culture. • On the abscissa (X) axis the expression of fluorescent proteins is represented.

• In the ordinate (Y) axis, the days of post-transfection (4, 7, 10 and 14) in which the expression of the fluorescent protein in the culture was analyzed, in the absence (-EPO) and presence (+ EPO) of recombinant human erythropoietin and depending on the vector used: PKLR (use of the LSinRPKCG vector) or PGK (use of the LSinPGKEG vector). CN are non-transfected cells. In brackets, the CNV detected in each culture is represented for each vector.

CD34 + cells purified from umbilical cord blood were transduced in vitro with the LSinPGKEG and LSinRPKCG lentiviral vectors and cultured in the presence of different combinations of growth factors (SCF, 300 ng / mL, thrombopoietin [TPO], 100 ng / mL and Flt3L, 100 ng / mL) and in the absence or presence of recombinant human erythropoietin (5LVmL). Four, seven, ten and fourteen days post-infection, the expression of the fluorescent protein in the culture was analyzed, in the absence and presence of recombinant human erythropoietin. The presence of human erythropoietin induces fluorescent protein expression in CD34 + cultures transduced with the LSinRPKCG vector containing the erythroid specific RPK promoter (PKLR).

Figure 4

Analysis of the activity of the RPK promoter in human primary hematopoietic cells, transduced with lentiviral vectors carried the green fluorescent protein directed by the RPK promoter and cultured in vitro. CD34 + cells purified from umbilical cord were transduced in vitro with lentiviral vectors

LSinPGKEG and LSinRPKCG and cultured in vitro in semi-solid medium enriched with growth factors (H4434, StemCell Technologies) to obtain granulomacrophage and erythroid colonies. The panel on the left shows representative micrographs of erythroid colonies (BFU-E) that are not transduced (CN) or transduced with the LSinRPKCG (PKLR) or LSinPGKEG (PGK) lentiviral vectors. In CN the colonies were negative for the expression of the green fluorescent protein (control). Transduction with the lentiviral vectors LSinRPKCG (PKLR) and LSinPGKEG (PGK) generated positive colonies of fluorescent green protein expression. On the right panel representative micrographs of granulomachlophage colonies (CFU-GM) from human primary cells without transduction are shown. and transduced with the LSinRPKCG and LSinPGKEG lentiviral vectors respectively. Unlike what happened with the erythroid colonies, the granulomachophageal colonies only showed green fluorescence when they were transduced with the lentiviral vector LSinPGKEG (PGFK), but never with the LSinRPKCG vector (PKLR), demonstrating the specificity of the erythroid tissue of the LSinRPKCG vector.

Figure 5

The figure shows the transduction efficiency of human hematopoietic repopulating cells after transduction of CD34 + umbilical cord blood with LSinPGKEG and LSinRPKCG lentiviral vectors and transplantation in NOD / SCID immunodeficient mice.

On the axis of abscissa (X) it is represented, in descending order:

• Mouse type. • VCN (mean number of viral DNA integrations per cell evaluated by quantitative real-time PCR).

• Type of promoter.

In the ordinate (Y) axis, the percentage of colonies positive to the vector is represented depending on whether they are granulomacrophagic (CFU-GM), erythroid (BFU-E) or total (CFCs) colonies, in which qPCR was detected by Proviral DNA of each vector 90 days after transplantation. The number of copies of viral DNA per cell (VCN) of the Human cells obtained from the marrow of each mouse have also been indicated. It is observed that the percentage of colonies positive to the DNA of the transgene is the same in all types of colonies.

Figure 6

The figure shows the activity of the PKLR promoter in vivo in human hematopoietic cells, after transduction of umbilical cord blood CD34 + cells with the LSinPGKEG and LSinRPKCG lentiviral vectors and transplantation in NOD / SCID immunodeficient mice.

to. In the ordinate axis (Y) the percentage of fluorescent colonies is represented and in the abscissa axis (X) the type of mouse and the promoter used are shown in descending order. Thus, the diagram shows the percentage of granulomacrophage colonies (CFU-GM, white bars) and erythroids (BFU-E, gray bars) positive for the expression of the green fluorescent protein, derived from human hematopoietic cells transduced with the LSinPGKEG vectors (PGK) and LSinRPKCG (PKLR) and transplanted in immunodeficient NOD / SCID mice. The analysis was performed at 60 (60 D) and 90 (90 D) days post-transplant.

b. The panel shows representative micrographs of granulomacrophage, erythroid and mixed colonies (granulomacrophages-erythroids) from mice transplanted with CD34 + cells transduced with the LSinRPKCG vector that has specific erythroid activity. The figure shows the specific and differential capacity of the promoter to drive the expression of the fluorescent protein only in erythroid colonies

Detailed description of the invention

In the present invention, a lentiviral vector has been developed, comprising the sequences that confer specific expression in RPK promoter erythroid tissue. (SEQ ID NO: 1), which directs the expression of the CopGreen marker gene (LSinRPKCG) (Figure 1). The results obtained indicated that the vectors containing the aforementioned sequences of the RPK promoter constitute a tool capable of directing the stable, moderate and specific expression of erythroid tissue of transgenes, in mammalian cells both in vitro and in vivo, limiting the circumscribed problem to the vectors present in the state of the art which, by comprising promoters that direct strong expression of the transgenes and in a ubiquitous manner, cause the transactivation of other genes, including oncogenes, in unnecessary cell lines, including in hematopoietic stem cells . Therefore, the use of vectors with promoters that direct a moderate, stable and tissue-specific expression of the transgene constitutes a safer tool for gene therapy. As comparative controls, vectors expressing the EGFP transgene were used under the control of the phosphoglycerate kinase promoter (LSinPGKEG; Figure 1).

Figure 2 shows representative histograms of the activity of the RPK promoter against the PGK promoter, evaluating the percentage of positive cells, the level of expression (measured by the MFI value of the EGFP transgene) and the relative efficiency of the transgene by estimating of the Transcription Index (TI) (35), in human and murine cell lines of hematopoietic differentiation to myelomonocytic or erythroid lineages. As can be seen, while the PGK promoter induces strong fluorescent protein expression in all cell lines, the RPK erythroid promoter induces transgene expression specifically in human erythroid differentiation cell lines (K562 and HEL) and not in the of myelomonocytic differentiation (HL60) or in non-hematopoietic cells (HELA). It is also observed that the expression from the RPK promoter is more moderate and is maintained over time, obtaining similar results after 3 months in culture. It was also found that the RPK promoter, in addition to being tissue specific, was species specific since the expression in murine erythroid differentiation cells (MEL cells) was much lower than that obtained in human cells. As with human cell lines, in murine non-erythroid differentiation lines (WEHI and 3T3 cells) there was no protein expression fluorescent from the RPK promoter. As additional information, the specificity of the RPK promoter was verified by quantifying the presence of transgene messenger RNA, and more specifically, by determining the Transcription Index (IT) that provides information on the activity of a particular promoter in a specific cell type, regardless of the number of copies of the transgene present in the cell and the number of cells evaluated. As can be seen in Figure 2b, the activity of the promoter increases as the phenotype of the erythroid cell line studied is more differentiated and is null in the case of non-erythroid lines. In murine cell lines, IT shows even more the minimal activity of the human promoter.

Overall, it has been proven that RPK promoter sequences that confer expression specificity to erythroid tissue can be used in lentiviral vectors, conferring a stable, moderate and tissue-specific expression of the transgenes to which it directs. Expression from the RPK promoter is undetectable in non-erythroid cells and very low in cells of non-human origin.

The next step in the development of the invention was to study whether the results that had been obtained using established cell lines were also demonstrated in primary hematopoietic cells. For this, primitive human progenitors were obtained from umbilical cord cells obtained with the prior informed consent of the pregnant mothers. Primitive cells, CD34 + were obtained by immunomagnetic purification. Once purified, the cells were infected in vitro with the LSinPGKEG and LSinRPKCG lentiviral vectors and maintained in culture for 14 days in the presence of different combinations of growth factors. At different days after infection, an analysis was performed by flow cytometry, observing that human erythropoietin induced the expression of the fluorescent protein in cells infected with the LSinRPKCG vector.

To confirm the differential activity of the RPK promoter in human erythroid cells, CD34 + cells infected with the LSinPGKEG lentiviral vectors and LSinRPKCG were grown in semi-solid medium in the presence of growth factors that allowed the growth of granulomacrophage colonies (CFU-GM) and erythroids (BFU-E). At 10 days of culture the colonies were analyzed and the expression of the fluorescent protein was determined in the different types of colonies (Figure 4). Whereas in cultures of cells infected with the control vector (PGK) fluorescent colonies could be detected in all types of identified colonies, in cultures of cells infected with the LSinRPKCG vector (PKLR) only erythroid colonies expressed the fluorescent protein. In no case could a CFU-GM colony be expressed expressing the fluorescent protein.

In an in vivo model (Figure 5), after transduction of CD34 + umbilical cord blood cells with the LSinPGKEG and LSinRPKCG lentiviral vectors, the cells were transplanted into immunodeficient NOD / SCID mice, and then they were cultured in vitro such as shown in Figure 4. The activity of the RPK promoter in human hematopoietic cells showed the same erythroid lineage specificity observed in cell line assays and in vitro cultures of human CD34 + cells (Figure 6).

Together, we can conclude that the results obtained demonstrate the specificity of the activity of the hRPK promoter in erythroid lineage cells, inducing stable and moderate transgene expression, both in cell lines and in human hematopoietic primary cells. These observations show the utility of the regulatory sequence described to direct the expression of transgenes of interest to human erythroid cells.

Thus, in a first aspect the present invention relates to the use of regulatory sequences comprising the PKLR gene promoter in the production of vectors (hereinafter vectors of the invention), with the exception of vectors comprising adeno-associated viruses, for be used in the preparation of pharmaceutical compositions intended for somatic gene therapy in erythroid tissue. In a preferred embodiment of the invention, the regulatory sequence is SEQ ID NO: 1 and wherein said therapy is performed "in vivo" in mammals or "in vitro" in cultures of mammalian cells, characterized in that the vector produced comprises an integrative virus and more specifically a Lentivirus virus.

A second aspect of the present invention relates to vectors, with the exception of vectors comprising adeno-associated viruses, characterized in that they comprise at least one regulatory sequence which, in turn, comprises the gene promoter.

PKLR, to be used in the preparation of pharmaceutical compositions intended for somatic gene therapy in erythroid tissue. In a preferred embodiment of the invention the vector would consist of an integrative virus and more specifically a Lentivirus virus.

A third aspect of the present invention relates to cells (hereinafter cells of the invention) transduced by the vectors of the invention.

A fourth aspect of the present invention relates to the use of the vectors of the invention for the preparation of pharmaceutical compositions intended for use in somatic gene therapy.

A fifth aspect of the invention relates to the use of the cells of the invention for the preparation of pharmaceutical compositions intended for use in somatic gene therapy.

A sixth aspect of the invention relates to pharmaceutical compositions comprising the vectors of the invention and pharmaceutically acceptable carriers.

The last aspect of the invention relates to pharmaceutical compositions comprising the cells of the invention, and pharmaceutically acceptable carriers.

The examples are presented below. The detailed exposition of the embodiments, examples and figures that follow are provided by way of illustration and are not intended to be limiting of the present invention. EXAMPLES OF EMBODIMENT OF THE INVENTION

Example 1. Construction of vectors

In the present example, third generation self-inactivating lentiviral vectors were used. Specifically, the LSinPGKEG transfer vectors (pRRLsinlδ.pptPGKeGFPWpre; EGFP, enhanced green fluorescence protein; Clontech) and LSinRPKCG, the packaging vectors pMDLg-pRRE and pRSV-REV and the envelope vector pMD2-VSVG (pMD2-VSVG vector) ). To generate the LSinRPKCG lentiviral vector (pRRLsinlδ.ppthRPKCopGreenWpre), a 601 base pair fragment of the PKLR gene was amplified by PCR and cloned into the pGEMT-EASY cloning vector. 40 clones were sequenced to ensure that the sequence after amplification and cloning remained unchanged. The resulting plasmid pGEMT-PKLR, containing the 601 base pair fragment of the PKLR gene was used to generate a lentiviral vector in which the expression of the CopGreen protein was directed by the promoter sequence of 601 base pairs of the gene. PKLR For this, the 601 base pair fragment of the PKLR gene was cloned into the vector pRRLsinlδ.pptCMVeGFPWpre (18,19), thus replacing the CMV promoter and the EGFP protein with the PKLR promoter and the CopGreen protein (Figure 1). Both the green fluorescent protein EGFP from Clontech and the CopGreen protein from Evrogen have the same characteristics in terms of MFI when expressed under the same promoter in the same number of copies.

Example 2. Production of recombinant Lentivirus supernatant

Supernatants containing the lentiviral vectors were produced by transient cotransfection of the transfer vectors, pRRLsinlδ.ppthPGKeGFPWpre, or pRRLsinl8.ppthRPKCopGreenWpre (9 mg), the packaging vectors pMDLg-pRRE (5.85 mgV) (p5RS-RE) (5.85 mg) 2.25 mg) and the envelope vector pMD2-VSVG (3.15 mg) in 10 cm plates with 293T cells (ATCC) at 60-70% confluence. For transient transduction with these vectors the protocol was carried out as previously described (36,37). The transfective reagent Polyfect (Qyagen) was used for transient transfection with the transfer vectors, following the manufacturer's instructions. In both cases, 1 mM sodium butyrate (Sigma) was added to favor viral production. Supernatants that were recovered 24 and 48 hours after transduction were filtered through 0.45 mm filters and concentrated by ultracentrifugation at 18000 rpm and 4 ° C for 90 minutes.

The titre of the lentiviral supernatants was determined in HEL cells by FACS analysis and expression of the fluorescent proteins in an EPICS XL flow cytometer (Coulter Electronics, Hialeah, FL, USA). We obtained titers greater than 107 v / ml with both vectors.

Example 3. Transduced cell lines

The human non-hematopoietic cell lines used were HeLa epithelial cells and the human hematopoietic cell lines used were the idel HL60 myeloid cell line and the HEL erythroid cell line and the ide K562 myeloid line.

The non-hematopoietic murine cell lines used were NIH 3T3 murine fibroblasts from the ATCC (Rockville, MD, USA). The hematopoietic murine cell lines used were WEHI myeloid murine cells and MEL erythroid murine cells.

HeLa, NIH 3T3, and MEL cells were grown in Dulbecco-modified Eagle's Medium (DMEM, Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Cambrex), antibiotics ( 100 U / ml penicillin and 50 μg / ml streptomycin) and 2 mM L-Glutamine. HEL and WEHI cells were grown in RPMI 1640 (medium of the Roswell Park Memorial Institute, GIBCO-BRL Laboratories), supplemented with 10% FBS, antibiotics and 2 mM L-Glutamine. HL60 and K562 cells were grown in Dulbecco's medium modified by Iscove (IMDM; GIBCO-BRL, Grand Island, NY), supplemented with 20% FBS and antibiotics (100 U / ml penicillin and 50 mg / ml streptomycin).

Example 4. Murine models used

NOD / LtSz-scid / scid (NOD / SCID) mice (deficient in Fc receptors, complement function and the function of B and T lymphocytes and natural killers) were used as receptors for human hematopoietic cells. Mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were handled in sterile conditions and kept in micro-isolators. Before the transplant, the mice from 6 to 8 weeks were irradiated in their entire body with 2.5 to 3.0 Gy of X-rays (the dosage rate was 1.03 Gy / min using a MG X-ray device -324 from Philips, Hamburg, Germany, at 300 kV, 1O mA).

All experimental procedures with experimental animals were carried out in accordance with Spanish and European regulations (RD 223/88 and OM 13-10-89 of the Spanish Ministry of Agriculture, Fisheries and Food, for the protection and use of animals in scientific research; and the European Convention ETS-123, for the use and protection of vertebrate animals used in experimentation and for other scientific purposes).

Example 5. Cell Transduction

Cell lines were transduced by adding different volumes of concentrated Lentivirus supernatants to a known number of cells depending on the multiplicity of infection (MOI) used. The cells were centrifuged 1 hour and 30 minutes at 2500 rpm and 37 ° C after 2 and 24 hours after infection in order to promote transduction efficiency.

Example 6. Analysis by flow cytometry Fluorescent protein positive cells were analyzed by flow cytometry in an EPICS XL flow cytometer (Coulter Electronics, Hialeah, FL).

In vitro or NOD / SCID mouse bone marrow cultured human cells were labeled with anti-human CD45-PECy5 monoclonal antibodies (Clone J33,

Immunotech, Marseille, France), to identify the human reconstitution of these animals, and in combination with anti-human CD34-PECy5 (Clone 8Gl 2; Becton

Dickinson Immunocytometry, San José, CA), CD33-PE anti-human (P67.6; Becton

Dickinson), CD71-PE anti-human (Becton Dickinson), or CD 19-PE anti-human (J4.119; Immunotech) to recognize human primitive cells, myeloid ides cells or B lymphoid cells, respectively.

Finally, the cells were washed, resuspended in PBA with 2 μg / mL propidium iodide (PI), and analyzed on an EPICS XL flow cytometer (Coulter Electronics, Hialeah, FL). A minimum number of 104-105 viable cells was achieved.

Example 7. Human samples and selection of CD34 positive cells

Umbilical cord (CB) blood cells were obtained after a normal delivery in which the gestation period was completed and with the mother's informed consent. Samples were collected at dawn and processed for the next 12 hours after delivery. Mononuclear cells (MN) were obtained by depositing the blood on Ficoll-Hypaque (1,077 g / ml; Pharmacia Biotech, Uppsala, Sweden), and centrifuging at 40Og for 30 minutes. Then the intermediate layer was collected between the Ficoll and the plasma, which was washed three times, and resuspended in the middle of

Dulbecco modified by Iscove (IMDM; Gibco-BRL, Grand Island, NY). To purify the CD34 + cells, the MN fraction was subjected to immunomagnetic separation using the CD34 progeniture cell isolation kit

VarioMACS (Myltenyi Biotech, Auburn, CA), following the manufacturer's recommendations. CD34 + cells were cryopreserved in liquid nitrogen, with 10% dimethylsulfoxide (DMSO) and 20% fetal bovine serum (FBS) until they were used.

Example 8. Liquid culture of human hematopoietic cells CD34 +

The purified cells were washed and resuspended in StemSpam serum-free medium (Stem CeIl Technologies Inc., Vancouver, BC, Canada) supplemented with antibiotics (100 U / ml penicillin and 50 mg / ml streptomycin), 300 ng / ml stem cell factor (SCF, kindly provided by Amgen, Thousand Oaks, CA), 100 ng / ml of thrombopoietin (TPO, kindly assigned by Kirin Brewery, Tokyo, Japan) and 100 ng / ml of the FLT3 ligand (FLT3-L , kindly provided by Amgen, Thousand Oaks, CA). For the induction of erythroid differentiation in some cultures, 5 LVmL of human erythropoietin (hEPO, kindly provided by Amgen, Thousand Oaks, CA) was added. Cultures were maintained at 37 ⁰ C under 5% CO2. At 4, 7, 10 and 14 days of culture, aliquots were extracted and analyzed by flow cytometry.

Example 9. Transduction, culture and transplantation in NOD / SCID mice

After thawing, the CD34 + cells were washed in IMDM supplemented with 10% FBS and antibiotics (100 U / ml penicillin and 50 mg / ml streptomycin). The cells were then resuspended for pre-stimulation for 2 hours with serum-free StemSpan medium (Stem CeIl Technologies Inc., Vancouver, BC, Canada) supplemented with antibiotics (100 U / ml penicillin and 50 mg / ml streptomycin), 300 ng / ml of stem cell factor (SCF, kindly provided by Amgen, Thousand Oaks, CA), 100 ng / ml of thrombopoietin (TPO, kindly assigned by Kirin Brewery, Tokyo, Japan) and 100 ng / ml of FLT3L.

These CD34 positive cells were then transduced with the supernatants of the LSinPGKEG and LSinRPKCG Lentiviruses for 24 hours. During this time the cells were centrifuged after 2 and 20 hours after transduction for 1 hour at 2500 rpm and 37 ° C. They were then washed again and 1 to 3x10 ⁶ cells per mouse were transplanted into subletically irradiated NOD / SCID receptor mice. After the transplant, bone marrow samples from these animals were periodically analyzed by flow cytometry to analyze the degree of implantation of the human transplant and the cell type in which the transgene was being expressed. These bone marrow samples were aspirated from a femur by puncture through the knee joint, according to previously described procedures (38).

The data in the examples are presented in the form of the mean ± standard deviation of the mean. The significance of the differences between groups of data was determined using the Student's t-test. Statistical analysis of the data was performed using the Statgraphics Plus program (Maugistics Inc., Rockville, MD, USA).

REFERENCES

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Claims

1. Use of regulatory sequences that comprise the promoter of the PKLR gene in the production of vectors, with the exception of vectors comprising adeno-associated viruses, to be used in the preparation of pharmaceutical compositions intended for somatic gene therapy in erythroid tissue.

2. Use of regulatory sequences comprising the promoter of the PKLR gene in the production of vectors, with the exception of vectors comprising adeno-associated viruses, to be used in the preparation of pharmaceutical compositions intended for somatic gene therapy in erythroid tissue, according to claim 1, wherein the regulatory sequence is SEQ ID NO: 1.

3. Use of regulatory sequences comprising the promoter of the PKLR gene in the production of vectors, with the exception of vectors comprising adeno-associated viruses, to be used in the preparation of pharmaceutical compositions intended for somatic gene therapy in erythroid tissue, according to claim 1 or 2, wherein said therapy is performed "in vivo" in mammals.

4. Use of regulatory sequences that comprise the promoter of the PKLR gene in the production of vectors, with the exception of vectors comprising adeno-associated viruses, to be used in the preparation of pharmaceutical compositions intended for somatic gene therapy in erythroid tissue, where said therapy is performed "in vitro" in mammalian cell cultures.

5. Use according to any of claims 1 to 4, characterized in that the vector produced comprises an integrative virus.

6. Use according to claim 5, characterized in that the vector produced comprises a Lentivirus virus.

7. Vector, with the exception of vectors comprising adeno-associated viruses, characterized in that it comprises at least one regulatory sequence which, in turn, comprises the promoter of the PKLR gene, to be used in the preparation of pharmaceutical compositions intended for gene therapy somatic in erythroid tissue.

8. Vector according to claim 7, characterized by comprising an integrative virus.

9. Vector, according to any of claims 7 and 8, characterized in that it comprises a Lentivirus virus.

10. Vector according to claim 7, wherein the regulatory sequence consists of the promoter region represented by SEQ ID NO: 1.

11. Vector according to claim 10, which is an integrative vector.

12. Vector according to claim 11, wherein the integrative vector is a lentiviral vector.

13. Vector according to claim 12, wherein the lentiviral virus has been produced from the LSinRPKCG transfer vector (pRRLsinl 8.ppthRPKCopGreenWpre).

14. Vector according to claim 11 or 12, which is a recombinant lentivirus.

15. Vector transduced cells of claims 7 to 14.

16. Use of the vectors of claims 7 to 14 for the preparation of pharmaceutical compositions intended for use in somatic gene therapy.

17. Use of the cells of claim 15, for the preparation of pharmaceutical compositions intended for use in somatic gene therapy.

18. Pharmaceutical compositions comprising vectors of claims 7 to 14 and pharmaceutically acceptable carriers.

19. Pharmaceutical compositions comprising the cells of claim 15, and pharmaceutically acceptable carriers.