ES2325711B1 - 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. - Google Patents

Abstract

Expression vectors comprising the promoter of the human PKLR gene and its use for the preparation of compositions Pharmaceuticals for somatic gene therapy with expression specific in erythroid cells. The present invention relates to the use of promoter regulatory sequences of the PKLR gene in the production of vectors, with the exception of vectors that include adeno-associated viruses, to be used in the preparation of pharmaceutical compositions intended for therapy somatic gene in erythroid tissue.

Description

Expression vectors comprising the promoter of the human PKLR gene and its use for the preparation of compositions Pharmaceuticals for somatic gene therapy with expression specific in erythroid cells.

Field of the Invention

The present invention is encompassed within the field of genetic engineering. Specifically, the invention is refers to the use of the promoter of the human gene that regulates expression of the R form of pyruvate kinase (hRPK) in the production of vectors, to be used in the elaboration of compositions Pharmaceuticals for somatic gene therapy. Through the use of the PKLR gene promoter, the generation of vectors in which the transgene is specifically expressed and of moderate form in erythroid differentiation line cells and more specifically, in the final stages of differentiation towards the erythrocyte.

Background of the invention

The development of gene therapy as generalized procedure for the treatment of diseases depend on the positive balance between the clinical benefit and the risk associated with this new therapeutic intervention. Although it is already The efficacy of gene therapy to produce improvement is evident clinic in patients with inherited disorders (1-6), relative reservations have recently emerged to 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 promoters viral (9,10).

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

Human RPK is encoded by the PKLR gene, with an approximate extension of 12 kb. Present a total of 12 exons and 11 introns encoding the RPK isoform, and the isoform LPK produced in the liver parenchyma. The initial two exons are specific for each isoform, 1 (R) for the RPK and the 1 (L) for LPK, specific expression in liver. He transcriptional control depends on the specific activity of the promoters flanking each exon 1 (the R and the L), allowing their differential expression in erythroid (RPK) and liver tissue (LPK) respectively. The 5 'region flanking the first exon (1R) presents two CAC boxes, four GATA motifs and a new element transcription regulator called PKR-RE1 with the motive (CTGTC) located between 250 current base pairs above the beginning 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 and absolute specificity tissue specificity, only in erythroid tissue, such as consequence of the characteristics described for their regions promoters and regulators.

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

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

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 the specific promoter activity in erythroid cells.

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

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

On the other hand, within the documentation of patents, two documents must be mentioned. WO document 02/29103, refers to a system for diagnosis and evaluation of the progression of liver cancer, hepatocellular carcinoma or metastasis, of a liver tumor, in a patient by means of detection of the expression level of two or more genes (one of them would be the human PKLR gene) in a sample of liver tissue. Similarly, WO 02/28999, relates to a method of granulocyte activation detection 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). Said method allows tracking of various states of different diseases, as well as assess pharmacotoxicity.

On the other hand, the document must be cited European patent EP 1 193 272, corresponding to a vector of a adeno-associated virus useful in gene therapy. 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 here proposed. The specific liver promoter would not regulate the PKLR gene expression in erythroid cell lines.

WO document should also be mentioned 89/02469, concerning an enhancer element of Transcription, specific to human erythroids. Said element provides an improvement in genetic constructs for transfection of erythroid lineage cells. Such constructions will be of utility 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) that that of the present invention, therefore, 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 gene of the β-globin, whose homology with the PKLR gene is void

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 one

Scheme of the lentiviral vectors generated:

one.

LSinRPKCG: carries the sequences specific to the PKR promoter directing protein expression CopGreen fluorescent.

2.

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

Figure 2

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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 cell lines immortalized, transduced with the LSinPGKEG lentiviruses and LSinRPKCG.

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On the axis of abscissa (X) they are represented, in Descending order:

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The VCN: average number of viral DNA integrations per cell evaluated by PCR Quantitative in real time.

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

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Cell lines used:

I.

K562: line of human origin of differentiation myelomonocytic and erythroid.

II.

HEL: line of human origin of differentiation erythroid

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 differentiation line of origin myelomonocytic

SAW.

3T3: murine origin line of differentiation to fibroblast

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Cell Origin: H (human) and M (murine).

The ordinate axis (Y) represents:

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

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

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In the graph below: promoter activity by evaluating the index of transcription (IT, see detail in description of the invention) by quantitative real-time PCR.

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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 promoter RPK in liquid culture.

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On the abscissa axis (X) it represents the expression of fluorescent proteins.

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On the ordinate axis (Y) you they represent the days of post-transfection (4, 7, 10 and 14) in which protein expression was analyzed fluorescent in culture, in absence (-EPO) and presence (+ EPO) of recombinant and vector-based human erythropoietin used: PKLR (use of LSinRPKCG vector) or PGK (use of the LSinPGKEG vector). CN are not cells Transfected In brackets the CNV detected in Each crop for each vector.

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CD34 + cells purified from umbilical cord blood were transduced in vitro with the LSinPGKEG and LSinRPKCG lentiviral vectors and cultured in the presence of the following combination of growth factors: SCF (300 ng / mL) + thrombopoietin [TPO] ( 100 ng / mL) + F1t3L (100 ng / mL), both in the absence and in the presence of recombinant human erythropoietin (5 U / mL). 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).

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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 . Purified CD34 + cells from umbilical cord were transduced in vitro with the LSinPGKEG and LSinRPKCG lentiviral vectors and cultured in vitro in semi-solid medium enriched with growth factors (H4434, StemCell Technologies) to obtain granulomachophageal and erythroid colonies.

The panel on the left shows micrographs representative of erythroid colonies (BFU-E) no transduced (CN) or transduced with lentiviral vectors LSinRPKCG (PKLR) or LSinPGKEG (PGK). In CN the colonies were negative expression of fluorescent green protein (control). Transduction with LSinRPKCG lentiviral vectors (PKLR) and LSinPGKEG (PGK) generated expression-positive colonies of green fluorescent protein The panel on the right shows  Representative micrographs of granulomachophageal colonies (CFU-GM) from human primary cells untranslated and transduced with lentiviral vectors LSinRPKCG and LSinPGKEG respectively. Unlike what it happened with the erythroid colonies, the colonies granulomacrophages only showed green fluorescence when were transduced with the lentiviral vector LSinPGKEG (PGFK), but never with the LSinRPKCG (PKLR) vector, demonstrating the Erythroid tissue specificity of the LSinRPKCG vector.

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Figure 5

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

In the axis of abscissa (X) it is represented, in Descending order:

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Mouse type.

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VCN (average number of viral DNA integrations per cell evaluated by PCR quantitative in real time).

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Type of promoter.

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The ordinate axis (Y) represents the percentage of positive colonies to the vector depending on whether they are granulomacrophagic colonies (CFU-GM), erythroids (BFU-E) or totals (CFCs), in which it was detected by qPCR the proviral DNA of each vector 90 days after transplantation. The number of copies of viral DNA per cell (VCN) of the cells Human obtained from the marrow of each mouse has 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.

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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.

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

b.

He panel shows representative micrographs of colonies granulomacrofágicas, erythroids and mixed (granulomacrofágicas-erythroids) from mice transplanted with CD34 + cells transduced with the vector LSinRPKCG presenting specific erythroid activity. In the figure the specific and differential capacity of the promoter for conduct fluorescent protein expression only in colonies erythroids

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).

Histograms are shown in Figure 2 representative of the activity of the RPK promoter against the promoter PGK, 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 the Index of Transcription (TI) 35, in human and murine cell lines of hematopoietic differentiation to myelomonocytic lineages or erythroid As you can see, while the PGK promoter induces strong expression of the fluorescent protein in all cell lines, the RPK erythroid promoter induces the expression of specifically transcribed in human cell lines of erythroid differentiation (K562 and HEL) and not those of differentiation myelomonocytic (HL60) or in nonhematopoietic cells (HELA). Be note, moreover, that the expression from the RPK promoter is more moderate and is maintained over time, obtaining Similar results after 3 months in culture. I also know verified that the RPK promoter, in addition to being tissue specific, it was species specific since the expression in cells of Murine erythroid differentiation (MEL cells) was much lower than that obtained in human cells. As with the lines Cells of human origin, in murine lines of differentiation no erythroid (WEHI and 3T3 cells) there was no protein expression fluorescent from the RPK promoter. As additional data is checked the specificity of expression of the RPK promoter by quantification of the presence of transgene messenger RNA, and more specifically, by determining the Index of Transcription (IT) that provides information on the activity of a certain 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. How can you check in Figure 2b the activity of the promoter increases the more differentiated is the phenotype of the erythroid cell line studied and it is null in the case of non-erythroid lines. On the lines murine cell phones IT further highlights the minimum Human promoter activity.

Globally, it has been proven that the sequences of the RPK promoter that confer tissue specificity expression Erythroid can be used in lentiviral vectors, giving these a stable, moderate and specific expression of tissue of the transgenes he directs. The expression from of 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 and LSinRPKCG lentiviral vectors are cultured in semi-solid medium in the presence of factors of growth that allowed the growth of colonies granulomacrophages (CFU-GM) and erythroids (BFU-E). At 10 days of culture, the colonies and it was determined, in different types of colonies, the Fluorescent protein expression (Figure 4). Meanwhile in cultures of cells infected with the control vector (PGK) are they were able to detect fluorescent colonies in all types of identified colonies, in cultures of cells infected with the LSinRPKCG (PKLR) vector only the erythroid colonies expressed the fluorescent protein It was not possible to observe a CFU-GM colony expressing protein fluorescent.

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 promoter activity hRPK in erythroid lineage cells, inducing an expression of stable and moderate transgene, both in cell lines and in human hematopoietic primary cells. These observations show the utility of the regulatory sequence described for direct the expression of transgenes of interest to erythroid cells human.

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 mammalian cell cultures, characterized in that the vector produced comprises a integrative virus and more specifically a Lentivirus virus.

A second aspect of the present invention is refers to vectors, with the exception of vectors that include adeno-associated viruses, characterized by they comprise at least one regulatory sequence that, in turn, comprises the promoter of the PKLR gene, to be used in the preparation of pharmaceutical compositions intended for therapy somatic gene 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 is refers to cells (hereinafter cells of the invention) transduced by the vectors of the invention.

A fourth aspect of the present invention is refers to the use of the vectors of the invention for the elaboration of pharmaceutical compositions intended for use in somatic gene therapy.

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

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

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

Then we proceed to expose the examples. The detailed exposition of the embodiments, examples and of the 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 Vector construction

In the present example vectors were used third-party auto-inactivating lentivirals generation. Specifically, the vectors of LSinPGKEG transfer (pRRLsinl8.pptPGKeGFPWpre; EGFP, protein of enhanced green fluorescence; Clontech) and LSinRPKCG, the pMDLg-pRRE and packaging vectors pRSV-REV and the envelope vector pMD2-VSVG (18,19). To generate the lentiviral vector  LSinRPKCG (pRRLsinl 8.ppthRPKCopGreenWpre), a fragment of 601 5 'base pairs of the PKLR gene was amplified by PCR and it was cloned into the cloning vector pGEMT-EASY. 40 clones were sequenced to ensure that the sequence after of the amplification and cloning permanence without modifications. He 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 PKLR gene. For this, the 601 fragment base pairs of the PKLR gene was donated in the vector pRRLsinl8.pptCMVeGFPWpre (18,19), thus replacing the CMV promoter and the EGFP protein by the PKLR promoter and the CopGreen protein (Figure 1). Both the green fluorescent protein EGFP from Clontech like Evrogen CopGreen protein have the same MFI characteristics when expressed under the same promoter in equal number of copies.

Example 2 Production of recombinant Lentivirus supernatant

Supernatants containing vectors Lentivirals were produced by transient cotransfection of transfer vectors, pRRLsinl8.ppthPGKeGFPWpre, or pRRLsinl8.ppthRPKCopGreenWpre (9 mg), the vectors of pMDLg-pRRE packaging (5.85 mg) and pRSV-REV (2.25 mg) and the envelope vector pMD2-VSVG (3.15 mg) in 10 cm plates with cells 293T (ATCC) at 60-70% confluence. For the transient transduction with these vectors the protocol as previously described (36,37).

For transient transfection with transfer vectors the transfection reagent was used Polyfect (Qyagen), following the manufacturer's instructions. In both cases 1 mM sodium butyrate (Sigma) was added to favor viral production Supernatants that recovered 24 and 48 hours after transduction they were filtered through filters 0.45 mm and concentrated by ultracentrifugation at 18000 rpm and 4 ° C for 90 minutes.

The title of the lentiviral supernatants is 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 titles higher than 107 v / ml with both vectors.

Example 3 Transduced Cell Lines

Human non-hematopoietic cell lines used were HeLa epithelial cells and cell lines human hematopoietic used were the line the line of HL60 myeloid cells and the HEL erythroid cell line and the Myeloid line K562.

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

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

Example 4 Murine models used

As hematopoietic cell receptors human mice were used NOD / LtSz-scid / scid (NOD / SCID) (deficient in Fc receptors, the function of complement and function of B and T lymphocytes and killers natural). The mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were handled in conditions sterile and kept in micro-insulators. Before the transplant, mice 6 to 8 weeks were irradiated in the your entire body with 2.5 to 3.0 Gy of X-rays (the speed of Dosage was 1.03 Gy / min using an x-ray machine MG-324 from Philips, Hamburg, Germany, at 300 kV, 10 mA).

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

Example 5 Cell transduction

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

Example 6 Flow Cytometry Analysis

Protein-positive cells fluorescent were analyzed by flow cytometry in a flow cytometer EPICS XL (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 human reconstitution of these animals, and in combination with CD34-PECy5 anti-human (Clone 8G12; Becton Dickinson Immunocytometry, San Jose, 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 cells or B lymphoid cells, respectively.

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

Example 7 Human samples and selection of CD34 positive cells

Cord blood cells were obtained umbilical (CB) after a normal delivery in which the gestation period and with the informed consent of the mother. Samples were collected at dawn and processed during Next 12 hours after delivery. Cells were obtained mononuclear (MN) depositing blood on Ficoll-Hypaque (1,077 g / ml; Pharmacia Biotech, Uppsala, Sweden), and centrifuging at 400 g for 30 minutes. Then the intermediate layer between the Ficoll and the plasma was collected, 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 is underwent immunomagnetic separation using the kit Isolation of CD34 VarioMACS progenitor cells (Myltenyi Biotech, Auburn, CA), following the recommendations of the maker.

CD34 + cells were cryopreserved in liquid nitrogen, with 10% dimethylsulfoxide (DMSO) and serum 20% fetal bovine (FBS) until the time of use.

Example 8 Liquid culture of human hematopoietic cells CD34 +

The purified cells were washed and resuspended in serum-free medium StemSpam (Stem Cell 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 assigned by Amgen, Thousand Oaks, CA), 100 ng / ml of thrombopoietin (TPO, kindly provided by Kirin Brewery, Tokyo, Japan) and 100 ng / ml of FLT3 ligand (FLT3-L, kindly assigned by Amgen, Thousand Oaks, CA). For induction of differentiation Erythroid in some cultures 5 U / mL of erythropoietin was added human (hEPO, kindly provided by Amgen, Thousand Oaks, CA). The cultures were maintained at 37 ° C in a 5% CO2 atmosphere. A 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 will washed in IMDM supplemented with 10% FBS and antibiotics (100 U / ml penicillin and 50 mg / ml streptomycin). Then the cells were resuspended for pre-stimulation for 2 hours with serum-free StemSpan medium (Stem Cell Technologies Inc., Vancouver, BC, Canada) supplemented with antibiotics (100 U / ml of penicillin and 50 mg / ml streptomycin), 300 ng / ml factor stem cells (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 Lentiviruses and LSinRPKCG for 24 hours. During this time the cells will centrifuged 2 and 20 hours after transduction for 1 hour at 2500 rpm and 37 ° C. Then they washed again and transplanted from 1 to 3x10 6 cells per mouse in mice NOD / SCID receivers subletually irradiated. After the transplant bone marrow samples were periodically analyzed for these animals 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 are aspirated a femur by puncturing through the joint of the knee, 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 data groups was determined using the Student's t-test. The statistical analysis of the data is performed using the Statgraphics Plus program (Maugistics Inc., Rockville, MD, USA).

References

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<120> EXPRESSION VECTORS THAT UNDERSTAND THE PROMOTER OF THE HUMAN PKLR GEN AND ITS USE FOR THE ELABORATION OF PHARMACEUTICAL COMPOSITIONS INTENDED FOR SOMATIC GENE THERAPY WITH SPECIFIC EXPRESSION IN ERYTHROID CELLS

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Claims (12)

1. Use of regulatory sequences that include the PKLR gene promoter in vector production, with the exception of vectors that include viruses adeno-associates, to be used in the preparation of pharmaceutical compositions intended for therapy somatic gene in erythroid tissue. 2. Use of regulatory sequences that include the PKLR gene promoter in vector production, with the exception of vectors that include viruses adeno-associates, to be used in the preparation of pharmaceutical compositions intended for therapy somatic gene in erythroid tissue according to claim 1, where the regulatory sequence is SEQ ID NO: 1. 3. Use according to any of claims 1 to 2, characterized in that the vector produced comprises an integrative virus. 4. Use according to claim 3, characterized in that the vector produced comprises a Lentivirus virus. 5. 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 therapy somatic gene in erythroid tissue. 6. Vector according to claim 5, characterized by comprising an integrative virus. 7. Vector, according to any of claims 5 and 6, characterized by comprising a Lentivirus virus. 8. Cells transduced by vectors of the claims 5 to 7. 9. Use of the vectors of the claims 5 to 7 for the preparation of pharmaceutical compositions intended to be used in somatic gene therapy. 10. Use of the cells of claim 8, for the preparation of pharmaceutical compositions intended to be used in somatic gene therapy. 11. Pharmaceutical compositions comprising vectors of claims 5 to 7 and vehicles pharmaceutically acceptable. 12. Pharmaceutical compositions comprising the cells of claim 8, and pharmaceutically carriers acceptable.