Recombinant lentiviral vector, host cell transduced with lentiviral vector, methods for transduction and uses thereof

FIELD: gene engineering.

SUBSTANCE: vector of present invention includes expression cassette containing transgene located under promoter control and central polypurine tract (cPPT) located upstream from cassette. Said vector provides delivery of desired transgenes into target cells and high level expression thereof in such cells. Also disclosed are host cell transduced with lentiviral vector, methods for transduction and uses thereof.

EFFECT: new agent for gene therapy.

26 dwg, 2 tbl, 4 ex

 

Background of the invention

The present invention claims priority of the provisional application for the grant of a U.S. patent serial No. 60/326593, filed October 2, 2001, the full text of which is included here as a reference.

1. The scope of the invention

The present invention relates to improved lentiviruses vectors and their application in gene delivery of desired transgenes into target cells and their expression in these cells at a high level, especially in differentiated blood shoots originating from modified lentiviruses vector of hematopoietic stem cells (hHSC).

2. Description related field

Gene therapy by transduction of hematopoietic stem cells (hHSC) represents a promising approach for the treatment of certain congenital and acquired lymphohematopoietic violations. However, stable manipulation of genes in hHSC with long-term maintenance of populations using existing systems for gene delivery did not provide effective, compatible with therapeutic realities. For example, oncoretrovirus vectors derived from a virus, murine leukemia, Malone (MLV), although are very attractive due to the fact that they integrate a portable product in the chromosomes of target cells can not provide what randaccio hHSC, which had not previously treated with inducers of proliferation (Kohn et al., 1991; Mazurier et al., 1998). Indeed, nuclear transport complex pre-MLV integration requires the destruction of the nuclear envelope, which occurs during mitosis (Roe et al., 1993; Lewis and Emerman, 1994). Unfortunately, hHSC, taken from bone marrow (BM), cord blood (UCB) or mobilized peripheral circulation, for the most part do not share and lose their pluripotency after long-term stimulation and proliferation (Bhatia et al., 1997; Dao et al., 1997; Dorrell et al., 2000). However, in the last message shows that a significant proportion of pluripotent cells and cells capable of long-term implementation entities without the excess weight with diabetes/severe combined immunodeficient (NOD/SCID), also called cells, supporting the population in SCID (SRC), can be supported, subject transduction and can even multiply using the specific conditions of stimulation (Dorrell et al., 2000; Dao et al., 1998; Piacibello et al., 1999; Ueda et al., 2000).

Lentiviruses are a subset of retroviruses that can infect non-dividing cells due caryophyllum properties of complex pre-integration, which enable them to be active import through nucleopore. Accordingly, the lentiviral transfer vectors derived from human immunodeficiency virus type 1 (HIV-1), can mediate efficient delivery, integration and long-term transgene expression in limitationsa cells in vitro and in vivo (Naldini et al., 1996a; Naldini et al., 1996b; Blomer et al., 1997). In particular, based on HIV vectors can carry out efficient transduction of hematopoietic CD34+human cells in the absence of stimulation by cytokines (Akkina et al., 1996; Sutton et al., 1998; Uchida et al., 1998; Miyoshi et al., 1999; Case et al., 1999). These cells are capable of long-term survival in mice NOD/SCID (Miyoshi et al., 1999). From the bone marrow of these primary recipients may re-formed the population of secondary mice with translotsirovannoi cells, which is confirmed by indirect lentivectors genetic modification of very primitive hematopoietic precursors, most likely true stem cells. Because none of the other currently available systems for gene delivery does not possess such ability, lentiviral transfer vectors provide a previously untapped basis for the study of hematopoiesis and gene therapy of congenital and acquired disorders of lymphohemopoietic through genetic modification HSC.

Demonstration of this important point, however, was carried out using early generation lentiviral transfer vector-based, not suitable for therapeutic applications, or because they could not answer the Tr is the requirements of Biosafety (Akkina et al., 1996; Sutton et al., 1998; Uchida et al., 1998), or because they induced the levels of transgene expression, which were unacceptably low (Miyoshi et al., 1999; Case et al., 1999; An et al., 2000). Accordingly, there is a significant need to develop improved lentiviruses for use as vectors transduction, which is capable of efficient transduction of hematopoietic cells, in particular hematopoietic precursor cells, and which is capable of expression of desired transgenes at a high level.

The optimal approach to gene therapy of stem cells should lead to efficient transduction of HSC, and, given the plasticity of stem cells to the limited expression of therapeutic genes in a particular sprouts Mature blood cells. Lentiviral transfer vectors of the third generation currently represent the most optimized tools for gene delivery in HSC person who is not in the cell cycle. Moreover, provided samoanaliziruyuschayasya design (SIN) for the application of tissue-specific promoters without interference to the upstream LTR.

The invention

The present invention relates to the development of improved lentiviral transfer vector-based, which meet the requirements of Biosafety and that can be stimulated, if required for the induction of high ur the init expression of the transgene in a manner specific tissue or germ stem cells. In addition, the present invention relates to the control of transgene expression in the subject transduction cell by activation or repression of transcription, which is the result of contacting the promoter or enhancers with factors in the regulation of transcription.

Accordingly, the present invention relates to carriers for gene transfer, which turned out to be particularly well adapted for transduction of hematopoietic progenitor cells (HPC) of a person and for expression of the transgene in specific differentiated blood shoots or under the control of specific transcription factors. These factors contribute to the further application of lentiviral transfer vector-based for genetic manipulation of hematopoietic stem cells, and, in particular, their use in research and therapeutic applications. Some examples of the examined cell types cover of immature blood cells and Mature blood cells, neutrophils, monocytes/macrophages and granulocytes.

However, the person skilled in the art will understand that the invention is not limited to transduction of hematopoietic cells and that lentiviral transfer vectors according to the invention can be used for specific cells of transgene expression in other cell is types. Some examples of other examined cell types cover completely differentiated cells, such as neurons, lung cells, muscle cells, liver cells, pancreatic cells, endothelial cells, cardiocyte, skin cells, stromal cells of bone marrow and cells of the eyes. In addition, it also addresses such stem cells as cells of the pancreatic ducts, the precursors of nerve cells and mesoderm stem cells.

Thus, the present invention generally relates to improved vectors, which are designed so that they allow transfection and transduction of hematopoietic precursor cells of the person, or stem cells (hHSC), and provide in these cells the expression of desired transgenes high level. In addition, the present invention relates to the limited expression data of desired transgenes in the sense that the expression is regulated to achieve expression in specific descending sprouts HSC or in response to activators of transcription. The vectors of the present invention can also be samonasraivayuschiesya lentivector in the sense that they may contain specific characteristics "samonatyagivayuscheysya" design that makes the data vectors are safe for use in chelovechnye characteristics samonatyagivayuscheysya, or SIN-designs may include modification of the LTR of the vector, so that prevents the reconstruction of replication competent lentiviruses genome. Particularly preferred implementation of such SIN-design includes a deletion of nucleotides at the 3'-region of LTR U3.

Lentivector of the present invention first provides an effective means for achieving a controlled, specific cell types and high-level expression of desired transgenes in the differentiated progeny of genetically modified hHSC. HSC man is hard to put transduction, because, being in estimulando condition, they are relatively resistant to transduction with pre-existing vector systems. Lentiviral transfer vectors of the present invention have the ability to infect non-dividing cells due caryophyllum properties of their complex pre-integration, which ensures their active import through nucleopore. Moreover, the preferred lentiviral transfer vectors of the present invention can mediate efficient delivery, integration and appropriate or long-term expression of transgenes in limitationsa cells in vitro and in vivo, even in the absence of stimulation by cytokines. Stem cells transduced preferred lentivectors for infusion is he to the invention, capable of long-term engraftment, for example, in mice NOD/SCID. But more importantly, more preferred lentivector of the present invention have a very necessary features that provide controlled, with high-level expression of transgenes in specific germs of human progenitor cells and in Mature, differentiated cell types, thus answering the requirements of the Biosafety person.

Therefore, viral vectors of the present invention can, in General, be described as recombinant vectors that contain at least lentivirinae genes gag, pol and rev, or those genes that are required for the production of viruses that enables the production of the vector in the right quantities with the use of available productive cell lines. To comply with important security needs for humans, the preferred vectors of the present invention do not contain any other active antivirusnik genes, such as vpr, vif, vpu, nef, tat. These genes may be deleted or inactivated in a different way. Preferred is that the only active lentivirusnye genes present on the vector, are above the genes gag, pol and rev.

The most preferred combination antivirusnik genes and Karka is a (ie long terminal repeats or LTRS)used when receiving lentivectors of the present invention, is that which proceeds from human immunodeficiency virus (HIV), and, more specifically, of HIV-1. Thus, the genes gag, pol and rev preferably are HIV genes and, more preferably, genes of HIV-1. However, for some applications of the present invention can be used genes gag, pol and rev and LTR region from other lentiviruses, including genes and LTR HIV-2, human immunodeficiency virus monkeys (SIV), human immunodeficiency virus cats immunodeficiency virus bovine virus infectious anemia horses, virus arthritis-encephalitis goats and the like. Such designs can be used, for example, when you want to modify some cells derived not from man. However, based on HIV vector frames (i.e. LTR HIV genes gag, pol and rev HIV) are generally preferred in most aspects of the present invention, as it is based on HIV designs are the most effective for transduction of hematopoietic precursor cells of the person.

Viral vectors of the present invention also contain expressing cassette comprising a transgene under the control of a promoter that is active in terms of providing detectable transcription of trance is in the cell. In a preferred implementation of a promoter active in ensuring transcription of the transgene in hematopoietic cell-ples. A more preferred embodiment include promoters that are active in ensuring transcription in specific cell types or descending the germ cell precursors. A further preferred embodiment are promoters that are subject to control through the activation or suppression factors control transcription or activators and repressor substances.

Examples of promoters that can be preferably used in connection with the present invention, cover gp91-phox, gp47-phox, CD11b, EF1-α, PGK, the promoter of the beta-globin, the promoters of MHC class II, coagulation factor IX, insulin, PDX1 promoter, promoters CD11, CD4 and CD2. Of them, particularly preferred is the promoter of gp91-phox. The promoter gp91-phox is an example of a promoter that provides controlled expression that is restricted to a particular desired cell types, as it contributes to the expression of the transgene primarily in monocytes and granulocytes and because its activity can be modulated by contact promoter with activators, in particular, interferon-gamma (IFN-gamma). Anyway, the embodiment of the present invention, however, is not limited the above promoters, if the promoter is active in the predecessor, blood or other cell that you want to apply as a target, or it responds to control transcription.

To determine whether to use a particular promoter selected promoter tested in the design of in vitro selected cell-precursor, and, if the promoter can provide transgene expression detected with a signal-to-noise ratio, it is, in General, may be used according to the present invention. The required signal-to-noise ratio is about 10 to 200, more preferred the ratio signal/noise is approximately from 40 to 200, and even more preferred the ratio signal/noise is approximately from 150 to 200. One way of testing such a promoter, described here in more detail below, is the application of the transgene, which generates a signal, such as green fluorescent protein (GFP).

The present invention also relates to increased transduction efficiency by including in the vector a Central polypurine tract (cPPT). The transduction efficiency can be approximately 20%, 30%, 40%, 50%, 60%, 70%, or up to and including 80% transduction. In the preferred implementation of the cPPT have higher promoter sequence. Example cPPT is a nucleotide sequence described by SEQ ID NO:1.

Yes inasia preferred aspects of the invention relate to multiple unique cloning sites. Unique cloning sites are parts of the sequences that are recognized by enzymes that are unique within the vector sequence. Some of these areas, clustered together, provide multiple unique cloning sites. These sections preferably have between cPPT and the promoter, or higher cPPT, although they may be there, where it will be convenient to move polynucleotides in vector or from it. For example, data of multiple unique cloning sites easily provide an introduction to the vector elements of the sequence, which are advanced and favorable embodiment of the invention.

The above promoters can include additional elements required for transcription and, thus, to be part of a transcriptional cassette. The transcriptional cassette is defined as containing one or more promoter elements, connected with enhancers and/or areas of control locus in order to guarantee powerful or limited tissue expression of the transgene. One or more enhancers can be located in the vector where they are most active in relation to modulation of transgene expression. To achieve a high level of expression is of ranchera in the target differentiated cell sprouts enhancers can also be a specific target of differentiated shoots. Specific sprouts enhancers include HS-plots. HS-areas known for beta-globin, CD2 and gp91, but can be identified by the additional HS-plots or plots of HS-type. For example, this enhancer GATA-1 erythroblasts. The availability of the sequence of the human genome will greatly facilitate the identification of such elements that are considered part of the present invention.

Especially preferred group enhancer and insulator elements are those that are localized in the field of control locus (LCR) and can be identified as hypersensitive to DNase areas. Coordinated enhancer activity data HS-sites, believed to be responsible for the activity of the chromatin opening domain, which thus facilitates the accessibility of the chromatin factor(s) transcription stimulates protein-protein interactions between the factors binding to the enhancer and promoter are required to define the boundary of the domain. HS-sections present in the CIS-position relative to the cassette, the promoter-gene lead to high-level, independent of the site of integration of the expression. These elements may be in the amount of one or more above or below the transgene cassette. In the most preferred implementation of the invention HS-elements have so that they fit snugly against cPPT element above and below it and completely above promoter. Therefore, data HS-sections can be put in the position of multiple unique cloning sites described above. Under the fit assumes that the specified element, for example, cPPT element that is the first functionally significant element encountered in the scan vector sequence from the borders of the underlying item, such as a promoter element.

For some applications, for example, in the case of promoters that are only moderately active in cells subject to transduction, will need to involve posttranscriptional regulatory sequence located so as to facilitate the expression of the transgene. One type of posttranscriptional regulatory sequence inside expressing cassette is intron, which can serve to stimulate gene expression. However, introns, located thus can exhibit lentivirusnyi RNA transcript for normal cellular mechanisms of splicing and processing. Thus, in a specific implementation, you may need to localize the intron-containing transgenes in the orientation opposite to that of vector genome transcripts.

The preferred method of amplification of transgene expression is the use of posttranscriptional regulatory element, which is not Rel is referring to the events of splicing, such as the item, posttranscriptional processing of herpes simplex virus, posttranscriptional regulatory element of the hepatitis B virus (HPRE) or any of hepatitis North American forest marmot (WPRE), which contains additional acting in CIS-position element, not found in HPRE. A regulatory element located within the vector so that it is part of the RNA transcript of the transgene, but behind the stop codon translational unit of the transgene. Found that the use of such regulatory elements are particularly preferably in the context of moderate promoters, but may be contraindicated in the case of very high efficiency promoters.

Particularly preferable to apply lentivector of the present invention LTR region, which has reduced the promoter activity relative to the wild-type LTR, because such designs provide "samoanaliziruyuschayasya" (SIN) characteristic Biosafety. Samonasraivayuschiesya vectors are vectors, in which the products are full-sized vector RNA in transduced cells is strongly reduced or even terminated. This characteristic greatly reduces the risk that there will be competent in terms of replication recombinants (RCR). Moreover, it reduces the risk that the cellular coding sequences, localizo is installed in close proximity to the site of integration of the vector, will be correctly expressed. Moreover, SIN-design reduces the possibility of mutual interference between the LTR and the promoter, which directs expression of the transgene. It is therefore particularly suitable is to identify the full potential of the internal promoter.

Seminative preferably achieved by introducing a deletion in the U3 region of the 3'LTR of the vector DNA, i.e. the DNA used for the production of vector RNA. Thus, during reverse transcription, this deletion is transferred to the 5'-LTR proviral DNA. You want to eliminate the number sequence U3, sufficient for a strong reducing or eliminating the transcriptional activity of the LTR, with a strong reduction or cancellation of full-sized products of vector RNA in transduced cells. However, in General, you want to preserve those elements of the LTR, which are involved in polyadenylation viral RNA, functions, distributed between the U3, R and U5. Accordingly, you want to eliminate as many transcriptional significant reasons of LTR as possible while maintaining the determinants of polyadenylation. In the case of lentivectors based on HIV, found that these vectors carry a significant deletions U3, including destruction of TATA-box LTR (for example, deletions from -418 to -18), without a significant reduction in the titers of the vector. These deletions result in LTR region, there is TSS in a transcriptionally inactive state, so as transcription ability LTR reduced by approximately 90% or below. In the preferred implementation of the LTR transcription is reduced by approximately 95%-99%. Thus, the LTR can be from about 90%, 91%, 92%, 93%, 94%, 95% 96% 97%, 98% transcriptionally inactive until trascrizione inactive for approximately 99%.

I believe that lentivector of the present invention can be used to deliver any desired transgene, depending on the application. In the case of delivery of hematopoietic precursor cells, typically, you should choose the transgene, which will give these cells the desired function, including, for example, genes Globino, hematopoietic growth factors, which include erythropoietin (EPO), interleukins such as interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-12 (IL-12), and so on) and colony-stimulating factors (such as granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor or colony stimulating factor stem cells), specific platelet integrin αIIbβ), genes multidrug resistance genes gp91-phox or gp47, defective in patients with chronic Wegener (CGD), virus genes, giving the cells resistance to infection that is their pathogens, as the human immunodeficiency virus, the genes encoding coagulation factors VIII or IX, the mutant hemophilia, ligands involved in mediated T-cell mediated immune responses, such as receptors, T-cell antigen receptors of B-cell antigens (antibodies), as well as combinations of receptors on T - and B-cell antigens alone or in combination with single-chain antibodies such as ScFv, tumor necrosis factor (TNF), IL-2, IL-12, gamma-interferon, CTLA4, B7 and the like, genes expressed in tumor cells, such as Melana, MAGE genes (such as MAGE-1, MAGE-3), P198, P1A, gp100, etc.

In the preferred implementation of the transgene to be introduction for treatment is a gene gp91-phox (Dinauer et al. 1987). In additional preferred implementation of the transgene is a gene gp91-phox, functionally linked to a promoter gp91-phox, introduced for the treatment of CGD. In the most preferred implementation of the promoter gp91-phox provides the expression of gp91-phox in monocytes and granulocytes and, in addition, provides modulation of the expression of gp91-phox under the action of the activator of IFN-gamma. In additional preferred implementation of the posttranscriptional regulatory element WPRE feature in the vector to enhance gene expression of gp91-phox. In likewise preferred implementation of the transgene to be introduction for the treatment, represents a gene gp47-phox.

The main application of these transgenes is the delivery of the desired transgene in hematopoietic cells on some possible reasons. They may cover, of course, without limitation, treatment mielosupression and neutropenia, which may be the result of chemotherapy or immunosuppressive therapy, or infections, such as AIDS, genetic disorders, tumors and the like.

Consider a typical genetic disorders of hematopoietic cells cover sickle cell anemia, thalassemia (including beta-thalassemia), hemoglobinopathy, a disease of Glanzmann, lysosomal storage disorders such as Fabry disease, Gaucher disease, Niemann-pick disease and syndrome Wiskott-Aldrich), syndromes severe combined immunodeficiency (SCID), leukocyte adhesion deficiency (LAD), as well as diseases resulting from lack of system products secreted protein, for example, coagulation factor VIII and/or IX. In such cases, it may require the introduction of transgenes, such as genes Globino (including beta-globin), alpha-galactosidase A, glucocerebrosidase, sphingomonadaceae-1, cytokine receptor CD18-integranova subunit, hematopoietic growth factors, which include erythropoietin (EPO), interleukin (oceanliteracy-1, interleukin-2, interleukin-3, interleukin-6, interleukin-12, and so on) and colony-stimulating factors (such as granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor or colony stimulating factor stem cells), specific platelet integrin αIIbβ), genes multidrug resistance genes gp91-phox or gp47-phox, virus genes, giving the cells resistance to infections such pathogens like human immunodeficiency virus, the genes encoding coagulation factors VIII or IX, the mutant hemophilia, ligands, involved in mediated T-cell mediated immune responses, such as receptors, T-cell antigen receptors of B-cell antigens (antibodies), as well as combinations of receptors on T - and B-cell antigens separately and/or in combination with single-chain antibodies (ScFv), IL-2, IL-12, TNF, IFN-γ, CTLA4, B7 and the like, genes expressed in tumor cells, such as Melana, MAGE genes (such as MAGE-1, MAGE-3), P198, P1A, gp100, etc.

A typical malignant tumors are those that are of hematopoietic origin, for example, arise from myeloid, lymphoid or erythroid sprouts, or their precursor cells. Typical myeloid disorders include, without limitation, acute promanager the th leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML). Lymphoid malignancies that can be treated using lentivectors of the present invention, include, without limitation, acute lymphoblastic leukemia (ALL), which includes B-cell ALL and T-cell ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and macroglobulinemia waldenstrom (WM). Additional forms of malignant lymphomas considered as candidates for treatment using a lentiviral transfer vector-based according to the present invention, include, without limitation, nahodkinskuju lymphoma and its variants, peripheral T-cell lymphoma, T-cell leukemia/lymphoma adult (ATL), cutaneous T-cell lymphoma (CTCL), both granular lymphocytic leukemia (LGF) and Hodgkin's disease.

In other implementations the present invention relates to cells-the hosts, who were transpulmonary one of the above lentivectors. I believe that lentivector of the present invention can be used for transduction of essentially any cell. Typical cells include, without limitation, CD4+T-cell lymphocytic cell peripheral blood mononuclear cell peripheral blood hematopoietic stem cells, CL is woven cord blood, the fibroblast, cell, brain cell lung, liver cell, a muscle cell, a cell of the pancreas, endothelial cell, a cardiac cell, a skin cell, a stromal cell bone marrow and cell eye, cell, pancreatic duct, nerve cell-predecessor, mesoderm stem cells and the like. The transduced cells can, in addition, by descent to refer to cells of a Primate, mouse, pig or human, or to be obtained from another species.

For the production of viral particles can be any cell that is compatible with the expression antivirusnik genes gag and pol, or any cell that can be designed to support this expression. For example, can be used such cells-producers, as cells 293T and HT1080 cells.

As noted above, lentivector according to the invention, undoubtedly, can be used, in particular, by transduction of hematopoietic precursor cells of human or hematopoietic stem cells derived from bone marrow, peripheral blood or umbilical cord blood, as well as transduction of CD4+T-cells, B - or T-lymphocytes of peripheral blood mononuclear cells of peripheral blood dendritic cells and macrophage cells. Particularly preferred targets are CD34+cells, including those that highlight the Lena from mobilized peripheral blood.

In another implementation of the present invention relates to a method transduction of hematopoietic stem cells, comprising contacting a population of human cells that contains hematopoietic stem cells, with one of the following lentivectors under conditions suitable for carrying out the transduction of hematopoietic precursor cells of human rights in the specified population data vector. Stem cells can be transpulmonary in vivo or in vitro, depending on the final application. Even in the context of human gene therapy, such as gene therapy of human stem cells can be transducible stem cells in vivo or, alternatively, to transducible them in vitro with subsequent infusion transductional stem cells to a subject person. In one aspect of this invention, a stem cell can be isolated from the human body, for example, patient-man, using methods well-known to specialists in this field, and can be transducible, as described above. Transduced stem cells are then injected back the same or a different person.

In the treatment of the subject-person directly by introducing the vector to the subject, the treatment is usually carried out by intravenous injection of the vector. When cells, such as CD34 cells, dendritic cells, cells of the peripheral blood or tumor cells transducers ex vivo, the vector particles incubated with cells, using doses, in General, about 1 to 50 multiplicity of infection (MOI), which also corresponds to 1·105-50·105units transduction of the viral vector in 105cells. This interval, no doubt, refers to the quantities of the vector corresponding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 and 50 MOI. Usually the number of vector can be expressed in terms of units transduction (TU) HeLa. Other methods of introducing the vector include intraarterial, endoscopic, in the area of corruption, percutaneous, subcutaneous, intramuscular, intrathecal, intraorbitally, intradermal, intraperitoneal, transtracheal, subcuticular by nutritivos injection, by inhalation or intranasal spray, endotracheal path and the like. In the implementation, related to methods for treating tumors/cancer vectors according to the invention, expressing the vector can be delivered by direct injection into the tumor or into the vascular bed of the tumor.

A preferred example of gene therapy ex vivo is a patient suffering from chronic disease Wegener (CGD), CD34+cells which can be isolated from bone marrow or peripheral blood and t is andalready ex vivo by lentivectors, expressing the gene gp91-phox under the control of the promoter of gp91-phox, back before implantation. A similar approach can be used in the treatment of patients suffering from thalassemia, for example, beta-thalassemia, where cells can be transpulmonary lentivectors expressing beta-globin under the control of the beta-globingo or other suitable promoter. Similarly, discusses lentivector of the present invention, expressing the appropriate combination of gene and promoter for the treatment of leukocyte adhesion deficiency (LAD).

In the case of patients suffering from severe combined immunodeficiency (SCID), the inventors consider a similar approach using lentivectors according to the invention, expressing the gene defective in a patient, for example, the gene encoding the common gamma chain interlacing receptor is functionally linked to a suitable promoter, providing specificity in relation to the appropriate tissue or cells and functional control. For the genetic treatment of HIV infection, the authors of the present invention involve intracellular immunization, where the cells attach resistance to the HIV virus through the introduction of antiviral genes. In the implementation of intracellular immunization against HIV target lentivectors according to the invention comprise hematopoietic precursors, D4 +T-cells peripheral blood monocytes. As is clear to a person skilled in the art, such methods intracellular immunization can also be used for other virus infections. For immunotherapy of malignant tumors tumor cells or antigenpresenting cells, such as dendritic cells, construct genetically using lentivectors according to the invention. For methods of treatment of malignant tumors of some transgenes that can be used in lentivector structures according to the invention are those that can inhibit and/or destroy and/or prevent proliferation, and/or to mediate apoptosis of cancer/tumor cells and/or genes, such as TNF.

Described here lentivector can also be used in vivo by direct injection into the blood or in a specific organ. For example, in one implementation for the treatment of Parkinson's disease can be injected into the brain of lentivectors expressing originating from glial cells nerve growth factor (GDNF). In another example, assume the introduction into the portal vein of lentivector expressing coagulation factor VIII, for the correction of hemophilia A. in Another example, assume intravenous or intramuscular injection lentivector of the present invention, expr shirousagi dystrophin gene, for the treatment of Duchenne muscular dystrophy. In a further preferred example, lentivector expressing gp91-phox, is administered for the treatment of chronic disease Wegener (CGD). In a particularly preferred implementation of lentivector expressing gp91-phox under the control of the promoter of gp91-phox, can be injected for the treatment of CGD. So, an ordinary person skilled in the art it is obvious intensive application lentivector structures of the present invention in terms of the methods of gene therapy.

As used in the specification or the claims, in the application of the word "containing", words in the singular means one item or several. Used herein, the expression "another" means, at least a second item or more.

Other objects, features and advantages of the present invention will be understood from the following detailed description. However, it should be understood that the detailed description and specific examples, though, and indicate preferred embodiment of the invention, are for illustration purposes only, therefore, the experts in this field from this detailed description will become apparent, various changes and modifications within the essence and scope of the invention.

Brief description of drawings

The following drawings form part of this specificity is AI and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more drawings in combination with the detailed description presented here is executing.

Figa. Lentivector containing the promoter of gp91-phox. Schematic maps lentiviral transfer vector-based, containing the promoter gp91-phox (1540 n) (pHPP91-GFP) and WPRE sequence (pWPP91-GFP).

Figv. Model of regulation of the promoter of gp91-phox. Transcriptional repressor CDP compete for the binding of activating transcription factors in the four elements. DNA-binding activity of CDP is subject to negative regulation during the final development of phagocytes, which provides the interaction of activators of transcription from the promoter of gp91-phox (Luo W, Skalnik D G. JBC, 271: 18203, 1996).

Figa. Induced by interferon-(expression of GFP in the happening of UCB CD34+monocytes, driven by the promoter of gp91-phox. CD34+cells from UCB were transducible lentivectors pWPT-GFP and pWPP91-GFP (MOI 10). Cells were differentiated in vitro in the presence of GM-CSF (granulocyte-macrophage colony-stimulating factor) in monocytes (CD14+cells) for 3 weeks. Differentiated cells stimulated IFN-((1000 u/ml) for 6 days and were labeled conjugated with PE monoclonal antibodies. The expression of GFP in the PE-positive populations were analyzed using FACS. Numbers indicate percentage of cells in the quadrants.

Figv. The Indus is dummy interferon-(expression of GFP in the happening of UCB CD34 +granulocytes, driven by the promoter of gp91-phox. CD34+cells from UCB were transducible lentivectors pWPT-GFP and pWPP91-GFP (MOI 10). Cells were differentiated in vitro in the presence of G-CSF (granulocyte colony-stimulating factor) in monocytes (CD15+cells) for 3 weeks. Differentiated cells stimulated IFN-((1000 u/ml) for 6 days and were labeled conjugated with PE monoclonal antibodies. The expression of GFP in the PE-positive populations were analyzed using FACS. Numbers indicate percentage of cells in the quadrants.

Figa. Expression of GFP in the bone marrow of mice NOD/SCID, which transplanted transduced by lentivectors CD34+UCB cells. Not transduced CD34+UCB cells were injected intravenously sublethal irradiated mice NOD/SCID (375 THD) (aged 8-10 weeks). After 8 weeks, the bone marrow cells obtained from the thighs of the transplanted mice were labeled conjugated with PerCP antibody against human CD45 labeling the implanted cells, then identified the specific germs of human cells using conjugated with PE antibodies against CD34 (hematopoietic precursor cells), CD19 (B-cells), CD33 (neutrophils), CD14 (monocytes), CD15 (granulocytes), CD42b (megakaryocytes) and glycophorin a (erythroblast). The expression of GFP was analyzed identified as CD45+ and PE-positive cells. Values oznachaet the proportion of cells in the quadrants.

Figv. Expression of GFP in the bone marrow of mice NOD/SCID, which transplanted transduced by lentivectors CD34+UCB cells. Transduced by lentivectors pHPT-GFP (MOI 10) CD34+UCB cells were injected intravenously sublethal irradiated mice NOD/SCID (375 THD) (aged 8-10 weeks). After 8 weeks, the bone marrow cells obtained from the thighs of the transplanted mice were labeled conjugated with PerCP antibody against human CD45 labeling the implanted cells, then identified the specific germs of human cells using conjugated with PE antibodies against CD34 (hematopoietic precursor cells), CD19 (B-cells), CD33 (neutrophils), CD14 (monocytes), CD15 (granulocytes), CD42b (megakaryocytes) and glycophorin a (erythroblast). The expression of GFP was analyzed identified as CD45+ and PE-positive cells. Numbers indicate percentage of cells in the quadrants.

Figs. Expression of GFP in the bone marrow of mice NOD/SCID, which transplanted transduced by lentivectors CD34+UCB cells. Transduced by lentivectors pHPP91-GFP (MOI 10) CD34+UCB cells were injected intravenously sublethal irradiated mice NOD/SCID (375 THD) (aged 8-10 weeks). After 8 weeks, the bone marrow cells obtained from the thighs of the transplanted mice were labeled conjugated with PerCP antibody against human CD45 labeling the implanted cells, then identifying what was aziraphale specific germs of human cells using conjugated with PE antibodies against CD34 (hematopoietic precursor cells), CD19 (B lymphocytes), CD33 (neutrophils), CD14 (monocytes), CD15 (granulocytes), CD42b (megakaryocytes) and glycophorin a (erythroblast). The expression of GFP was analyzed identified as CD45+ and PE-positive cells. Numbers indicate percentage of cells in the quadrants.

Fig.3D. Expression of GFP in the bone marrow of mice NOD/SCID, which transplanted transduced by lentivectors CD34+UCB cells. Transduced by lentivectors pWPT-GFP (MOI 10) CD34+UCB cells were injected intravenously sublethal irradiated mice NOD/SCID (375 THD) (aged 8-10 weeks). After 8 weeks, the bone marrow cells obtained from the thighs of the transplanted mice were labeled conjugated with PerCP antibody against human CD45 labeling the implanted cells, then identified the specific germs of human cells using conjugated with PE antibodies against CD34 (hematopoietic precursor cells), CD19 (B-cells), CD33 (neutrophils), CD14 (monocytes), CD15 (granulocytes), CD42b (megakaryocytes) and glycophorin a (erythroblast). The expression of GFP was analyzed identified as CD45+ and PE-positive cells. Numbers indicate percentage of cells in the quadrants.

File. Expression of GFP in the bone marrow of mice NOD/SCID, which transplanted transduced by lentivectors CD34+UCB cells. Transduced by lentivectors pWPP91-GFP (MOI 10) CD34+UCB cells were injected intravenously sublethal on the obtained mice NOD/SCID (375 THD) (aged 8-10 weeks). After 8 weeks, the bone marrow cells obtained from the thighs of the transplanted mice were labeled conjugated with PerCP antibody against human CD45 labeling the implanted cells, then identified the specific germs of human cells using conjugated with PE antibodies against CD34 (hematopoietic precursor cells), CD19 (B-cells), CD33 (neutrophils), CD14 (monocytes), CD15 (granulocytes), CD42b (megakaryocytes) and glycophorin a (erythroblast). The expression of GFP was analyzed identified as CD45+ and PE-positive cells. Numbers indicate percentage of cells in the quadrants.

Figure 4. WPRE saves the GFP expression directed by the promoter of gp91-phox in myeloid cells in vivo. Data from FIGU, 3C and 3D; background subtracted fluorescence of control cells. Neu - neutrophils, Mo - monocytes, Gr - granulocytes.

Figa, 5B, 5C and 5D. Lentiviral transfer vectors carrying the GFP marker under the control of the constitutive promoter EF-1-alpha (5A, 5B) or control mielodepresivnogo promoter gp91-phox (5C, 5D). Vectors 5B and 5D contain WPRE sequence.

Figa, 6B and 6C. Design lentiviruses vector carrying a fragment of the promoter of gp91-phox (500 n) and the multiple cloning sites (MCS) and the insertion of gp91-phox-specific enhancers, starting with pHPT-GFP. In the first stage, the fragment size of 500 npprovea human gp91-phox was generated by PCR with the use of the cation fragment size 1540 n / a as matrix (sequence MCS-I was included in the 5'-terminal direct primer), and have been built into the XhoI-BamHI pHPT-GFP instead of the short promoter EF-1 (intermediate vector pHP500-GFP). Then PCR fragment containing the MCS-II, cPPT, MCS-I and a fragment of gp91-phox 500 i.e. created using vector pHP500-GFP as template (sequence MCS-II was added to the 5'-direct primer), were built in areas ClaI-BamHI vector pHOX-GFP with the formation of the vector pHPX-GFP (pigv). gp91-phox-Specific enhancers were built sequentially in both MCS above or below the cPPT sequence with the formation of the vector pHPHS-GFP (figs). Finally, the vector pWPHS-GFP was created by insertion of the WPRE sequence in the vector pHPHS-GFP. Position amplified by PCR elements of HS (+1 transcription start gp91-phox) in the DNA sequence of the human genome: HS-12 (-11503, -13244), HS-14 (-13244, -14715), HS-26 (-25345, -26529), HS-27 (-26529, -27656), HS-28 (-27657, -28893) (figs). Described fragments slightly larger than calculated due to the introduction of restriction sites at the ends to facilitate cloning (HS-12, HS-14; gta for obtaining 5' SnaBI after cloning in this site in pHPX-GFP; HS-26 HS-27, HS-28 SalI; gcgtcgac and XhoI; ctcgagcggc).

7. Insert cPPT element in lentiviral transfer vectors. Central polypurine tract (cPPT) was obtained from vector pRRLsinb.hPGK.EGFP (see Follenzi A, Ailles, L. E., Bakovic, S., Geuna, M., Naldini, L. (2000) Gene Transfer by Lentiviral Vecotrs is Limited by Nuclear Traslocation and Rescued by HIV-1 pol Sequences. Nat. Genet. 25: 217-22.). The NotI fragment-EcorRV containing cPPT element, cloned in areas NotI-ClaI vector pHOX-GFP with the formation of pHPT-GFP. Sites NotI and ClaI were placed in both fragments, ligating, so NotI was not saved in pHPT-GFP. A ClaI site was preserved, but was methylated at the expense of the dam. Therefore, this plasmid vector for use of the site ClaI must be grown in dam(-) bacteria.

Description of the illustrative implementations

Although lentiviral transfer vectors represent a great potential for gene therapy, especially for transduction of hematopoietic stem cells (hHSC), developed so far, the vectors did not meet the standards of Biosafety and while inefficient in terms of transgene expression. For example, although containing a CMV-promoter derived from HIV vectors can induce high levels of transgene expression in the Central nervous system (Naldini et al., 1996a; Naldini et al., 1996b; Blomer et al., 1997) and provide an initial demonstration that the pluripotent hemopoietic precursors can be efficiently transpulmonary this tool for gene delivery, they, for the most part, useless in the sense of transfer of therapeutic genes in most cells lymphohemopoietic because these targets their transcriptional activity is too small (Miyoshi et al., 1999; Case et al., 1999; An et al., 2000). Modern Lentini asnie vectors contain multiple attenuated virulence genes of HIV, that removes the potential reconstruction of the wild-type virus by recombination (Zufferey et al., 1997; Dull et al., 1998). Samoanaliziruyuschayasya design makes these vectors even more biologically safe by removing the transcriptional elements of HIV (Zufferey et al., 1998). However, this can have a negative impact on transgene expression, obviously, by reducing the efficiency of polyadenylation (DeZazzo et al., 1991; Valsamakis et al., 1991; Brown et al., 1991; Cherrington and Ganem, 1992; Valsamakis et al., 1992; Gilmartin et al., 1992).

The present invention overcomes these and other deficiencies in this area, and refers to the development of improved vectors derived from HIV, which is optimized in terms of Biosafety and increased gene expression. Thus, in the embodiment of the present invention human cells can transducerless originating from HIV lentivectors that contain elements that prevent the formation of competent in terms of replication of recombinant (RCR), and further contain an internal promoter element, which induces high levels of transgene expression in hematopoietic precursors and differentiated in vitro shoots blood cells and primary T cells. For example, using the vectors of this invention can be transducible CD34+-human cells, and t is the train of other hematopoietic sprouts person.

Promoter elements described vectors contain a promoter gp91-phox, the promoter of beta-thalassemia, promoters gp47-phox and CD4, the EF1 promoter or promoter CD11b, though, as you can understand the person skilled in the art, can be used in almost any promoter element.

The promoter gp91-phox was active in securing expression in specific cell types, namely, differentiated granulocytes and monocytes. Moreover, the promoter of gp91-phox can be activated by contact promoter with activators, such as IFN-gamma. Also discusses the analysis of engraftment and re-population of mice NOD/SCID data vectors, to confirm the stability of expression from these promoters in vivo.

The element that prevents RCR in lentivector of the present invention, is samoanaliziruyuschayasya (SIN) design. It is achieved by deletion of the main part of U3 3'-terminal LTR vector plasmid, which leads to the formation samonatyagivayuscheysya (SIN) configuration (Zufferey et al., 1998). This deletion prevents potential interference between the LTR and the internal promoter elements. However, SIN can induce a decrease in the expression of the transgene, especially in not very strong promoters, such as the PGK promoter. The invention further relates to methods of preserving levels of the transgene in lentivector design is x, who do not have strong promoters, by inserting into a vector of other regulatory elements, such as posttranscriptional regulatory element of the hepatitis C virus is the North American forest marmot (WPRE) or a regulatory element of the hepatitis B virus (HPRE), directly above deletirovanie 3' LTR. Insertion of the WPRE element does not affect the specificity of expression of the promoter elements.

Additional benefits can be achieved by functional inclusion in the vectors HS-elements. For example, the inclusion of the HS-series enhancer elements in pHPX-GFP can lead to higher gene expression and a smaller variation in the level of expression due to silencemag activity of HS. See, for example, May, et al. (2000) "Therapeutic haemaglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin", Nature 406: 82-86, included here as a reference. May, et al. (2000) describe the HS-elements included in lentivector above promoter of the beta-globin, to control higher and also less variable level of expression of the cDNA of beta-globin.

Lentivector according to this invention can effectively transducible the germ cells of some human blood, including CD34+cells, using conditions under which inefficient based on MLV vectors. Moreover, it was also demonstrated that human CD34+cells can effectively who actively to transducerless with a relatively low MOI, although the efficiency of gene transfer reaches about 60-70% of transduced cells. For example, MOI of 10 was used for optimal transduction, which is substantially lower than described in previous studies, where it was changed in the range 60-300 and 1000-3000 (Miyoshi et al., 1999; Case et al., 1999). This may in part be due to an increased likelihood of meeting the vector-target because the methods of the present invention utilizes the effect on CD34+cells vector particles in a small volume (105cells in 200 μl) and for about 6-24 hours

Thus, the present invention relates to the events of HIV vectors that are safe, highly effective and very efficient in terms of transgene expression in hematopoietic cells mammals, and other derivatives blood cells, even in samonatyagivayuscheysya configuration. Moreover, these vectors provide for specific cell types, expression and controlled expression in differentiated cells. Therefore, these vectors provide the right tools for the genetic treatment of diseases such as hereditary and acquired lymphohematopoietic violations for gene therapy of malignant tumors, especially hematological malignancies, for l the treatment and prevention of HIV infection, and also for the study of hematopoiesis mediated through lentivectors modification of human HSC person.

1. Lentiviral transfer vectors and gene therapy

Lentiviruses are complex retroviruses that, in addition to the normal retroviral genes gag, pol and env, contain other genes with a regulatory or a structural function. Higher complexity allows the virus to modulate its life cycle, as in the case of latent infection. Some examples of lentivirus cover the human immunodeficiency viruses HIV-1, HIV-2 and human immunodeficiency virus monkeys: SIV. Lentiviral transfer vectors were obtained by multiple attenuation of virulence genes of HIV, for example, genes env, vif, vpr, vpu and nef were deleterule, making vector biologically safe.

Lentiviral transfer vectors provide great advantages for gene therapy. They are stably integrated into the chromosomes of target cells that is required for long-term expression. Further, they do not tolerate viral genes, thus avoiding the problem of obtaining transduced cells, which can be destroyed by cytotoxic T-cells. Moreover, they are characterized by a relatively large capacity cloning, sufficient for most prospective clinical applications. In addition, lentiviruses, in contrast to other retroviruses, the way the s to transducible non-dividing cells. This is very important in the context of gene therapy tissue, such as blood, brain, liver, lungs and muscles. For example, vectors derived from HIV-1, provide efficient delivery in vivo and ex vivo, integration and stable expression of transgenes in cells, such as neurons, hepatocytes, and myocytes (Blomer et al., 1997; Kafri et al., 1997; Naldini et al., 1996; Naldini et al., 1998).

Lentivirusnyi genome and the proviral DNA contains three genes found in retroviruses: gag, pol and env, which are flanked by two sequences of the long terminal repeats (LTR). Gene gag encodes the internal structural proteins (matrix, capsid and nucleocapsid); pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase, protease and integrase; and the env gene encodes viral envelope glycoproteins. 5'- and 3'LTR are used to provide transcription and polyadenylation of the RNA virion. LTR contain all the other sequences acting in CIS-direction required for virus replication. Lentiviruses contain additional genes, including vif, vpr, tat, rev, vpu, nef and vpx.

To the 5'-LTR adjacent sequences necessary for reverse transcription of the genome (the tRNA binding site of the primer) and for the effective encapsidate viral RNA into particles (the plot Psi). If sequence necessary for encapsidation (or packaging of retroviral RNA infections in the ion virions), lost from the viral genome, CIS-defect prevents encapsidation genomic RNA. However, the resulting mutants capable of directing the synthesis of all proteins of the virion.

In this area known lentiviral transfer vectors, see Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., (1998), Ramezani et al., (2000), all included here as a reference. Also see U.S. patent№5994136; 6013516; 6165782; 6207455; 6218181; 6218186; and 6277633; all of them are included here as a reference. In General, the data vectors based on the plasmid or virus, and they are configured so that the sequences are needed to enable the alien nucleic acid, for selection and transfer of nucleic acid into the cell host.

Two components are involved in the formation based on virus system gene delivery: first, the packaging elements, covering structural proteins and enzymes required for the formation of infectious particles, and secondly, the vector itself, i.e. genetic material that is transferred. In the design of both of these components can be introduced elements of Biosafety. Thus, the packing unit based on HIV vectors of the first generation consisted of all the proteins of HIV-1, in addition to the envelope proteins (Naldini et al., 1998). Subsequently, it was shown that the deletion of four additional viral genes, which are responsible for violentest is, including vpr, vif, vpu and nef, did not change the applicability of the vector system (Zufferey et al., 1997). It was also shown that Tat, the main transactivator HIV is not essential to the formation of a fully effective vector (Dull et al., 1998). Thus, the packaging system of the third generation based on HIV lentiviral transfer vector-based contain only three genes of the parent virus: gag, pol and rev that cancels the possibility of reconstruction of wild-type virus by recombination.

This system was further improved by removing from the vector transcriptional units HIV (Zufferey et al., 1998). In this work it was demonstrated that the introduction of deletions in the U3 region of the 3'-LTR DNA used for the production of vector RNA, generated samonasraivayuschiesya (SIN) vectors. During reverse transcription, this deletion is transferred to the 5'-LTR proviral DNA. A sufficient portion of the sequence has been removed, including the deletion of the TATA-box, which stopped the transcriptional activity of the LTR, preventing the production of full-sized vector RNA in transduced cells. However, this does not affect the titers of the virus or the properties of the virus in vitro or in vivo.

The present invention relates to certain improvements of existing lentivectors as described above and in other parts of this specification. The introduction of lentivector, predostavlyauschie packaging heterologous gene, such as the genes for the treatment of human blood and lymphohemopoietic, this invention provides producing cell, which releases the infectious viral particles carrying the foreign gene of interest.

The env gene may be derived from any virus, including retroviruses. Preferably, env is amphotropic envelope protein, which provides the transduction of cells of humans and other species. Examples derived from retrovirus env genes include, without limitation: virus murine leukemia, Malone (MoMuLV or MMLV), the virus murine sarcoma Harvey (HaMuSV or HSV)virus mammary tumor mouse (MuMTV or MMTV), leukosis virus monkey Gibbon (GaLV or GALV), human immunodeficiency virus (HIV) and sarcoma virus of Rausch (RSV). They may also use other env genes, such as protein G (VSV G) of vesicular stomatitis virus (VSV), one of the hepatitis viruses and flu.

Although protein VSV G is preferred by the env gene, since VSV G gives the recombinant virus is a wide interval owners, VSV G can harm the cell-master. So, when using this gene as VSV G, preferred is the use of inducible promoter system, so that the expression of VSV G can be adjusted to minimize toxicity to the host, when the expression of VSV G is not required. For example, adjustable tetracycli the MD gene expressing the system by Gossen & Bujard, (1992), can be used to provide induced expression of VSV G, when transformed cells are removed tetracycline. Thus, transactivator tet/VP16 is present in the first vector and the coding sequence of the VSV G clone below promoter, operator controlled sequences of tet on another vector.

The vector that provides the viral nucleic acid sequence of the env, functionally associated with regulatory sequences, such as promoter or enhancer. The regulatory sequence can be any eukaryotic promoter or enhancer, including, for example, EF1α, PGK, promoter-enhancer element of the virus murine leukemia, Malone, enhancer of human cytomegalovirus, the promoter cowpox virus P7.5 or similar (see also the examples listed in tables 1 and 2 below). In some cases, such as promoter-enhancer element of the virus murine leukemia, Malone, promoter-enhancer elements are located within the LTR sequences or adjacent to them. Preferably, the regulatory sequence is not endogenous to lentivirus from which the constructed vector. Thus, if the vector created from SIV, a regulatory sequence of SIV found in SIV LTR, will be replaced with a regulatory element is m, which is not happening from the FAQ.

You can continue to target the recombinant virus by binding to envelope protein with the antibody or specific ligand for targeting the receptor specific cell type. When inserting a sequence of interest (including regulatory region) in the viral vector, along with another gene which encodes the ligand for a specific receptor on the target cell, for example, the vector becomes a specific target. Retroviral vectors can be made specific target by inserting, for example, glycolipid, or protein. Targeting is often performed by using antigennegative part of the antibodies or recombinant molecule type antibodies, such as single-chain antibody to target the retroviral vector. Specialists in this area known specific ways to achieve delivery of the retroviral vector to a specific target, or they can easily identify them without undue experimentation.

Heterologous or foreign nucleic acid sequence, such as a polynucleotide sequence encoding this gene as the gene for the treatment of congenital or acquired hematopoietic disorders mentioned herein, functionally linked to a regulatory nucleic acid sequence. Prefer the Ino, the heterologous sequence is associated with a promoter, which results in a chimeric gene.

Marker genes can be used in the analysis of the presence of the vector and, thus, to confirm infection and integration. The presence of the marker gene ensures the selection and growth of only those host cells which Express a paste. Normal genes selection encode proteins that provide resistance to antibiotics and other toxic substances, for example, histidinol, puromycin, hygromycin, neomycin, methotrexate and markers on the cell surface.

The recombinant virus according to the invention is capable of carrying a nucleic acid sequence into a cell of the mammal. The term "nucleic acid sequence" refers to any nucleic acid molecule, preferably DNA, as discussed in detail here. The nucleic acid molecule may be selected from a variety of sources, including DNA, cDNA, synthetic DNA, RNA, or combinations thereof. Such nucleic acid sequence may contain genomic DNA, which may include naturally occurring introns and may not contain them. Moreover, such genomic DNA may be obtained in Association with promoter regions, poly-A sequences or associated with other sequences in the Genomic DNA can be extracted and purified from a suitable cell means, well known in this field. Alternatively, RNA messenger (mRNA) can be isolated from the cells and used for the production of cDNA by reverse transcription, or other means.

Vectors administered by transfection or infection in the packaging cell line. Packaging cell line produces viral particles which contain the vector genome. Methods of transfection or infection are well known to the person skilled in the art. After co-transfection packing vectors and vector transfer in packaging cell line, the recombinant virus isolated from the culture medium and titrated with standard methods used by the experts in this field. Thus, the packing design can be typed in a line of human cells by transfection with calcium phosphate, lipofection or electroporation, mainly together with the dominant breeding marker, such as neomycin, DHFR, glutamylcysteine or ADA, followed by selection in the presence of a suitable medicinal product and the selection of clones. Breeding marker gene can be physically connected in a design with packing genes.

Known stable cell line, which is configured for packing functions for expression suitable for packing cells. For example, smatest U.S. No. 5686279; and Ory et al., (1996)described the packaging cells. Packing cells with introduced them lentiviruses vector form cells-producers. Cells-producers, therefore, are cells or cell lines that can produce or release Packed infectious viral particles, bearing interest of a therapeutic gene. These cells, in addition, may be dependent on the attachment, and this means that these cells grow, survive or optimally support being attached to a surface, such as glass or plastic. Cells-producers can also be a neoplastic transformed cells. Some examples of dependent attachment of cell lines used as packaging cells lentiviruses vector, that vector competent in terms of replication, are either HeLa or 293 cells and PERC.6.

In some applications, especially those where the virus is to be used for applications in gene therapy, it is preferable that the vector was characterized by a lack of replication (or defect replication) in order to avoid uncontrolled proliferation of the virus in susceptible to treatment of the subject. In such cases, choose a mammalian cell lines, which were constructed by modifying the genome of the cell product is a, to encode the inherent virus functions, or through co-infection of cells-producer helper virus for protein expression, the replacement effect sequences that have been deleted from the viral genome. For example, vectors derived from HIV-1, can be used cell line packaging of HIV-1, PSI422, as described in Corbeau, et al. (1996). Similarly, where the viral vector must be produced by a retrovirus, can be used derived from 293 packing retroviruses human cell line (293GPG), capable of producing high titers of retroviral particles, as described in Ory, et al. (1996). When production is minimal vector systems design cell-producer (by modifying the viral genome or by using a helper virus or Comedy) to Supplement the parental virus that provides replication and packaging into virions in cell line-producers.

Lentiviral transfer vectors wrap Naldini et al., (1996)was used for infection of human cells by stopping growth in vitro and for transduction of neurons after direct injection into the brain of adult rats. The vector was effective in the transfer of marker genes in vivo into neurons, and was achieved long-term expression in the absence of detectable pathology. Animals were analyzed ten months after a single injects and vector and it shows no decline in the average level of transgene expression and the absence of signs of pathology tissue or immune response (Blomer et al., 1997).

2. SIN-design

SIN-design improves Biosafety lentiviral transfer vector-based. A large part of the LTR of HIV consists of a sequence of U3. The U3 region contains an enhancer and promoter elements, which modulate the baseline and induced the expression of the HIV genome in infected cells and in response to the activation of the cells. Some of these promoter elements are essential for virus replication. Some of enhancer elements highly conserved viral isolates and used as a critical virulence factors in viral pathogenesis. Enhancer elements may act by affecting the speed of replication in different cellular targets of the virus (Marthas et al., 1993).

Since viral transcription starts at the 3'end of the U3 region of the 5'-LTR, those sequences that are not part of the viral mRNA and its copies from the 3'LTR act as a matrix to obtain both LTR in the integrated provirus. If the 3'copy of the U3 region in the retroviral vector design is changed, the vector RNA is still produced with intact 5'-LTR cells producers, but can not be regenerated in the target cells. Transduction of such, etc) the RA leads to inactivation of both LTR in the virus progeny. Thus, the retrovirus is seminariruumis (SIN), and such vectors are known as SIN-transfer vectors.

SIN-design is described in more detail in Zufferey et al., 1998, and U.S. patent No. 5994136, both source included here as a reference. However, as described here there are limits deletions in the 3'LTR. First, the 5'end of the U3-region performs other essential function when the transfer vector, which is required for integration (terminal dinucleotide + sequence att). Thus, the terminal dinucleotide and the att sequence can be a 5'end border sequence U3, which can be removed. In addition, some loosely defined region can affect the activity of the downstream polyadenylation site in the R-region. Excessive deletion of the U3 sequence of the 3'LTR can reduce the polyadenylation vector transcripts with adverse consequences both for the titer of the vector in cells-producers, and for expression of the transgene in target cells. On the other hand, limited deletions may not violate the transcriptional activity of the LTR in transduced cells.

Described here lentiviral transfer vectors can carry a deletion of the U3 region of the 3'LTR, extending from nucleotide -418 to-18. This is the most extensive deletion, and it is extended to the TATA-box, violating any of transcr plannow LTR activity in transduced cells. The titer of the vector in cells-producers, as well as transgene expression in target cells in such vectors are not exposed to. Thus, this construction provides exceptional increase security of the vector.

Vectors SIN-type with such extensive deletions U3-region can be generated for retroviral vectors based on murine virus leukemia (MLV) or the virus, spleen necrosis (SNV), without impairing the efficiency of transduction.

Deletion of the nucleotide sequence from -418 to -18 stops transcriptional activity of the LTR, cancelling, thus, production of a full-sized vector RNA in transduced cells. However, in the unfolding of the HIV lentivector no properties in vitro or properties in vivo was not broken due to SIN-designs.

3. Posttranscriptional regulatory elements (PRE)

The increase of transgene expression may be required for some implementations, especially those that use lentivirinae design of the present invention with moderate promoters.

One type of PRE intron is located inside expressing cassette, which can stimulate gene expression. However, introns can be cut during the lifecycle events lentivirus. Therefore, if introns are used as PRE, they should put is in the opposite orientation relative to the genomic transcript of the vector.

Posttranscriptional regulatory elements that have no relation to the events of splicing, provide the advantage that they are not removed during the virus life cycle. Some examples are elements posttranscriptional processing of herpes simplex virus, posttranscriptional regulatory element of the hepatitis B virus (HPRE) and hepatitis North American forest marmot (WPRE). Of these the most preferred is WPRE, because it contains additional acting in CIS-direction of the element, not found in HPRE (Donello et al., 1998). This regulatory element is located inside the vector, so it is included in the RNA transcript of the transgene, but outside of the stop codon translational unit of the transgene. As demonstrated in the present invention and Zufferey et al., 1999, WPRE element is a suitable tool to stimulate and enhance gene expression of desired transgenes in the context of a lentiviral transfer vector-based.

WPRE characterized and described in U.S. patent No. 6136597 included here as a reference. As described here, WPRE is an element of export of RNA that mediates efficient transport of RNA from the nucleus to the cytoplasm. It enhances transgene expression by inserting acting in CIS-direction nucleic acid sequence, so that the given element and transgenderists in one transcript. The presence of the WPRE in the sense orientation, as shown, increases the expression of the transgene in 7-10 times. Retroviral vectors carrying sequences in the form of cDNA instead of the full, containing introns of genes, because the introns basically cut within the sequence of events leading to the formation of retroviral particles. Introns mediates the interaction of the primary transcript by splicing apparatus. Since the processing RNA splicing apparatus facilitates their export to the cytoplasm due to the connection between the devices splicing and transport, often cDNA expressed inefficiently. Thus, the inclusion of the WPRE vector leads to enhanced expression of transgenes.

4. Promoters and enhancers

"Promoter" is a control sequence that is a region of nucleic acid sequence, which is controlled by the initiation and rate of transcription. It may contain genetic elements that can bind regulatory proteins and molecules, such as RNA polymerase and other transcription factors to initiate the specific transcription of the nucleic acid sequence. The expression "functionally located", "functionally linked", "controlled by" and "under transcriptional control" mean that a promoter is in the correct functionalname location and/or orientation relative to the nucleic acid sequence to control transcription initiation and/or expression of this sequence.

The promoter, in General, contains sequence that functions with the location of the start sector for the synthesis of RNA. Her best-known example is the TATA box, but in some promoters lacking the TATA-box, such as, for example, the promoter of the gene terminal deoxynucleotidyltransferase mammal and promoter, SV40 late genes, another element adjacent to the start sector, facilitates the accurate determination of the place of initiation. Additional promoter elements to regulate the frequency of transcription initiation. They are usually localized in the region 30-110 i.e. above the start sector, although some promoters have been shown to contain functional elements below the start sector. To place the coding sequence under the control of promoters include the 5'-end of the site of transcription initiation in the transcriptional reading frame "below" (i.e. in the 3'direction) the chosen promoter. "Upstream" promoter stimulates transcription of DNA and promotes the expression of the encoded RNA.

The spacing between promoter elements is often changeable, so that the function of the promoter is saved when its elements are turned or shifted relative to each other. In the tk promoter, the spacing between promoter elements can be increased by 50 N.P., until, until you start to decrease its activity. Could be the cost from the promoter, it turns out that the individual elements can function to activate transcription of the cooperative or independently. The promoter may be used in connection with the "enhancer", which means acting in CIS-towards a regulatory sequence that is involved in activation of transcription of the nucleic acid sequence or enhancer may not be.

The promoter may be associated in nature with a sequence of nucleic acid, which can be obtained by separating the 5'-non-coding sequences, localized above the coding segment and/or exon. Such a promoter may be referred to as "endogenous". Also, the enhancer may be in the nature associated with a sequence of nucleic acid below or above this sequence. Alternatively, some advantage can be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. Recombinant or heterologous enhancer also applies to the enhancer, not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include the step in the promoters or enhancers of other genes and promoters or enhancers, isolated from any other virus, or prokaryotic, or eukaryotic cells, and promoters or enhancers, not "found in nature", i.e. containing different elements of different areas of regulation of transcription and/or mutations that alter expression. For example, promoters that are most commonly used in the construction of recombinant DNA, include a promoter system β-lactamase (penitsillinazy), operons lactose and tryptophan (trp). In addition to the synthetic products of the nucleic acid sequences of promoters and enhancers, sequences can be produced using recombinant cloning and/or technology for nucleic acid amplification, including PCR™in combination with the compositions described herein (see U.S. patent No. 4683202 and 5928906, each of which is included here as a reference). Moreover, it is understood that they can also be used control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like. The control sequence covering promoters, enhancers and other elements of the control/modulation of locus or transcription, also referred to as "transcription cassette".

Naturally, it is important to use the promoter and/or enhancer, that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ or organism selected for expression. Specialists in the field of molecular biology, basically, you know the use of promoters, enhancers, and combinations of cell types for expression of the protein (see, for example, Sambrook et al., 1989, included here as a reference). Used promoters can be constitutive, dependent on tissue, induced and/or may be used in conditions suitable for directing high level expression of the introduced DNA segment, such as those preferred for gene therapy or applications such as large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

The use of cytoplasmic expression system, T3, T7 or SP6 is another possible implementation. Eukaryotic cells can support transcription in the cytoplasm with other bacterial promoters, if given a suitable bacterial polymerase, as part of a complex delivery or as an additional design of gene expression.

Table 1 lists non-limiting examples of elements/promoters that may be used in the context of the present invention for regulating the expression of RNA. In table 2 provide the aulani non-limiting examples of inducible elements, which constitute the region nucleic acid sequence that can be activated in response to a specific stimulus.

Omitz et al., 1987
Table 1

The promoter and/or enhancer
The promoter/enhancerLinks
Heavy chain immunoglobulinBanerji et al., 1983; Gilles et al., 1983; Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al., 1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.; 1990
Light chain immunoglobulinQueen et al., 1983; Picard et al., 1984
T-cell receptorLuria et al., 1987; Winoto et al., 1989; Redondo et al.; 1990
HLA DQ a and/or DQ βSullivan et al., 1987
β-interferonGoodbourn et al., 1986; Fujita et al., 1987; Goodbourn et al., 1988
Interleukin-2Greene et al., 1989
The receptor for interleukin-2Greene et al., 1989; Lin et al., 1990
MHC class II 5Koch et al., 1989
MHC class II HLA-DraSherman et al., 1989
β-actinKawamoto et al., 1988; Ng et al.; 1989
Muscle creatine kinase (MCK)Jaynes et al., 1988; Horlick et al., 1989; Johnson et al., 1989
Prealbumin (transthyretin)Costa et al., 1988
Elastase I
Metallothionein (MTII)Karin et al., 1987; Culotta et al., 1989
CollagenasePinkert et al., 1987; Angel et al., 1987
AlbuminPinkert et al., 1987; Tronche et al., 1989, 1990
α-fetoproteinGodbout et al., 1988; Campere et al., 1989
γ-globinBodine et al., 1987; Perez-Stable et al., 1990
β-globinTrudel et al., 1987
c-fosCohen et al., 1987
c-HA-rasTriesman, 1986; Deschamps et al., 1985
InsulinEdlund et al., 1985
Neural cell adhesion molecule (NCAM)Hirsh et al., 1990
α1-antitripsinLatimer et al., 1990
Histone H2B (TH2B)Hwang et al., 1990
Collagen mouse and/or type IRipe et al., 1989
Glucoseregulated proteins (GRP94 and GRP78)Chang et al., 1989
Growth hormone ratLarsen et al., 1986
Serum amyloid A (SAA) personEdbrooke et al., 1989
Troponin I (TN I)Yutzey et al., 1989
Growth factor platelet origin (PDGF)Pech et al., 1989
Muscular dystrophy DuchenneKlamut e al., 1990
SV40Banerji et al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al., 1988
PolyomaSwartzendruber et al., 1975; Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndell et al., 1981; G. Gallina et al., 1983; de Villiers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbell and/or Villarreal, 1988
RetrovirusesKriegler et al., 1982, 1983; Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al., 1988; Celander et al., 1988; Chol et al., 1988; Reisman et al., 1989
The human papilloma virusCampo et al., 1983; Lusky et al., 1983; Spandidos and/or Wilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987
Hepatitis b virusBulla et al., 1986; Jameel et al., 1986; Shaul et al., 1987; Spandau et al., 1988; Vannice et al., 1988
The human immunodeficiency virusMuesing et al., 1987; Hauber et al., 1988; Jakobovits et al., 1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddock et al., 1989
CD11bHickstein et al., 1992
The leukosis virus monkey GibbonHolbrook et al., 1987; Quinn et al., 1989

Table 2

Induced
The inductorLinks
MT II Herbology ester (TFA)heavy metalsPalmiter et al., 1982; Haslinger et al., 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989
MMTV (virus mammary tumor mouse)GlucocorticoidsHuang et al., 1981; Lee et al., 1981; Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta et al., 1985; Sakai et al., 1988
β-interferonPoly(rI)x

Poly(rc)
Tavernier et al., 1983
Adenovirus 5 E2E1AImperiale et al., 1984
CollagenaseHerbology ester (TPA)Angel et al., 1987
StromelysinHerbology ester (TPA)Angel et al., 1987b
SV40Herbology ester (TPA)Angel et al., 1987b
Murine MX geneInterferon, Newcastle disease virusHug et al., 1988
Gene GRP78A23187Resendez et al., 1988
α-2-macroglobulinIL-6Kunz et al., 1989
VimentinSerumRittling et al., 1989
MHC Class I Gene H-2κbInterferonBlanar et al., 1989
HSP70E1A, Large T-antigen SV40Taylor et al., 1989, 1990a, 1990b
Herbology ester-TPAMordacq et al., 1989
The tumor necrosis factorPMAHensel et al., 1989
Gene thyroid-stimulating hormone αThyroid hormoneChatterjee et al., 1989

The distinctive features of tissue-specific promoters or members, as well as analyses to characterize their activity is well known to specialists in this field. Non-limiting examples of such areas cover the human LIMK2 gene (Nomoto et al., 1999), gene somatostatinomas receptor 2 (Kraus et al., 1998), gene binds retinoic acid protein appendages of testicles mouse (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), murine alpha-2-(XI)collagen (Tsumaki, et al., 1998), gene dopamine receptor D1A (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997) and the molecule-1 adhesion of platelets to endothelial cells (Almendro et al., 1996).

Lentiviral transfer vectors of the present invention designed initially for cell transformation therapeutic gene under the control of the regulated eukaryotic promoters. Although it is preferred promoter gp91-phox, can also be used by the promoter and other regulatory signaling elements described above in tables 1 and 2. In addition, also can use any combination of prom is Thor/enhancer (according to the database of eukaryotic promoters EPDB) to direct the expression of the structural genes, coding interested in therapeutic gene, which is used in the context of lentivirusnye vectors of the present invention. Alternatively, a tissue-specific promoter for gene therapy of malignant tumors or for sighting effect on the tumor can be used with lentivirusnye vectors of the present invention for the treatment of malignant tumors, especially malignant hematological diseases.

Usually promoters and enhancers that control transcription of protein coding genes in eukaryotic cells composed of multiple genetic elements. The cellular phone can combine and integrate regulatory information, which carries each element that allows different genes to form various, often complex profiles transcriptional regulation. Activation or repression of promoter and enhancer elements can be carried out by contact of these elements with appropriate transcriptional activator or repressor substances, such as described in figv for promoter gp91-phox and disclosed in Luo and Skalnik (1996) J. Biol. Chem. 271: 18203-210, and Luo and Skalnik (1996) J. Biol. Chem. 271: 23445-23451 included here as a reference. In respect of the promoter gp91-phox, the activity of interferon-gamma in modulating the transcription and expression of the expression cassette I have is example as such promoter or enhancer elements and factors that interact with them, can be used in the embodiment of the present invention.

The enhancers were first identified as genetic elements that increase the transcription promoter is localized in a remote position on the same DNA molecule. This ability to operate at a great distance rarely occurred in classical studies the regulation of transcription in prokaryotes. Subsequent work showed that DNA with enhancer activity is organized much like the promoters. That is, they consist of many separate elements, each of which is associated with one or more transcriptional proteins. See, for example, the model of regulation of the promoter gp91-phox presented on figv. Typical enhancers considered in this invention, are hypersensitive to DNase elements and their homologues, as described in the publication Lien LL, Lee Y, Orkin SH, (1997) "Regulation of the myeloid-cell-expressed human gp91-phox gene as studied by transfer of yeast artificial chromosome clones into embryonic stem cells: suppression of a variegated cellular pattern of expression requires a full complement of distant cis elements," Mol Cell Biol. 17(4): 2279-90, specially fully included here as a reference. Under the influence of these enhancer elements, the gene expression may increase (due to enhancer activity HS) and be less than VA is iabeling (due silencemag activity HS).

Analogues HS-elements gp91-phox active in other promoter-enhancer systems. See, for example, the publication May C, Rivella S, Callegari J, Heller G, Gaensler K M, Luzzatto L, Sadelain M, (2000) Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. Nature 406(6791): 82-6, specially fully included here as a reference, where a similar HS-elements of the beta-globin built into lentivector above promoter of the beta-globin to direct the expression of the cDNA of beta-globin.

Promoters and enhancers are characterized by the same General function of the activation of transcription in the cell. They often overlap and are adjacent to each other, and often they seem to have a similar modular organization. Data combined considerations indicate that the enhancers and promoters are homologous objects and that proteins are activators of transcription, bind to these sequences can interact with cellular transcription apparatus is fundamentally the same way. The main difference between enhancers and promoters is practical. Enhancer region as a whole must be able to stimulate transcription at a distance; this may not be binding to the promoter region or of its constituent elements. On the other hand, the promoter must have one or more elements, which direct the initiation of synthesis of P Is in the specific area and in a particular orientation, while the enhancers do not have such specific features. Without this practical differences between enhancers and promoters are very similar objects. The design elements that control the transcription and expression may therefore be composed of various elements, systematized so that feature ways to control increased the usefulness and effectiveness.

The signal, which may be applicable, represents the polyadenylation signal (hGH, BGH, SV40). The use of elements of the internal binding sites of the ribosome (IRES) was used to create multigene, or polycistronic mRNA. IRES elements are able to bypass the model of the scanning ribosome 5'-methylated cap dependent translation at internal sites (Pelletier and Sonenberg, 1988). Described IRES-elements of two representatives of the family of picornaviruses (polio and encephalomyocarditis) (Pelletier and Sonenberg, 1988), as well as the IRES from a mammalian mRNA (Macejak and Sarnow, 1991). IRES elements are able to communicate with heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by IRES, with the formation of a polycistronic mRNA. In the presence of the IRES element, each open reading frame is available to the ribosomes for effective broadcast. Multiple genes can effectively expression is to be cleansed and purified using a single promoter/enhancer for transcription of one mRNA.

In any case, it is clear that the promoters are DNA elements that are at the location of the above gene leads to the expression of this gene. A large part of transgenes, which transform using lentiviral transfer vector-based according to the present invention, functionally arranged below the promoter element.

Specific signal initiation may also be required for efficient translation of coding sequences. These signals cover the initiating codon ATG or adjacent sequences. Exogenous control signals broadcast, including the initiating ATG codon, may be necessary. An ordinary person skilled in the art can easily identify them and provide the necessary signals. It is well known that the initiating codon must be in frame with the open reading frame of the desired coding sequence to ensure translation of the entire insert. Signals exogenous control broadcast initiator codons can be natural or synthetic. The efficiency of expression may be enhanced by the inclusion of suitable enhancer elements, transcription.

5. Brief description of sequence listings

SEQ ID NO:1 provides the nucleotide sequence of the Central element poliprinolov tract HIV taken from HIV, which is reported Follenzi, A, Ailles, LE, Bakovic, S., Geuna, M., Naldini, L. (2000) "Gene Transfer by Lentiviral Vecotrs is Limited by Nuclear Translocation and Rescued by HIV-1 pol Sequences," Nat. Genet. 25:217-22. SEQ ID NOS:2-6 provide the nucleotide sequence encompassing areas, hypersensitive to DNase, and are used as enhancers in some implementations of the invention. Cm. Lien LL, Lee Y, and Orkin SH, "Regulation of the myeloid-cell-expressed human gp91-phox gene as studied by transfer of yeast artificial chromosome clones into embryonic stem cells: suppression of a variegated cellular pattern of expression requires a full complement of distant cis elements," Mol. Cell. Biol. 17(4): 2279-90 (1997).

SEQ ID NOS:7-16 provide the nucleotide sequence of PCR primers used to obtain HS-elements of SEQ ID NOS:2-6. Sequences of PCR primers and HS-elements based on the sequence of the human genome, published in the framework of the project "human Genome" (contig NT-011844; http://www.ncbi.nlm.nih.gov/genome/guide/human/). Provisions amplified by PCR HS-elements (+1 - start transcription gp91-phox) in the DNA sequence of the human genome are: HS-12 (-11503, -13244), HS-14 (-13244, -14715), HS-26 (-25345, -26529), HS-27 (-26529, -27656), HS-28 (-27657, -28893). Used fragments of slightly greater than calculated due to the inclusion at the ends of the restriction sites to facilitate cloning (HS-12, HS-14; gta to restore the 5'-end SnaBI after cloning in this section pHPX-GFP; HS-26, HS27, HS-28; SalI; gcgtcgac and XhoI; ctcgagcggc).

Other sequences included in specific is sushestvennee of the present invention, cover those that encode gp91-phox and their homologues, including the nucleotide sequence disclosed under the inventory number of the GenBank NM000397 for gp91-phox (Dinauer et al., 1987), SEQ ID NO:18 and the polypeptide sequence SEQ ID NO:19. Also included in the concrete implementation of the promoters of gp91-phox encoded by the nucleotide sequence under the inventory number of the GenBank M66390, SEQ ID NO:17 and the CD11b promoter encoded by the nucleotide sequence with the inventory number of the GenBank M82856, SEQ ID NO:20.

6. Nucleic acids

One implementation of the present invention is the transfer of nucleic acids encoding therapeutic gene, especially gene, which provides treatment of disorders of the blood and lymphohemopoietic, such as acquired or congenital disorders described above. In one implementation nucleic acids encode full-length, essentially a full-sized, or functionally equivalent form of this gene.

Thus, in some implementations of the present invention the treatment of disorders of the blood and lymphohemopoietic covers introduction lentiviruses vector according to the invention, containing expressing the design of therapeutic nucleic acid into a cell of hematopoietic origin. It is assumed that hematopoietic cells capture the design and expre serout therapeutic polypeptide, encoded by the nucleic acid, restoring, so the normal phenotype of the cells.

The nucleic acid may be obtained by any method known to the person skilled in the art. Non-limiting examples of a synthetic nucleic acid, in particular, synthetic oligonucleotide cover a nucleic acid obtained by chemical synthesis in vitro using phosphocreatine, hospitaI or phosphoramidite chemistry and solid-phase methods, such as described in EP 266032, included here as a reference, or through deoxynucleoside-H-postnataly intermediates as described by Froehler et al., 1986, and in U.S. patent No. 5705629, each of which is included here as a reference. A non-limiting example of the produced enzyme nucleic acid encompasses such acid produced by enzymes in amplification reactions such as PCR (see, for example, U.S. patent No. 4683202 and 4682195, each of which is incorporated here by reference), or the synthesis of oligonucleotides described in U.S. patent No. 5645897 included here as a reference. Non-limiting example of biologically produced nucleic acid comprises the production of recombinant nucleic acids in living cells (see, for example, Sambrook et al. 1989, included here as a reference).

Nucleic acid can be clean n the polyacrylamide gels, on gradient centrifugation in cesium chloride, or any other means known to the ordinary person skilled in the art (see, for example, Sambrook et al. 1989, included here as a reference).

The term "nucleic acid" generally refers to at least one molecule or chain DNA, RNA or a derivative or mimetic comprising at least one nucleic acid base, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine "A," guanine "G," thymine "T" and cytosine "C") or RNA (e.g. A, G, uracil "U" and C). The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide". The term "oligonucleotide" refers to at least one molecule with a length of from about 3 to 100 nucleic bases. The term "polynucleotide" refers, at least one molecule with a length of more than about 100 nucleic bases. These definitions generally include at least one single-stranded molecule, but in specific implementations also include at least one additional circuit, which in part, on the merits or fully complementary to at least one single-stranded molecule. Thus, the nucleic acid may include at least one double-stranded molecule or, at the very the least, one of triplex molecule that contains one or more complementary circuits, or "complements" the specific sequence containing the chain of this molecule.

In a specific implementation "gene" refers to nucleic acid that is transcribed. As used here, "gene segment" means a segment of a nucleic acid of the gene. In some aspects, the gene comprises regulatory sequences involved in transcription or products or compositions mRNA. In a specific implementation transcribed gene contains sequences that encode a protein, polypeptide or peptide. In other specific aspects of gene contains a nucleic acid and/or encoding encodes a polypeptide or peptide sequence of the gene that is defective or mutant with impaired blood or lymphohemopoietic. In the framework described here, the terms "isolated gene" may contain transcribed(s) of nucleic acid(s) - acid(s), regulatory sequences, coding sequences, or the like, selected essentially separately from other such sequences, such as naturally occurring genes, regulatory sequences, sequences encoding the polypeptide or peptide, etc. In this respect, the term "gene" is used simply is that for the reference nucleic acid, containing a nucleotide sequence that is transcribed, and she complementary sequence. In a particular aspect of the transcribed nucleotide sequence contains at least one functional unit, and encoding a protein, polypeptide and/or peptide. As it is clear to experts in the field, this functional term "gene" encompasses both genomic sequences, RNA sequences or cDNA, or designed smaller segments of nucleic acids, including nucleic acid segments are not transcribed part of the gene, including, without limitation, transcribed promoter or enhancer region of the gene. Smaller engineered gene segments of nucleic acids can be Express or may be adapted for the expression of proteins, polypeptides, domains, peptides, fusion proteins, mutants and/or the like, using the technology of manipulation of nucleic acid. Thus, the shortened gene" refers to nucleic acid sequence that lacks plot consecutive residues of nucleic acids.

Different segments of nucleic acid can be designed based on the specific nucleic acid sequence and can be of any length. By assigning a follower of the spine of the numerical values, where, for example, the first residue is a 1, the second residue is 2, etc, can be created with an algorithm defining all segments of nucleic acids:

from n to n+y,

where n is an integer from 1 to the last number of the sequence, and y represents the length of a segment of nucleic acid minus one, where n+y does not exceed the last number of the sequence. Thus, for a 10-Clinica segments of nucleic acids correspond to bases 1 to 10, 2 to 11, 3 to 12... and/or so forth. For 15-Clinica segments of nucleic acids correspond to bases 1 to 15, from 2 to 16, from 3 to 17... and/or so forth. For 20-Clinica segments of nucleic acids correspond to bases 1 to 20, from 2 to 21, from 3 to 22... and/or so forth.

Nucleic acid(s) - acid(s) according to the present invention, regardless of the length of the sequence itself, may(may) be combined with other nucleic acid sequences, including, without limitation, promoters, enhancers, polyadenylation signals, lots of enzymes, multiple cloning sites, coding segments, and the like, to create one or more structures of nucleic acids. The total length of the structures of nucleic acids can greatly vary. Thus, can be used behold the cops nucleic acid almost any length to total length, which preferably is limited by the ease of obtaining or application in accordance with the required method of recombinant nucleic acids.

The term "vector" is used to refer to a carrier nucleic acid molecule, which can be integrated sequence of nucleic acid for introduction into a cell where it can be replicated. The vectors of the present invention is based on lentiviruses as described above and in other parts of the specification. Molecules of nucleic acid carried by the vectors according to the invention, encode a therapeutic genes and will be used for carrying out the methods of gene therapy. The specialist in this area is equipped with everything necessary for constructing such a therapeutic vector using standard recombinant methods (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both source included as a reference).

The term "expressing vector" refers to any type of genetic constructs containing nucleic acid encoding a RNA capable transcribed. In some cases, the RNA molecules are then translated into a protein, polypeptide or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expressing the vectors may contain different "to stalnye sequence", which refer to nucleic acid sequences necessary for the transcription and possibly translation operatively linked coding sequence in a particular cell host. In addition to control sequences that govern transcription and translation, vectors and expressing the vectors may contain nucleic acid sequence that also perform other functions, and are described below.

A. Multiple cloning sites

The vectors of the present invention may include a multiple cloning site (MCS), which is a region of nucleic acid that contains multiple sites of restriction enzymes, each of which can be used in conjunction with standard recombinant technology to the splitting of the vector (see, for example, Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, incorporated here as a reference). "Cleavage with restriction enzymes" means the catalytic splitting of molecules of the nucleic acid enzyme, which functions only in a certain position in the molecule of nucleic acid. Most of these restriction enzymes are commercially available. The use of such enzymes is largely understandable to experts in this field. Often, the vector Lin is arisaema or fragmented using a restriction enzyme, which cuts the molecule in the MCS to provide the possibility of exogenous sequences to lagerbuchse with the vector. "Ligation" refers to the process of education fosfolipidnyh relations between the two nucleic acid fragments, which can be adjacent to each other or not. Methods involving restriction enzymes and ligation reaction, well known to experts in the field of recombinant technology.

Century Sites of splicing

Most transcribed eukaryotic RNA molecules are RNA splicing to remove introns from the primary transcript. For vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression (see, for example, Chandler et al., 1997, included here as a reference).

C. Signals termination

The vectors or constructs of the present invention typically will include at least one terminal signal. "Terminal signal" or "terminator" is in the DNA sequences involved in specific termination of RNA transcript RNA polymerase. Thus, in some implementations considered signal termination, which completes the formation of the RNA transcript. Those who mentor may be necessary in vivo to achieve the required levels of mRNA.

In eukaryotic systems, the termination region may also include certain DNA sequences that allow site-specific cleavage of a new transcript in order to make available the site of polyadenylation. This gives a signal of certain endogenous polymerase to add an area approximately 200 residues (poly A) to the 3'-end of the transcript. RNA molecules modified with the help of the late poly A, apparently, are more stable and are translated more efficiently. Thus, in other implementations related to eukaryotes, it is preferable that the terminator has included the signal for the cleavage of RNA, and most preferably termination signal contributed to polyadenylated mRNA. The terminator and/or elements of the polyadenylation site can serve to increase the levels of mRNA and to minimize end-to-end reading from the cassette in the other sequences.

Terminators considered for use in the invention include any transcription terminator described herein or known to the ordinary person skilled in the art, including, without limitation, for example, the sequence termination of genes, such as, for example, the terminator bovine growth hormone or viral sequence termination, such as, for example, terminate the p SV40. In some implementations the signal termination may be the lack of consistency suitable for transcription or translation, for example, by shortening the sequence.

D. polyadenylation Signals

When the expression of eukaryotic gene it usually includes the polyadenylation signal for the proper polyadenylation of the transcript. It is assumed that the nature of the polyadenylation signal is not crucial for the successful practice of the invention, and can be applied to any such sequence. Some examples include the SV40 polyadenylation signal or polyadenylation signal of bovine growth hormone, suitable and, as you know, a well-functioning in various target cells. The polyadenylation can increase the stability of the transcript or can facilitate cytoplasmic transport.

E. Point of replication initiation

In order to implement the vector according to the invention in a cell host, it may contain one or more sites of replication initiation (often called "ori"), which is a specific sequence of nucleic acid, in which replication is initiated. Alternatively, you can use an offline can replicate the sequence (ARS), if the host-cell is yeast.

F. Markers, is going to selection and screening

In some implementations of the invention, the cells transduced by lentivectors of the present invention, can be identified in vitro or in vivo by including a marker in expressing vector. These markers give identifiable changes Translationum cells, making it easy to determine contains expressing vector cells. Mainly suitable for the selection marker is the marker, giving the property that allows the selection. Positive suitable for breeding marker is one in which the presence of the marker allows selection, while suitable for negative selection marker is one in which its presence prevents its selection. An example of a positive right for the selection marker is a marker of resistance to the drug.

Usually the inclusion of marker selection for drug facilitates the cloning and identification of transformants, for example, genetic constructions, which give resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are applicable, suitable for breeding markers. In addition to markers, giving a phenotype that allows for the differentiation of transformants based on the implementation of the conditions shall be the also other types of markers, includes appropriate for screening markers like GFP, whose basis is a colorimetric analysis. Alternatively, you can use these suitable for screening enzymes as thymidine kinase of herpes simplex virus (tk) or chloramphenicolchloramphenicol (CAT). The experts also know how to apply immunological markers, possibly in combination with FACS analysis. Suppose that the marker is not important, provided that it is able to be expressed simultaneously with the nucleic acid that encodes a gene product. Further examples of markers suitable for breeding and screening, well-known specialists in this field.

7. Cell-hosts

Used herein, the terms "cell", "cell line" and "cell culture" can be used interchangeably. All of these terms also include their offspring, which covers any or all of the next generation. It is clear that all progeny may not be identical due to deliberate or inadvertent changes. In the context of the expression of the heterologous nucleic acid sequence, "a host cell" means a prokaryotic or eukaryotic cell, and it includes any transformable organisms that are capable of replicating a vector and/or to Express gets rological nucleic acid, encoded vector according to this invention. A host cell can be used and is used as a recipient for vectors. A host cell may be "transtitional" or "transformed," which means the process by which exogenous nucleic acid is transferred or injected into the cell host. Transformed cells include primary cell of the subject and her offspring. Used herein, the terms "designed" and "recombinant" cells or cells of the owners is intended to refer to cells, which was introduced exogenous nucleic acid sequence, such as, for example, lentivector according to the invention, carrying therapeutic gene construct. Therefore, recombinant cells differ from naturally occurring cells that do not contain recombinante introduced nucleic acids.

In some implementations considered that the sequence of RNA or protein can be co-expressed with other selected sequences of RNA or protein in the same cell as the host. Joint expression can be achieved through co-transfection of a host cell by two or more different recombinant vectors. Alternatively, a single recombinant vector can be designed to contain multiple different is cnie coding region for RNA, which could then be expressed in the cell-master, transtitional one vector.

Cell owners may be derived from prokaryotes or eukaryotes, depending on whether the desired result is replication of the vector or expression of part or all encoded by vector sequences of nucleic acids. Numerous cell lines and cultures are available for use as a host cell, and may be obtained through the American type culture collection (ATCC), which is the organization that serves as an archive for living cultures and genetic materials (www.atcc.org). Some examples of host cells used in this invention include, without limitation, cells, packing viruses, cells producing virus, 293T cells, hematopoietic precursor cells human hematopoietic stem cells human cells CD34+the cells CD4+and the like.

A. Tissues and cells

The fabric may include cell owner or cells that are transformed or which will be in contact with the composition for delivering a nucleic acid, and/or additional agent. The fabric can be a part of the body or may be selected from the body. In some implementations, the fabric and its constituent cells may include, without limitation the, blood (for example, blood cells (such as hematopoietic precursor cells human hematopoietic stem cells human cells CD34+the cells CD4+), lymphocytes and other cells of the germ blood), bone marrow, brain, stem cells, blood vessels, liver, lungs, bone, breast, cartilage, neck, colon, cornea, embryonic tissue, endometrium, endothelial, epithelial, esophagus, fascia, fibroblasts, fabric follicles, ganglia cells, glial cells, goblet cells, kidney, lymph node, muscle, neuron, ovaries, pancreas, peripheral blood, prostate, skin, small intestine, spleen, stomach, testicles.

B. Organisms

In some implementations a host cell or tissue may include at least one organism. In some implementations, the organism can be a human body, the primacy or mouse. In other implementations the body can be any eukaryotic or even prokaryotes (e.g., eubacteria, archaea), as should be clear to the person skilled in the art (see, for example, web page http://phylogeny.arizona.edu/tree/phylogeny.html). Some lentivector according to the invention can use the control sequences that allow them to be replicated and/or expressed in prokaryotic and eukaryotic cells. Specialists in D. the authorized area should be further understood conditions, when incubated all of the above host cells for their maintenance and replication of the vector. Also clear and well-known are the methods and conditions that allow large-scale production of lentivectors according to the invention, as well as the production of nucleic acids, encoded by lentivectors, and related polypeptides, proteins or peptides, some of which is therapeutic genes or proteins that will be used for gene therapy.

C. Suitable for injection compositions and pharmaceutical compositions

For carrying out gene therapy using lentiviral transfer vector-based compositions of the present invention, in General, should ensure that contact needs in this cell with lentiviruses a vector containing a therapeutic gene. The next cell will be introduced into the body, such as man, in need of gene therapy. Methods of introduction, of course, will vary depending on the location and nature of the disease and will include, for example, intravenous, intraarterial, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, nutritherapy, intraperitoneal, intratumoral route of administration, perfusion and lavage. Sometimes the cells will be released from organisms to face interaction is the influence with lentivectors ex vivo and then re-implanted.

Introduction structures antivirusnik nucleic acids according to the invention can be performed using a syringe or any other method used for injection of the solution, as expressing the design can pass through the needle of a certain size required for injection. Was recently described a new needleless injection system (U.S. patent No. 5846233)having a nozzle defining the limits of the sealed chamber for storing the solution, and a power device for extrusion of the solution through the nozzle to the place of delivery. Also for use in gene therapy has been described syringe system, which allows the repeated introduction of predefined quantities of the exact solution at any depth (U.S. patent No. 5846225).

Solutions of nucleic acids in the form of free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for use in injection include sterile aqueous solutions or the var the var and sterile powders for the preparation intended for immediate reception of sterile solutions or dispersions, suitable for injection (U.S. patent No. 5466468, specifically fully included here as a reference). In all cases the form must be sterile and must be fluid to the extent that it is easy to enter the syringe. It must be stable under conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol and the like), suitable mixtures and/or vegetable oil. The proper fluidity can be maintained, for example, through the use of a coating such as lecithin, maintaining the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be performed using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In most cases, the preferred will be the inclusion isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the compositions for injection can be performed through the use of agents that slow down the absorption, for example, aluminum monostearate and the same is Athyn.

For parenteral administration in an aqueous solution, for example, the solution, if necessary, must be appropriately buffered and first make a liquid diluent isotonic with sufficient amount of salt or glucose. These particular aqueous solutions are mainly suitable for intravenous, intraarterial, intramuscular, subcutaneous, intratumoral, and intraperitoneal administration. In this connection, sterile aqueous medium, which may be used are well known to specialists in this field in the light of the present description. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of fluid for hypodermoclysis or enter into the proposed site of infusion, (see, for example, "Remington''s Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some changes dosages will inevitably have a place, depending on the conditions of treatment of the patient. In any case, the person responsible for the introduction, will determine the appropriate dose for the individual patient. In addition, for the introduction of man, the drugs must meet the standards of sterility, progenote, General safety and purity at the request of the FDA Office of Biologics standards.

Sterile suitable for injection solutions are prepared by combining the active compounds in the required amount ACC is cstuuyxm solvent with various other above ingredients, subsequent, on-demand, sterilization by filtration. Dispersion, typically prepared by combining the various sterilized active ingredients into a sterile vehicle, which contains the basic dispersion medium and the required other of the above ingredients. In the case of sterile powders for the preparation of sterile solutions suitable for injection, the preferred methods of preparation are the means of vacuum drying and freeze drying, which lead to the formation of a powder of the active ingredient together with any additional necessary ingredient of its solution, pre-sterilized by filtration.

Described herein compositions can be prepared in a neutral or salt form. Pharmaceutically acceptable salts include additional acid salts and salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic acid and the like. Salts formed with the free carboxyl groups can also be obtained from inorganic bases, such as, for example, hydroxides of sodium, potassium, ammonium, calcium or iron, and such organic bases as Isopropylamine, trimethylamin is, histidine, procaine and the like. In the manufacture of the composition of the solutions will be introduced in a way compatible with the measured composition and in such a quantity that is therapeutically effective. The compositions are easily incorporated into a variety of dosage forms, such as suitable for injection solutions, capsules, releasing the drug, and the like.

Used herein, the term "carrier" includes any and all solvents, dispersion media, media, coatings, diluents, antibacterial and antifungal agents, isotonic and slow absorption agents, buffers, media solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in this field. Except when the conventional medium or agent is incompatible with the active ingredient, considered their use in therapeutic compositions. Supplementary active ingredients can also be added to the composition.

The phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular substances and compositions that do not cause allergic or similar severe reactions when introducing them to the person. The preparation of an aqueous composition that contains the protein as an active ingredient, well onata in this area. Typically, such compositions are prepared as suitable for injection or in the form of liquid solutions or suspensions; also it is possible to prepare solid forms suitable for dissolution or obtain a suspension in the liquid phase before the introduction.

The terms "contacting" and "exposed"applied to the cell, are used herein to describe the process by which therapeutic lentiviral transfer vector is delivered to the target cell.

For gene therapy of isolated available solid tumors specifically considered intratumoral injection or injection into the vascular system of the tumor. Local, regional or systemic injection may also be suitable. For tumors > 4 cm, the quantity that should be entered, will be approximately 4-10 ml, preferably 10 ml), whereas tumors <4 cm will be used approximately 1-3 ml (preferably 3 ml). Multiple injections, which are delivered as a single dose, contain amounts of from about 0.1 to 0.5 ml of Viral particles mainly can be contacted through the introduction of multiple injections into the tumor, separated by intervals of about 1 see System introduction it is preferable for conditions such as hematological malignancy.

Long call is giving can also be used there, where it's appropriate. Preferred is delivered through a syringe or catheter. Such prolonged perfusion may occur during the period of time from approximately 1-2 hours to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, up to approximately 1-2 weeks or longer after starting treatment. Typically, the dose of therapeutic composition through continuous perfusion will be equivalent to the dose that is given by means of single or multiple injections, introduced over a period of time during which the perfusion was carried out.

The treatment can also be changed, and it often depends on the type of disease and location of the diseased tissue, and factors such as overall health and age of the patient. Clinicians are best suited to make such decisions based on the known efficacy and toxicity (if it occurs) therapeutic compositions based lentiviral transfer vector-based according to the present invention.

Treatment may include various "single dose". A single dose is defined as the dose contains a predetermined quantity of therapeutic compositions containing the lentiviral transfer vector of the present invention. The number that you want to enter, and a particular way of introduction and composition of well-known experts in the clinical field. There is no need to enter a single dose as a single injection, but it may include continuous perfusion for a set period of time. A single dose of the present invention can easily be described on the basis of units transduction (T.U.) lentivectors determined by titration of vector in cell lines such as HeLa or 293. Single doses ranging from 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013T.U. and above.

8. EXAMPLES

The following examples are included to demonstrate preferred implementations of the invention. Professionals in this field should understand that the methods described in the following examples, are proposed by the inventors of the procedures that appropriately function in the embodiment of the invention, and, therefore, they should be viewed as components of the preferred ways of its realization. However, experts in this field should, in the light of this description, to understand that you can make many changes in certain implementations, which are described and at the same time provide the same or similar results without deviating from the essence and scope of the invention.

A. Materials and methodology used in the examples 1-3

1. Obtaining vector

Obtaining occurring is from HIV vectors, falsely classified with shell G protein of vesicular stomatitis virus (VSV), conducted by the transit cooperative transfection of three plasmids in the epithelial cell line 293T, as described by Naldini et al 1996a. Used originating from HIV packaging design was pCMVΔR8.91, which encodes the precursor Gag and Pol HIV-1 regulatory proteins Tat and Rev (Zufferey et al., 1997). G VSV expressed from pMD.G.

The plasmid vector HIV received from the source main circuit pHR' (Naldini et al., 1996a) with the following modifications. Samonasraivayuschiesya vectors were obtained from the previously described vector SIN-18, which contains a deletion in the U3 region of the 3'LTR from nucleotide -418 to nucleotide -18, removes all transcriptionally active sequences (Zufferey et al., 1998). In brief, plasmids pHR'SIN received, as set forth below; fragment Cloned-XbaI containing preparedby the site and the 3'LTR were removed from the plasmid pHR' and was subcloned into the corresponding sites of pUC18. This plasmid was completely digested EcoRV and partially PvuII and ligated within itself. A plasmid carrying a deletion 400 nucleotides from U3, restored. In the EcoRI site of the deletion plasmids were inserted linker XhoI, and the fragment Xhol-XbaI was cloned back into plasmid CMVlacZ pHR', split the corresponding enzymes. All other plasmids SIN-18 was obtained by replacing the genes reporters (code is their luciferase, GFP and Neo) for lacZ. Used plasmid vector pHR' differed from that originally described (Naldini et al., 1996) a deletion of the XhoI-kpni restriction sites, 118 removes nucleotides from the sequence encoding Nef above poliprinolov site, and deletion 1456 nucleotides of the human sequence below the 3'LTR. This human sequence remained from the original cloning of proviral genome HXB2. Two deletions had no effect on the titers of vectors or transgenic expression in dividing cells 293T.

To obtain the vector pHR-EF1-GFP-W-SIN was made to insert EF1-alpha by inserting the cassette ClaI-BamHI containing the insert EF1-GFP, plot ClaI-BamHI pHR'-GFP-W-SIN (Zufferey et al. 1999). Derivatives of this vector had some functional differences. Functional differences are the promoter EF1-alfa-short, the loxP site in the 3'LTR and cPPT. The loxP site in the 3'LTR duplicitous to 5'LTR in transduced cells and in the presence optional expressed Cre recombinase, enables removal of the United provirus. However, the plot loxP and Cre mediated excitation is not required for activity protomer gp91-phox and will not be required and will not be used in any potential application of vectors in clinical trials, based on hematopoietic stem cells. The difference between EF1-alpha, presents the pHR-EF1-GFP-(+/-)W-SIN, and promoter EF1-alpha shortened in pHPT-GFP and pWPT-GFP is that the shorter version does not contain introns, which makes the vector less.

The map shown in figa-5D depicting plasmid construction pHPT-GFP, pWPT-GFP, pHPP91-GFP and pWPP91-GFP.

After transit transfection of plasmids by the calcium phosphate in 293T cells, the supernatant was collected, concentrated by ultracentrifugation using a gradient of sucrose, resuspendable in the serum-free Cellgro medium(SCGM (Cellgenix, Germany) and filtered through a filter SpinX 0.45 µm. Viral mother solution was stored at -70°C and titers were determined by transduction and analysis by flow cytometry the expression of GFP in HeLa cells as described previously (Zufferey et al., 1997). Titers ranged between 5×107and 108units transduction (TU) in HeLa Jr. on

2. Purification and transduction of cells CD34+

Samples of umbilical cord blood (CB) was obtained according to the established leadership, and cells CD34+was purified as described (Arrighi et al., 1999). Briefly, mononuclear cells CB, obtained after zentrifugenbau in a gradient of Ficoll-Paque (Pharmacia, Uppsala, Sweden), were incubated on ice with Dynabeads M450 against CD34 (Dynal, Norway)as described by the manufacturer. After several cleaning to remove unbound cells, CD34+was removed from the beads by incubating for 15 minutes at a temperature of 37°C pre is the Arat "Detach-a-bead", included in the set. Cells were immediately washed and analyzed using flow cytometry. The percentage of treated cells CD34+was 89±7,0. For transduction of 105cells were grown in 96-well tablets in 100 ál Cellgro medium (SCGM, supplemented with antibiotics (Gibco BRL, Life Technologies LTD, Paisley, Scotland, U.K.), 10-4M dithiothreitol (Fluka Biochemika, Buchs, Switzerland) and TPO (10 ng/ml), SCF (50 ng/ml), Flt3L (50 ng/ml). After incubation over night 106(usually) or 105-5×106(for analysis of response to dose) units transduction (TU) vector in HeLa added to the cell and brought volume up to 200 μl with Cellgro medium (SCGM containing TPO. After 24 hours the cells were washed, diluted in 400 ál Cellgro medium (SCGM, supplemented with antibiotics, 10-4M dithiothreitol and TPO (10 ng/ml), SCF (50 ng/ml), Flt3L (50 ng/ml) for 3 days. Cells were either directly analyzed for GFP expression and CD34, or cultivated next 3 growth factors.

3. Cytokines

All cytokines were a recombinant human material and were obtained from Peprotech (London, U.K.).

4. Antibodies and immunoreagents

All antibodies were obtained from Becton Dickinson Pharmingen (USA). mIgG against CD34, treated Dynabead M450, were obtained from Dynal A/S (Oslo, Norway).

5. Differentiation in vitro and stimulation of INF-gamma

Differentiation was performed in vitro in the presence of GM-CSF and SCF for differentiation of MES is Titov and G-CSF or G-CSF for 3 weeks and SCF for the differentiation of granulocytes. Differentiated cells stimulated with INF-((1000 u/ml) for 6 days, were labeled conjugated with PE monoclonal antibodies using FACS analyzed the expression of GFP positive for PE population. Numbers indicate percentage of cells in the quadrants.

6. Analysis by flow cytometry

Cells were analyzed as described (Arrighi et al, 1999), on the FACScalibur instrument (Becton-Dickinson) with minor modifications. FL-1 was used for GFP, FL-2 to PE-labeled Mabs, FL-3 Mabs labeled with PercP. Prior to analysis, the cell suspension was brought to 0.5% paraformaldehyde. Data were analyzed using WINMDI, written by J. Trotter of the Scripps Institute (La Jolla, CA)and CellQuest program (Becton-Dickinson).

B. Examples 1-4

Example 1: Transduction of hematopoietic precursor cells and person-based HIV lentivectors containing expressing cassette under the control of the promoter of gp91-phox, leading to limited expression in monocytes and granulocytes.

Lentiviral transfer vectors pWPP91-GFP (WPRE) and pHPP91-GFP (without WPRE) was created by replacing the promoter EF-1 (fragment 1540 n subunit gp91-phox promoter(1) NADPH-oxidase phagocyte in antivirusnik vectors pWPT-GFP and pHPT-GFP, respectively. As pWPT-GFP and pHPT-GFP, are SIN (samonasraivayuschiesya) lentivirusnye vectors and to increase transduction efficiency of target cells contain a Central serial is a major polypurine plots (cPPT) (figure 1 and 5). Recombinant lentivector received and concentrated 100 (previously described standard methods and used for transduction of hematopoietic CD34+of progenitor cells/stem cells (HSC). Cm. Salmon P, Kindler V, Ducrey O, Chapuis B, Zubler RH, Trono D., "High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors", Blood 96(10): 3392-8 (2000), included here as a reference.

A high percentage of transduction UCB HSC, up to 80%, was achieved using lentivector, in which the Central polypurine plot was placed above expressing cassette EF1-alpha-GFP. A large number of GFP-positive cells persisted after differentiation of transduced HSC in vitro (cytokine cocktail) or in vivo (transplantation mice NOD/SCID).

Transduction of HSC by lentivector bearing cartridge gp91-phox-GFP, which is followed by their differentiation in vitro (in the cytokine cocktail) or in vivo (transplantation sublethal irradiated mice NOD/SCID)resulted in expression of GFP, limited exclusively to Mature monocytes and granulocytes. The expression of GFP under the promoter of gp91-phox in Mature neutrophils was increased by inserting WPRE without loss of specificity.

The promoter gp91-phox, passed in HP/SC through lentivector, with WPRE or without him, showed their physiological reactivity against INF- (Mature neutrophils in vitro (figure 2).

Example 2: the Elements of enhancer HS cause higher and less heterogeneous expression.

An important result of genetic treatment of various lymphohematopoietic disorders is the level of expression of a supposedly therapeutic transgene in the appropriate number of differentiated cells. In order to obtain a higher and more homogeneous gene expression in a lentiviral transfer vector cloned sequence gp91-specific enhancer located at 30 n above gene gp91-phox in the four areas of hypersensitivity (HS) to DNase.

First, in order to reduce the size of the viral genome, a fragment of the promoter gp91 value in 1540 N.P. was replaced by a functionally equivalent fragment size of 0.5 tisp (Skalnik et al. 1991), forming the vector pHP500-GFP (figa). To facilitate cloning has introduced multiple unique restriction sites allowing the introduction of enhancers on both plots cPPT (which should retain its Central position and functionality after cloning enhancer), forming the vector pHPX-GFP (pigv).

Fragments that overlap 4 individual field HS was obtained by PCR using genomic DNA as template. The sequence of primers for PCR were based on the sequence of the human genome, published in the project"human Genome" (contig NT_011844; http://www.ncbi.nlm.nih.gov/genome/guide/human/). Cm. SEQ ID NOS:2-16. Siting gp91-phoxHS was based on published data. Cm. Lien LL, Lee Y, and Orkin SH (1997). Sequence approximately 1-1,5 tysp flanking a defined area, amplified and cloned in lentivector, getting ultimately pHPHS-GFP. Cm. SEQ ID NOS: 2-16. There was the option of WPRE sequences (pWPHS-GFP, figs). When HSC transduction and differentiation of the inclusion of elements of HS in this and other configurations will lead to a higher full expression and/or less heterogeneous expression of the transgene.

Example 3: treatment of chronic Wegener (CGD)

Chronic Wegener strongly correlates with the absence of gene gp91-phox, which encodes a subunit of NADPH (Dinauer et al., 1987). Gene gp91-phox, coupled with the X-chromosome, defective, almost 60% of patients with CGD. For an overview, see Malech HL, "Progress in gene therapy for chronic granulomatous disease", J. Infect. Dis. 179 (Suppl.2):S 318-25, 1999. One approach to treatment will include the transduction of the respective cells so that at least one functional copy of the gp91-phox was introduced to the patient. As discussed above, this type of treatment may include the selection of cells from a patient, the transduction ex vivo, and then move these cells back into the patient.

Cells CD34 distinguish patients with CGD and transducers vector of the present invention, which is carries a functional copy of the gene gp91-phox, for example, gene gp91-phox encoded by the nucleotide sequence consisting of nucleotide SEQ ID NO:18, or any nucleotide sequence that encodes a functional polypeptide amino acid sequence SEQ ID NO:19. The corresponding expression of the polypeptide gp91-phox receive under the control of the promoter of gp91-phox, is introduced into the vector. Increased expression can be obtained by inclusion of the WPRE element and enhancers HS, as described above.

Cells are implanted into a test system, such as a mouse SCID-NOD, or patients indicated. Further evaluation of the effectiveness of this approach will be achieved by transplantation of transduced cells in the corresponding lines of knockout mice.

Example 4: Therapeutic approaches to deficiency of leukocyte adhesion (LAD)

One approach to treatment will include the transduction of the respective cells so that at least one functional copy of myelomonocytic leukocyte integrin was introduced to the patient. As discussed above, this type of treatment may include the selection of cells from a patient, the transduction ex vivo, and then move these cells back into the patient.

Cells CD34 distinguish patients with LAD and transducers vectors of the present invention, which have a functional copy of the gene integrin. Relevant expr is the Russia of integrin polypeptide receive under the control of the CD11b promoter, introduced in the vector. The CD11b promoter selected from promoters encoded by polynucleotide sequence SEQ ID NO:20. Cm. Hickstein, et al. 1992. Increased expression can be obtained by inclusion of the WPRE element and enhancers HS, as described above.

All described and claimed here compositions and/or methods may be received and executed without undue experimentation in light of the present description. While the composition and method according to this invention is described in terms of preferred implementations, the person skilled in the art it is clear that the compositions and/or methods and stages or sequences of the stages of the method described here can be applied variations without deviating from the concept, nature and scope of the invention. More specifically, it is clear that some means related chemically and physiologically, can replace the tools described here to achieve the same or similar results. It is assumed that all such substitutions and modifications are understood by the experts in this field, consistent with the essence, scope and concept of the invention as defined by the attached claims.

Links

The following references are specifically incorporated here by reference to the extent to which they provide methodological or other details supplementary to those shown here.

The patent is SHA No. 4682195

U.S. patent No. 4683202

U.S. patent No. 5466468

U.S. patent No. 5645897

U.S. patent No. 5686279

U.S. patent No. 5705629

U.S. patent No. 5846225

U.S. patent No. 5846233

U.S. patent No. 5925565

U.S. patent No. 5928906

U.S. patent No. 5935819

U.S. patent No. 5994136

U.S. patent No. 6013516

U.S. patent No. 6136597

U.S. patent No. 6165782

U.S. patent No. 6207455

U.S. patent No. 6218181

U.S. patent No. 6218186

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1. Recombinant lentiviral transfer vector containing:

(a) expression cassette containing the transgene positioned under the control of the promoter, which has activity against stimulation detectable transcription of the transgene in human cells; and

(b) the Central polypurine tract (srrt)above expressing cassette.

2. Lentiviral transfer vector according to claim 1, where the transgene is under the control of a cell-specific, tissue-specific or idolising promoter.

3. The vector according to claim 1, where SRT contains the nucleotide sequence of SEQ ID NO:1, and preferably additionally includes multiple unique cloning sites located above and/or below srrt.

4. The vector according to claim 1, where the vector is capable of transduction from about 20 to 80% of the cells.

5. The vector according to claim 1, where the vector is capable of transduction from about 60 to 80% of the cells.

6. The vector according to claim 1, additionally containing multiple unique cloning sites below or above srrt.

7. The vector according to claim 1, further defined as savingthrow the different vector (SIN).

8. The vector according to claim 1, where the LTR region is given, essentially, in a transcriptionally inactive state by deletion of the U3 region of the 3'end LTR.

9. The vector of claim 8, in which delegated the nucleotides in positions from -418 to -18 relative to the boundaries of the U3-R region.

10. The vector according to claim 1, additionally containing at least one enhancer sequence.

11. The vector of claim 10, where the enhancer sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

12. The vector according to claim 1, where the promoter is able to stimulate the expression of the transgene in the signal-to-noise ratio of about 10 to 200, preferably from about 40 to 200 and more preferably from about 150 to 200.

13. The vector according to claim 1, where the promoter is able to stimulate the expression of the transgene in response to the transcriptional activator, where the transcriptional activator is preferably IFN-gamma.

14. The vector according to claim 1, where the promoter is a promoter of gp91-phox, a promoter gp47-phox, the CD11b promoter, an EF1 promoter-α, PGK promoter, the promoter of the beta-globin, the promoter of MHC class II, the promoter of coagulation factor IX promoter of insulin, PDX1 promoter, the promoter CD4 and CD2 promoter.

15. The vector according to claim 1, where the promoter is able to stimulate transgene expression in specific cell types.

16. The vector according to item 15, where specific cell types selected from a group is s, including Mature blood cells, neutrophils, monocytes, granulocytes, neurons, lung cells, muscle cells, liver cells, pancreatic cells, endothelial cells, heart cells, skin cells, stromal cells in the bone marrow and cells of the eyes.

17. The vector according to claim 1, where the transgene is a gp91-phox, gp47-phox, erythropoietin, interleukin, colony stimulating factor, integrin α11bβ, the multidrug resistance, antiviral gene, the gene encoding the coagulation factor VIII gene encoding coagulation factor IX; T-cell receptor antigen, b-cell receptor antigen single-chain antibody (ScFv), TNF, IFN-γ, CTLA4, B4, Melana, MAGE, marker gene, a luciferase or GFP.

18. The vector according to claim 1, further containing a sequence of posttranscriptional regulation, located so that it stimulates the expression of the transgene.

19. Vector on p, where the sequence posttranscriptional regulation represents an intron located in expressing cassette, or the element of posttranscriptional regulation.

20. The vector according to claim 19, where the element posttranscriptional regulation is the element posttranscriptional regulation of hepatitis North American forest marmot (WRPE) or item posttranscriptional regulation of hepatitis b virus (HPRE).

1. A host cell, translationa vector according to any one of claims 1 to 20, where a host cell is a hematopoietic cell-precursor.

22. A host cell according to item 21, where hematopoietic cell-precursor is a hematopoietic cell-ples.

23. A host cell according to item 21, where hematopoietic cell-precursor is a CD34+cell.

24. The way in vivo transduction of hematopoietic stem cells, comprising contacting a population of human cells that includes hematopoietic stem cells with a vector according to any one of claims 1 to 20.

25. The method according to paragraph 24, where the population of hematopoietic precursor cells contain CD34+cells.

26. The method according to paragraph 24, where the population of cells is treated to stimulate cell proliferation without significant loss of pluripotency of stem cells.

27. The method according to paragraph 24, where transgene expression is carried out using activator, which induces the promoter.

28. The use of cells according to item 21 to obtain drugs for infusion human or animal.

29. The use of hematopoietic stem cells, transduced in accordance with the method according to any of PP-27 to obtain drugs for infusion human or animal.



 

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