Mediated virus amplified dna transfer

 

(57) Abstract:

The invention relates to medicine and concerns mediated virus amplified DNA transfer and method of improving the efficiency of transduction of hematopoietic and other cells under the action of the retrovirus. The method involves infection of cells in the presence of fibronectin or its fragments. Fibronectin and its fragments significantly enhance retrovirus-mediated gene transfer into cells, in particular hematopoietic cells, including commitirovannah precursor cells and primary hematopoietic stem cells. The present invention also provides improved methods of somatic gene therapy-based enhanced gene transfer, populations of hematopoietic cells and new designs intended to enhance retrovirus-mediated transfer of DNA into cells, and their use. The advantage of the invention is to develop an efficient transfer of genetic material into mammalian cells without dangerous consequences and limitations. 18 C. and 70 C.p. f-crystals, 4 tab., 11 Il.

This invention was made with government support provided by the National Institute of health on CLASS="ptx2">

The technical field to which the invention relates

The object of the present invention are methods of increasing the efficiency of transduction of cells by viruses and, in particular, how to strengthen mediated virus gene transfer into cells with fibronectin and/or fragments of fibronectin.

Prior art

Progress in the understanding of the molecular basis of many human diseases and the improvement of gene transfer technology have made possible the development of procedures of somatic gene therapy for the treatment of serious genetic diseases. Currently using gene therapy can treat diseases caused by a defect or deficiency of an enzyme or other protein that requires precise control of the level of these components, and bone marrow diseases of man.

For example, one disease that can be treated with gene therapy is adenosylmethionine (ADA) deficiency, which causes severe combined immunodeficiency. Patients suffering from adenosylmethionine insufficiency, bone marrow cells contain little of the enzyme or it does not. However adenosylmethionine n is Southsea selective effect compared to diseased cells and usually restore the bone marrow of the patient.

Bone marrow cells are a good target for somatic gene therapy, because the tissue of the bone marrow can easily be used to perform in vitro tests and it contains repopularize cells. An alternative has been demonstrated that umbilical cord blood contains a large number of primary precursor cells. Successful gene transfer into hematopoietic stem cells, long-lived repopularize cells can cure different diseases by receiving the progeny of these cells.

Gene transfer and long-term gene expression in repopularised stem cells was achieved in models obtained by a number of scientists in studies on mice. However, when performing in vivo experiments on larger animals, such as dogs and primates, the success has been very limited, due mainly to the low efficiency of infection of primary hematopoietic stem cells. The use of current technology of gene transfer is further complicated in the treatment of people on several factors, which include the low number of stem cells present in the bone marrow of an adult, the lack of acceptable methods of cleaning such cells and nebtek and large animals includes the study of bone marrow cells, it was found that the most successful treatments have included co-cultivation of target cells with lines retroviral cells-producers. In addition, the majority approved by Management under the control over products and medicines of experiments on gene transfer in humans are recombinant retroviral vectors designed for transduction genes. Recombinant retroviral vectors are highly desirable for gene therapy, as they now carry, accurately and stably integrate exogenous DNA into cellular DNA. These vectors contain exogenous DNA is used for gene transfer, and then modified to eliminate viral pathogenesis. Due to this modification the production of viruses is achieved by use of a retroviral packaging cells. However, for clinical gene therapy more desirable cell-free transduction in relation to issues of Biosafety and quality control. Unfortunately, efficient gene transfer in these hematopoietic cells, like stem cells, is impossible without cocultivation with virusprotection cells.

Recently it was found that e is different cells during infection. Stromal cells are a major component of the hematopoietic microenvironment. Hematopoietic microenvironment consists of an organized network of macrophages, stromal cells, endothelial cells, adipocytes and complex extracellular matrix, composed of a number of specific adhesion molecules. Molecules of complex extracellular matrix, such as laminin, collagen, thrombospondin, proteoglycans, glycosaminoglycans and fibronectin form a connecting the centers for hematopoietic cells and growth factors. Still not clear on the mechanism underlying such a stimulating action of stromal cells by retroviral infection, but it is already known that the physiological regulation of proliferation and differentiation of hematopoietic cells occurs when these cells are in direct contact with the cells of the haematopoietic microenvironment.

Efficient gene transfer into long-lived repopularize hematopoietic stem cells and other cells remains problematic, preventing widespread use in the present methods of gene transfer for the treatment of diseases associated with blood-forming process, and other diseases. There is an urgent need in anichini, inherent in previously used methods. The present invention is directed to satisfying these needs.

Summary of the invention

One preferred embodiment of the present invention provides a method of increasing the frequency of transduction of hematopoietic cells by retroviral vector. This method involves infection of viable hematopoietic cells recombinant retroviral vector with a defect replication in the presence of essentially pure fibronectin and/or its fragments, effectively increasing the frequency of cell transduction with a retrovirus. Fibronectin and/or its fragments can be obtained from natural materials or to synthesize artificial means (for example, to create genetically using methods of recombinant or chemical synthesis) or obtained from a combination of natural and synthetic materials. Furthermore, it is obvious that the polypeptide or polypeptide of fibronectin used in this invention may include mutations, forming a natural amino acid sequence of fibronectin, which nevertheless represents a functional polypeptides having adhesional">

Another preferred embodiment of the present invention provides a method of obtaining transduced hematopoietic cells, which includes infection of viable hematopoietic cells by recombinant retrovirus with a defect replication containing exogenous DNA in the presence of immobilized fibronectin immobilized fragments of fibronectin or immobilized mixture in amounts sufficient to increase the frequency of cell transduction with a retrovirus.

Another preferred embodiment of the present invention provides an improved method of cell transplantation. This method includes the stage of obtaining viable hematopoietic cells from an animal donor; infection of hematopoietic cells by the vector recombinant retrovirus with the aim of obtaining transduced viable hematopoietic cells, and the infection takes place in the presence of fibronectin and/or its fragment in immobilized form, contributing to an increase in the frequency of transduction; and the introduction of transduced viable hematopoietic cells of the animal to the recipient in the form of cell transplant. In tropidosternum of the embodiment of the present invention provides a method of obtaining transducing cells of umbilical cord blood for the purpose of transplantation. This method involves infection of hematopoietic cells derived from umbilical cord blood, recombinant retroviral vector with a defect replication in the presence of a sufficient amount of immobilized fibronectin and/or its fragments with the aim of increasing the frequency of transduction of hematopoietic cells under the action of the retroviral vector. The present invention also includes viable populations of transduced cells from umbilical cord blood obtained in accordance with this method, and methods of transplantation of cells, which include the introduction of the animal populations of transduced cells as a cell transplant.

In accordance with particular aspects of the above embodiments of the present invention used fibronectin or a fragment must contain a first amino acid sequence, which provides a retrovirus binding activity of the binding region of heparin P fibronectin, and a second amino acid sequence, which provides linking cell activity area CS-1 fibronectin. Simultaneous use of these two areas of binding of fibronectin has provided a very significant increase in Tr is tvline the present invention provides a method of creating a genetically engineered design to increase the frequency of transduction predetermined target cells under the action of the retroviral vector. This method includes a step covalent binding of the ligand that binds the target cell with a polypeptide containing the amino acid sequence, which provides a retrovirus binding activity of the binding region of heparin P fibronectin. The present invention also includes methods of using these genetically engineered structures to increase the frequency of transduction of predefined target cells by retroviral vector and transplant procedure using transduced cells.

Another preferred embodiment of the present invention provides a method to localize the amount of virus, comprising culturing medium containing the virus in the presence of the necessary immobilizovannogo number of fibronectin or fragments of fibronectin, providing the virus binding region of heparin binding of fibronectin to locate the number of the virus.

Other preferred variants of implementation of the present invention provides for culture transduced viable hematopoietic and other cells, containing pure and/or immobilized fibronectin or e is in the description.

The aim of the present invention is the creation of effective retrovirus infection of mammalian cells.

Another objective of the present invention is to provide methods of gene transfer using retroviral vectors that do not require cocultivation.

Another objective of the present invention is to provide improved methods and cultures of cells for autologous and/or allogeneic transplantation of cells.

These and other objectives, advantages and features of the present invention will become obvious from the following description of the invention.

Description of drawings

Fig. 1 - schematic representation of molecules fibronectin, including fragments of chymotrypsin.

Fig. 2 - the picture of the effectiveness of infection commiteeman precursor cells of human rights in the presence of fragments of fibronectin using TKNEO vector, as described in example 1, below.

Fig. 3 - comparison of the efficiency of infection with various commiteeman hematopoietic precursor cells of human rights in the presence of fragments of fibronectin using TKNEO vector, as in mice, the transplanted with bone marrow cells that were transpulmonary through (i) cocultivation (columns 2-4), (ii) infection nedostatochnoi fluid in the presence of immobilized fragments of fibronectin (columns 5 to 7) and infection supernatant based on bovine serum albumin (columns 8 to 10), as further described in example 7, below. A control sample for adenozindezaminazy person shown in columns 1 and 12, and for adenozindezaminazy mice in column 11.

Fig. 5 - image of the retrovirus binding fragments of fibronectin, as described in example 8, below.

Fig. 6 - the image that the binding of a retrovirus with fragments of fibronectin depends on the dose, as described in example 8, below.

Fig. 7 is a schematic representation illustrating different recombinant fragments of fibronectin used in examples 9 to 11, see below.

Fig. 8 - picture link retrovirus with different fragments of fibronectin, including several recombinant fragments as described in example 9, below.

Fig. 9 is an image of how the heparin inhibits the binding of a retrovirus is vnesti infection with retrovirus hematopoietic cells of mice in the presence of different fragments of fibronectin, as described in example 10, below.

Fig. 11 - comparison of the availability of adenoidectomies person in mice which were transplanted bone marrow cells transduced through (i) cocultivation, (ii) infection supernatant using different fragments of fibronectin and (iii) infection supernatant using bovine serum albumin as described in example 11, below.

Description of the preferred embodiments of the invention

For more clear understanding of the principles of the present invention is described below some of the options for its implementation. However, it should be remembered that they in no way limit the scope of the invention, and it is assumed that the experts in this field should be obvious changes, modifications and applications of the principles of the present invention, shown here to illustrate.

As mentioned above, the present invention provides methods of increasing the frequency of transduction of viable cells under the action of a virus such as a retrovirus. The present invention predusmatrivayuthih vectors, methods of obtaining transduced cells, as well as methods and materials for obtaining autologous and other cellular grafts.

One distinctive feature of the present invention is the discovery that fibronectin and its fragments containing the region of cell adhesion to CS-1 fibronectin, greatly enhance retrovirus-mediated gene transfer in these cells, like hematopoietic cells, for example Commition precursor cells and hematopoietic stem cells or initiating cells are long-lived culture, with the fibronectin receptor and, thus, capable of binding to fibronectin or fragments. Very favorable is the fact that the increase in efficiency does not require cocultivation with producing the virus cells. Other distinctive features of the present invention is based on the discovery linking viruses region of fibronectin, located in the region of the binding of heparin P. This linking viruses region can be used for localization of viral particles in many applications, including, for example, a wide range of genetically engineered structures designed to deliver virus to the aspects of the present invention contain exogenous DNA and are not pathogenic, that is, they have the defect replication. These vectors efficiently transferred accurately and stably integrate exogenous DNA into host cells such as animal cells, in particular mammalian cells. For example, in accordance with the present invention the nucleotide sequence comprising a number of bases from the coding sequence of the gene of interest can be included in a recombinant retroviral vector to transfer the gene under the control of an appropriate promoter. Such a promoter is usually an exogenous promoter. In this regard, the exogenous DNA can include DNA, obtained by natural or artificial means, and may consist of parts derived from heterologous sources, and these parts can be a natural or chemically synthesized molecules and can be joined by ligation or other known methods. As mentioned above, the introduced nucleotide sequence should be under the control of the promoter and is read in the forward direction. In other words, the sequence of the promoter is usually in the opposite direction (i.e. in the 5' direction) from the coding sequence. Good the successive enhancers), which interact with the promoter and initiating codon of transcription with the aim of obtaining a transcription of the exogenous coding sequence. The phrase "under control" implies the existence of such elements, which are required for obtaining a transcription of the introduced gene. In addition, recombinant DNA preferably includes termination sequence located in the forward direction from the introduced coding sequence.

You can use retroviral vectors, which include exogenous DNA, representing the selectivity of the marker or with other selective advantage. For example, the vectors may contain one or more exogenous genes that are resistant to different selective agents, including antibiotics such as neomycin. Representative vectors that can be used in this invention include, for example, the vector N/ZipTKNEO (TKNEO) (titer: 1105G418rcolony forming units/ml on cell N1H 3N3), vector ZipPGK-hADA and vector ZipPGK-mADA, which were previously described by Moritz and others (1993) in the journal J. Exp. Med. 178:529. In the TKNEO vector neotestamentaria sequence is expressed in the form of semantic orientation (relative to 5' long forging marker which provides resistance to neomycin, which facilitates the identification of transduced cells. In the vector ZipPGK - hADA cDNA of adenozindezaminazy person ("hADA") is expressed in the sense orientation relative to the 5' long terminal repeat through the promoter phosphoglycerides person (PGK). It contains only one expressed genetic sequence and has no breeding token dominant. Vector ZipPG-K-mADA (PGK-mADA) is identical to the vector ZipPGK-hADA except that the cDNA adenozindezaminazy man replaced by DNA adenozindezaminazy mice (mADA). These and other viral vectors and methods for their preparation are well known and their use in the present invention may not cause difficulties for professionals in this field.

Viral vectors used in this invention have the ability to communicate with the amino acid sequence of the binding region of heparin P fibronectin, including fibronectin person. Since the present invention is not limited to theoretical calculations, it can be assumed that co-localization of virus and target cells through binding of the virus and cells to the appropriate functional areas contributes took what sequence region of the binding of heparin-P and, therefore, to ensure the effective achievement of the objectives of the present invention can easily be ascertained by standard procedures similar to those described in the following examples 8 and 9. That is, these analyses allow us to determine the degree of binding of viral particles with immobilized polypeptides containing a binding region of heparin P in order to resist leaching of the immobilized polypeptide matrix. Briefly, virus-containing supernatant can be incubated in the coated well with the immobilized polypeptide including a binding region of heparin P fibronectin. This hole is then intensively washed with buffer representing a physiological solution, after which the well is incubated for target cells for the virus to determine its level of infective activity. Determine the reduction in infective activity or titer compared to the original virus-containing supernatant liquid and compare the indicator with the same control series (obtained, for example, using wells coated with bovine serum albumin). Significantly higher titers in the hole-containing region of heparin P, compared to the zebrette. To facilitate screening viral vector may contain a gene of breeding marker, as described above.

Fragments of fibronectin, intended for use in this invention can be of natural or synthetic origin and may be derived from natural materials with a sufficiently high degree of purity, for example, as described previously, Ruoslahti and others (1981) in the journal J. Biol. Chem. 256: 7277; Patel, Lodish (1986) in the journal J. Cell Biol. 102:449; and Bernardi and others (1987) in the journal J. Cell Biol. 105:489. In this regard, the link in this invention on pure fibronectin or fragments means that they do not contain other proteins, together with which usually occurs fibronectin. Pure fibronectin or fragments intended for use in this invention can also be obtained by recombinant, for example, as described in U.S. patent N 5198423, issued March 30, 1993, Taguchi and others with the transfer of rights to a patent Takara Shuso company, Ltd., Kyoto, Japan. In particular, recombinant fragments identified in the following examples, H-271, H-296, CH-271, CH-296 and C-CS1, as well as methods for their preparation, are described in detail in the opposed patent. Fragment C247, espoli fragments or fragments, from which they can be obtained by standard methods, obtained by culturing E. coli cells, a registered research Institute of fermentation Agency of industrial science and technology, Japan, under the names PERM P-10721 (H-296), FERM BP-2799 (C-277, associated with H-271 using methionine), FERM BP-2800 (C-277, associated with H-296 using methionine) and FERM BP-2264 (H-271) and also described in U.S. patent N 5198423. In addition, useful information about used in this invention the fragments of fibronectin, or the source materials for these fragments contained in the work Kimizuka and others, published in the journal J. Biochem., 110, 284-291 (1991), which are described in more detail above recombinant fragments; in the journal EMBO J. 1755-1759 (1985), which describes the structure of the gene of fibronectin person; and in the journal Biocnemistry, 25, 4936-4941 (1986), which shows the region of the heparin binding of human fibronectin. It was found that fragments of fibronectin containing as the area of cell adhesion to CS-1 and the binding region of heparin P, such as 30 or 35 KD fragment (fibronectin 30/35) and in the form of different recombinant fragments, consider the examples below, significantly increase effektivnosti to understand the polypeptide or polypeptides related to fibronectin and used in this invention comprise the amino acid sequence, providing linking cell activity in the field of cell adhesion to CS-1 fibronectin, as well as the amino acid sequence of the binding region of heparin P fibronectin, which provides the binding of the virus. It is quite clear that the desired binding activity of cells and viruses may be provided as a native amino acid sequences of these functional areas of fibronectin and amino acid sequences that differ from the native sequence, but have similar binding activity of cells and viruses. Such amino acid sequences show significant homology to the corresponding native sequences may include sequences in which amino acids have been deleted, substituted and/or modified, while obtaining amino acid sequence with the desired binding characteristics of cells or viruses.

Developed biotechnology has reached such a level that can be easily prospie amino acid sequence can then be tested by standard methods to determine the desired binding activity of cells or viruses. For example, the binding activity of the mutant viruses or modified forms area of the heparin binding of fibronectin can be tested as described below, in particular, in examples 8 and 9, by culturing viruses, washing and analysis of viral titers, which allows to determine the degree of infection compared to the control sample. Taking into account these materials analyses linking will be for experts in the field of conventional experimental work.

Linking cells with modified or mutant forms of the field of cell adhesion to CS-1 fibronectin or other binding cells polypeptides can be analyzed using known methods. For example, such procedures include methods described in the journal Nature 352: 438-441 (1991). In short, the plastic cups were covered connecting the cells with the polypeptide and designed for analyzing a population of cells were placed in a medium for a time from 30 minutes to 2 hours. After incubation, the cells that are not associated with protein, were isolated, counted and analyzed in relation to viability. Cells associated with the polypeptide, also was isolated using trypsin or buffer for dissociation of cells (e.g., Gibco), counted and testelets, these cells were cultured for 12-14 days, and then were evaluated by colony-forming characteristics of the cells. Then we calculated the percentage of adhesive cells and compared this result with the control sample, obtained using bovine serum albumin. Significant binding of target cells analyzed with the polypeptide indicates that the combination of the polypeptide and cells suitable for achieving the objectives of the present invention and the polypeptide can be attached to the retrovirus binding fragment of fibronectin with the formation of the structure of the present invention, designed to increase the infection of target cells by a viral vector.

In accordance with the most characteristic aspects of the present invention binds the virus polypeptide used to enhance transduction under the action of retroviral vectors, must include (i) a first amino acid sequence, which corresponds to Ala1690- Thr1960the field of heparin binding of human fibronectin, which is represented by the formula (ID number sequence 1, see the end of the description).

or sufficiently similar and the sequence which corresponds to one of the binding fibronectin IIICS person (the region of connection of the cells CS-1), which is represented by the formula (ID number sequence 2, see below):

or sufficiently similar to the amino acid sequence possessing the ability to bind hematopoietic cells, in particular, the primary precursor cells and/or Dolgorukaya repopularize (stem) cells.

As noted above, it is obvious that the scope of the present invention includes modifications and/or mutations of these native sequences, if the derived amino acid sequence sufficiently similar to the native sequence and has the ability to bind the virus (in the case of the field of binding heparin P), as well as the ability to bind target cells (in the case of the field of CS-1).

One aspect of the present invention provides a method of somatic gene therapy, which includes in vitro cell therapy and subsequent transplantation of target cells of the host, which is also known as "grafting" the owner of transduced target cells. Blood or other cells can be taken from the person of the bone marrow, the peripheral blood circulatory system of the human donor or cord blood. Received hematopoietic cells may not necessarily be processed using standard methods to enrich their stem cells and/or primary cells predecessors. Hematopoietic cells can then be incubated accordingly, for example, on tablets with tissue cultures. Optionally, in this step, the unbound (adherent - negative) mononuclear cells with a low density can be pre-stimulate to infection by retroviruses. Pre-stimulation, known in this area and used in this invention is a process of influence on the cells simulating growth factors to infection by retroviruses. It has been proven that such pre-stimulation improves the transduction of hematopoietic cells by retroviruses.

After pre-stimulation of the cells can be collected and incubated with fibronectin or fragments, as described in this invention, resulting in increased frequency of transduction of cells under the action of retroviruses. The cells are preferably incubated with purified and/or nerastvorim virus, for example, a retrovirus containing the gene, serving to eliminate the failure or defect of an enzyme or other protein in the cells, in the presence of fibronectin or its fragment in a quantity sufficient to increase the purity of the transduction of cells by the virus. Received transduced hematopoietic cells can then be entered, for example, intravenous animal to the recipient, preferably autologous to the donor, but has also allogenic grafts, which is especially true when used as a graft cells in umbilical cord blood, as described below.

The methods of the present invention can be used in the field of labeling gene or gene therapy to treat various diseases, including diseases of the bone marrow, including, for example, various forms of cancer and leukemia, diseases associated with deficiencies or defects in the protein, and for modification of hematopoietic cells to reduce their resistance to other therapeutic methods such as chemotherapy. Typical diseases for which treatment you can use this invention, are adenosylmethionine failure, for example caused her severe combined immunodot.

In accordance with one preferred embodiment of the present invention, the cells used for cell transplantation are obtained from cord blood of a man. Thus, cord blood can be collected and enrich viable primitive cells-precursors and/or stem cells, for example, by obtaining a population of unrelated mononuclear cells with low plotnostyu. This population is then optionally pre-stimulated and incubated in the presence of the retroviral vector and immobilized and/or purified fibronectin or its fragments to increase transduction efficiency of cells under the action of the vector. In this regard, it was found that transduction of primitive hematopoietic and/or stem cells derived from umbilical cord blood, increases significantly in the presence of fibronectin or its fragments, even if fibronectin does not form part of a complex extracellular matrix of the umbilical cord blood and even if primitive precursor cells and stem cells from umbilical cord blood have characteristics that are different from the indicators of bone marrow. In particular, stem cells from umbilical cord blood were characterized by Kai inventors opening, what a primitive precursor cells derived from umbilical cord blood, effectively transducers in the presence of fibronectin or its fragments, allows the use of convenient and significantly enriched in stem cells, the source of hematopoietic cells. In addition, evidence of successful transplantation, many patients allogeneic transplants of umbilical cord blood, enriched primitive cells-precursors and stem cells, makes the umbilical cord blood is the preferred source of hematopoietic cells. Cm. Kohli-Kumer and others, Brit. Haematol. 85:419-422 (1993); Broxmeyer and other Blood Cell 17:313-329 (1991); Gluckman etc., Br. J. Haematol. 45:557 (1980); Heidelberg; Sptinger - Verlag, pp. 60-68 (1968); Wagner and others, Blood, 79: 1874-1881 (1992) and Wagner and others, Blood, 82-86a (abstract).

Collected transduced hematopoietic or other cells can optionally be subjected to testing to determine transduction efficiency and gene expression. For example, significant improvements retrovirus-mediated gene transfer achieved as a result of application of the present invention are the following examples describe some of the tests indicating high effektnosti, hematopoietic cells of mice infected with the retrovirus PGK-hADA, characterized by high levels moved cDNA of adenozindezaminazy. Similarly, colonies of progenitor cells of a person infected with the virus PGK-mADA, was characterized by levels of adenozindezaminazy mice, which are 10-fold the levels of endogenous protein adenozindezaminazy person. Therefore, in order to accurately analyze the transfer efficiency, colony precursor cells believed translotsirovannoi only if the expression of the transferred adenozindezaminazy mice was equal to or exceeded the levels of endogenous adenozindezaminazy person. High levels of expression of Neogene obtained from TKNEO vector were detected by G418 drug resistance when performing analysis on neotestamentaria (product of Neogene) activity.

As mentioned above, the methods of the present invention can be accomplished without the need of cocultivation in the presence of retroviral cells-producers. Thus, in accordance with one aspect of this invention retrovirus-mediated gene transfer can be carried out in the absence of any cells except cu is asmide retroviral vector, and collect the supernatant. Containing retrovirus supernatant can then be used to infect hematopoietic cells in the presence of fibronectin and/or its fragments, which are preferably immobilized, that is deposited on the substrate, which is the infection, or in contact with the environment, designed to infection. In this regard, any cell producers, which form retroviruses with high titer and without helper viruses are considered suitable for use in this invention. They include, for example, packaging cells, such as Psi-2, C2, PA12, PA317 and GP+envAM12, and many other well-known line of packaging cells.

In accordance with other features of the present invention in a strong binding of the virus with amino acids in heparin binding of fibronectin can be used to create delivery systems used for viral therapy in the cells of various types. Thus, a polypeptide comprising a binding region of a retrovirus from fibronectin, can be covalently linked to any ligand, which gives this design specificity against target cells. This is the target (Kasahara N. , A, M. Dozy and G. W. Kan, Science, I. 266, pp. 1373-1376 (1994) and Valsesia-Wittmann, S., A. Drynda, G. Deleage, M. Auailley, J. M. Heard, O. Danos, G. and Verdier F. Z. Cosset, J. Virol. so 68, page 4609 - 4619 (1994). The specificity of the design of the target cells can be achieved through the use of ligand, including, for example,

1) protein that provides cell adhesion,

2) hormones or cytokines,

3) monoclonal antibodies specific to the target cells,

4) carbohydrates that bind target cells (G Aswel and others, Annu, Rev. Biochem., vol 51, pp. 531-554 (1982)),

5) metabolites to target cells or

6) functional polypeptides that bind target cells. Design efficiency for gene delivery can be improved to include more areas of the binding of the virus heparin P, resulting in an increasing number of viral particles delivered to the target cells. For example, it was found that binding of the cell region of human fibronectin, which corresponds to Pro1239- Ser1515as described in U.S. patent N 5198423, provides binding to cells, including cells KSS and B16-F10 (Kimizuka etc., J. Biochem., so 110, pages 285-291 (1991)). In addition, it was found that heparin Clause itself provides binding to fibroblasts, cells of the endo is retrovirus of fibronectin and to the predefined target cells, designed to infection by the retrovirus.

Typical applications in the system hematopoietic cells also include the design of erythropoietin or G-CSF, which are attached to the retrovirus binding of fibronectin to deliver virus respectively to the erythroid or granulocyte cells predecessors. Another application in accordance with the present invention is the connection region or regions of a retrovirus binding with a ligand which is specific or mainly performs the binding of cancer cells. For example, it was found that in vitro and even in vivo the growth of cancer cells in the breast can be influenced by substances, which bind to receptors on target cells, such derived release of luteinizing hormone Emmons, and other Hum. Reprod., 9:1364- 1379 (1994), estrogen, Tolcher A. C., Oncol. 8:39-43 (1994) or antiestrogens, Howell, A. and others, Lancet,

345: 29-30 (1995), POCs or antiprogestogens, Klijn. F. G., and others, Annex 9 to the journal Hum. Reprod, 1:181-189 (1994), Griffiths K., and others , Semin. Oncol. , 21:672-687 (1994), which serve as ligands in the structures of the present invention, containing one or more regions of the binding of viruses from fibronectin. As can be seen from either the of odara use structures with CodeDOM, and (cancerous) cells of the liver can be affected by using structures containing alphalinoleic high density or parts thereof. And, finally, construction of a monoclonal antibody and region of the retrovirus binding fibronectin allow you to purposefully affect any cells and organs, for which the intended antibody. Thus it is possible to purposefully influence a number of mammalian cells, focusing on the capacity region of the retrovirus binding of fibronectin to bind and localize viral vectors.

Another preferred implementation of the present invention includes obtaining a design that can be used to enhance viral transduction of target cells. Linking the virus amino acid sequence of the binding region of heparin P fibronectin attached to the ligand, which is linked to a cell-target. As mentioned above, the ligand can be, for example, the polypeptide of fibronectin or other protein (including providing a cell adhesion protein, in particular laminin, collagen, vitronectin, osteopontin or thrombospondin), hormone, metabolite, antibodies (including monoclonal antibodies), or any other ligand, is tructio can be used in immobilized form in the same way as used fibronectin polypeptides specifically consider the examples below.

Such designs and approaches on the basis of purposeful influence on cells in vitro can be used, as mentioned above, as well as during in vivo targeted transfer retrovirus taking into account various factors such as the stability and specificity of the design and interaction of retroviral constructs under physiological conditions. Specificity can also be improved by changing the delivery system with the aim of localizing the delivery of the structure only to the target cells, for example, using catherization portal vein for targeted impact on liver cells.

Mediated by retrovirus transfer of DNA can be performed using sets, specially designed for the practical implementation of the methods of the present invention. Therefore, another aspect of the present invention provides kits that include a certain amount of pure polypeptide or above design, which enhances the transduction of target cells under the action of retroviruses, together with the artificial substrate, which can be made we are on an artificial substrate. In the case of procedures infection intended for hematopoietic cells, kits may also include growth factors hematopoietic cells for preliminary simulation cells. In addition, these kits may include recombinant retroviral vectors described above for the implementation of transduction. These kits should include sterile packaging, which safely isolates the various components from each other, preventing them from mixing during the work with this set. For example, for this purpose are typically used molded plastic products with multiple compartments intended for separate storage of the components included in the kit.

For a fuller and deeper understanding of the essence of the present invention provides the following typical examples. It is obvious that these examples are illustrative and in no way limit the scope of the invention.

EXAMPLE 1

Gene transfer into bone marrow cells using a vector TKNEO

1.1. Obtaining supernatant from virus

Cells-producers GP+Env 12 AM (see Markowitz and others (1988) Virology 167: 400) containing the retroviral plasmid vector TKNEO, were cultured in the medium Dolbit calf (Hyclone, Logan, PCs Utah), 100 units/ml penicillin and 100 micrograms/ml streptomycin (P/S provided by the firm "Gibco"). Virus-containing supernatant was collected during the night by adding 10 ml of medium, Dulbecco modified by the method of Claim containing 20% serum amniotic calf, confluent tablets. The collected medium was filtered through the 0.45-micron filters (Gelman Sciences, Ann arbor, pieces Michigan) and stored until use at a temperature of -80oC.

1.2 Obtaining fragments of fibronectin

Fibronectin was purified from human plasma (Lifesource Glenview, PCs Illinois), as described earlier in Ruoslahti and other, Methods Enzymol. 82: 803-831 (1982), except that a column of gelatin and agarose were washed in 1 M urea solution prior to elution of fibronectin with 4 M urea solution. Purified fibronectin was subjected to slow dialysis at a temperature of 4oC using 10 mmol of 3-(cyclohexylamino)-1-propanesulfonic acid, 150 mmol NaCl, 2 mmol CaCl2with a pH of 11.0 and stored in aliquot at a temperature of -80oC. the Region of connection of the cells of chymotrypsin (CS-1) and fragments of heparin binding of fibronectin was purified as described above (Ruoslahti and others (1982), ibid, Peterhost (30 kD, 35kD) and 42kD) were obtained in 1 M solution of NaCl eluate from the column with heparin and agarose. For further purification of these heparin binding fragments 1 M solution of NaCl eluate dialyzed over night at a temperature of 4oC using 10 mmol Tris-HCl, pH 7, and was passed through the anion-exchange column (2 ml fast flow diethylaminoethyl-sepharose (Farmacia Fine Chemicals, Uppsala, Sweden)/mg protein), which was balanced 10 mmol Tris-HCl, pH to 7.0. Fragments 30/35 kD collected in the form of unbound fraction, and the fragment 42kD was suirable from the column using 100 mmol NaCl. From 500 mg of fibronectin was obtained approximately 26 mg of fragments 30/35 kD) and 4 mg of fragment 42kD. Fragment 42kD, not fragments 30/35kD recognized by the antibody in binding of fibrin in the determination by the method of Western blokirovaniya. In addition, a fragment of 42kD associated with a column with immobilized fibrin and separate.

For use in the process of infection fragments of fibronectin was immobilized on 35 or 100 mm Petri dishes (Falcon, Lincoln Park, sh. New Jersey) at a concentration of 75 pmoles/cm2as described by Patel and Logichem (1986), see above. In the control cups similarly inflicted 2% (not containing fibranet what I retrovirus

A bone marrow sample taken from healthy adult donors was collected in tubes containing sterile, without preservative heparansulfate sodium, in accordance with procedures approved by the Institutional Board of the medical College of the University pc. Indiana. Mononuclear cells with low density was obtained by centrifugation on Ficoll-Hypaque (density of 1.077 g/ml, Pharmacia, Piscataway, PCs, NJ) for 45 minutes at a temperature of 25oC. Plastic bound cells were removed from bone marrow cells with a low density by an additional incubation on tablets with tissue culture for 4-16 hours at a temperature of 37oC with 5% CO2in the environment Dulleck, modified by way of Lawsuits, with the addition of 2% serum amniotic calf.

Unbound mononuclear cell low-density pre-stimulated to infection by the retrovirus, as described Luckey and others (1992), Blood 80-396, within 48 hours at a temperature of 37oC with 5% CO2in the environment of Dulbecco modified by the method of Claim containing 20% amniotic calf serum, 100 units/ml of recombinant interleukin 6 people, 100 ng/ml of growth factor recombinant human stem cells (the 1 of 106cells/ml in Petri dishes. Pre-stimulated cells were collected by intensive pipetting to remove cells, weakly adhering to the plastic.

Pre-stimulated cells (5 to 105cells/ml) were incubated for 6 hours on tablets, on which was applied bovine serum albumin (control tablets), fibronectin or fragments (subjected to ultraviolet irradiation for best binding proteins with plastic tablet), and then infected the virus-containing supernatant liquid in the presence of growth factors (described above) and 7.5 micrograms/ml polybrene (Aldrich Chemical, Milwaukee, items of Wisconsin). Virus-containing supernatant was replaced (including growth factors and 5.0 micrograms/ml polybrene) after 2 hours, after which cells were incubated for another 12-24 hours. At each change of medium was again added unbound cells.

In accordance with the procedure of infection unbound cells were decanted, and the associated hematopoietic cells were collected from cultures using buffer for dissociation of cells (without enzyme/on the basis of phosphate buffered saline solution, Gibco) in accordance with the instructions shotley were cultured in methylcellulose predecessor for a count of clonogenic assays or long-lived cultures of bone marrow.

1.4. Long-lived culture of the bone marrow.

Analyses initiating cells long-lived culture (stem cell) was performed according to previously described methods when making minor changes. Sutherland and other Blood, 74:1563 (1989). Briefly, this method can be described as follows: 0.5 to 1106infected cells were sown in long-lived culture of bone marrow on confluent irradiated (as described above) allogeneic fibroblasts of human bone marrow in 5 ml of medium, Dulbecco modified according to the method Claims, which contained 10% of amniotic calf serum, 10% horse serum (Sigma), a mixture of penicillin/streptomycin, 1 10-5M solution of hydrocortisone (decision Upjohn, Kalamazoo, pieces Michigan,) and 320 momola sodium chloride for 6-hole tablets, filled with tissue culture (Costar, Cambridge, pieces Massachusetts). Long-lived culture of bone marrow were incubated at the temperature of the 33oC in 5% CO2and removed weekly 50% protection and unbound cells. After five weeks of initiating cells are long-lived crops destroyed, using the buffer for the dissociation of the cells to remove the associated hematopoietic cells from fibroblasts bone marrow. Unrelated initiating cells long-lived culture.

1.5. Count of clonogenic assays in methylcellulose

Analyses on the methylcellulose was performed as described Taxonom and others, Proc. Natl. Acad. Sci., USA, T. 89, page 7350 (1992) with minor adjustment. Briefly, this method can be described as follows: 2-5 104infected cells in the bone marrow of an adult were cultured with 5 units/ml erythropoietin (Epo, Amgen), 100 ng/ml of growth factor recombinant human stem cells, 10 ng/ml recombinant interleukin-3 (Genzyme, Cambridge, units mA) in 1 ml of 2.4% methylcellulose medium Dulbecco modified by the method of Claims (Fluka, Ronkonkoma, pc. new York), which contained 25% of amniotic calf serum, 10% human plasma, 10-5M solution of batmancatherine (Sigma) and a mixture of penicillin/streptomycin. Cultures were incubated at temperature 37oC in an atmosphere consisting of 5% CO2and 95% air, and on the 13th day under the inverted microscope was assessed colonies (> 50 cells) on the content of granulocytes and macrophages (granulocyte-macrophage colony-forming unit), the content of myeloid and erythroid cells (colony-forming unit-mix) or on the content of erythroid elements (Eritrea is fitiavana virus TKNEO analyzed by determining the percentage of colonies in methylcellulose, resistant to 1.5 mg/ml (dry powder, Gibco) G418 on the 13th day. In each experiment produced pseudoceratina by incubating bone marrow on line packaging cells GP+EnvAM 12 without creating a recombinant virus. Culture of these cells, pseudoceratina 1.5 mg/ml G418, consistently demonstrated the formation of < 1% of background colonies.

1.7 the Efficiency of gene transfer in commitirovannah precursor cells

The transduction efficiency was compared by infection of bone marrow cells during cultivation on cups, covered with fragments of fibronectin 30/35 or bovine serum albumin. When creating these conditions there was no difference in the number of colonies obtained after infection without performing selection. In Fig. 2 shows the percentage of G418 colonies after infection. A higher percentage of G418r colonies was observed on fragments of fibronectin 30/35 for precursor cells of all types, including those that have been separated from precursor cells with a limited line of differentiation (BOEE and CFU-GM) and multiple line differentiation (CFU-mix). Infection all commiteeman precursor cells increased by 9 times to frag the genes in initiating cells are long-lived culture

Gene transfer in initiating cells are long-lived culture was estimated using TKNEO vector. Gene transfer in the colony, received from the originating cells of long-lived culture, was discovered only after infection fragments of fibronectin 30/35 (16% of the colonies of G418' compared to 0% of the colonies G-418r when using fragments of fibronectin 30/35 compared to bovine serum albumin).

1.9. The influence of the specificity of the fragments of fibronectin 30/35 on the efficiency of infection of hematopoietic cells

To determine the specificity of the higher efficiency of gene transfer observed when using fragments of fibronectin 30/35, produced infection TKNEO on tablets coated with bovine serum albumin, fragments of fibronectin 30/35, intact fibronectin, MD 115 fragment of fibronectin with a Central region of the binding of cells containing tetrapeptide sequence RGDS and 42 KD C-terminal fragment of fibronectin (42FN), characterized by the presence of the binding region of heparin P, but no sequence CS-1 (Fig. 1). Infection on bovine serum albumin allowed to obtain 31% Boe G418r, 11% of CFU-GM G418r and 00% of CFU-mix G418r. In Central the higher the infection of Boe (6,0 1%) was observed on fibronectin 42 (Fig. 3). However, intact fibronectin was increased gene transfer in all commitirovannah precursor cells. The percentage of G418r colonies after infection on intact fibronectin was less than the fragments of fibronectin 30/35 in all lines of differentiation, including Bee (162 compared to 244%), CFU-GM (52 compared to 204%) and CFU-mix (61 compared to 91; accordingly intact fibronectin compared with fragments of fibronectin 30/35).

EXAMPLE 2

Gene transfer into bone marrow cells using a vector PGK-mADA

2.1. General procedures

The supernatant from virus PGK-mADA was obtained in the same way as described for TKNEO vector in example 1. Chymotrypsinogen fragments of fibronectin (Fig. 1) was obtained analogously to example 1, it was followed the procedure of infection with retrovirus as described in example 1. Analyses initiating cells long-lived culture (stem cells) and analyses on the methylcellulose was performed in accordance with example 1.

2.2. Analysis of infection with retrovirus

The efficiency of infection by vector PGK-mADA was determined using the protein analysis by performing electrophoresis isoenzyme of adenozindezaminazy. Analysis of individual p is (1989) Proc. Natl. Acad. Sci. , USA, I. 86, pp. 8892. To improve the accuracy of analysis transfer efficiency translotsirovannoi considered only those colonies that demonstrated the formation of adenozindezaminazy mice at the same or higher level as compared with the endogenous adenoidectomies person. For analysis of pools of colonies were isolated from the culture in methylcellulose, collected in a 1.5 ml microtube (Rainin, Woburn, pieces Massachusetts), washed with warm medium and phosphate buffered saline, centrifuged and stored at -20oC. For analysis adenozindezaminazy cells were literally 5-Microlitre buffer for lizirovania by repeating cycles of freezing and thawing, after which he was assigned isoenzyme electrophoresis as described above.

2.3. The efficiency of gene transfer in commitirovannah precursor cells

The transduction efficiency was compared by infection of bone marrow cells during cultivation on cups, covered with fragments of fibronectin 30/35 or bovine serum albumin. In these conditions there was no difference in the number of colonies obtained after infection without performing selection. As shown in table 1, eff the fibronectin 30/35, than bovine serum albumin. As expected in the case of a vector with a higher titer (~1107viruses/ml), the transduction efficiency commiteeman precursor cells was very high. From table 1 it is evident that infection of bone marrow fragments of fibronectin 30/35 by PGK-mADA allowed to reach nearly 100% transduction commiteeman precursor cells in two separate experiments.

2.4. The efficiency of gene transfer in initiating cells are long-lived culture

In four independent experiments, performed using vector PGK-wADA, a significant part of the colonies precursor cells, isolated after 5 weeks (i.e. colony-initiating cells long-lived culture), was characterized by the expression of a gene of adenozindezaminazy mice. Expression ranged from 2/12 (17%) to 6/6 (100%) of the analyzed colonies (table 2). Expression of the introduced gene adenozindezaminazy mice was superior or at least equal to the value of the activity of endogenous adenozindezaminazy person all colonies were considered positive. The efficiency of infection for PGK-mADA was higher than for TKNEO. As can be seen from table 2, infection of the bone marrow on f is predecessors in two separate experiments.

2.5. The influence of the specificity of the fragments of fibronectin 30/35 on the efficiency of infection of hematopoietic cells

The efficiency of gene transfer in initiating cells are long-lived culture was higher in fragments of fibronectin 30/35. Because of the relatively small size of these secondary colonies formed by the originating cells of long-lived culture, the ability to perform protein analysis in a separate colonies was limited. After infection vector PGK-mADA on bovine serum albumin, intact fibronectin and the fragment of fibronectin 42 expression of adenozindezaminazy mice in the colonies formed by the originating cells of long-lived culture, was 0/6, 0/3 0/4 and, while the same indicator for colonies infected with fragments of fibronectin 30/35, was 3/5. In addition, when several colonies formed by the originating cells of long-lived culture, were combined prior to analysis in two additional experiments, expression of adenozindezaminazy mice was detected only after infection fragments of fibronectin 30/35 and to a much lesser extent on intact fibronectin, while it was absent when using fragmento brain using vector PGK-hADA

3.1. The General procedure

The supernatant from virus PGK-hADA was obtained in the same way as described for TKNEO vector in example 1. Chymotrypsinogen fragments of fibronectin (Fig. 1) received in accordance with the description given in example 1 was followed the procedure of infection by the retrovirus in example 1. Analyses initiating cells long-lived culture and analyses on the methylcellulose was performed analogously to example 1.

3.2. Analysis of infection with retrovirus

For analysis of pools of colonies were isolated from the culture in methylcellulose, collected in a 1.5 ml microtube (Raini, Woburn, pieces Massachusetts), washed with warm medium and phosphate buffered saline, centrifuged and stored at -20oC. For analysis adenoidectomies cells were literally 5 microliter buffer for lizirovania by repeating cycles of freezing and thawing, after which he was assigned isoenzyme electrophoresis, as described above.

EXAMPLE 4

Gene transfer into cells of umbilical cord blood using vector TKNEO

4.1. The General procedure

The supernatant from virus TKNEO and chymotrypsinogen fragments of fibronectin (Fig. 1) were obtained as described in primasia heparin, collected cord blood of normal, full-term newborns in accordance with procedures approved by the Institutional Board of the medical College of the University pc. Indiana, and used it instead of bone marrow cells. Analyses initiating cells long-lived culture (stem cells) and analyses on the methylcellulose was performed in accordance with example 1.

4.2. The efficiency of gene transfer in commitirovannah precursor cells

The level of infection on the fragments of fibronectin 30/35 increased more than four times compared with the use of bovine serum albumin in three separate experiments (table 3).

EXAMPLE 5

Gene transfer into cells of umbilical cord blood using vector PGK-mADA

5.1. The General procedure

The supernatant from virus PGK-mADA and chymotrypsinogen fragments of fibronectin (Fig. 1) were obtained as described in example 1. Performed procedure infection by the retrovirus in example 1 except that in tubes containing heparin, collecting the umbilical cord blood of normal, full-term newborns in accordance with procedures approved by the Institutional Board of the medical College piece Indie the example 1.

5.2. The efficiency of gene transfer in initiating cells are long-lived culture

When using vector PGK-mADA with higher titer analysis of colonies formed by the originating cells of long-lived culture showed high expression of the introduced cDNA of adenozindezaminazy mice only in cultures derived from umbilical cord blood, infected supernatant liquid fragments of fibronectin 30/35. A small expression of adenozindezaminazy mice was detected in colonies formed by the originating cells of long-lived culture, in the control cups with bovine serum albumin.

The results given in examples 4 and 5 show that high efficiency of infection when using fragments of fibronectin 30/35 can also be achieved with the use of progenitor cells, cord blood and stem cells.

EXAMPLE 6

Gene transfer into cells of umbilical cord blood using vector PGK-hADA

The supernatant from virus PGK-hADA and chymotrypsinogen fragments of fibronectin (Fig. 1) was obtained in the same way as described for TKNEO vector in example 1. Performed procedure infection by the retrovirus in example 1 except tog is availa able scientific C with the procedures approved by the Institutional Board of the medical College of the University pc. Indiana, and used it instead of bone marrow cells. Analyses initiating cells long-lived culture and analyses on the methylcellulose was performed in accordance with example 1.

EXAMPLES 7-11

Retroviral vectors and line precursor cells for examples 7-11

For examples 7-11 used two lines forming a retrovirus packaging cells: anatropous cell line GP + E86 (Markowitz, D., S. Goff, and A. Bank, J. Virol., 62, pp. 1120-1124 (1988) and amphitropous cell line GP + env AM12 (Markowitz, D., S. Goff, and A. Bank, Virology, I. 167, pp. 400-406 (1988)). Used in the studies described here retroviral vectors and clones of cells-producers are shown in table 4.

All cell lines were cultured in modified according to the method of Dulbecco environment Needle (Gibco, Grand island, pc. NY) containing 10% amniotic calf serum (Hyclone, Logan, PCs Utah), 100 units/ml penicillin and 100 μg/ml of streptomycin (Gibco), except that cells EAL2a were grown in modified according to the method of Dulbecco environment Needle F12 (Gibco) with 10% serum amniotic calf. Virus-containing supernatant was harvested by adding 10 ml of alpha-minimum podderjivaetsa, each of which contained 10% of amniotic calf serum and a mixture of penicillin/streptomycin order to merge 10 cm of tablets during the night. The collected medium was filtered through the 0.45-micron filters (Gelman Scienses Ann arbor, pieces Michigan) and stored until use at a temperature of -80oC.

EXAMPLE 7

Transduction of primary hematopoietic cells of mice

7.1. Experimental part

For research using cells of mice bone marrow was taken from the femoral and tibial bones of mice C3H/ / HeJ aged 6-8 weeks 2 days after injection of 150 mg/kg 5-fluorouracil (SoloPack Laboratories, Franklin Park, PCs Illinois) (B. Lim, J. F. Aperley, S. H. Orhin, and D. A. Williams, Proc. Natl. Acad. Sci, USA, 86 so p. 8892-8896 (1989)). The cells were pre-stimulated at a concentration of 5 105cells/ml in the medium, Dulbecco modified according to the method Claims, which contained 20% of amniotic calf serum and a mixture of penicillin/streptomycin 100 mg/ml of growth factor recombinant stem cells in rats (Amgen Thousand-oaks, PCs California) and 100 units/ml of recombinant interleukin-6 person (Pepro Tech Inc., Rock hill, , new Jersey) within 48 hours (Luskey B. D. , M. Rosenblatt, K. Zsebo, and D. A. Williams, Blood, I. 80, pages 396-402 (1992)). Then compare effectssuch procedures of infection:

1) infection supernatant;

2) infection supernatant fragments of fibronectin 30/35;

3) soultrane on cells-producers of NRN-5. Therefore, 100 mm bacteriological cups were covered with 2.5 µg/cm2fragments of fibronectin 30/35 (equivalent to 75 mmol/cm2) dissolved in 5 ml of phosphate buffered saline (Gibco), and then for 1 hour and kept at room temperature and ultraviolet light in the cups with the lid open and back in one hour - in cups with the lid closed. After blocking with 2% bovine serum albumin (Fraction V; Boehringer Mannheim, Indianapolis, PCs Indiana) for 30 minutes at room temperature cups washed once balanced salt Hanks solution, which was added 2.5% (volume ratio) of 1 M solution of HEPES (Gibco). For infection the supernatant cups covered only bovine serum albumin. 5 106pre-stimulated donor cells were incubated with 10 ml of virus-containing supernatant obtained from cells of NRN-5, which was added 100 units/ml of recombinant interleukin 6 people, 100 ng/ml of growth factor, recombinantly the Jay supernatant liquid with the virus. For cocultivation cell NRN-5 in 4 ml of medium were incubated with 10 μg/ml of mitomycin for 2 hours at a temperature of 37oC, washed, and treated with trypsin and were sown in 100 ml cups with tissue culture at a concentration of 3 to 106cells in 10 ml of alpha-minimal supportive environment with 20% amniotic calf serum and a mixture of penicillin/streptomycin. The next day, within 48 hours was added 5 106pre-stimulated bone marrow cells with 100 units/ml of recombinant interleukin 6 people, 100 ng/ml of growth factor recombinant stem cells of rats and 4 µg/polybrene. In accordance with the procedure of infection unbound cells were decanted, and the adhesion of hematopoietic cells were isolated from the cultures using a buffer for dissociation of cells (without enzyme /on the basis of phosphate buffered saline solution, Gibco) following the manufacturer's instructions. The bound cells were added to the fraction unbound cells, washed twice and suspended in 1 ml of a mixture of balanced salt solution Hanks and HEPES. All cells derived from 5 106pre-stimulated cells were injected with the tail vein of three mice recipient, the whole body which p / 396-402 (1992)). Transduction of hematopoietic stem cells was analyzed by examination of mice that underwent rehabilitation therapy, in relation to the expression of the introduced cDNA of adenozindezaminazy person. Isozyme analysis of adenozindezaminazy performed in transplanted mice by examining blood cells in the peripheral blood system in the presence of protein adenozindezaminazy person using cellulose acetate when performing in situ enzyme assay (B. Lim, D. A. Williams and S. H. Orkin, Mol. Cell. Biol. so 7 p. 3459-3465 (1987)). The study was begun 4 months after transplantation and repeat it every month.

7.2. Results

Recovery of long-lived bone marrow cells in mice using genetically modified hematopoietic stem cells is usually considered sufficient to determine the efficiency of transduction of stem cells 4 months after transplantation. Spent 7 months isozyme analysis recipients transducing bone marrow showed that:

1) expression of the cDNA adenozindezaminazy person was under the infection of coculture or supernatant fragments of fibronectin 30/35 and was absent in the group, expressii were comparable for groups infected by the method of cocultivation and using fragments of fibronectin 30/35. As shown in Fig. 4, columns 2 - 4, three mice that were transplanted bone marrow, transpulmonary cocultures on the cells of NRN-5, was easily detected adenosylmethionine person. Similar levels of adenozindezaminazy person were detected in three mice that were transplanted hematopoietic cells transduced by infection supernatant with fragments of fibronectin 30/35 (Fig. 4, columns 5-7). In contrast, adenoidectomies man was not detected in three mice that were transplanted hematopoietic cells transduced by infection the supernatant liquid bovine serum albumin (Fig. 4, columns 8 to 10). The control results for adenozindezaminazy person listed in columns 1 and 12, and for adenozindezaminazy mice in column 11 of Fig. 4. Area, which shows the results for mice, columns 2-10, shows that were put equal amounts of protein. These data show that transduction of long-lived regenerating hematopoietic stem cells by infection supernatant on fragmentary supernatant without fragments of fibronectin 30/35.

EXAMPLE 8

The mechanism of improvement of transduction by binding to retroviral vectors with fragments of fibronectin 30/35

8.1. Experimental part

To determine whether higher transduction result of colocalization virus and hematopoietic cells, we analyzed particles of recombinant retrovirus in relation to binding fragments of fibronectin 30/35. Therefore, the tablets covered with fragments of fibronectin 30/35, within 30 minutes pre-incubated with the supernatant fluid containing the virus TKNEO, then intensively washed. The viral titer of the supernatant was determined using cells NIH/3T3 in accordance with standard methods (D. Markowitz , S. Goff, and A. Bank., J. Virol., 62, pp. 1120-1124 (1988)). The 3T3 cells were placed at a concentration of 1000 cells/well on 6-well plate with tissue culture and grown overnight. To each well containing the 7.5 µg/ml polybrene, was sequentially added a solution of the supernatant liquid with virus and incubated for 2.5 hours at a temperature of 37oC, after which was added 2 ml of medium. After 24 hours the medium was replaced with medium containing G418 (0.75 mg/ml, dry powder, Gibco), and incubated tablets for 10-12 days. Resistant G41 is bauleni virus-containing supernatant liquid, used as infectious particles (CFU)/ml of the supernatant liquid. We evaluated/"titrated" the number of retroviral particles remaining on 35 mm tablets, covered with fragments of fibronectin 30/35 or bovine serum albumin, after pre-incubation with virus-containing supernatant liquid and intensive washing by adding 1000 cells NIH/3T3 35 mm bacteriological Cup together with polybrene. Twenty-four hours in culture has introduced a medium containing 0.75 mg/ml G418 (dry powder), after which the cells were continued to incubate for 10-12 days. After incubation produced quantitative determination of the presence of the adhesion of the virus by determining the number of resistant to G418 colonies NIH-3T3.

To establish whether the virus fragments of fibronectin 30/35 dose, repeated the above experiments with increasing concentrations of fragments of fibronectin 30/35, applied to the Cup. Therefore, 35 mm bacteriological cups were covered with 1, 4, 10 and 20 µg/cm2fragments of fibronectin 30/35, as described above. Diluted in the ratio 1:50 strain of the virus TKNEO, whose title before it was 1104infectious particles/ml, for 30 MiNC was added 2000 cells NIH-3T3. The selection was made in accordance with the description above, and 10 days after selection made calculations resistant to G418 colonies of cells NIH-3T3.

8.2. Results

In Fig. 5 shows the results of one of three typical experiments. When you use containing TKNEO supernatant titers of retrovirus, measured as the number of colonies G419r in cells NIH/3T3, decreased by more than 3 log (4 of 103to 0) on tablets coated with bovine serum albumin, whereas the tablets covered with fragments of fibronectin 30/35, the reduction in titer was only 1 logarithm. These data show that happens quantitative binding of a retrovirus with fragments of fibronectin 30/35 and is not binding cups coated with bovine serum albumin (control Cup). In Fig. 6 shows that more resistant to G418 colonies were found when virus-containing supernatant was incubated on tablets covered with fragments of fibronectin 30/35 with higher concentrations. Therefore, the virus fragments of fibronectin 30/35 depends on the dose.

EXAMPLE 9

The binding of the virus with recombinant fragments of fibronectin
4infectious particles/ml Fragments of fibronectin was used with a concentration of 120-130 pmoles/cm2(equivalent to 4 mg/cm2C-274, H-271, H-296, C-CS1, fragments of fibronectin 30/35 and 8 µg/cm2for CH-271 and CH-296). The tablet was coated, was added to the virus, tablets slowly washed for 24 hours was injected cells NIH/3T3, and then they were grown in selection medium for 10 days, after which colonies were stained and made calculations.

9.2. Results

In Fig. 8 shows, 271 and CH-296. In addition, this figure shows that the number of related viruses comparable to these recombinant fragments and fragments of fibronectin 30/35, although in this work the fragment H-271 was characterized by the highest level of binding of the virus. Common to all these 5 fragments of fibronectin are 12-14 repetitions of type III, which includes the binding site of heparin with high affinity (Ruoslahti E., Ann. Rev. Biochem., so 57, pp. 375-413 (1988) and Kimizuka F. , Y. Taguchi, Y. Ohdate, Y. Kawase, T. Shimojo, K. Hashino, I. Kato, K. Sekiguchi, and K. Titani, J. Biochem.,

so 110, pages 284-291 (1991)), probably located in the repeat 13 (Kimisuka, F., Y. Taguchi, Y. Ohdate, Y. Kawase, T. Shimojo, K. Hashino, I. Kato, K. Sekiguchi, and K. Titani, J. Biochem., so 110, pages 284-291 (1991)). This suggests that the binding of the virus occurs at this famous site of adhesion. Evidence has been obtained in the pre-incubation cups coated with 4 µg/cm2CH-271 with increasing concentrations (10-1000 μg/ml) of heparin sulfate, highly charged molecule, which, as you know, inhibits the binding of cells with the binding site of heparin. As can be seen from Fig. 9, the number of resistant to G418 colonies is reduced after pre-incubation CH-271 with heparin sulfate in increasing concentrations. These data suggest that h is defined near the site of CS-1 in carboxykinase areas of fibronectin.

EXAMPLE 10

Transduction of hematopoietic cells with a recombinant fragment of fibronectin

10.1. Experimental part

To analyze whether the above-described higher transduction of hematopoietic cells with fragments of fibronectin 30/35 be observed in recombinant fragments of fibronectin, we evaluated the transduction efficiency infections supernatant in vitro by assays of colony forming cells with high proliferative potential. Using vectors EAL2a, we compared different recombinant fragments of fibronectin and fragments of fibronectin 30/35 with infection the supernatant liquid bovine serum albumin in relation to the effects on the transduction of hematopoietic cells using growth resistant to G418 colonies as an indicator of gene transfer. In addition, we compared the ability of viral particles that are already associated with fragments of fibronectin (compared to the viruses in the supernatant), transducible hematopoietic cells. From 0.5 to 1 106pre-stimulated bone marrow cells were incubated in 35 mm Petri dishes coated with fibronectin, 1-2 ml of virus-containing EAL2a radosevich cells with a virus, associated with the fragment of fibronectin, 35 mm Cup, coated with fibronectin, were incubated with virus-containing supernatant liquid and washed three times with 2 ml of phosphate buffered saline. After this was added from 0.5 to 1106pre-stimulated bone marrow cells in 2 ml of medium supplemented with growth factors and polybrene. After 22 hours the cells were collected and were analyzed by colony forming cells with high proliferative potential with 1.5 mg/ml G418 and without it, as previously described (Bradley T. R., and D. Metcalf, Aust. J., Exp. Biol. Med. Sci., so 44, pp. 287-293 (1966)). These cultures were incubated for 14 days in 7% CO2when the temperature of the 33oC, and then calculated the efficiency of gene transfer in the form of a percentage resistant to G418 colonies.

10.2 Results

Transduction of primary hematopoietic cells during infection the supernatant (Fig. 10), was significantly higher than when the infection on bovine serum albumin for all fragments that included the binding site of heparin and at least one more active area of cell adhesion (solid columns). The transduction efficiency of recombinant fragments CH-271, H-296, CH-296 and C-CS1 was similar deistvie all other cases transduction strongly reduced. These data show that increasing the transduction of primary hematopoietic cells, previously demonstrated fragments of fibronectin 30/35, can be repeated with recombinant fragments of fibronectin. This further suggests that the virus is directly related to the fibronectin capable of genetic transduction of hematopoietic cells. Finally, these data confirm that the presence of both CS-1 site and the binding site of heparin is useful to enhance transduction of hematopoietic (stem) cells-precursors.

EXAMPLE 11

Recovery of long-lived bone marrow cells of mice in the transduction of cells of mice donor to recombinant fragments of fibronectin

11.1 Experimental

We repeated the above in vitro studies (from example 10) for primitive hematopoietic precursor cells for comparison of infection supernatant to bovine serum albumin with fragments of fibronectin 30/35 and recombinant fragments of fibronectin with cocultures, producing bone marrow transplantation to assess the impact on the recovery of hematopoietic stem cells. Mice poluchili cDNA of adenozindezaminazy person. After 1 month using isozyme analysis adenozindezaminazy determined the efficiency of gene transfer from the peripheral blood circulatory system.

11.2. Results

In Fig. 11 clearly shows that the fragment of fibronectin H-296, containing the binding site of heparin and CS-1 the plot shows the same results as the fragments of fibronectin 30/35 and coculture. Fragments that do not have these sites are less effective in transduction of transplantable hematopoietic cells. These data show that colocalization primitive hematopoietic pillar cells and retrovirus associated with recombinant fragments of fibronectin, including CS-1 site and the binding site of heparin, effectively transducer transplanted hematopoietic cells.

Although this invention has been illustrated in detail and considered in the above description, it should be considered as illustrative and not limit the invention. It is understandable that presented only the preferred embodiments of the present invention and it is highly desirable that the patent has been protected all changes and modifications that are included in the scope of the materials, that is equivalent to their inclusion and full statement separately.

1. The way to increase the frequency of transduction of viable hematopoietic cells with a recombinant retroviral vector, replication defective, including infection with viable hematopoietic cells recombinant retroviral vector, replication defective, in the presence of essentially pure recombinant fibronectin fragments essentially pure recombinant fibronectin or mixtures thereof in order to increase the frequency of transduction of hematopoietic cells using a retroviral vector.

2. The method according to p. 1, wherein the hematopoietic cells are characterized by a deficiency or defect in protein and recombinant retroviral vector to include an exogenous gene that encodes a protein.

3. The method according to p. 2, wherein the exogenous gene is the gene encoding adenoidectomies.

4. The method according to p. 3, wherein the exogenous gene is the gene encoding adenoidectomies person.

5. The method according to p. 1, characterized in that the cells infect retroviral vector in the presence of a recombinant fragment of fibronectin containing s is now the sequence which provides a retrovirus-binding activity domain of heparin-II.

6. The method according to p. 1, wherein the hematopoietic stem cells include human cells.

7. The method according to p. 6, wherein the hematopoietic cells are neslepties mononuclear cells with a low density.

8. The method of obtaining transduced hematopoietic cells, including infection with viable hematopoietic cells in culture with a recombinant retrovirus replication defective, in the presence of immobilized recombinant fibronectin immobilized recombinant fragments of fibronectin or immobilized mixture, with the aim of obtaining transduced hematopoietic cells.

9. The method according to p. 8, characterized in that it includes the collection of transduced hematopoietic cells.

10. The method according to p. 8, wherein the hematopoietic cells are characterized by a deficiency or defect in protein and recombinant retroviral vector includes an exogenous gene that encodes a protein.

11. The method according to p. 8, wherein the hematopoietic cells are characterized by a deficiency or defect of an enzyme, and an exogenous gene assetsbefore human cells, characterized by a deficiency or defect of an enzyme, and the exogenous gene is a human gene that encodes the enzyme.

13. The method according to p. 11, wherein the hematopoietic cells have adenosylmethionine insufficiency and exogenous gene encodes adenoidectomies.

14. The method according to p. 12, wherein the hematopoietic cells have adenosylmethionine insufficiency and exogenous gene encodes adenoidectomies.

15. The method according to p. 12, characterized in that infect cells with retrovirus in the presence of immobilized recombinant fragment of fibronectin containing the amino acid sequence, which provides linking cell activity domain CS-1 and amino acid sequence, which provides retroviruses activity domain of heparin-II.

16. The method according to p. 15, wherein the hematopoietic stem cells include human cells.

17. The method according to p. 16, wherein the hematopoietic cells are neslepties mononuclear cells with a low density.

18. The method according to p. 1, characterized in that the recombinant fragment of fibronectin viber uchumi stage: obtain viable hematopoietic cells from an animal donor; infection of viable hematopoietic cells recombinant retroviral vector, replication defective, containing exogenous DNA, with the aim of obtaining transduced viable hematopoietic cells, and the infection is produced in the presence of immobilized amount of recombinant fibronectin and/or its fragment can increase the efficiency of transduction of cells using a retroviral vector; and the introduction of transduced viable hematopoietic cells of the animal to the recipient in the form of cell transplant.

20. The method according to p. 19, characterized in that the specified cell transplantation is intended for the treatment of disorders of hematopoietic bone marrow of the animal recipient.

21. The method according to p. 19, characterized in that the specified cell transplantation is intended for treatment failure or defect of the protein of the animal recipient.

22. The method according to p. 20, wherein the animal is a donor and an animal recipient is the man.

23. The method according to p. 22, wherein the animal is a recipient is autologous donor.

24. The way to improve efficiency mediated by retrovirus is renosa genes in the presence of immobilized recombinant fibronectin, immobilized recombinant fragments of fibronectin or immobilized mixture.

25. The method according to p. 24, wherein the hematopoietic stem cells include mammalian cells.

26. The way to increase the frequency of transduction of hematopoietic cells with a recombinant retroviral vector, replication defective, including infection with viable populations of hematopoietic cells enriched in hematopoietic stem cells, the recombinant retroviral vector, replication defective, in the presence of effective immobilized amount of recombinant polypeptide containing the first amino acid sequence, which provides a retrovirus-binding activity domain of heparin-II fibronectin, and a second amino acid sequence, which provides linking cell activity domain CS-1 fibronectin to increase the frequency of transduction of hematopoietic cells by retroviral vector.

27. The method according to p. 26, wherein the hematopoietic cells are characterized by a deficiency or defect in protein and recombinant retroviral vector includes an exogenous gene that encodes a protein.

29. The method according to p. 28, wherein the exogenous gene is the gene encoding adenoidectomies person.

30. The method according to p. 27, wherein the hematopoietic cells are neslepties mononuclear cells with a low density.

31. The method according to p. 26, characterized in that the infection is carried out in the presence of recombinant polypeptide containing (1) a first amino acid sequence represented by the formula (see the graphical part)

or substantially similar amino acid sequence having the ability to bind retroviruses; and (II) a second amino acid sequence represented by the formula

Asp Gly Leu Pro Gln Leu Val Thr Leu Pro His Pro Asn Leu His Gly Pro Glu Iie, Leu Asp Val Pro Ser Thr

or substantially similar amino acid sequence having the ability to bind primitive hematopoietic cells.

32. Cultural environment for retrovirus-mediated gene transfer, including retroviral supernatant; and immobilized amount of recombinant polypeptide containing (I) a first amino acid sequence, which provides a retrovirus-binding activity domain heparin is Yunosti domain CS-1 fibronectin.

33. Culture medium for p. 32, characterized in that the retroviral supernatant includes recombinant retroviral vector comprising an exogenous gene for correction of the deficiency or defect of protein in hematopoietic cells.

34. Culture medium for p. 33, characterized in that said exogenous gene is a gene adenozindezaminazy person.

35. Culture medium for p. 32, characterized in that the immobilized amount of recombinant polypeptide effective to increase the frequency of transduction of hematopoietic cells from umbilical cord blood using retroviral supernatant.

36. Culture medium for p. 32, characterized in. the specified recombinant polypeptide is selected from the group consisting of CH-296 and H-296.

37. The way the transplantation of cells, including the introduction of the animal as a cell transplant viable hematopoietic cells transduced by retrovirus-mediated gene transfer in the absence of retroviral cells-producers and in the presence of immobilized amount of recombinant polypeptide containing (I) a first amino acid sequence to the second sequence, which provides linking cell activity domain CS-1 fibronectin, and specified immobilized amount of recombinant polypeptide effective to increase the frequency of transduction of hematopoietic cells by retroviral vector.

38. The method of cell transplantation on p. 37, characterized in that the said viable hematopoietic cells enriched in hematopoietic stem cells.

39. The method of cell transplantation on p. 38, characterized in that the specified viable hematopoietic cells are hematopoietic cells enriched in hematopoietic stem cells of a person.

40. The method of cell transplantation on p. 39, characterized in that the said hematopoietic cells are essentially homogeneous population of hematopoietic cells, which nasophilia mononuclear cells with a low density.

41. The method of cell transplantation on p. 39, characterized in that the said hematopoietic cells traduzioni recombinant retroviral vector containing an exogenous gene, with the aim of correction of the deficiency or defect of the protein in the cells.

42. Method transplantation cleto the sector, containing gene adenozindezaminazy person, with the purpose of correcting adenosylmethionine deficiency in the cells.

43. The method of cell transplantation on p. 37, characterized in that said recombinant polypeptide is selected from the group consisting of CH-296 and CH-271.

44. The method of obtaining transduced blood cells of the umbilical cord suitable for transplantation of cells, including infection with viable hematopoietic cells obtained from the blood of the umbilical cord recombinant retroviral vector, replication defective, in the presence of effective immobilized amount of recombinant polypeptide containing (I) a first amino acid sequence, which provides a retrovirus-binding activity domain of heparin-II fibronectin, and (II) a second amino acid sequence, which provides linking cell activity domain CS-1 fibronectin, and referred to the immobilized amount of recombinant polypeptide effective to increase the frequency of transduction of hematopoietic cells by a recombinant retroviral vector.

45. The method according to p. 44, wherein the specified hematopoietic glue hematopoietic stem cells.

46. The method according to p. 45, characterized in that the said hematopoietic cells are essentially homogeneous population of hematopoietic cells obtained from the blood of the umbilical cord, which are nasophilia mononuclear cells with a low density.

47. The method according to p. 45, wherein the hematopoietic cells are characterized by a deficiency or defect in protein and recombinant retroviral vector comprises a human gene that encodes a protein.

48. The method according to p. 47, wherein the exogenous gene is the gene encoding adenoidectomies.

49. The way the transplantation of cells, including the introduction of the animal as a cell transplant viable hematopoietic cells from the blood of the umbilical cord transduced by retrovirus-mediated gene transfer in the presence of immobilized amount of recombinant polypeptide containing (I) a first amino acid sequence, which provides a retrovirus-binding activity domain of heparin-II fibronectin and (II) a second amino acid sequence, which provides linking cell activity domain CS-1 fibronectin, and the decree of the AI blood stem cells umbilical cord retroviral vector.

50. The method of cell transplantation on p. 49, characterized in that said animal is man, as specified hematopoietic cells are hematopoietic cells enriched in hematopoietic stem cells of a person.

51. The method of transplantation of cells by p. 50, characterized in that the said hematopoietic cells are essentially homogeneous population of hematopoietic cells, which nasophilia mononuclear cells with a low density.

52. The method of transplantation of cells under item 50, wherein the specified hematopoietic cells transpulmonary recombinant retroviral vector containing an exogenous gene, with the aim of correction of the deficiency or defect of the protein in the cells.

53. The method of cell transplantation on p. 52, characterized in that the said hematopoietic cells transpulmonary recombinant retroviral vector containing the gene of adenozindezaminazy person, with the purpose of correcting adenosylmethionine deficiency in the cells.

54. The way to increase the frequency of transduction of hematopoietic cells by recombinant retroviral vector, replication defective, including infitsirovanie the main immobilized amount of recombinant polypeptide, containing the first amino acid sequence represented by the formula (see the graphical part)

or substantially similar amino acid sequence having the ability to bind retroviruses;

and a second amino acid sequence represented by the formula

Asp Gly Leu Pro Gln Leu Val Thr Leu Pro His Pro Asn Leu His Gly Pro Glu Ile Leu Asp Val Pro Ser Thr

or substantially similar amino acid sequence having the ability to bind primitive hematopoietic cells.

55. The method according to p. 54, wherein the hematopoietic cells are characterized by a deficiency or defect in protein and recombinant retroviral vector includes an exogenous gene that encodes a protein.

56. The method according to p. 55, wherein the exogenous gene is the gene encoding adenoidectomies.

57. The method according to p. 56, wherein the exogenous gene is the gene encoding adenoidectomies person.

58. The method according to p. 55, wherein the hematopoietic stem cells include human cells, as indicated by the exogenous gene is a human gene.

59. The method according to p. 58, wherein the hematopoietic cells are neslepties Mononoke the wasp gene, including retroviral supernatant; and an effective amount of immobilized recombinant polypeptide, which increases the frequency of transduction of hematopoietic cells with the indicated recombinant polypeptide contains a first amino acid sequence represented by the formula (see the graphical part)

or substantially similar amino acid sequence having the ability to bind retroviruses; and

second amino acid sequence represented by the formula

Asp Gly Leu Pro Gln Leu Val Thr Leu Pro His Pro Asn Leu His Gly Pro Glu Ile Leu Asp Val Pro Ser Thr

or substantially similar amino acid sequence having the ability to bind primitive hematopoietic cells.

61. Cultural environment under item 60, wherein the retroviral supernatant includes recombinant retroviral vector comprising an exogenous gene to correct the deficiency or defect of protein in hematopoietic cells.

62. Culture medium for p. 61, characterized in that said exogenous gene is a gene adenozindezaminazy person.

63. The way the transplantation of cells, including the introduction of the animal as a cell transplant is in the absence of retroviral cells-producers and in the presence of effective immobilized amount of recombinant polypeptide, which increases the frequency of transduction of hematopoietic cells, and mentioned recombinant polypeptide contains a first amino acid sequence represented by the formula (see the graphical part)

or substantially similar amino acid sequence having the ability to bind retroviruses; and a second amino acid sequence represented by the formula Asp Gly Leu Pro Gln Leu Val Thr Leu Pro His Pro Asn Leu His Gly Pro Glu Ile Leu Asp Val Pro Ser Thr

or substantially similar amino acid sequence having the ability to bind primitive hematopoietic cells.

64. The method of cell transplantation on p. 63, characterized in that the said viable hematopoietic cells enriched in hematopoietic stem cells.

65. The method of cell transplantation on p. 64, wherein the specified viable hematopoietic cells are hematopoietic cells enriched in hematopoietic stem cells of a person.

66. The method of transplantation of cells by p. 65, characterized in that the said hematopoietic cells are essentially homogeneous population of hematopoietic cells, which nasophilia mononuclear is shown hematopoietic cells transpulmonary recombinant retroviral vector, including exogenous gene, with the aim of correction of the deficiency or defect of the protein in the cells.

68. The method of transplantation of cells by p. 67, characterized in that the said hematopoietic cells transpulmonary recombinant retroviral vector containing the gene of adenozindezaminazy person, with the purpose of correcting adenosylmethionine deficiency in the cells.

69. The method of localization of retrovirus, including the incubation medium containing the retrovirus in effective contact with the immobilized amount of recombinant polypeptide comprising the amino acid sequence, which provides a retrovirus-binding activity domain of heparin-II fibronectin.

70. The method according to p. 69, characterized in that said recombinant polypeptide comprises the amino acid sequence represented by the formula (see the graphical part)

or substantially similar amino acid sequence, providing retrovirus-svyazyvayuschuyu activity domain of heparin-II fibronectin.

71. The way to create designs that are useful for improving retrovirus-mediated transfer of DNA to a pre-defined the target cell, including: the selection of the ligand, which is Benanti the polypeptide, comprising the amino acid sequence, which provides a retrovirus binding activity domain of heparin-II fibronectin.

72. The method according to p. 71, characterized in that the ligand is a protein cell adhesion, a hormone, a cytokine, a monoclonal antibody, a carbohydrate, a metabolite, or a polypeptide protein other than fibronectin.

73. The method according to p. 72, characterized in that the specified cell target is a malignant cell, the specified ligand is a hormone.

74. The method according to p. 73, characterized in that the specified cell is a target cell is breast carcinoma, as specified ligand is a hormone that stimulates luteinizing hormone, estrogen, progestogen or antiprogestogen.

75. The way to increase the frequency of transduction of a population of viable target cells with the retrovirus, including infection of the cells with retrovirus in the presence of effective immobilized number of constructs containing a ligand that specifically binds to cells covalently attached to the recombinant polypeptide that binds retrovirus, with the indicated recombinant polypeptide contains amino acid ol CLASS="ptx2">

76. The method according to p. 75, characterized in that the ligand is a protein cell adhesion, a hormone, a cytokine, a monoclonal antibody, a carbohydrate, a metabolite, or a polypeptide protein other than fibronectin.

77. The method according to p. 75, characterized in that said recombinant polypeptide is a recombinant polypeptide having the amino acid sequence represented by the formula (see the graphical part)

or substantially similar amino acid sequence, providing a retrovirus-binding activity.

78. Design, increasing retrovirus-mediated gene transfer to a pre-defined the target cell, comprising a ligand that specifically binds to a specified cell target, covalently attached to the recombinant polypeptide comprising the amino acid sequence, which provides a retrovirus-binding activity domain of heparin-II fibronectin.

79. Design by p. 78, characterized in that said polypeptide is a recombinant polypeptide having the amino acid sequence represented by the formula (see the graphical part)

or substantially similar to the amino on p. 79, characterized in that the ligand is a protein cell adhesion, a hormone, a cytokine, a monoclonal antibody, a carbohydrate, a metabolite, or a polypeptide, a protein other than fibronectin.

81. The set, designed to perform retrovirus-mediated transfer of DNA into hematopoietic cells, including:

(a) essentially pure recombinant polypeptide containing (I) a first amino acid sequence domain of heparin-II human fibronectin, which provides a retrovirus-binding activity, and (II) a second amino acid sequence, which provides linking cell activity domain CS-1 fibronectin person;

(b) an artificial substrate that is designed for incubation retroviral vector in contact with blood cells of the person; and (C) growth factors, hematopoietic cells, intended for pre-stimulation of hematopoietic cells.

82. Set on p. 81, characterized in that the essentially pure recombinant polypeptide immobilized on a specified artificial substrate.

83. Set on p. 81, characterized in that it also includes:

(d) recombinant retroviral vector, pregnaancy essentially pure polypeptide (a) comprises a recombinant polypeptide, having the amino acid sequence represented by the formula (see the graphical part)

or substantially similar amino acid sequence, having a retrovirus-binding activity.

85. Set on p. 84, characterized in that the essentially pure recombinant polypeptide immobilized on a specified artificial substrate.

86. Set on p. 85, characterized in that it also includes (d) a recombinant retroviral vector designed for transduction of hematopoietic cells.

87. The method of obtaining viable populations of primitive hematopoietic cells, comprising contacting the cell population containing viable primitive hematopoietic cells with an effective immobilized amount of recombinant polypeptide, which provides linking cell activity domain CS-1 fibronectin to bind primitive hematopoietic cells with the indicated immobilized amount of recombinant polypeptide.

88. The method according to p. 87, characterized in. the specified recombinant polypeptide has the amino acid sequence represented by the formula

Asp Gly Leu Pro Gln Leu Val Thr Leu Pro His Pro is th binding ability of primitive hematopoietic cells.

 

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