Described are compositions and methods relating to gene therapy, particularly as applied to hematopoietic progenitor (HP) cells, to transduced cells and methods of obtaining them, and to methods of using them to provide prolonged engraftment of modified hematopoietic cells in human subjects. The invention particularly relates to ex vivo gene therapy of HP cells for treatment or prevention of HIV infection.

Description of the Invention

SUMMARY OF THE INVENTION

This invention provides a composition suitable for administration to a human subject comprising a pharmaceutically acceptable carrier and at least 1.63.times.10.sup.6 CD34.sup.+ hematopoietic cells per kg of body weight of the human subject to whom the composition is to be administered, at least 0.52.times.10.sup.6 of such CD34.sup.+ hematopoietic cells being transduced by a viral construct which expresses an anti-HIV agent.

This invention also provides a method of inserting into hematopoietic cells of a human subject a gene of interest comprising: a) mobilizing CD34.sup.+ hematopoietic progenitor cells into the blood of the human subject; b) isolating leukocytes from the subject by apheresis; c) isolating CD34.sup.+ hematopoietic cells from the isolated leukocytes by an immunoselective method; d) subjecting the CD34.sup.+ hematopoietic cells of step c) to a transduction process with a gene of interest in the presence of an agent that colocalizes the cells with a transduction vector; e) determining the total number of CD34.sup.+ hematopoietic cells after step d), and if the total number is at least 1.63.times.10.sup.6 cells per kg of body weight of the human subject, then proceeding to step f), and if the total number of CD34.sup.+ hematopoietic cells after step d) is less than 1.63.times.10.sup.6 cells per kg of body weight of the human subject, then performing at least steps b)-d) and combining the CD34.sup.+ hematopoietic cells; and f) delivering to the subject the CD34.sup.+ hematopoietic cells, thereby inserting into hematopoietic cells of the human subject a gene of interest.

This invention further provides a use of the composition comprising a pharmaceutically acceptable carrier and at least 1.63.times.10.sup.6 CD34.sup.+ hematopoietic cells per kg of body weight of a human subject to whom the composition is to be administered, at least 0.52.times.10.sup.6 CD34.sup.+ of such cells per kg being transduced with a viral construct which expresses an anti-HIV agent, for the manufacture of a medicament for the treatment of the human subject infected with HIV.

This invention yet further provides a kit comprising a) an amount of an agent capable of mobilizing hematopoietic progenitor bells in a human subject; b) a culture medium including at least one cytokine acceptable for culturing CD34.sup.+ hematopoietic cells; c) a retroviral vector comprising nucleotides having a sequence that in a cell gives rise to a ribozyme having the sequence 5'-UUA GGA UCC UGA UGA GUC CGU GAG GAC GAA ACU GGC UCC-3' (SEQ ID NO.:1) (Rz2); and d) tissue culture vessels coated on their inside with a recombinant fibronectin fragment. A package comprising the kit and instructions for its use is also provided by this invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a composition comprising a pharmaceutically acceptable carrier and at least 1.63.times.10.sup.6 CD34.sup.+ hematopoietic cells per kg of body weight of the human subject to whom the composition is to be administered, at least 0.52.times.10.sup.6 of such CD34.sup.+ hematopoietic cells being transduced with a viral construct which expresses an anti-HIV agent. Alternatively, the composition comprises at least about 1.7.times.10.sup.6 CD34.sup.+ hematopoietic cells per kg, at least about 0.5.times.10.sup.6 of such cells per kg being transduced with the viral construct. The composition is suitable for administration to a human subject. The human subject may be an adult.

The viral construct may be a retroviral construct. The composition may also be substantially free of cytokines, or substantially free of virus.

This invention also provides a composition where at least 5.times.10.sup.6 CD34.sup.+ hematopoietic cells per kg of body weight of a human subject to whom the composition is to be administered are transduced; or comprising at least 9.37.times.10.sup.6 CD34.sup.+ hematopojetic cells per kg of body weight of a human subject, wherein at least 5.times.10.sup.6 of such CD34.sup.+ hematopoietic cells are transduced; or comprising at least about 10.times.10.sup.6 CD34.sup.30 hematopoietic cells per kg of body weight where at least 5.times.10.sup.6 such cells are transduced; or where the anti-HIV agent is an RNA molecule; or where the anti-HIV agent is an RNAi molecule; or where the anti-HIV agent is an antisense molecule; or where the antiHIV agent is a ribozyme. The ribozyme may comprisenucleotides having the sequence 5'- UUA GGA UCC UGA UGA GUC CGU GAG GAC GAA ACU GGC UCC -3' (SEQ ID NO.:1) (Rz2)

In the composition, the transduced CD34.sup.+ cells are capable of engraftment, and of giving rise to progeny cells for at least 12 months, in the subject. The cells may be in a primary cell culture.

Also disclosed is a composition comprising a pharmaceutically acceptable carrier and at least 1.63.times.10.sup.6 CD34.sup.+ hematopoietic cells per kg of body weight of the subject to whom the composition is to be administered, at least 0.52.times.10.sup.6 of such CD34.sup.+ hematopoietic cells being transduced with a viral construct which expresses an anti-HIV agent, wherein the composition is produced by a process comprising the steps of: (a) isolating CD34.sup.+ hematopoietic cells from the subject; (b) culturing the CD34.sup.+ hematopoietic cells with at least one cytokine; (c) transducing the CD34.sup.+ hematopoietic cells with the viral construct which expresses the anti-HIV agent in the presence of an agent which enhances colocalization of the cells and the viral construct; (d) washing the CD34.sup.+ hematopoietic cells, and (e) mixing the CD34.sup.+ hematopoietic cells with a pharmaceutically acceptable carrier, to thereby obtain the composition. The composition is suitable for administration to a human subject.

In the composition, the culturing of step (b) may be performed in the presence of at least one cytokine, at least two cytokines or only two cytokines. Step (c) may be performed in the presence of a recombinant fibronectin fragment.

This invention also provides a composition comprising a pharmaceutically acceptable carrier and at least 1.63.times.10.sup.6 CD34.sup.+ hematopoietic cells per kg of body weight of the human subject to whom the composition is to be administered, at least 0.52.times.10.sup.6 CD34.sup.+ of such CD34.sup.+ hematopoietic cells being transformed with a gene of interest not found in the CD34.sup.+ cells prior to transformation. The composition is suitable for administration to a human subject. In this composition, the numbers of cells can be as defined above. The subject may be an adult. In this composition, the gene of interest may express an RNA agent.

This invention yet also provides a composition comprising a pharmaceutically acceptable carrier and at least 1.63.times.10.sup.6 CD34.sup.+ hematopoietic cells per kg of body weight of a human subject to whom the composition is to be administered, at least 0.52.times.10.sup.6 CD34.sup.+ of such CD34.sup.+ hematopoietic cells being transformed with a gene of interest not found in the CD34.sup.+ cells prior to transformation, wherein the composition is produced by a process comprising the steps of: (a) isolating CD34.sup.+ hematopoietic cells from the subject; (b) culturing the CD34.sup.+ hematopoietic cells with at least one cytokine; (c) transforming the CD34.sup.+ hematopoietic cells with a vector which encodes a gene of interest in the presence of an agent which enhances colocalization of the cells and the vector; (d) washing the CD34.sup.+ hematopoietic cells, and (e) mixing the CD34.sup.+ hematopoietic cells with a pharmaceutically acceptable carrier, to thereby obtain the composition. The composition is suitable for administration to a human subject. In this composition, the numbers of cells can be as defined above. The subject may be an adult. In this composition, the gene of interest may express an RNA agent.

This invention further provides a method of inserting into hematopoietic cells of a human subject a gene of interest comprising: a) mobilizing CD34.sup.+ hematopoietic progenitor cells into the blood of the human subject; b) isolating leukocytes from the subject's blood by apheresis; c) isolating CD34.sup.+ hematopoietic cells from the isolated leukocytes by an immunoselective method; d) subjecting the CD34.sup.+ hematopoietic cells of step c) to a transduction process with a gene of interest in the presence of an agent that colocalizes the cells with a transduction vector; e) determining the total number of CD34.sup.+ hematopoietic cells after step d), and if the total number is at least 1.63.times.10.sup.6 cells per kg of body weight of the human subject, then proceeding to step f), and if the total number of CD34.sup.+ hematopoietic cells after step d) is less than 1.63.times.10.sup.6 cells per kg of body weight of the human subject, then performing at least steps b)-d) and combining the CD34+hematopoietic cells; and f) delivering to the subject the CD34.sup.+ hematopoietic cells, thereby inserting into hematopoietic cells of the human subject a gene of interest. The human subject may be an adult.

In the method, the agent that colocalizes the cells with a transduction vector may be a fragment of fibronectin.

In the method, step f) may be performed without myeloablation. Step a) of mobilizing hematopoietic progenitor cells in the subject may be performed by administering to the subject an amount of a cytokine sufficient to mobilize the hematopoietic progenitor cells. In the step of isolating the leukocytes from the subject's blood, apheresis may be performed at least twice.

In the method, the step of subjecting the CD34.sup.+ hematopoietic cells to a transduction process with a gene of interest is performed in the presence of a recombinant fibronectin fragment, which may be recombinant fibronectin fragment CH-296.

In the method, the gene of interest may encode an anti-HIV agent. The anti-HIV agent may-be an RNA molecule; or an RNAi molecule; or an antisense molecule; or a ribozyme. The ribozyme may comprise nucleotides having the sequence 5'- UUA GGA UCC UGA UGA GUC CGU GAG GAC GAA ACU GGC UCC -3' (SEQ ID NO.:1) (Rz2)

In an embodiment of the method, in step e), if the total number of CD34.sup.+ hematopoietic cells after step d) is less than 1.63.times.10.sup.6 cells per kg of body weight of the human subject, then further including a step of cryogenically storing the CD34.sup.+ hematopoietic cells from step d), repeating steps a)-d), and combining any cryogenically stored cells with the cells from step d). The specific number of cells to be obtained may be increased as described above.

In the method, all or almost all of the CD34.sup.+ hematopoietic cells of step e) are delivered to the subject, for example at least 90% of the total number.

The method may further comprise a step of culturing the isolated CD34.sup.+ hematopoietic cells of step c) in the presence of at least two cytokines or a cytokine mixture.

The cytokine mixture may comprise one or more cytokines selected from the group consisting of stem cell factor (SCF) megakaryocyte growth and development factor (MGDF), Flt-3 ligand (FL, sometimes abbreviated Flt-3), interleukin 3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF) and thrombopoietin (TPO) The cytokine mixture may further comprise one or more cytokines selected from the group consisting of interleukin 1 (IL-1), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 15 (IL-15), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), erythropoietin (EPO), leukemia inhibitory factor (LIF), transforming growth factor beta (TGF-.beta.), macrophage inhibitory protein 1 (MIP-1), tumor necrosis factor (TNF) and stromal cell-derived factor 1 (SDF-1).

In a further embodiment of the method, the cytokine mixture comprises one cytokine selected from a first group and one cytokine selected from a second group, wherein the first group consists of SCF, MGDF, FL, IL-3, GM-CSF, TPO, IL-1, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15, G-CSF, M-CSF, EPO, LIF, TGF-.beta., MIP-1, TNF and SDF-1, and wherein the second group consists of MGDF, FL, GM-CSF, TPO, IL-1, IL-4, IL-5, IL-7, IL-9, IL-11, IL-12, IL-15, G-CSF, M-CSF, EPO, LIF, TGF-.beta., MIP-1, TNF and SDF-1.

This invention further provides a method of inserting into hematopoietic cells of a human subject a gene of interest comprising: a) mobilizing CD34.sup.+ hematopoietic progenitor cells into the blood of the subject; b) isolating leukocytes from the subject's blood by apheresis; c) isolating CD34.sup.+ hematopoietic cells from the isolated leukocytes by an immunoselective method; d) determining the total number of CD34.sup.+ hematopoietic cells after step c), and if the total number is at least 1.63.times.10.sup.6 cells per kg of body weight of the human subject, then proceeding to step e), and if the total number of CD34.sup.+ hematopoietic cells after step c) is less than 1.63.times.10.sup.6 cells per kg of body weight of the human subject, then performing steps b)-c) and combining the CD34.sup.+ hematopoietic cells; e) subjecting the CD34.sup.+ hematopoictic cells of step c) to a transduction process with a gene of interest in the presence of an agent that colocalizes the cells with a transduction vector; and f) delivering to the subject the CD34.sup.+ hematopoietic cells, thereby inserting into hematopoietic cells of the human subject a gene of interest. The relevant specifics of this method may be varied as discussed for the previous methods.

The invention further provides a method of inserting into hematopoietic cells of a human subject a gene that expresses a ribozyme comprising nucleotides having the sequence 5'-UUA GGA UCC UGA UGA GUC CGU GAG GAC GAA ACU GGC UCC-3' (SEQ ID NO.:1) (Rz2) comprising: a) mobilizing CD34.sup.+ hematopoietic progenitor cells into the blood of the subject by administering to the subject an amount of a cytokine sufficient to mobilize the hematopoietic progenitor cells; b) isolating leukocytes from the subject's blood by apheresis, which is performed at least twice; c) isolating CD34.sup.+ hematopoietic cells from the isolated leukocytesby an immunoselective method; d) culturing the isolated CD34.sup.+ hematopoietic cells of step c) for about one day in a culture medium in the presence of a cytokine; e) subjecting the CD34.sup.+ hernatopoietic cells of step dy to a transduction process with a retrovirus comprising a vector that gives rise in the cell to a ribozyme comprising nucleotides having the sequence 5'- UUA GGA UCC UGA UGA GUC CGU GAG GAC GAA ACU GGC UCC -3' (SEQ ID NO. :1) (Rz2) in the presence of a recombinant fibronectin fragment; f) determining the total number of CD34.sup.+ hematopoletic cells after step e), and if the total number is at least 1.63.times.10.sup.6 cells per kg of body weight. of the human subject, then proceeding to step g), and if the total number of CD34.sup.+ hematopoietic cells after step e) is less than 1.63.times.10.sup.6 cells per kg of body weight of the human subject, then again performing steps b)-e) and combining the CD34.sup.+ hematopoietic cells; and g) delivering to the subject, without myeloablation, the CD34.sup.+ hematopoietic cells, thereby inserting into hematopoietic cells of the human subject a gene that expresses the ribozyme. The relevant specifics of this method may be varied as discussed for the previous methods.

Also provided is a method of preparing the compositions described above, comprising: a) mobilizing CD34.sup.+ hematopoietic cells into the blood of the subject; b) isolating leukocytes from the subject's blood by apheresis; c) isolating the CD34.sup.+ hematopoietic cells from the isolated leukocytes by an immunoselective method; d) subjecting the CD34.sup.+ hematopoietic cells of step c) to a transduction process with a gene of interest in the presence of an agent that colocalizes the cells with a transduction vector; and e) determining the total number of CD34.sup.+ hematopoietic cells after step d), and if the total number of CD34+hematopoietic cells after step d) is less than 1.63.times.10.sup.6 cells per kg of body weight of the human subject, then again performing steps b)-d) and combining the CD34.sup.+ hematopoietic cells.

Also provided is a use of a composition comprising a pharmaceutically acceptable carrier and at least 1.63.times.10.sup.6 CD34.sup.+ hematopoietic cells per kg of body weight of a human subject to whom the composition is to be administered, at least 0.52.times.10.sup.6 CD34.sup.+ of such cells per kg being transduced with a viral construct which expresses an anti-HIV agent, for the manufacture of a medicament for the treatment of the human subject infected with HIV.

Also provided isa kit comprising elements for use in carrying out the described methods. A specific embodiment of a kit comprises a) an amount of an agent capable of mobilizing hematopoietic progenitor cells in a human subject; b) a culture medium including at least one cytokine acceptable for culturing CD34.sup.+ hematopoietic cells; c) a retroviral vector comprising nucleotides having a sequence that in a cell gives rise to a ribozyme having the sequence 5'- UUA GGA UCC UGA UGA GUC CGU GAG GAC GAA ACU UGC UCC-3' (SEQ ID NO.:1) (Rz2); and d) tissue culture vessels coated on their inside with a recombinant fibronectin fragment.

Yet further provided is a package comprising the described kits and instructions for the use of the kits.

In a further embodiment of the described method, the total combined time taken for the steps of culturing and transducing the CD34.sup.+ hematopoietic cells is not more than about three days, that is, the time during which the cells are in a culture medium at 37.degree. C. in the presence of added cytokines (at normal levels) is not more than about three days. Alternatively, the time during which the cells are in culture media in the presence of more than one cytokine is not more than three days. The transduction of the cells may be performed in the presence of a recombinant fibronectin fragment CH-296 or an equivalent agent.

The compositions and methods of this invention can be used to treat any of a variety of diseases in which there is a genetic aspect. Of particular interest are diseases of the blood or immune systems. These include hemoglobinopathies, defects of leukocyte production or function including cancers, immune deficiencies such as HIV, viral infections, lysosomal storage diseases and stem cell defects such as Fanconi's anemia, chronic granulomatous disease, Gaucher's disease, G6PD deficiency etc. They also include infectious diseases such as AIDS/HIV infection or acquired disease such as cancers or cardiovascular diseases.

The present invention relates to gene therapy, particularly as applied to hematopoietic progenitor (HP) cells, to transduced cells and methods of obtaining them, and to methods of using them to provide prolonged engraftment of modified hematopoietic cells in human subjects. The invention particularly relates to ex vivo gene therapy of HP cells for treatment or prevention of HIV infection. The invention provides compositions of transduced HP cells that comprise sufficient numbers of totipotent cells capable of providing therapeutic benefit. In one embodiment, this invention provides compositions of transduced human HP cells and methods of gene therapy against HIV in order to give rise, in human subjects, to protected T-lymphocytes.

In the context of viral infection, particularly HIV infection, significant therapeutic benefit is provided by the invention through increased long term survival of modified T-lymphocytes in the human subject and thereby increased numbers of T-lymphocytes and improved immune function, leading to lower viral replication and viral load.

In a further embodiment, the transduced human HP cells of the composition or system are capable of long-term engraftment when infused into a patient, giving rise to differentiated hematopoietic cells for at least 12 months after infusion, preferably at least 24 months and even more preferably at least 30 months after infusion. In a further embodiment, the transduced human HP cells are capable of long-term engraftment when infused into an autologous subject. In a further embodiment, the transduced human HP cells are capable of long-term engraftment when infused into a subject without myeloablation.

Another embodiment provides a composition or system comprising transduced human HP cells in sufficient numbers that, when delivered into a human subject, provide long term engraftment at a level such that at least 0.01% gene-modified cells of at least one cell type can be detected in the blood or bone marrow for example, by biopsy. It is preferred that the cell type be T-lymphocytes or macrophages/monocytes. Preferably, the level of gene-modified cells is at least 0.1%, more preferably at least 1% and most preferably at least 10%. It is preferred that the transduced cells are delivered into an autologous subject. It is preferred that the transduced cells are delivered in the absence of myeloablation. It is preferred that long term engraftment occurs for at least 12 months, more preferred at least 24 months, even more preferred, at least 30 months. It is preferred that the transduced gene is for treatment of diseases other than SCID, for example cancers and infectious diseases. It is more preferred that the transduced gene is for treatment or prevention of HIV infection.

The HP cells for transduction were preferably obtained from one subject. The CD34.sup.+ purity of the transduced human HP cells (% CD34.sup.+) should be at least 65%, preferably at least 90% and more preferably at least 95%. The percentage transduction should be at least about 10%, preferably at least about 30% and more preferably at least about 50%.

In a further embodiment, the transduced human HP cells are derived from CD34.sup.+ cells isolated from the blood of a human subject after mobilization of HP cells into the peripheral blood. Mobilization can be achieved by the use of cytokines, preferably one or more from the group consisting of granulocyte colony-stimulating factor (G-CSF), conjugated G-CSF, pegylated G-CSF and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytokine(s) may further comprise stem cell factor (SCF), interleukin 3 (IL-3), or stromal cell-derived factor-1 (SDF-1, Lataillade et al 2000) or similar acting cytokines. Mobilization may be assisted by the use of a short course of chemotherapy with agents such as cyclophosphamide. More preferably, mobilization is carried out using G-CSF or pegylated G-CSF. The cytokine(s) may be administered daily at an amount of at least about 10 .mu.g per kg of weight of the subject and more preferably at about 30 .mu.g per kg. The CD34.sup.+ cells may be collected by apheresis on days 3, 4, 5, 6 or later after beginning cytokine treatment. Preferably, apheresis is carried out at least twice. The CD34.sup.+ cells may be selected by any of the clinical grade devices known in the art such as the Isolex 300i cell selection system or the CEPRATE SC Stem Cell Concentration System.

In a further embodiment, the CD34.sup.+ cells are treated prior to transduction with a cytokine mixture, preferably comprising MGDF and SCF, or essentially MGDF and SCF, to induce entry into cell cycle, preferably at concentrations of about 100 ng/ml and 50 ng/ml, respectively. It is preferred that cell cycle induction occur in the absence of added cytokines IL-3, IL-6 or SCF, or the combination of the three of these.

The transduced human HP cells contain an introduced gene which may encode one or more proteins or RNA molecules, for example antisense molecules, RNAi molecules, RNA decoys or ribozyme RNA (ie. RNA agents). The introduced gene may be any introduced gene provided that the encoded protein or RNA or both alter the properties of the transduced human HP cells in a desired way compared to the non-transduced HP cells. In one embodiment, the introduced gene, when expressed, provides resistance to the transduced HP cells or to differentiated progeny of these cells against viral infection, preferably resistance against HIV infection. More preferably, the introduced gene encodes antisense or ribozyme RNA capable of inhibiting HIV-1 replication in cells.

Types of ribozymes which may be directed against viral infection such as HIV-1 infection or against non-viral diseases include the hammerhead, hairpin, RNAse P, hepatitis delta virus (HDV), intervening sequence ribozymes of the Group I or Group II type, or catalytic motifs selected by in vitro selection methods. The ribozymes are preferably hammerhead or hairpin ribozymes, more preferably hammerhead ribozymes. Such ribozymes are capable of cleaving RNA molecules associated with the disease.

The invention includes the use of multiple ribozymes (eq. Ramezani et al 2002), for example a ribozyme with multiple catalytic domains, or a combination of types of ribozymes. This should reduce the likelihood of viral resistance in the case of treatment of virus infection. It is also preferred that the ribozyme cleavage site(s) is highly conserved in the viral target RNA, as is the case for the Rz2 cleavage site.

Any combination of the above is also possible, providing more than one mechanism of effect.

The transduced human HP cells of the composition or system are transduced by DNA or a plasmid or viral transfer vector. It is desired that the introduced gene is integrated into the cell genome, after reverse transcription if appropriate. Preferably, the cells are transduced with a retroviral vector, for example a murine retroviral vector or a lentiviral vector. More preferably, the retroviral vector is derived from LNL6 (Bender et al. 1987) or other oncoretroviral vector. In a particular embodiment, the cells are transduced with RRz2.

The introduced gene is expressed in the transduced human HP cells or progeny cells from a promoter. The promoter may be constitutively expressed or inducible, for example being expressed preferentially under favorable conditions or circumstances. The gene may be transcribed by RNA polymerase II (RNA pol II promoters) or by RNA polymerase III.

In another embodiment of the invention, the composition is formulated to be ready for delivery into a human subject. The great majority of cells should be viable for example greater than 95% and preferably greater than 98%. The volume of the composition is preferably from about 10 ml to about 1000 ml, more preferably from about 100 ml to about 500 ml. The composition comprises a pharmaceutically acceptable carrier which is preferably a buffered salts solution comprising a protein agent such as an albumin or gelatine and/or a sugar such as glucose, which agents may act to stabilize the cells. The carrier may contain anticoagulant agents such as sodium citrate. The carrier may comprise a plasma expander, well known in the art. In further aspects, the composition is sterile (bacterial, fungal, mycoplasma), detectably free of bacteria, endotoxin, mycoplasma, HIV p24 antigen or replication-competent retrovirus, substantially free of free transducing vector, or any combination of these. In a further aspect, the composition is substantially free of added cytokines. The composition is administered to the subject by parenteral means, preferably by infusion or injection on one or more occasions.

The invention also provides methods for gene therapy of hematopoietic cells, particularly hematopoietic progenitor cells, using the compositions as described herein. The invention also provides methods of treatment or prevention of genetic or infectious diseases, for example HIV infection. The methods may comprise the use of the CH-296 fragment of human fibronectin (RetroNectin.TM.) or equivalent, or one or more debulking steps to remove unwanted cells, or one or more washing steps.

Gene therapy can be carried out ex vivo or in vivo. The methods described here preferably apply to the ex vivo approach but could also be applied to in vivo approaches (for example, Newbound et al., 2001). The invention can be performed for subjects already having disease, or prophylactically to reduce the occurrence or prevent disease.

HP cells for use in the methods of the invention can be obtained from peripheral blood, bone marrow, umbilical cord blood, or from stem cells that give rise to hematopoietic cells. They are preferably obtained from peripheral blood after mobilization. HP cells can be mobilized into the peripheral blood by administering one or more cytokines, with or without administration of a chemotherapeutic agent. The cytokines may be selected from the group consisting of G-CSF, pegylated G-CSF, conjugated G-CSF, GM-CSF and any combination of the above. The cytokines may further comprise one or more selected from the group consisting of SCF, FL and IL-3.

The methods of the invention are capable of providing at least 0.01% of gene-modified hematopoietic cells long term in a patient in the absence of myeloablation.

The parameters and characteristics of each of the embodiments described above are interchangeable when applicable to each other, and are therefore not repeated. Thus, for example, any parameter or characteristic of the first embodiment may be employed in the other embodiments of the invention.
 

Claim 1 of 18 Claims

1. A composition for engraftment of transduced autologous CD34.sup.+ hematopoietic progenitor cells in a human subject which comprises a pharmaceutically acceptable carrier and a minimum dose of 0.52.times.10.sup.6 transduced CD34.sup.+ hematopoietic progenitor cells per kg body weight of the subject in a total CD34.sup.+ cell population of at least 1.63.times.10.sup.6 CD34.sup.+ hematopoietic progenitor cells per kg body weight of the subject, the transduced cells comprising a RRz2 vector encoding a ribozyme and the number of transduced cells being determinable by performing competitive PCR on such transduced CD34.sup.+ hematopoietic cells, wherein the ribozyme is encoded by DNA nucleotides having the sequence 5'-TTA GGA TCC TGA TGA GTC CGT GAG GAC GAA ACT GGC TC-3' (SEQ ID NO: 3).

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.