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WO2002031110A2 - Therapie genique aux cellules souches hematopoietiques - Google Patents

Therapie genique aux cellules souches hematopoietiques Download PDF

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Publication number
WO2002031110A2
WO2002031110A2 PCT/IB2001/002739 IB0102739W WO0231110A2 WO 2002031110 A2 WO2002031110 A2 WO 2002031110A2 IB 0102739 W IB0102739 W IB 0102739W WO 0231110 A2 WO0231110 A2 WO 0231110A2
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Prior art keywords
cells
thymus
cell
patient
lhrh
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PCT/IB2001/002739
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English (en)
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WO2002031110A3 (fr
Inventor
Richard Boyd
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Monash University
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Publication date
Priority claimed from AUPR0745A external-priority patent/AUPR074500A0/en
Priority claimed from US09/966,576 external-priority patent/US20020071833A1/en
Priority to JP2002534480A priority Critical patent/JP2004511495A/ja
Priority to BR0114639-4A priority patent/BR0114639A/pt
Priority to AU2002216320A priority patent/AU2002216320C1/en
Priority to APAP/P/2003/002800A priority patent/AP2003002800A0/en
Application filed by Monash University filed Critical Monash University
Priority to CA002462073A priority patent/CA2462073A1/fr
Priority to KR10-2003-7005255A priority patent/KR20030082542A/ko
Priority to IL15541301A priority patent/IL155413A0/xx
Priority to EP01986714A priority patent/EP1357978A4/fr
Priority to AU1632002A priority patent/AU1632002A/xx
Publication of WO2002031110A2 publication Critical patent/WO2002031110A2/fr
Publication of WO2002031110A3 publication Critical patent/WO2002031110A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/09—Luteinising hormone-releasing hormone [LHRH], i.e. Gonadotropin-releasing hormone [GnRH]; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/46—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present disclosure is in the field of gene therapy.
  • this invention is in the field of modifying a patient's immune system through stimulation of the thymus along with gene therapy of hematopoietic stem cells (HSC) or bone marrow.
  • HSC hematopoietic stem cells
  • the major function of the immune system is to distinguish “foreign” antigens from “self and respond accordingly to protect the body against infection.
  • the sequence of events involves dedicated antigen presenting cells (APC) capturing foreign antigen and processing it into small peptide fragments which are then presented in clefts of major histocompatibility complex (MHC) molecules on the APC surface.
  • the MHC molecules can either be of class I expressed on all nucleated cells (recognized by cytotoxic T cells (Tc)) or of class II expressed primarily by cells of the immune system (recognized by helper T cells (Th)).
  • Th cells recognize the MHC H/peptide complexes on APC and respond; factors released by these cells then promote the activation of either of both Tc cells or the antibody producing B cells which are specific for the particular antigen.
  • Tc cells the MHC H/peptide complexes on APC and respond; factors released by these cells then promote the activation of either of both Tc cells or the antibody producing B cells which are specific for the particular antigen.
  • the importance of Th cells in virtually all immune responses is best illustrated in HIV/AIDS where their absence through destruction by the virus causes severe immune deficiency eventually leading to death. Inappropriate development of Th (and to a lesser extent Tc) can lead to a variety of other diseases such as allergies, cancer and autoimmunity.
  • T and B lymphocytes The ability to recognize antigen is encompassed in a plasma membrane receptor in T and B lymphocytes. These receptors are generated randomly by a complex series of rearrangements of many possible genes, such that each individual T or B cell has a unique antigen receptor. This enormous potential diversity means that for any single antigen the body might encounter, multiple lymphocytes will be able to recognize it with varying degrees of binding strength (affinity) and respond to varying degrees. Since the antigen receptor specificity arises by chance, the problem thus arises as to why the body doesn't "self destruct" through lymphocytes reacting against self antigens. Fortunately there are several mechanisms which prevent the T and B cells from doing so - collectively they create a situation where the immune system is tolerant to self.
  • the most efficient form of self tolerance is to physically remove (kill) any potentially reactive lymphocytes at the sites where they are produced (thymus for T cells, bone marrow for B cells). This is called central tolerance.
  • An important, additional method of tolerance is through regulatory Th cells which inhibit autoreactive cells either directly or more likely through cytokines. Given that virtually all immune responses require initiation and regulation by T helper cells, a major aim of any tolerance induction regime would be to target these cells. Similarly, since Tc's are very important effector cells, their production is a major aim of strategies for, e.g., anti-cancer and anti-viral therapy. THE THYMUS
  • the thymus is arguably the major organ in the immune system because it is the primary site of production of T lymphocytes. Its role is to attract appropriate bone marrow- derived precursor cells from the blood, and induce their commitment to the T cell lineage including the gene rearrangements necessary for the production of the T cell receptor for antigen (TCR). Associated with this is a remarkable degree of cell division to expand the number of T cells and hence increase the likelihood that every foreign antigen will be recognized and eliminated.
  • TCR T cell receptor for antigen
  • a strange feature of T cell recognition of antigen is that unlike B cells, the TCR only recognizes peptide fragments physically associated with MHC molecules; normally this is self MHC and this ability is selected for in the thymus.
  • T cells will develop, however, which can recognize by chance, with high affinity, self MHC/peptide complexes. Such T cells are thus potentially self-reactive and could cause severe autoimmune diseases such as multiple sclerosis, arthritis, diabetes, thyroiditis and systemic lupus erythematosis (SLE). Fortunately, if the affinity of the TCR to self MHC/peptide complexes is too high in the thymus, the developing thymocyte is induced to undergo a suicidal activation and dies by apoptosis, a process called negative selection. This is called central tolerance. Such T cells die rather than respond because in the thymus they are still immature.
  • the most potent inducers of this negative selection in the thymus are APC called dendritic cells (DC). Being APC they deliver the strongest signal to the T cells; in the thymus this causes deletion, in the peripheral lymphoid organs where the T cells are more mature, the DC cause activation.
  • DC dendritic cells
  • thymic function including both trophic (thyroid stimulating hormone or TSH, and growth hormone or GH) and atrophic effects (leutinizing hormone or LH, follicle stimulating hormone or FSH, and adrenocorticotropic hormone or ACTH)
  • TSH thyroid stimulating hormone
  • GH growth hormone
  • atrophic effects leutinizing hormone or LH, follicle stimulating hormone or FSH, and adrenocorticotropic hormone or ACTH
  • thymus atrophy The precise target of the hormones and the mechanism by which they induce thymus atrophy is yet to be determined. Since the thymus is the primary site for the production and maintenance of the peripheral T cell pool, this atrophy has been widely postulated as the primary cause of an increased incidence of immune-based disorders in the elderly. In particular, deficiencies of the immune system illustrated by a decrease in T-cell dependent immune functions such as cytolytic T-cell activity and mitogenic responses, are reflected by an increased incidence of immunodeficiency, autoimmunity and tumor load in later life (Hirokawa, 1998). [0009] The impact of thymus atrophy is reflected in the periphery, with reduced thymic input to the T cell pool resulting in a less diverse T cell receptor (TCR) repertoire.
  • TCR T cell receptor
  • the thymus essentially consists of developing thymocytes interspersed within the diverse stromal cells (predominantly epithelial cell subsets) which constitute the microenvironment and provide the growth factors and cellular interactions necessary for the optimal development of the T cells.
  • the present disclosure concerns methods of gene therapy utilizing genetically modified HSC, lymphoid or myeloid progenitor cells, epithelial stem cells, or combinations thereof (the group and each member herein referred to as "GM cells"), delivered to a reactivating thymus.
  • GM cells genetically modified progenitor cells
  • the atrophic thymus in an aged (post- pubertal) patient is reactivated.
  • This reactivated thymus becomes capable of taking up HSC and bone marrow cells (preferably genetically modified and/or exogenous) from the blood and converting them in the thymus to both new T cells and DC.
  • the present disclosure provides a method for treating a T cell disorder in a patient, the method comprising disrupting sex steroid mediated signaling to the thymus in the patient and transplanting into the patient bone marrow or HSC.
  • the T cell disorder is one that has a defined genetic basis.
  • the T cell disorder is selected from the group consisting of viral infections such as by human immunodeficiency virus (HIN), T cell functional disorders, and any other disease or condition that reduces T cells numerically or functionally, directly or indirectly.
  • HSC are genetically modified to create resistance to HIN in the T cells formed during and after thymic reactivation.
  • the HSC are modified to include a gene whose product will interfere with HIN infection, function and/or replication in the T cell.
  • the present disclosure provides methods for preventing infection by an infectious agent such as HIN.
  • GM that have been genetically modified to resist or prevent infection, activity, replication, and the like, and combinations thereof, of the infectious agent are injected into a patient concurrently with thymic reactivation.
  • the present disclosure provides for the reactivation of the thymus by disrupting sex steroid mediated signaling.
  • castration is used to disrupt the sex steroid mediated signaling.
  • chemical castration is used.
  • surgical castration is used. Castration reverses the state of the thymus to its pre-pubertal state, thereby reactivating it.
  • sex steroid mediated signaling to the thymus is blocked by the administration of agonists or antagonists of LHRH, anti-estrogen antibodies, anti-androgen antibodies, or passive (antibody) or active (antigen) anti-LHRH vaccinations, or combinations thereof ("blockers").
  • the blocker(s) is administered by a sustained peptide-release formulation. Examples of sustained peptide-release formulations are provided in WO 98/08533, the entire contents of which are incorporated herein by reference.
  • genetically modified HSC are transplanted into the patient, in a preferred embodiment just before, at the time of, or soon after reactivation of the thymus, creating a new population of genetically modified T cells.
  • Figure 1 A and B Changes in thymocyte number pre- and post-castration.
  • Thymus atrophy results in a significant decrease in thymocyte numbers with age.
  • cell numbers have increased to young adult levels.
  • numbers have significantly increased from the young adult and they are stabilized by 4 weeks post-castration.
  • *** Significantly different from young adult (2 month) thymus, p ⁇ .001
  • FIG. 1 (A) Spleen numbers remain constant with age and post- castration.
  • the B:T cell ratio in the periphery also remains constant (B), however, the CD4:CD8 ratio decreases significantly (p ⁇ 0.001) with age and is restored to normal young levels by 4 weeks post-castration.
  • FIG. 3 Fluorescence Activated Cell Sorter (FACS) profiles of CD4 vs. CD8 thymocyte populations with age and post-castration. Percentages for each quadrant are given above each plot. Subpopulations of thymocytes remain constant with age and there is a synchronous expansion of thymocytes following castration.
  • FACS Fluorescence Activated Cell Sorter
  • Figure 4 Proliferation of thymocytes as detected by incorporation of a pulse of
  • Figure 5 A-D Effects of age and castration on proliferation of thymocyte subsets.
  • A Proportion of each subset that constitutes the total proliferating population — The proportion of CD8+ T cells within the proliferating population is significantly increased.
  • B Percentage of each subpopulation that is proliferating — The TN and CD8 Subsets have significantly less proliferation at 2 years than at 2 months. At 2 weeks post-castration, the TN population has returned to normal young levels of proliferation while the CD8 population shows a significant increase in proliferation. The level is equivalent to the normal young by 4 weeks post-castration.
  • C Overall TN proliferation remains constant with age and post- castration.
  • Figure 9 A-C Changes in thymus (A), spleen (B) and lymph node (C) cell numbers following irradiation and castration on the same day. Note the rapid expansion of the thymus in castrated animals when compared to the non-castrate group at 2 weeks post- treatment.
  • HSV-1 Herpes Simplex Niras-1
  • Figure 12 A-C V ⁇ lO expression on CTL (cytotoxic T lymphocytes) in activated LN (lymph nodes) following HSV-1 inoculation. Note the diminution of a clonal response in aged mice and the reinstatement of the expected response post-castration.
  • Figure 13 A-C Castration restores responsiveness to HSV-1 immunization,
  • mice HSV-1 and cultured for 3 days.
  • CTL assays were performed with non-immunized mice as control for background levels of lysis (as determined by 51 Cr-release). Results are expressed as mean of 8 mice, in triplicate ⁇ 1 SD.
  • Aged mice showed a significant (p ⁇ O.Ol , *) reduction in CTL activity at an E:T ratio of both 10:1 and 3:1 indicating a reduction in the percentage of specific CTL present within the lymph nodes. Castration of aged mice restored the CTL response to young adult levels.
  • Figure 15 A and B Analysis of CD4 + T cell help and V ⁇ TCR response to
  • A At two weeks, thymus cell number of castrated mice was at normal levels and significantly higher than that of noncastrated mice (*p ⁇ 0.05). Hypertrophy was observed in thymuses of castrated mice after four weeks. Noncastrated cell numbers remain below control levels.
  • B CD45.2 + cells - CD45.2+ is a marker showing donor derivation. Two weeks after reconstitution donor-derived cells were present in both castrated and noncastrated mice. Four weeks after treatment approximately 85% of cells in the castrated thymus were donor-derived. There were no donor-derived cells in the noncastrated thymus.
  • Figure 18 FACS profiles of CD4 versus CD8 donor derived thymocyte populations after lethal irradiation and fetal liver reconstitution, followed by surgical castration. Percentages for each quadrant are given to the right of each plot. The age matched control profile is of an eight month old Ly5.1 congenic mouse thymus. Those of castrated and noncastrated mice are gated on CD45.2 + cells, showing only donor derived cells. Two weeks after reconstitution subpopulations of thymocytes do not differ between castrated and noncastrated mice.
  • A Donor-derived myeloid dendritic cells — Two weeks after reconstitution DC were present at normal levels in noncastrated mice. There were significantly more DC in castrated mice at the same time point. (*p ⁇ 0.05). At four weeks DC number remained above control levels in castrated mice.
  • B Donor-derived lymphoid dendritic cells — Two weeks after reconstitution DC numbers in castrated mice were double those of noncastrated mice. Four weeks after treatment DC numbers remained above control levels.
  • A Total cell number — Two weeks after reconstitution bone marrow cell numbers had normalized and there was no significant difference in cell number between castrated and noncastrated mice. Four weeks after reconstitution there was a significant difference in cell number between castrated and noncastrated mice (*p ⁇ 0.05).
  • B CD45.2 cell number. There was no significant difference between castrated and noncastrated mice with respect to CD45.2+ cell number in the bone marrow two weeks after reconstitution. CD45.2 4* cell number remained high in castrated mice at four weeks. There were no donor-derived cells in the noncastrated mice at the same time point.
  • T cell number Numbers were reduced two and four weeks after reconstitution in both castrated and noncastrated mice.
  • Donor derived myeloid dendritic cells Two weeks after reconstitution DC cell numbers were normal in both castrated and noncastrated mice. At this time point there was no significant difference between numbers in castrated and noncastrated mice.
  • Donor-derived lymphoid dendritic cells Numbers were at normal levels two and four weeks after reconstitution. At two weeks there was no significant difference between numbers in castrated and noncastrated mice.
  • (A) Total cell number Two weeks after reconstitution cell numbers were decreased and there was no significant difference in cell number between castrated and noncastrated mice. Four weeks after reconstitution cell numbers were approaching normal levels in castrated mice.
  • T cell number — -Numbers were reduced two and four weeks after reconstitution in both castrated and noncastrated mice.
  • B Donor derived (CD45.2 + ) myeloid dendritic cells — two and four weeks after reconstitution DC numbers were normal in both castrated and noncastrated mice. At two weeks there was no significant difference between numbers in castrated and noncastrated mice.
  • C Donor-derived (CD45.2 + ) lymphoid dendritic cells — umbers were at normal levels two and four weeks after reconstitution. At two weeks there was no significant difference between numbers in castrated and noncastrated mice.
  • A Total cell numbers — Two weeks after reconstitution cell numbers were at normal levels and there was no significant difference between castrated and noncastrated mice. Four weeks after reconstitution cell numbers in castrated mice were at normal levels.
  • B CD45.2 cell number — There was no significant difference between castrated and noncastrated mice with respect to donor CD45.2 cell number in the lymph node two weeks after reconstitution. CD45.2 cell number remained high in castrated mice at four weeks. There were no donor-derived cells in the noncastrated mice at the same point.
  • A T cell numbers were reduced two and four weeks after reconstitution in both castrated and noncastrated mice.
  • B Donor derived myeloid dendritic cells were normal in both castrated and noncastrated mice. At four weeks they were decreased. At two weeks there was no significant difference between numbers in castrated and noncastrated mice.
  • C Donor- derived lymphoid dendritic cells — Numbers were at normal levels two and four weeks after reconstitution. At two weeks there was no significant difference between numbers in castrated and noncastrated mice.
  • Figure 26 The phenotypic composition of peripheral blood lymphocytes was analyzed in human patients (all >60 years) undergoing LHRH agonist treatment for prostate cancer. Patient samples were analyzed before treatment and 4 months after beginning LHRH agonist treatment. Total lymphocyte cell numbers per ml of blood were at the lower end of control values before treatment in all patients. Following treatment, 6/9 patients showed substantial increases in total lymphocyte counts (in some cases a doubling of total cells was observed). Correlating with this was an increase in total T cell numbers in 6/9 patients. Within the CD4 + subset, this increase was even more pronounced with 8/9 patients demonstrating increased levels of CD4 T cells. A less distinctive trend was seen within the CD8 + subset with 4/9 patients showing increased levels, albeit generally to a smaller extent than CD4 + T cells.
  • Figure 27 Analysis of human patient blood before and after LHRH-agonist treatment demonstrated no substantial changes in the overall proportion of T cells, CD4 or CD8 T cells, and a variable change in the CD4:CD8 ratio following treatment. This indicates the minimal effect of treatment on the homeostatic maintenance of T cell subsets despite the substantial increase in overall T cell numbers following treatment. All values were comparative to control values.
  • Figure 28 Analysis of the proportions of B cells and myeloid cells (NK, NKT and macrophages) within the peripheral blood of human patients undergoing LHRH agonist treatment demonstrated a varying degree of change within subsets. While NK, NKT and macrophage proportions remained relatively constant following treatment, the proportion of B cells was decreased in 4/9 patients.
  • Figure 29 Analysis of the total cell numbers of B and myeloid cells within the peripheral blood of human patients post-treatment showed clearly increased levels of NK (5/9 patients), NKT (4/9 patients) and macrophage (3/9 patients) cell numbers post-treatment.
  • B cell numbers showed no distinct trend with 2/9 patients showing increased levels; 4/9 patients showing no change and 3/9 patients showing decreased levels.
  • Figure 30 A and B The major change seen post-LHRH agonist treatment was within the T cell population of the peripheral blood. In particular there was a selective increase in the proportion of na ⁇ ve (CD45RA + ) CD4+ cells, with the ratio of na ⁇ ve
  • CD45RA + memory (CD45RO + ) in the CD4 + T cell subset increasing in 6/9 of the human patients.
  • Figure 31 Decrease in the impedance of skin using various laser pulse energies. There is a decrease in skin impedance in skin irradiated at energies as low as 10 mJ, using the fitted curve to interpolate data.
  • Figure 32 Permeation of a pharmaceutical through skin. Permeability of the skin, using insulin as a sample pharmaceutical, was greatly increased through laser irradiation.
  • Figure 33 Change in fluorescence of skin over time after the addition of 5- aminolevulenic acid (ALA) and a single impulse transient to the skin. The peak of intensity occurs at about 640 nm and is highest after 210 minutes (dashed line) post-treatment.
  • ALA 5- aminolevulenic acid
  • Figure 34 Change in fluorescence of skin over time after the addition of 5- aminolevulenic acid (ALA) without an impulse transient. There is little change in the intensity at different time points.
  • ALA 5- aminolevulenic acid
  • Figure 35 Comparison of change in fluorescence of skin after the addition of 5-aminolevulenic acid (ALA) and a single impulse transient under various peak stresses. The degree of permeabilization of the stratum corneum depends on the peak stress.
  • ALA 5-aminolevulenic acid
  • the present disclosure comprises methods for gene therapy using genetically modified hematopoietic stem cells, lymphoid progenitor cells, myeloid progenitor cells, epithelial stem cells, or combinations thereof (GM cells). Previous attempts by others to deliver such cells as gene therapy have been unsuccessful, resulting in negligible levels of the modified cells.
  • the present disclosure provides a new method for delivery of these cells which promotes uptake and differentiation of the cells into the desired T cells.
  • the modified cells are injected into a patient whose thymus is being reactivated by the methods of this invention.
  • the modified stem and progenitor cells are taken up by the thymus and converted into T cells, dendritic cells, and other cells produced in the thymus.
  • Each of these new cells contains the genetic modification of the parent stem/progenitor cell.
  • the recipient's thymus may be reactivated by disruption of sex steroid mediated signaling to the thymus. This disruption reverses the hormonal status of the recipient.
  • a preferred method for creating disruption is through castration. Methods for castration include but are not limited to chemical castration and surgical castration.
  • GM cells are transplanted into the patient. These cells are accepted by the thymus as belonging to the patient and become part of the production of new T cells and DC by the thymus.
  • the resulting population of T cells contain the genetic modifications that had been inserted into the stem/progenitor cells.
  • a preferred method of reactivating the thymus is by blocking the direct and/or indirect stimulatory effects of LHRH on the pituitary, which leads to a loss of the gonadotrophins FSH and LH.
  • gonadotrophins normally act on the gonads to release sex hormones, in particular estrogens in females and testosterone in males; the release is blocked by the loss of FSH and LH.
  • the direct consequences of this are an immediate drop in the plasma levels of sex steroids, and as a result, progressive release of the inhibitory signals on the thymus.
  • the degree and kinetics of thymic regrowth can be enhanced by injection of CD34 + hematopoietic cells (ideally autologous).
  • This invention may be used with any animal species (including humans) having sex steroid driven maturation and an immune system, such as mammals and marsupials, preferably large mammals, and most preferably humans.
  • a less permanent version of castration is through the administration of a chemical for a period of time, refe ⁇ ed to herein as "chemical castration.”
  • chemical castration A variety of chemicals are capable of functioning in this manner.
  • the patient's hormone production is turned off.
  • the castration is reversed upon termination of chemical delivery.
  • a patient is infected with HIN.
  • the preferred procedure for treating this patient includes the following steps, which are provided in more detail below:
  • sex steroid mediated signaling to the thymus can be disrupted in a range of ways well known to those of skill in the art, some of which are described herein.
  • inhibition of sex steroid production or blocking of one or more sex steroid receptors within the thymus will accomplish the desired disruption, as will administration of sex steroid agonists or antagonists, or active (antigen) or passive (antibody) anti-sex steroid vaccinations.
  • Inhibition of sex steroid production can also be achieved by administration of one or more sex steroid analogs. In some climcal cases, permanent removal of the gonads via physical castration may be appropriate.
  • the sex steroid mediated signaling to the thymus is disrupted by administration of a sex steroid analog, preferably an analog of luteinizing hormone-releasing hormone (LHRH).
  • a sex steroid analog preferably an analog of luteinizing hormone-releasing hormone (LHRH).
  • LHRH luteinizing hormone-releasing hormone
  • SAx steroid analogs and their use in therapies and chemical castration are well known.
  • Such analogs include, but are not limited to, the following agonists of the LHRH receptor (LHRH-R): Buserelin (Hoechst), Cystorelin (Hoechst), Decapeptyl (trade name Debiopharm; Ipsen/Beaufour), Deslorelin (Balance
  • LHRH analogs also include, but are not limited to, the following antagonists of the LHRH-R: Abarelix (trade name Plenaxis; Praecis) and Cetrorelix (trade name; Zentaris).
  • LHRH-R LHRH receptor
  • This protocol will abolish or limit any spike of sex steroid production, before the decrease in sex steroid production, that might be produced by the administration of the agonist, i an alternate embodiment, an LHRH-R agonist that creates little or no sex steroid production spike is used, with or without the prior administration of an LHRH-R antagonist.
  • IL2 Interleukin 2
  • IL7 Interleukin 7
  • IL15 Interleukin 15
  • GCSF granulocyte colony stimulating factor
  • KGF keratinocyte growth factor
  • the compounds used in this invention can be supplied in any pharmaceutically acceptable carrier or without a carrier.
  • examples include physiologically compatible coatings, solvents and diluents.
  • the compositions may be protected such as by encapsulation.
  • the compositions may be provided with carriers that protect the active ingredient(s), while allowing a slow release of those ingredients.
  • Numerous polymers and copolymers are known in the art for preparing time-release preparations, such as various versions of lactic acid/glycolic acid copolymers. See, for example, U.S. Patent No. 5,410,016, which uses modified polymers of polyethylene glycol (PEG) as a biodegradeable coating.
  • Formulations intended to be delivered orally can be prepared as liquids, capsules, tablets, and the like. These compositions can include, for example, excipients, diluents, and/or coverings that protect the active ingredient(s) from decomposition. Such formulations are well known. [0076] hi any of the formulations, other compounds that do not negatively affect the activity of the LHRH analogs may be included. Examples are various growth factors and other cytokines as described herein.
  • the LHRH analog can be administered in a one-time dose that will last for a period of time. Preferably, the formulation will be effective for one to two months.
  • the standard dose varies with type of analog used. In general, the dose is between about 0.01 ⁇ g/kg and about 10 mg/kg, preferably between about 0.01 mg/kg and about 5 mg/kg. Dose varies with the LHRH analog or vaccine used, hi a preferred embodiment, a dose is prepared to last as long as a periodic epidemic lasts. For example, "flu season" occurs usually during the winter months.
  • a formulation of an LHRH analog can be made and delivered as described herein to protect a patient for a period of two or more months starting at the beginning of the flu season, with additional doses delivered every two or more months until the risk of infection decreases or disappears.
  • the formulation can be made to enhance the immune system.
  • the formulation can be prepared to specifically deter infection by flu viruses while enhancing the immune system.
  • This latter formulation would include GM cells that have been engineered to create resistance to flu viruses (see below).
  • the GM cells can be administered with the LHRH analog formulation or separately, both spatially and/or in time. As with the non-GM cells, multiple doses over time can be administered to a patient to create protection and prevent infection with the flu virus over the length of the flu season. DELIVERY OF AGENTS FOR CHEMICAL CASTRATION
  • Delivery of the compounds of this invention can be accomplished via a number of methods known to persons skilled in the art.
  • One standard procedure for administering chemical inhibitors to inhibit sex steroid mediated signaling to the thymus utilizes a single dose of an LHRH agonist that is effective for three months.
  • an LHRH agonist that is effective for three months.
  • a simple one-time i.v. or i.m. inj ection would not be sufficient as the agonist would be cleared from the patient' s body well before the three months are over.
  • a depot injection or an implant may be used, or any other means of delivery of the inhibitor that will allow slow release of the inhibitor.
  • Examples of more useful delivery mechanisms include, but are not limited to, laser irradiation of the skin, and creation of high pressure impulse transients (also called stress waves or impulse transients) on the skin, each method accompanied or followed by placement of the compound(s) with or without carrier at the same locus. A preferred method of this placement is in a patch placed and maintained on the skin for the duration of the treatment.
  • One means of delivery utilizes a laser beam, specifically focused, and lasing at an appropriate wavelength, to create small perforations or alterations in the skin of a patient. See U.S. Pat. No.
  • the laser beam has a wavelength between 0.2 and 10 microns. More preferably, the wavelength is between about 1.5 and 3.0 microns. Most preferably the wavelength is about 2.94 microns.
  • the laser beam is focused with a lens to produce an irradiation spot on the skin through the epidermis of the skin. In an additional embodiment, the laser beam is focused to create an irradiation spot only through the stratum corneum of the skin.
  • abbreviations mean a hole created in the skin. Such a hole can vary in depth; for example it may only penetrate the stratum corneum, it may penetrate all the way into the capillary layer of the skin, or it may terminate anywhere in between.
  • alteration means a change in the skin structure, without the creation of a hole, that increases the permeability of the skin. As with perforation, skin can be altered to any depth.
  • Several factors may be considered in defining the laser beam, including wavelength, energy fluence, pulse temporal width and irradiation spot-size. In a preferred embodiment, the energy fluence is in the range of 0.03-100,000 J/cm 2 .
  • the energy fluence is in the range of 0.03 - 9.6 J/cm .
  • the beam wavelength is dependent in part on the laser material, such as Er:YAG.
  • the pulse temporal width is a consequence of the pulse width produced by, for example, a bank of capacitors, the flashlamp, and the laser rod material.
  • the pulse width is optimally between 1 fs (femtosecond) and 1,000 ⁇ s.
  • the perforation or alteration produced by the laser need not be produced with a single pulse from the laser.
  • a perforation or alteration through the stratum corneum is produced by using multiple laser pulses, each of which perforates or alters only a fraction of the target tissue thickness.
  • the pulse repetition rate from the laser should be such that complete perforation is produced in a time of less than 100 ms.
  • the orientation of the target tissue and the laser can be mechanically fixed so that changes in the target location do not occur during the longer irradiation time.
  • the laser beam is focussed precisely on the skin, creating a beam diameter at the skin in the range of approximately 0.5 microns - 5.0 cm.
  • the spot can be slit-shaped, with a width of about 0.05-0.5 mm and a length of up to 2.5 mm.
  • the width can be of any size, being controlled by the anatomy of the area irradiated and the desired permeation rate of the fluid to be removed or the pharmaceutical to be applied.
  • the focal length of the focusing lens can be of any length, but in one embodiment it is 30 mm.
  • a pulse energy above approximately 100 mJ causes partial or complete ablation, while any pulse energy below approximately 100 mJ causes partial ablation or non-ablative alteration to the stratum corneum.
  • the threshold pulse energy required to enhance permeation of body fluids or for pharmaceutical delivery is reduced by a factor approximately equal to the number of pulses.
  • halving the spot area will result in halving the energy required to produce the same effect.
  • Irradiation down to 0.5 microns can be obtained, for example, by coupling the radiant output of the laser into the objective lens of a microscope objective, (e.g., as available from Nikon, Inc., Melville, NY), hi such a case, it is possible to focus the beam down to spots on the order of the limit of resolution of the microscope, which is perhaps on the order of about 0.5 microns.
  • the beam profile is Gaussian
  • the size of the affected irradiated area can be less than the measured beam size and can exceed the imaging resolution of the microscope.
  • Picosecond and femtosecond pulses produced by lasers can also be used to produce alteration or ablation in skin. This can be accomplished with modulated diode or related microchip lasers, which deliver single pulses with temporal widths in the 1 femtosecond to 1 ms range. (See D.
  • High pressure impulse transients e.g., stress waves (e.g., laser stress waves (LSW) when generated by a laser), with specific rise times and peak stresses (or pressures), can safely and efficiently effect the transport of compounds, such as those of the present disclosure, through layers of epithelial tissues, such as the stratum corneum and mucosal membranes.
  • stress waves e.g., laser stress waves (LSW) when generated by a laser
  • LSW laser stress waves
  • peak stresses or pressures
  • an epithelial tissue layer e.g., the stratum corneum
  • Exposure of the epithelial layer to the impulse transients enables the compound to diffuse through the epithelial layer.
  • the rate of diffusion in general, is dictated by the nature of the impulse transients and the size of the compound to be delivered.
  • the rate of penetration through specific epithelial tissue layers also depends on several other factors including pH, the metabolism of the cutaneous substrate tissue, pressure differences between the region external to the stratum corneum, and the region internal to the stratum corneum, as well as the anatomical site and physical condition of the skin.
  • the physical condition of the skin depends on health, age, sex, race, skin care, and history. For example, prior contacts with organic solvents or surfactants affect the physical condition of the skin.
  • the amount of compound delivered through the epithelial tissue layer will also depend on the length of time the epithelial layer remains permeable, and the size of the surface area of the epithelial layer which is made permeable.
  • the properties and characteristics of impulse transients are controlled by the energy source used to create them. See WO 98/23325, which is incorporated herein by reference. However, their characteristics are modified by the linear and non-linear properties of the coupling medium through which they propagate.
  • the linear attenuation caused by the coupling medium attenuates predominantly the high frequency components of the impulse transients. This causes the bandwidth to decrease with a co ⁇ esponding increase in the rise time of the impulse transient.
  • the non-linear properties of the coupling medium cause the rise time to decrease.
  • the decrease of the rise time is the result of the dependence of the sound and particle velocity on stress (pressure). As the stress increases, the sound and the particle velocity increase as well.
  • the rise time, magnitude, and duration of the impulse transient are chosen to create a non-destructive (i.e., non-shock wave) impulse transient that temporarily increases the permeability of the epithelial tissue layer.
  • the rise time is at least 1 ns, and is more preferably about 10 ns.
  • the peak stress or pressure of the impulse transients varies for different epithelial tissue or cell layers.
  • the peak stress or pressure of the impulse transient should be set to at least 400 bar; more preferably at least 1,000 bar, but no more than about 2,000 bar.
  • the peak pressure should be set to between 300 bar and 800 bar, and is preferably between 300 bar and 600 bar.
  • the impulse transients preferably have durations on the order of a few tens of ns, and thus interact with the epithelial tissue for only a short period of time. Following interaction with the impulse transient, the epithelial tissue is not permanently damaged, but remains permeable for up to about three minutes.
  • these methods involve the application of only a few discrete high amplitude pulses to the patient.
  • the number of impulse transients administered to the patient is typically less than 100, more preferably less than 50, and most preferably less than 10.
  • the time duration between sequential pulses is 10 to 120 seconds, which is long enough to prevent permanent damage to the epithelial tissue.
  • Impulse transients can be generated by various energy sources.
  • the physical phenomenon responsible for launching the impulse transient is, in general, chosen from three different mechanisms: (1) thermoelastic generation; (2) optical breakdown; or (3) ablation.
  • the impulse transients can be initiated by applying a high energy laser source to ablate a target material, and the impulse transient is then coupled to an epithelial tissue or cell layer by a coupling medium.
  • the coupling medium can be, for example, a liquid or a gel, as long as it is non-linear.
  • the coupling medium can include a surfactant that enhances transport, e.g., by prolonging the period of time in which the stratum corneum remains permeable to the compound following the generation of an impulse transient.
  • the surfactant can be, e.g., ionic detergents or nonionic detergents and thus can include, e.g., sodium lauryl sulfate, cetyl trimethyl ammonium bromide, and lauryl dimethyl amine oxide.
  • the absorbing target material acts as an optically triggered transducer.
  • the target material undergoes rapid thermal expansion, or is ablated, to launch an impulse transient.
  • metal and polymer films have high absorption coefficients in the visible and ultraviolet spectral regions.
  • the target material can be composed of a metal such as aluminum or copper; a plastic, such as polystyrene, e.g., black polystyrene; a ceramic; or a highly concentrated dye solution.
  • the target material must have dimensions larger than the cross-sectional area of the applied laser energy.
  • the target material must be thicker than the optical penetration depth so that no light strikes the surface of the skin.
  • the target material must also be sufficiently thick to provide mechanical support. When the target material is made of a metal, the typical thickness will be 1/32 to 1/16 inch. For plastic target materials, the thickness will be 1/16 to 1/8 inch.
  • Impulse transients can also be enhanced using confined ablation, hi confined ablation, a laser beam transparent material, such as a quartz optical window, is placed in close contact with the target material. Confinement of the plasma, created by ablating the target material by using the transparent material, increases the coupling coefficient by an order of magnitude (Fabro et al, J. Appl. Phys., 68:775, 1990).
  • the transparent material can be quartz, glass, or transparent plastic.
  • the transparent material is preferably bonded to the target material using an initially liquid adhesive, such as carbon-containing epoxies, to prevent such voids.
  • the laser beam can be generated by standard optical modulation techniques known in the art, such as by employing Q-switched or mode-locked lasers using, for example, electro- or acousto-optic devices.
  • Standard commercially available lasers that can operate in a pulsed mode in the infrared, visible, and/or infrared spectrum include Nd: YAG, Nd: YLF, C0 2 , excimer, dye, Ti:sapphire, diode, holmium (and other rare-earth materials), and metal-vapor lasers.
  • the pulse widths of these light sources are adjustable, and can vary from several tens of picoseconds (ps) to several hundred microseconds.
  • the optical pulse width can vary from 100 ps to about 200 ns and is preferably between about 500 ps and 40 ns.
  • Impulse transients can also be generated by extracorporeal lithotripters (one example is described in Coleman et al, Ultrasound Med. Biol., 15:213-227, 1989). These impulse transients have rise times of 30 to 450 ns, which is longer than laser-generated impulse transients.
  • the impulse transient is propagated in a nonlinear coupling medium (e.g., water) for a distance determined by equation (1), above.
  • the distance that the impulse transient should travel through the coupling medium before contacting an epithelial cell layer is approximately 5 mm.
  • An additional advantage of this approach for shaping impulse transients generated by lithotripters is that the tensile component of the wave will be broadened and attenuated as a result of propagating through the non-linear coupling medium. This propagation distance should be adjusted to produce an impulse transient having a tensile component that has a pressure of only about 5 to 10% of the peak pressure of the compressive component of the wave. Thus, the shaped impulse transient will not damage tissue.
  • the type of lithotripter used is not critical. Either an electrohydraulic, electromagnetic, or piezoelectric lithotripter can be used.
  • the impulse transients can also be generated using transducers, such as piezoelectric transducers.
  • the transducer is in direct contact with the coupling medium, and undergoes rapid displacement following application of an optical, thermal, or electric field to generate the impulse transient.
  • dielectric breakdown can be used, and is typically induced by a high-voltage spark or piezoelectric transducer (similar to those used in certain extracorporeal lithotripters, Coleman et al, Ultrasound Med. Biol, 15:213-227, 1989).
  • the transducer undergoes rapid expansion following application of an electrical field to cause a rapid displacement in the coupling medium.
  • impulse transients can be generated with the aid of fiber optics.
  • Fiber optic delivery systems are particularly maneuverable and can be used to irradiate target materials located adjacent to epithelial tissue layers to generate impulse transients in hard-to reach places.
  • These types of delivery systems when optically coupled to lasers, are prefe ⁇ ed as they can be integrated into catheters and related flexible devices, and used to irradiate most organs in the human body, hi addition, to launch an impulse transient having the desired rise times and peak stress, the wavelength of the optical source can be easily tailored to generate the appropriate absorption in a particular target material.
  • an energetic material can produce an impulse transient in response to a detonating impulse.
  • the detonator can detonate the energetic material by causing an electrical discharge or spark.
  • Hydrostatic pressure can be used in conjunction with impulse transients to enhance the transport of a compound through the epithelial tissue layer. Since the effects induced by the impulse transients last for several minutes, the transport rate of a drug diffusing passively through the epithelial cell layer along its concentration gradient can be increased by applying hydrostatic pressure on the surface of the epithelial tissue layer, e.g., the stratum corneum of the skin, following application of the impulse transient.
  • GENETIC MODIFICATION OF STEM OR PROGENITOR CELLS Genes Useful genes and gene fragments (polynucleotides) for this invention include those that affect genetically based diseases and conditions of T cells.
  • Such diseases and conditions include, but are not limited to, HIV infection/AIDS, T cell leukemia virus infection, and other lymphoproliferative diseases.
  • HIN/ AIDS a number of genes and gene fragments maybe used, including, but not limited to, the nef transcription factor; a gene that codes for a ribozyme that specifically cuts HIN genes, such as tat and rev (Bauer G., et al. (1997); the trans-dominant mutant form of HIN-1 rev gene, RevMlO, which has been shown to inhibit HIN replication (Bonyhadi et al. 1997); an overexpression construct of the HIN-1 rev-responsive element (RRE) (Kohn et al, 1999); any gene that codes for an R ⁇ A or protein whose expression is inhibitory to HIN infection of the cell or replication; and fragments and combinations thereof.
  • RRE HIN-1 rev-responsive element
  • stably expressible form means that the product (R ⁇ A and/or protein) of the gene or gene fragment ("functional fragment) is capable of being expressed on at least a semipermanent basis in a host cell after transfer of the gene or gene fragment to that cell, as well as in that cell's progeny after division and/or differentiation. This requires that the gene or gene fragment, whether or not contained in a vector, has appropriate signaling sequences for transcription of the DNA to RNA. Additionally, when a protein coded for by the gene or gene fragment is the active molecule that affects the patient's condition, the DNA will also code for translation signals.
  • Expression vectors are vectors that are capable of directing transcription of DNA sequences contained therein and translation of the resulting RNA.
  • Expression vectors are capable of replication in the cells to be genetically modified, and include plasmids, bacteriophage, viruses, and minichromosomes. Alternatively the gene or gene fragment may become an integral part of the cell's chromosomal DNA. Recombinant vectors and methodology are in general well-known.
  • Expression vectors useful for expressing the proteins of the present disclosure contain an origin of replication.
  • Suitably constructed expression vectors contain an origin of replication for autonomous replication in the cells, or are capable of integrating into the host cell chromosomes.
  • Such vectors may also contain selective markers, a limited number of useful restriction enzyme sites, a high copy number, and strong promoters. Promoters are DNA sequences that direct RNA polymerase to bind to DNA and initiate RNA synthesis; strong promoters cause such initiation at high frequency.
  • the expression vectors of the present disclosure are operably linked to DNA coding for an RNA or protein to be used in this invention, i.e., the vectors are capable of directing both replication of the attached DNA molecule and expression of the RNA or protein encoded by the DNA molecule.
  • the expression vector must have an appropriate transcription start signal upstream of the attached DNA molecule, maintaining the co ⁇ ect reading frame to permit expression of the DNA molecule under the control of the control sequences and production of the desired protein encoded by the DNA molecule.
  • Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors and specifically designed plasmids or viruses.
  • Hematopoietic stem cells are the prefe ⁇ ed cells for genetic modification.
  • lymphoid and myeloid progenitor cells and epithelial stem cells are also either autologous or nonautologous.
  • nonautologous (donor) cells are used, tolerance to these cells is created during the step of thymus reactivation.
  • the relevant genetically modified donor cells are transplanted into the recipient. These cells are accepted by the thymus as belonging to the recipient and become part of the production of new T cells and DC by the thymus.
  • the resulting population of T cells recognize both the recipient and donor as self, thereby creating tolerance for a graft from the donor. See copending patent application U.S.S.N. 09/ , which is incorporated herein by reference.
  • Standard recombinant methods can be used to introduce genetic modifications into the cells being used for gene therapy.
  • retroviral vector transduction of cultured HSC is one successful method (Belmont and Jurecic, 1997, Bahnson, A.B., et al, 1997).
  • Additional vectors include, but are not limited to, those that are adenovirus derived or lentivirus derived, and Moloney murine leukemia virus-derived vectors.
  • particle-mediated gene transfer such as with the gene gun (Yang and Ziegelhoffer, 1994), liposome-mediated gene transfer (Nabel et al, 1992), coprecipitation of genetically modified vectors with calcium phosphate (Graham and Van Der Eb, 1973), electroporation (Potter et al, 1984), and microinj ection (Capecchi, 1980), as well as any other method that can stably transfer a gene or oligonucleotide, preferably in a vector, into the HSC such that the gene will be expressed at least part of the time.
  • particle-mediated gene transfer such as with the gene gun (Yang and Ziegelhoffer, 1994), liposome-mediated gene transfer (Nabel et al, 1992), coprecipitation of genetically modified vectors with calcium phosphate (Graham and Van Der Eb, 1973), electroporation (Potter et al, 1984), and microinj ection (Capecchi, 1980), as well as any other method that can stab
  • the present disclosure provides methods for gene therapy through reactivation of a patient's thymus. This is accomplished by the administration of GM cells to a recipient.
  • the genetically modified cells may be HSC, epithelial stem cells, or myeloid or lymphoid progenitor cells.
  • the genetically modified cells are CD34 HSC, lymphoid progenitor cells, or myeloid progenitor cells.
  • the genetically modified cells are CD34 + HSC.
  • the genetically modified cells are administered to the patient and migrate through the peripheral blood system to the thymus. The uptake into the thymus of these hematopoietic precursor cells is substantially increased in the absence of sex steroids.
  • mice become integrated into the thymus and produce dendritic cells and T cells carrying the genetic modification from the altered cells.
  • the results are a population of T cells with the desired genetic change that circulate in the peripheral blood of the recipient, and the accompanying increase in the population of cells, tissues and organs caused by reactivation of the patient's thymus.
  • SMALL ANIMAL STUDIES Materials and Methods Animals [0125] CBA/CAH and C57B16/J male mice were obtained from Central Animal
  • Ages ranged from 4-6 weeks to 26 months of age and are indicated where relevant.
  • mice received vehicle alone injections.
  • thymuses were dissected and either a cell suspension made for FACS analysis, or immediately embedded in Tissue Tek (O.C.T. compound, Miles INC, Indiana), snap frozen in liquid nitrogen, and stored at -70°C until use.
  • CD8 B220 and Mac-1 collectively detected by anti-rat Ig-Cy5 (Amersham, U.K.), and the negative cells (TN) gated for analysis. They were further stained for CD25-PE (Pharmingen) and CD44-B (Pharmingen) followed by Streptavidin-Tri-colour (Caltag, CA) as previously described (Godfrey and Zlotnik, 1993). BrdU detection was then performed as described above.
  • Frozen thymus sections (4 ⁇ m) were cut using a cryostat (Leica) and immediately fixed in 100% acetone.
  • thymocyte subpopulations remained in the same proportions and, since thymocyte numbers increase by up to 100-fold post-castration, this indicates a synchronous expansion of all thymocyte subsets rather than a developmental progression of expansion.
  • the decrease in cell numbers seen in the thymus of aged animals thus appears to be the result of a balanced reduction in all cell phenotypes, with no significant changes in T cell populations being detected. Thymus regeneration occurs in a synchronous fashion, replenishing all T cell subpopulations simultaneously rather than sequentially.
  • Proliferation of thymocytes 15-20% of thymocytes are proliferating at 4-6 weeks of age. The majority (-80%) of these are DP with the TN subset making up the second largest population at -6% ( Figure 5A). Accordingly, most division is seen in the subcapsule and cortex by immunohistology (data not shown). Some division is seen in the medullary regions with FACS analysis revealing a proportion of SP cells (9% of CD4 T cells and 25% of CD8 T cells) dividing ( Figure 5B).
  • Figure 5B illustrates the extent of proliferation within each subset in young, old and castrated mice.
  • the decrease in DN proliferation is not returned to normal young levels by 4 weeks post-castration.
  • proliferation within the CD8+ T cell subset is significantly (p ⁇ 0.001) increased at 2 weeks post-castration and is returning to normal young levels at 4 weeks post-castration.
  • the decrease in proliferation within the DN subset was analyzed further using the markers CD44 and CD25.
  • the DN subpopulation in addition to the thymocyte precursors, contains ⁇ TCR+CD4-CD8- thymocytes, which are thought to have downregulated both co-receptors at the transition to SP cells (Godfrey & Zlotnik, 1993). By gating on these mature cells, it was possible to analyze the true TN compartment (CD3 " CD4 " CD 8 " ) and these showed no difference in their proliferation rates with age or following castration (Figure 5C).
  • the antigens recognized by these MAbs can be subdivided into three groups: thymic epithelial subsets, vascular-associated antigens and those present on both stromal cells and thymocytes.
  • MTS 10 medulla
  • MTS44 cortex
  • Adipose deposition severe decrease in thymic size and the decline in integrity of the cortico-medullary junction are shown conclusively with the anti-cytokeratin staining (data not shown).
  • the thymus is beginning to regenerate by 2 weeks post-castration. This is evident in the size of the thymic lobes, the increase in cortical epithelium as revealed by MTS 44, and the localization of medullary epithelium.
  • the medullary epithelium is detected by MTS 10 and at 2 weeks, there are still subpockets of epithelium stained by MTS 10 scattered throughout the cortex.
  • the markers MTS 20 and 24 are presumed to detect primordial epithelial cells (Godfrey, et al, 1990) and further illustrate the degeneration of the aged thymus. These are present in abundance at El 4, detect isolated medullary epithelial cell clusters at 4-6 weeks but are again increased in intensity in the aged thymus (data not shown).
  • the blood-thymus barrier is thought to be responsible for the immigration of T cell precursors to the thymus and the emigration of mature T cells from the thymus to the periphery.
  • the MAb MTS 15 is specific for the endothelium of thymic blood vessels, demonstrating a granular, diffuse staining pattern (Godfrey, et al, 1990). In the aged thymus, MTS 15 expression is greatly increased, and reflects the increased frequency and size of blood vessels and perivascular spaces (data not shown).
  • the thymic extracellular matrix containing important structural and cellular adhesion molecules such as collagen, laminin and fibrinogen, is detected by the mAb MTS 16. Scattered throughout the normal young thymus, the nature of MTS 16 expression becomes more widespread and interconnected in the aged thymus.
  • MHC II expression in the normal young thymus, detected by the MAb MTS 6, is strongly positive (granular) on the cortical epithelium (Godfrey et al, 1990) with weaker staining of the medullary epithelium.
  • the aged thymus shows a decrease in MHC II expression with expression substantially increased at 2 weeks post-castration.
  • 4 weeks post-castration expression is again reduced and appears similar to the 2 month old thymus (data not shown).
  • Thymocyte emigration Approximately 1% of T cells migrate from the thymus daily in the young mouse (Scollay et al, 1980). We found migration was occurring at a proportional rate equivalent to the normal young mouse at 14 months and even 2 years of age ( Figure 5) although significantly (p ⁇ 0.0001) reduced in number. There was an increase in the
  • the human patient received anti-T cell antibodies in the form of a daily injection of 15mg/kg of Atgam (xeno anti-T cell globulin, Pharmacia Upjohn) for a period of 10 days in combination with an inhibitor of T cell activation, cyclosporin A, 3mg/kg, as a continuous infusion for 3-4 weeks followed by daily tablets at 9mg/kg as needed.
  • Atgam xeno anti-T cell globulin, Pharmacia Upjohn
  • cyclosporin A 3mg/kg
  • This treatment did not affect early T cell development in the patient's thymus, as the amount of antibody necessary to have such an affect cannot be delivered due to the size and configuration of the human thymus.
  • the treatment was maintained for approximately 4-6 weeks to allow the loss of sex steroids followed by the reconstitution of the thymus.
  • T cell reactivity may also be combined with inhibitors of second level signals such as interleukins or cell adhesion molecules to enhance the T cell ablation.
  • second level signals such as interleukins or cell adhesion molecules to enhance the T cell ablation.
  • SEX STEROID ABLATION THERAPY [0163] The patient was given sex steroid ablation therapy in the form of delivery of an LHRH agonist. This was given in the form of either Leucrin (depot injection; 22.5mg) or Zoladex (implant; 10.8 mg), either one as a single dose effective for 3 months. This was effective in reducing sex steroid levels sufficiently to reactivate the thymus. In some cases it is also necessary to deliver a suppresser of adrenal gland production of sex steroids, such as Cosudex (5mg/day) as one tablet per day for the duration of the sex steroid ablation therapy.
  • a suppresser of adrenal gland production of sex steroids such as Cosudex (5mg/day) as one tablet per day for the duration of the sex steroid ablation therapy.
  • the patient's skin may be i ⁇ adiated by a laser such as an Er:YAG laser, to ablate or alter the skin so as to reduce the impeding effect of the stratum corneum.
  • a laser such as an Er:YAG laser
  • the operating parameters were as follows: The energy per pulse was 40, 80 or 120 mJ, with the size of the beam at the focal point being 2 mm, creating an energy fluence of 1.27, 2.55 or 3.82 J/cm 2 .
  • the pulse temporal width was 300 ⁇ s, creating an energy fluence rate of 0.42, 0.85 or 1.27 x 10 4 W/cm 2 .
  • an amount of LHRH agonist is applied to the skin and spread over the i ⁇ adiation site.
  • the LHRH agonist may be in the form of an ointment so that it remains on the site of i ⁇ adiation.
  • an occlusive patch is placed over the agonist in order to keep it in place over the i ⁇ adiation site.
  • a beam splitter is employed to split the laser beam and create multiple sites of ablation or alteration. This provides a faster flow of LHRH agonist through the skin into the blood stream.
  • the number of sites can be predetermined to allow for maintenance of the agonist within the patient's system for the requisite approximately 30 days.
  • B. Pressure Wave A dose of LHRH agonist is placed on the skin in a suitable container, such as aplastic flexible washer (about 1 inch in diameter and about 1/16 inch thick), at the site where the pressure wave is to be created. The site is then covered with target material such as a black polystyrene sheet about 1 mm thick.
  • target material such as a black polystyrene sheet about 1 mm thick.
  • the black polystyrene target completely absorbs the laser radiation so that the skin is exposed only to the impulse transient, and not laser radiation. No pain is produced from this procedure. The procedure can be repeated daily, or as often as required, to maintain the circulating blood levels of the agonist.
  • HSC granulocyte-colony stimulating factor
  • G-CSF donor granulocyte-colony stimulating factor
  • CD34 + donor cells are purified from the donor blood or bone ma ⁇ ow, preferably using a flow cytometer or immunomagnetic beading.
  • Donor-derived HSC are identified by flow cytometry as being CD34 + .
  • these HSC are expanded ex vivo with Stem Cell Factor.
  • a retroviral vector is constructed to contain the trans-dominant mutant form of
  • HJN-1 rev gene, RevMlO which has been shown to inhibit HIN replication (Bonyhadi et al. 1997).
  • Amphotropic vector-containing supernatants are generated by infection with filtered supernatants from ecotropic producer cells that were transfected with the vector.
  • the collected CD34 cells are prestimulated for 24 hours in LCTM media supplemented with IX- 3, IL-6 and SCF (lOng/ml each) to induce entry of the cells into the cell cycle.
  • Supernatants containing the vectors are then repeatedly added to the cells for 2-3 days to allow transduction of the vectors into the cells.
  • the patient is injected with the genetically modified HSC, optimally at a dose of about 2-4 x 10 6 cells/kg.
  • G-CSF may also be injected into the recipient to assist in expansion of the HSC.
  • the reactivated thymus takes up the genetically modified HSC and converts them into donor-type T cells and dendritic cells, while converting the recipient's HSC into recipient-type T cells and dendritic cells. By inducing deletion by cell death, or by inducing tolerance through immunoregulatory cells, the donor dendritic cells will tolerize any T cells that are potentially reactive with recipient.
  • T cell ablation and sex steroid ablation may be begun at the same time. T cell ablation is maintained for about 10 days, while sex steroid ablation is maintained for around 3 months.
  • the immunosuppressive therapy is gradually reduced to allow defense against infection. If there is no sign of rejection, as indicated in part by the presence of activated T cells in the blood, the immunosuppressive therapy is eventually stopped completely. Because the HSC have a strong self-renewal capacity, the hematopoietic chimera so formed will be stable theoretically for the life of the patient (as for normal, non-tolerized and non-grafted people).
  • peripheral blood lymphocytes were analyzed in patients (all >60 years) undergoing LHRH agonist treatment for prostate cancer (Fig 23). Patient samples were analyzed before treatment and 4 months after beginning LHRH agonist treatment. Total lymphocyte cell numbers per ml of blood were at the lower end of control
  • NK, NKT and macrophages B cells and myeloid cells within the peripheral blood of patients undergoing LHRH agonist treatment demonstrated a varying degree of change within subsets (Fig 25). While NK, NKT and macrophage proportions remained relatively constant following treatment, the proportion of B cells was decreased in 4/9 patients.

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Abstract

L'invention concerne des méthodes de thérapie génique utilisant des cellules souches hématopoïétiques, des cellules progénitrices lymphoïdes et/ou des cellules progénitrices myéloïdes. Les cellules sont génétiquement modifiées afin de produire un gène qui soit exprimé dans ces cellules et leur descendance après différenciation. Dans un mode de réalisation, les cellules contiennent un gène ou un fragment de gène qui confère aux cellules une résistance contre l'infection par le VIH et/ou la réplication de celui-ci. Les cellules sont administrées à un patient conjointement au traitement afin de réactiver le thymus du patient. Les cellules peuvent être autologues, syngéniques, allogéniques ou xénogéniques, étant donné que la tolérance à des cellules étrangères est créée chez le patient au cours de la réactivation du thymus. Dans un mode de réalisation préféré, les cellules souches hématopoïétiques sont CD34+. Le thymus du patient est réactivé par disruption de la signalisation induite par des stéroïdes sexuels vers le thymus. Dans un mode de réalisation préféré, cette disruption est créée par administration d'agonistes de LHRH, d'antagonistes de LHRH, d'anticorps du récepteur anti-LHRH, des vaccins anti-LHRH ou des combinaisons de ces derniers.
PCT/IB2001/002739 2000-10-13 2001-10-12 Therapie genique aux cellules souches hematopoietiques WO2002031110A2 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
AU1632002A AU1632002A (en) 2000-10-13 2001-10-12 Hematopoietic stem cell gene therapy
EP01986714A EP1357978A4 (fr) 2000-10-13 2001-10-12 Therapie genique aux cellules souches hematopoietiques
BR0114639-4A BR0114639A (pt) 2000-10-13 2001-10-12 Terapia genética de célula tronco hematopoiética
AU2002216320A AU2002216320C1 (en) 2000-10-13 2001-10-12 Hematopoietic stem cell gene therapy
APAP/P/2003/002800A AP2003002800A0 (en) 2000-10-13 2001-10-12 Hematopoietic stem cell gene therapy
JP2002534480A JP2004511495A (ja) 2000-10-13 2001-10-12 造血幹細胞遺伝子療法
CA002462073A CA2462073A1 (fr) 2000-10-13 2001-10-12 Therapie genique aux cellules souches hematopoietiques
KR10-2003-7005255A KR20030082542A (ko) 2000-10-13 2001-10-12 조혈 간세포 유전자 요법
IL15541301A IL155413A0 (en) 2000-10-13 2001-10-12 Hematopoietic stem cell gene therapy

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US79528600A 2000-10-13 2000-10-13
US79530200A 2000-10-13 2000-10-13
US09/795,286 2000-10-13
AUPR0745 2000-10-13
AUPR0745A AUPR074500A0 (en) 2000-10-13 2000-10-13 Treatment of t cell disorders
US09/795,302 2000-10-13
US75891001A 2001-01-10 2001-01-10
US09/758,910 2001-01-10
US09/966,576 US20020071833A1 (en) 1999-04-15 2001-09-26 Hematopoietic stem cell gene therapy
US09/966,576 2001-09-26
US96951001A 2001-10-01 2001-10-01
US09/969,510 2001-10-01

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004103271A3 (fr) * 2003-04-18 2005-11-24 Norwood Immunology Ltd Prevention de maladies et vaccination avant reactivation thymique
WO2004094649A3 (fr) * 2003-04-18 2005-12-01 Norwood Immunology Ltd Tolerance au transplant suite a la reactivation thymique
WO2009100309A2 (fr) 2008-02-08 2009-08-13 Medimmune, Llc Anticorps anti-ifnar1 ayant une affinité réduite pour le ligand fc
WO2012058768A1 (fr) 2010-11-05 2012-05-10 Zymeworks Inc. Conception d'anticorps hétérodimérique stable ayant des mutations dans le domaine fc

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WO1996014853A1 (fr) * 1994-11-10 1996-05-23 University Of Washington Implantation intrathymique de cellules souches
WO2000062657A2 (fr) * 1999-04-15 2000-10-26 Monash University Amelioration de l'immunite induite par les lymphocytes t

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WO1996014853A1 (fr) * 1994-11-10 1996-05-23 University Of Washington Implantation intrathymique de cellules souches
WO2000062657A2 (fr) * 1999-04-15 2000-10-26 Monash University Amelioration de l'immunite induite par les lymphocytes t

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BONYHADI M. ET AL: 'RevM10-expressing T cells derived in vivo from transduced human hematopoietic stem-progenitor cells inhibit human immunodeficiency virus replication' J. VIROL. vol. 71, no. 6, 1997, pages 4707 - 4716, XP002969707 *
FABRIS N. ET AL: 'Plasticity of neuro-endocrine-thymus interaction during aging' CELLULAR AND MOLEC. BIOL. vol. 43, no. 4, 1997, pages 529 - 541, XP002969786 *
GREENSTEIN B. ET AL: 'Regeneration of the thymus in old male rats treated with a stable analogue of LHRH' J. ENDOCR. vol. 112, 1987, pages 345 - 350, XP002969788 *
NIKOLIC B. ET AL: 'A novel application of cyclosporin A in nonmyeloablative pretransplant host conditioning for allogeneic BMT' BLOOD vol. 96, no. 3, August 2000, pages 1166 - 1172, XP002969706 *
PENIT C. ET AL: 'Cell proliferation and thymocyte subset reconstitution in sublethally irradiated mice: Compared kinetics of endogenous and intrathymically transferred progenitors' PROC. NATL. ACAD. SCI. USA vol. 86, 1989, pages 5547 - 5551, XP002969787 *
See also references of EP1357978A2 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004103271A3 (fr) * 2003-04-18 2005-11-24 Norwood Immunology Ltd Prevention de maladies et vaccination avant reactivation thymique
WO2004094649A3 (fr) * 2003-04-18 2005-12-01 Norwood Immunology Ltd Tolerance au transplant suite a la reactivation thymique
WO2004094599A3 (fr) * 2003-04-18 2005-12-29 Norwood Immunology Ltd Prvention contre une maladie et vaccination apres reactivation thymique
EP1620125A2 (fr) * 2003-04-18 2006-02-01 Norwood Immunology, Ltd. Tolerance au transplant suite a la reactivation thymique
EP1620125A4 (fr) * 2003-04-18 2009-11-04 Norwood Immunology Ltd Tolerance au transplant suite a la reactivation thymique
WO2009100309A2 (fr) 2008-02-08 2009-08-13 Medimmune, Llc Anticorps anti-ifnar1 ayant une affinité réduite pour le ligand fc
WO2012058768A1 (fr) 2010-11-05 2012-05-10 Zymeworks Inc. Conception d'anticorps hétérodimérique stable ayant des mutations dans le domaine fc

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JP2004511495A (ja) 2004-04-15
BR0114639A (pt) 2004-01-20
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AP2003002800A0 (en) 2003-06-30

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