WO1994015466A1 - Glandes endocrines de substitution obtenues par rearrangement genetique - Google Patents

Glandes endocrines de substitution obtenues par rearrangement genetique Download PDF

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Publication number
WO1994015466A1
WO1994015466A1 PCT/US1993/012662 US9312662W WO9415466A1 WO 1994015466 A1 WO1994015466 A1 WO 1994015466A1 US 9312662 W US9312662 W US 9312662W WO 9415466 A1 WO9415466 A1 WO 9415466A1
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Prior art keywords
cells
endocrine
peptide hormone
hormone
dna
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PCT/US1993/012662
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English (en)
Inventor
Theodore E. Spielberg
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Spielberg Theodore E
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Publication of WO1994015466A1 publication Critical patent/WO1994015466A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • 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

Definitions

  • SURROGATE ENDOCRINE GLANDS BY GENETIC REASSIGNMENT Background of the Invention Some endocrine disorders are characterized by the 5 failure of the intended gland to make a hormone that is required for normal biochemical and physiological functioning of the organism. Some examples of this glandular failure are hypothyroidism, (thyroid gland) , Addison's disease, (adrenal glands), hypopituitism 10 (pituitary gland) , and hypogonadism, (testes and ovaries) .
  • diabetes mellitus is characterized by an absolute failure to produce insulin in Type 1 or insulin dependent diabetes mellitus, or a relative failure to produce enough insulin to overcome 15 insulin resistance in Type 2 or non-insulin dependent mellitus.
  • the lack of insulin secretion is a result of the failure of the ⁇ cells in the islets of Langerhans in the pancreas.
  • Hybrid organ related approaches include intravenous shunts with islet cells separated from the blood via micro-porous membranes (U.S. Patent 4,925,555); macrocapsules of micro-porous membranes containing groups of islet cells; microcapsules of micro-porous membranes containing single cells; and various combinations of the above.
  • Another avenue of potential therapy has involved attempts to modify the immune system to prevent transplanted islets from being rejected.
  • immunoprivileged sites such as the thy us, testes, and anterior chamber of the eye
  • drugs that suppress rejection such as Cyclosporin, anti- lymphocytic globulin, FU408, etc
  • special culture techniques to rid islets of passenger lymphocytes and other antigen presenting cells techniques to ameliorate the effect of low lymphocyte MHC1 expression on ⁇ cell auto-immunity; and genetic modification in vitro of non- insulin producing foreign cells to produce insulin followed by transplantation of these cells.
  • Examples of in vitro genetic modifications include mouse pituitary tumor cells transfected with cDNA for human pro-insulin (1983) and, more recently, such cells transfected with GLUT-2C DNA (1991) .
  • Human hematopoietic progenitor cells have been transfected in vitro by a retroviral vector which conferred neomycin resistance (Hoch et al., Nature Vol. 320, 275 (1986)). None of the above-mentioned techniques have resulted in a cure for diabetes mellitus or even a new standard of treatment.
  • endocrine cells in a mammal are reprogrammed via genetic engineering techniques to produce a peptide hormone that they have not previously made.
  • the invention features a method of treating a mammal suffering from a disease characterized by insufficient production of a peptide hormone, by a) isolating from the mammal endocrine cells which have not previously produced the peptide hormone; b) transfecting the isolated endocrine cells with DNA encoding the peptide hormone using plasmids, retroviral vectors, microinjection, or liposomes; and c) transplanting the transfected cells into the mammal so that the cells produce the hormone in the mammal.
  • the invention features a method of treating a mammal suffering from a disease characterized by insufficient production of a peptide hormone, by either a) inserting, into the genome of a virus, DNA encoding the peptide hormone, the virus being a DNA virus which specifically infects an endocrine gland other than the endocrine gland which normally produces the peptide hormone; and then infecting the mammal with the virus so that the hormone is produced in the infected endocrine gland; or b) binding the DNA encoding the peptide hormone to a chemical that can be targeted specifically to the desired endocrine gland; or c) packaging the DNA in organ-targeting liposomes, as described, e.g., in Bangham (1992) Hospital Practice. 45 and Feigner et al., (1987) Proc. Natl. Acad. Sci. 84:
  • Advantages of the invention include the ability to restore the normal, physiologic secretions of hormones in hormonal and glandular deficiency states to patients suffering from endocrine diseases; the related ability to avoid the dependence on exogenous administration of hormones in deficiency states; and the ability to circumvent the problems of rejection and rejection preventing drugs in endocrine transplantation.
  • the invention allows the avoidance of the problems of rejection due to autoimmune reactions.
  • the invention also allows the utilization of the mammal's own excess endocrine capacity to manufacture the deficient hormone(s) and may provide a cost-efficient treatment modality.
  • the invention also provides a means to decrease long-term complications and the associated lethality currently associated with the disease.
  • transfected DNA encoding peptide hormones DNA which may include separately or together the associated regulatory DNA sequences such as promoters, enhancers, repressors, transcription factors, and antisense sequences.
  • a gene or set of genes is coupled with a vector (viral or plasmid) for gene transfer into a target endocrine cell.
  • a vector viral or plasmid
  • Alternative gene insertion techniques which may be used include fusion endocytosis and microinjection.
  • the new genetic combination of the gene and the vector is preferably injected intravenously into the bloodstream of the treated animal or human and transported to the target endocrine gland where the new gene is incorporated.
  • the target gland is reprogrammed in vivo to produce the desired hormone.
  • the target gland is not the original gland that has been damaged and has been responsible for the deficiency state.
  • Useful viral vectors include retroviruses as well as viruses that have a predilection for infecting specific tissues; these latter viruses may be used to target the gland to be reprogrammed.
  • these viruses may be used in their native configuration (they are sub-acute) , or in an attenuated state as genetic vectors. The viral infection procedures may be repeated as often as necessary.
  • calcium phosphate precipitation is a well known in vitro technique of gene insertion known as transfection.
  • the thyroid gland which specifically traps iodine and other monovalent ions such as perchlorate and pertechnate, these substances may be used as genetic vectors in vivo or in vitro and their use may also be repeated as often as necessary.
  • endocrine cells from the patient to be treated are harvested by biopsy and grown in a tissue culture. These endocrine cells are then transfected or transduced (chemical or viral) in vitro and are then returned to the same patient, avoiding rejection of non-self cells. Whether or not the transduction or transfection occurs in vitro or in vivo, since only the patient's own (self) endocrine cells are treated, they cannot be rejected or destroyed by the auto-immune mechanisms which destroyed the originally failed gland. In the case of Type 1 insulin dependent diabetes mellitus, the auto antibodies produced against ⁇ cells would have no destructive effect on transduced or transfected thyroid follicular, parathyroid, or other endocrine cells that are non-pancreatic. Those cells treated in vitro may be re-implanted within the target gland or in the abdominal cavity or the liver or the portal vein, or bone marrow, or in any other desired location within the body.
  • the transfected or transduced endocrine cells are not manufacturing and secreting their originally assigned hormone, but instead use their specialized hormone producing apparatus, including enzymes such as proteases, energy generating systems such as mitochondria, and intra-cellular structures such as golgi bodies, endoplasmic reticulxim, microsomes, vesicles, and specialized cell membranes, in the service of the reprogrammed gene and its intended reassigned hormone, thus producing and secreting this hormone at a rate and efficiency that is unique to a specialized endocrine gland.
  • enzymes such as proteases
  • energy generating systems such as mitochondria
  • intra-cellular structures such as golgi bodies, endoplasmic reticulxim, microsomes, vesicles, and specialized cell membranes
  • a reprogrammed or reassigned (transfected or transduced) endocrine gland can produce a hormone that is difficult to replace exogenously and in a physiological manner, while its originally programmed hormone that can be replaced physiologically and exogenously is used to both satisfy the organism's needs and shut off production of the original hormone in favor of the new programmed hormone.
  • the human pro- insulin cDNA and the GLUT-2 cDNA are genes that have been successfully transduced in AtT-20ins cells derived from tumors of the mouse pituitary gland.
  • transfection or transduction of these genes into the thyroid follicular cells of a diabetic patient constitutes a preferred embodiment, whether done in vivo or in vitro. Since regulation of glucose mediated pro-insulin takes place at the level of translation, the requirement for more tissue specific endocrine control is obviated.
  • an insulin producing surrogate ⁇ cell or surrogate endocrine pancreas gland has been created from a reprogrammed thyroid follicular cell.
  • either chemical or viral vectors may be used to insert the peptide hormone-encoding DNA into the cultured host endocrine cells.
  • Viral vectors suitable for in vitro and in vivo methods are discussed first.
  • suitable viral vectors are of two types: 1) retroviruses, and 2) DNA viruses with tropism for (i.e., capable of specifically infecting) particular endocrine cells.
  • Retroviral vectors have been used in a clinical setting (Rosenberg, et al N. Engl. J. Med 323:370 (1990)). In Vivo use of Viral Vectors
  • viral vectors are employed in the in vivo method of the invention, they must be engineered so that they specifically target the endocrine cells which are to express the hormone-encoding DNA.
  • This can be carried out in a number of ways, for example, nucleic acid encoding a ligand fusion in the envelope protein which is specific for a receptor found exclusively on the targeted endocrine cells may be added to the vector of choice, e.g. SV40 to create a targeted viral mutant (Shortle et al., Genetic Eng. 1:73, (1979)).
  • Suitable retroviral constructs, packaging cell lines, and delivery systems include, but are not limited to, one, or a combination of, the following: Moloney murine leukemia viral vector types; self inactivating vectors; double copy vectors; selection marker vectors; and suicide mechanism vectors.
  • retroviral engineering can be obviated by employing, in place of a retroviral vector, a viral vector which specifically infects the target endocrine cells.
  • a retroviral vector which specifically infects the target endocrine cells.
  • retrovirus type 1 and lymphocytic choriomeningitis (CCM) virus (Sprinivasuppa et al., Endocrinology Vol. 22 No. 2, 563 (1988); Klavinstris et al., Endocrinology Vol. 22 No. 2, 567 (1988)).
  • Gonadal cells are specifically infected by the mumps virus as well as coxsachie virus (an enterovirus) and dengue and bat salivary virus (Group 13 Arborviruses) (Riggs et al., N. Engl. J. of Med. Vol. 266, No. 19, 990 (1962)).
  • Adrenal cells are specifically infected with CMV virus in patients afflicted with the HIV virus (Tapper et al., Ann Intern. Med, 100:239 (1984)) and Green et al. Ann. Intern. Med, 101:497 (1984).
  • HSV-2 variants infect both adrenal and ovarian tissue (Podlech et al. Arch Virol, 110: (3-4) . 165, 1990). It is preferable to employ a virus which causes a sub-acute infection, although, in the case of more pathologic viruses, a standard attenuation method can be used. Pre-treating an animal with one virus may provide the ligand for the transfecting virus.
  • Baby Hamster Kidney (BHK-21) cells do not express a receptor for murine hepatitis virus (MHV-A59) . However, they become susceptible to infection when preinfected with influenza (Fuller et al. EMBO J. 4, 2 475).
  • Another in vivo technique used to create surrogate endocrine glands is through genetic surgery.
  • the gland to be transfected is surgically exposed, and the DNA encoding the peptide hormone is directly introduced into the cells of the exposed endocrine gland. This may be accomplished by multiple microinjections, or via microprojectiles — such as tungsten microprojectiles fired into cells (Klein et al.. Nature 327:70-73, 1987), or directly via chemical or viral vectors.
  • a pulsed electric current may be applied to the endocrine gland to be treated to create "pores" as in electroporation, to enhance the transfection process.
  • the desired cells are isolated from the subject, e.g., by standard biopsy, and cultured according to standard methods.
  • the vector which can be a retroviral vector, a specific viral vector, a chemical vector, or a plasmid vector, is used to transduce or transfect the cultured endocrine cells, using standard techniques.
  • Cells which have taken up the vector DNA, including the DNA encoding the peptide hormone, are selected using conventional techniques, and the selected cells are then re-introduced into the patient as described above.
  • Non viral methods for the therapeutic delivery of nucleic acid encoding pro-insulin and GLUT-2 in vitro may be administered to the patient using one or a combination of two or more of the following gene transfer techniques known to one skilled in the art: microinjection (Wolff et al..
  • Cells transfected with the desired peptide hormone-encoding DNA can be introduced into a human patient surgically, using techniques which are well- established for non-transfected cells.
  • in vitro transfection in endocrine cells include: Viral and human thyroid epithelial cells (Kennedy et al., J. Endo. 133, 477,
  • EXAMPLE II Non-viral method for the therapeutic delivery of nucleic acid encoding pro-insulin and GLUT-2 in vivo. It has recently been shown that a soluble DNA carrier system consisting of two covalently linked components: 1) a polycation, poly-L-lysine, that binds DNA, and 2) an asialoglycoprotein which can be targeted specifically to hepatocytes by cell surface receptors was successful in vivo for genetic transfection. Wu et al., J. of Biol. Chem. Vol. 264, No. 29, 16985, (1989).
  • Analogous combinations using the proinsulin cDNA and GLUT-2 cDNA bound to poly-L-lysine along with iodine or perchlorate or other monovalent anions that are specifically trapped by the thyroid gland may also be used for in vivo transfection.
  • Non-viral (chemical) methods may be combined with viral methods to enhance transfection, as in the combination of various polycation-ligand chemical conjugates, such as tranferrin-polylysine with a transcription and replication-defective adenovirus (called a defective virus particle in trans) for enhanced endocytosis (Cotten et al., Proc. Natl. Acad. Sci. USA 89:6094-6098, 1992).
  • various polycation-ligand chemical conjugates such as tranferrin-polylysine with a transcription and replication-defective adenovirus (called a defective virus particle in trans) for enhanced endocytosis (Cotten et al., Proc. Natl. Acad. Sci. USA 89:6094-6098, 1992).

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  • Health & Medical Sciences (AREA)
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  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

Procédé de traitement d'un mammifère souffrant d'une maladie caractérisée par une production insuffisante d'une hormone peptidique, ledit procédé consistant à a) isoler à partir du mammifère des cellules endocrines qui n'ont pas précédemment produit l'hormone peptidique; b) transfecter lesdites cellules avec l'ADN codant ladite hormone peptidique; et c) transplanter les cellules transfectées dans le mammifère, de sorte que les cellules produisent l'hormone chez ledit mammifère. Selon un autre mode de réalisation, la transfection est effectuée in vivo.
PCT/US1993/012662 1992-12-30 1993-12-29 Glandes endocrines de substitution obtenues par rearrangement genetique WO1994015466A1 (fr)

Applications Claiming Priority (2)

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US99884292A 1992-12-30 1992-12-30
US07/998,842 1992-12-30

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Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ARCHIVES OF VIROLOGY, Volume 110, issued 1990, J. PODLECH et al., "Colonization of Adrenal Glands and Ovaries of Mice by HSV-2 Variants I. Virological Studies", pages 165-177. *
CELL, Volume 35, issued December 1983, H-P.H. MOORE et al., "Expressing a Human Proinsulin cDNA in a Mouse ACTH-Secreting Cell. Intracellular Storage, Proteolytic Processing, and Secretion on Stimulation", pages 531-538. *
ENDOCRINOLOGY, Volume 122, No. 2, issued 1988, J. SRINIVASAPPA et al., "Virus-Induced Thyroiditis", pages 563-566. *
ENDOCRINOLOGY, Volume 122, No. 2, issued 1988, L.S. KLAVINSKIS et al., "Persistent Viral Infection of the Thyroid Gland: Alteration of Thyroid Function in the Absence of Tissue Injury", pages 567-575. *
ENDOCRINOLOGY, Volume 127, No. 6, issued 1990, F.E. DOMANN et al., "Restoration of Thyroid Function after Total Thyroidectomy and Quantitative Thyroid Cell Transplantation", pages 2673-2678. *
MOLECULAR AND CELLULAR BIOLOGY, Volume 7, No. 9, issued September 1987, A. FUSCO et al., "One- and Two-Step Transformations of Rat Thyroid Epithelial Cells by Retroviral Oncogenes", pages 3365-3370. *
ONCOGENE, Volume 4, No. 2, issued 1989, G. PORTELLA et al., "The Kirsten Murine Sarcoma Virus Induces Rat Thyroid Carcinomas In Vivo", pages 181-188. *
THE NEW ENGLAND JOURNAL OF MEDICINE, Volume 266, No. 19, issued 10 May 1962, S. RIGGS et al., "Viral Orchitis", pages 990-993. *

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