WO2002049664A1 - Administration directe d'un produit genique a l'hypothalamus - Google Patents

Administration directe d'un produit genique a l'hypothalamus Download PDF

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Publication number
WO2002049664A1
WO2002049664A1 PCT/US2001/050404 US0150404W WO0249664A1 WO 2002049664 A1 WO2002049664 A1 WO 2002049664A1 US 0150404 W US0150404 W US 0150404W WO 0249664 A1 WO0249664 A1 WO 0249664A1
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Prior art keywords
hypothalamus
leptin
feedback loop
mammal
controlled function
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PCT/US2001/050404
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English (en)
Inventor
Cecilia Lundberg
Richard C. Mulligan
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The Children's Medical Center Corporation
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Priority to AU2002231272A priority Critical patent/AU2002231272A1/en
Publication of WO2002049664A1 publication Critical patent/WO2002049664A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2264—Obesity-gene products, e.g. leptin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/02—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
    • 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

  • a distinguishing characteristic of the endocrine system is the feedback control of hormone production.
  • Feedback control for the hypothalamic-pituitary axis includes the interaction of hypothalamus, pituitary and target tissue (e.g., thyroid gland, adrenal gland, fat cells, hormones and gonad). Hormones produced by target tissue endocrine glands feed back on the hypothalamus and pituitary, thus regulating the levels of hormones produced by target tissue endocrine glands. Most hormones are under a feedback control, some by cations (calcium on parathyroid hormone), some by other hormones (somatostatin on insulin and glucagon) and some by osmolality or extracellular fluid volume (vasopressin, renin and aldosterone). See, e.g., Williams Textbook of Endocrinology, 9th edition, J.D. Wilson et al, editors, W.B. Saunders Co., Philadelphia (1998)).
  • the central nervous system is separated by a blood barrier from the general circulation, thereby permitting rigorous control of the microenviro ment required for complex neural signaling.
  • This blood brain barrier maintains the homeostatic environment of the brain so that it can function irrespective of fluctuations in the systemic concentrations of compounds in the body. Moreover, it protects the brain from toxic agents and degradation products present in the circulatory system. Paradoxically, this barrier, which normally protects the brain, may be the cause for inefficient drug delivery into the brain.
  • the present invention relates to novel methods of regulating or altering a specific hypothalamus-controlled function in a mammal in need of such regulation or alteration by direct delivery to the hypothalamus of the mammal an effective amount of a gene product associated with a feedback loop (e.g., negative feedback loop, positive feedback loop) regulating the hypothalamus-controlled function, or a nucleic acid sequence encoding the gene product associated with a feedback loop regulating the hypothalamus-controlled function.
  • a feedback loop e.g., negative feedback loop, positive feedback loop
  • the invention also relates to novel methods of treating a disease or disorder associated with a specific hypothalamus-controlled function in a mammal in need of such treatment by direct delivery to the hypothalamus of the mammal an effective amount of a gene product associated with a feedback loop regulating the hypothalamus-controlled function, or a nucleic acid sequence encoding a gene product associated with a feedback loop regulating the hypothalamus-controlled function.
  • hypothalamus-controlled function is meant a function that is controlled through a hypothalamic feedback mechanism. Hypothalamus-controlled functions include body weight, parturition, folliculogenesis, ovulation, spermatogenesis, cortisol production, mammary gland development, lactogenesis, epithelial secretion, thyroid hormone production, parathyroid hormone production, electrolyte balance, metabolism and insulin growth factor production.
  • gene product associated with a feedback loop regulating the hypothalamus-controlled function is meant a gene product having a receptor in the hypothalamus.
  • leptin is a gene product associated with a feedback loop regulating body weight
  • ACTH follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin are gene products associated with a feedback loop regulating parturition.
  • FSH and LH are gene products associated with a feedback loop regulating folliculogenesis, a feedback loop regulating ovulation or a feedback loop regulating spermatogenesis, respectively.
  • ACTH is a gene product associated with a feedback loop regulating cortisol production.
  • prolactin is a gene product associated with a feedback loop regulating mammary gland development, a feedback loop regulating lactogenesis or a feedback loop regulating epithelial secretion, respectively.
  • thyroid hormone-releasing hormone (TRH) and somatostatin are gene products associated with a feedback loop regulating thyroid hormone production.
  • parathyroid hormone releasing hormone is a gene product associated with a feedback loop regulating parathyroid hormone production.
  • growth hormone is a gene product associated with a feedback loop regulating insulin growth factor production.
  • the invention relates to methods of regulating or altering body weight in a mammal in need of such regulation or alteration by direct delivery to the hypothalamus of the mammal an effective amount of leptin, leptin analog, leptin fragment, leptin fusion protein, or a nucleic acid sequence encoding leptin, leptin analog, leptin fragment or leptin fusion protein.
  • the invention further relates to methods of treating obesity in a mammal in need of such treatment by direct delivery to the hypothalamus of the mammal an effective amount of leptin, leptin analog, leptin fragment, leptin fusion protein, or a nucleic acid sequence encoding leptin, leptin analog, leptin fragment or leptin fusion protein.
  • the methods of the invention obviate the need for the gene product to traverse the blood brain barrier and thus, provide more potent means to regulate or alter a specific hypothalamus-controlled function and to treat diseases and disorders associated with specific hypothalamus-controlled functions.
  • Figure 1 A is a graph showing the effect on body weight in animals injected with AAV-hOB intramuscularly (TM), intraventricularly (IN) and directly to the hypothalamus (IH).
  • Figure IB is a bar graph showing the effect on food intake in animals injected with AAV-hOB , IV and IH.
  • hypothalamus-controlled functions can be regulated or altered in a mammal by direct delivery to the hypothalamus of the mammal an effective amount of a gene product associated with a feedback loop (e.g., negative feedback loop, positive feedback loop) regulating the hypothalamus-controlled function.
  • a feedback loop e.g., negative feedback loop, positive feedback loop
  • hypothalamus-controlled functions can be regulated or altered in a mammal by direct delivery to the hypothalamus of the mammal an effective amount of a nucleic acid sequence encoding the gene product associated with a feedback loop regulating the hypothalamus-controlled function.
  • the invention also relates to novel methods of treating a disease or disorder associated with specific hypothalamus-controlled function in a mammal in need of such treatment by direct delivery to the hypothalamus (IH) of the mammal an effective amount of a gene product associated with a feedback loop regulating the hypothalamus-controlled function.
  • the methods of treating a disease or disorder associated with specific hypothalamus-controlled function in a mammal in need of such treatment entail direct delivery to the hypothalamus of the mammal an effective amount of a nucleic acid sequence encoding a gene product associated with a feedback loop regulating the hypothalamus-controlled function.
  • hypothalamus-controlled function is meant a function that is controlled through a hypothalamic feedback mechanism. Hypothalamus-controlled functions include body weight, parturition, folliculogenesis, ovulation, spermatogenesis, cortisol production, mammary gland development, lactogenesis, epithelial secretion of mammary epithelium, thyroid hormone production, parathyroid hormone production, electrolyte balance, metabolism and insulin growth factor production. Other hypothalamus-controlled functions are known in the art (see, e.g., Williams Textbook of Endocrinology, 9th edition, J.D. Wilson et al., editors, W.B. Saunders Co., Philadelphia (1998), the teachings of which are incorporated herein by reference).
  • gene product associated with a feedback loop regulating the hypothalamus-controlled function is meant a gene product having a receptor in the hypothalamus.
  • a gene product refers to a peptide (e.g., hormone) that can be expressed and is encoded by a gene that can be incorporated into a vector.
  • Such diseases and disorders include, but are not limited to, obesity, infertility, polycystic ovary symdrome, precocious puberty, hypothyroidism, hyperthyroidism, hypogonadism, hypothalamic hyposomatotropinemia, Addison disease, pituitary adenomas (e.g., GH-producing adenoma, prolactin-producing adenoma), hyperprolactinemia, hypoprolactinemia, acromegaly, Cushing's disease, prolactinoma, Graves disease, goiters, thyroiditis, hyperaldosteronism, hyperandrogenism, hyperaldosteronism, dysmenorrhea, premenstrual syndrome, amenorrhea, gynecomastia, hyperparathyroidism, hypercalcemia, osteogenesis imperfecta, etc.
  • the hormone leptin has been shown to be an afferent signal in a negative
  • Leptin is produced by adipocytes in response to increased trigyceride storage, and appears to affect body weight primarily through target cells in the hypothalamus.
  • plasma levels of leptin correlate positively with adipose tissue mass in normal humans and animals (Caro, J.F. et al, Lancet, 348:159-161(1996); Considine, RN. et al, ⁇ . Engl. J. Med., 334:292-295 (1996); Frederich, R.C. et al., Nat. Med., 1:1311-1314 (1995); Maffei, M. et al., Nat. Med.
  • the invention relates to methods of regulating or altering body weight in a mammal in need of such regulation or alteration by direct delivery to the hypothalamus of the mammal an effective amount of leptin, leptin analog, leptin fragment, leptin fusion protein, or a nucleic acid sequence encoding leptin, leptin analog, leptin fragment or leptin fusion protein.
  • the invention further relates to methods of treating obesity in a mammal in need of such treatment by direct delivery to the hypothalamus of the mammal an effective amount of leptin, leptin analog, leptin fragment, leptin fusion protein or a nucleic acid sequence encoding leptin, leptin analog, leptin fragment or leptin fusion protein.
  • ACTH is a gene product associated with a feedback loop regulating cortisol production.
  • prolactin is a gene product associated with a feedback loop regulating mammary gland development, a feedback loop regulating lactogenesis or a feedback loop regulating epithelial secretionof mammary epithelium, respectively.
  • thyroid hormone production is the hypothalamus-controlled function
  • TRH and somatostatin are gene products associated with a feedback loop regulating thyroid hormone production.
  • parathyroid hormone production is the hypothalamus-controlled function
  • PHRH is a gene product associated with a feedback loop regulating parathyroid hormone production.
  • insulin growth factor production is the hypothalamus-controlled function
  • GH is a gene product associated with a feedback loop regulating insulin growth factor production.
  • the invention also relates to methods of regulating or altering parturition in a mammal in need of such regulation or alteration by direct delivery to the hypothalamus of the mammal an effective amount of ACTH, ACTH analog,
  • methods of regulating or altering parturition in a mammal in need of such regulation or alteration comprise direct delivery to the hypothalamus of the mammal an effective amount of FSH, FSH analog, FSH fragment, FSH fusion protein, or a nucleic acid sequence encoding FSH, FSH analog, FSH fragment or FSH fusion protein.
  • methods of regulating or altering parturition in a mammal in need of such regulation or alteration comprise direct delivery to the hypothalamus of the mammal an effective amount of LH, LH analog, LH fragment, LH fusion protein, or a nucleic acid sequence encoding LH, LH analog, LH fragment or LH fusion protein.
  • the invention relates to methods of regulating or altering folliculogenesis, ovulation or spermatogenesis in a mammal in need of such regulation of alteration by direct delivery to the hypothalamus of the mammal an effective amount of FSH, FSH analog, FSH fragment, FSH fusion protein, or a nucleic acid sequence encoding FSH, FSH analog, FSH fragment or FSH fusion protein.
  • the methods of regulating or altering folliculogenesis, ovulation or spermatogenesis in a mammal in need of such regulation or alteration treatment comprise direct delivery to the hypothalamus of the mammal an effective amount of LH, LH analog, LH fragment, LH fusion protein, or a nucleic acid sequence encodmg LH, LH analog, LH fragment or LH fusion protein.
  • the invention further relates to methods of regulating or altering cortisol production in a mammal in need of such regulation or alteration by direct delivery to the hypothalamus of the mammal an effective amount of ACTH, ACTH analog, ACTH fragment, ACTH fusion protein, or a nucleic acid sequence encoding ACTH, ACTH analog, ACTH fragment or ACTH fusion protein.
  • the invention relates to methods of regulating or altering mammary gland development, lactogenesis or mammary gland epithelial secretion in a mammal in need of such regulation or alteration by direct delivery to the hypothalamus of the mammal an effective amount of prolactin, prolactin analog, prolactin fragment, prolactin fusion protein, or a nucleic acid sequence encoding prolactin, prolactin analog, prolactin fragment or prolactin fusion protein.
  • the invention relates to methods of regulating or altering thyroid hormone production in a mammal in need of such regulation or alteration by direct delivery to the hypothalamus of the mammal an effective amount of TRH, TRH analog, TRH fragment, TRH fusion protein, or a nucleic acid sequence encoding TRH, TRH analog, TRH fragment or TRH fusion protein.
  • methods of regulating or altering thyroid hormone production in a mammal in need of such regulation or alteration comprise direct delivery to the hypothalamus of the mammal an effective amount of somatostain, somatostatin analog, somatostatin fragment, somatostatin fusion protein, or a nucleic acid sequence encoding somatostatin, somatostatin analog, somatostatin fragment or somatostatin fusion protein.
  • the invention relates to methods of regulating or altering thyroid hormone production in a mammal in need of such regulation or alteration by direct delivery to the hypothalamus of the mammal an effective amount of TRH, TRH analog, TRH fragment, TRH fusion protein, or a nucleic acid sequence encoding TRH, TRH analog, TRH fragment or TRH fusion protein.
  • the invention further relates to methods of regulating or altering parathyroid hormone production in a mammal in need of such regulation or alteration by direct delivery to the hypothalamus of the mammal an effective amount of PHRH, PHRH analog, PHRH fragment, PHRH fusion protein, or a nucleic acid sequence encoding PHRH, PHRH analog, PHRH fragment or PHRH fusion protein.
  • the invention relates to methods of regulating or altering insulin growth factor production in a mammal in need of such regulation or alteration by direct delivery to the hypothalamus of the mammal an effective amount of growth hormone, growth hormone analog, growth hormone fragment, growth hormone fusion protein, or a nucleic acid sequence encoding growth hormone, growth hormone analog, growth hormone fragment or growth hormone fusion protein.
  • the invention also provides methods of regulating or altering hypothalamus- controlled functions in a mammal in need of such regulation or alteration by intraventricular (IN) delivery of an effective amount of a gene product associated with a feedback loop regulating the hypothalamus-controlled function.
  • IN intraventricular
  • hypothalamus-controlled functions can be regulated or altered in a mammal by TV delivery to the mammal of an effective amount of a nucleic acid sequence encoding the gene product associated with a feedback loop regulating the hypothalamus-controlled function.
  • the invention further provides methods of treating a disease or disorder associated with a specific hypothalamus-controlled function in a mammal in need of such treatment by IV delivery of an effective amount of a gene product associated with a feedback loop regulating the hypothalamus-controlled function.
  • the methods of treating a disease or disorder associated with specific hypothalamus-controlled function in a mammal in need of such treatment entail IN delivery of an effective amount of a nucleic acid sequence encoding a gene product associated with a feedback loop regulating the hypothalamus-controlled function.
  • regulating or “regulation” is meant the ability to control the rate and extent to which a process occurs.
  • regulating body weight refers to the ability to control the rate and extent to which body weight increases or decreases.
  • altering or “alteration” is meant the ability to change, modify or make different relative to a control or baseline.
  • mammalian refers to any vertebrate animal, including monotremes, marsupials and placental, that suckle their young and either give birth to living young (eutharian or placental mammals) or are egg-laying (metatharian or nonplacental mammals).
  • mammalian species include humans and primates (e.g., monkeys, chimpanzees), rodents (e.g., rats, mice, guinea pigs) and ruminents (e.g., cows, pigs, horses).
  • a gene product associated with a feedback loop regulating the hypothalamus-controlled function can be manufactured according to methods generally known in the art.
  • the gene product can be manufactured by chemical synthesis or recombinant technology or isolated from nature (see, e.g., Sambrook et al., Eds., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor University Press, New York (1989); and Ausubel et al, Eds., Current Protocols In Molecular Biology, John Wiley & Sons, New York, 1997).
  • the gene product associated with a feedback loop regulating the hypothalamus-controlled function can be intact protein or a functional or biologically active equivalent of the gene product.
  • a functional or biologically active equivalent of a particular gene product refers to a molecule which functionally resembles (mimics) the gene product.
  • a functional equivalent of a particular gene product can contain a "SILENT" codon or one or more amino acid substitutions, deletions or additions (e.g., substitution of one acidic amino acid for another acidic amino acid; or substitution of one codon encoding the same or different hydrophobic amino acid for another codon encoding a hydrophobic amino acid). See Ausubel et al., Eds., Current Protocols In Molecular Biology, John Wiley & Sons, New York, 1997).
  • gene product analogs, or derivatives, defined herein as proteins having amino acid sequences analogous to the endogenous gene product e.g., endogenous leptin, ACTH, FSH, LH, prolactin, TRH, somatostatin, PHRH, growth hormone.
  • Analogous amino acid sequences are defined herein to mean amino acid sequences with sufficient identity of amino acid sequence of an endogenous gene product to possess the biological activity of the gene product, but with one or more "SILENT" changes in the amino acid sequence.
  • the present invention also encompasses the administration of biologically active polypeptide fragments of a gene product associated with a feedback loop regulating the hypothalamus-controlled function.
  • Such fragments can include only a part of the full-length amino acid sequence of the gene product, yet possess biological activity.
  • Such fragments can be produced by carboxyl or amino terminal deletions, as well as internal deletions.
  • fusion proteins comprising a particular gene product as described herein, referred to as a first moiety, linked to a second moiety not occurring in the gene product.
  • the second moiety can be a single amino acid, peptide or polypeptide or other organic moiety, such as a carbohydrate, a lipid or an inorganic molecule.
  • the present invention further encompasses biologically active derivatives or analogs of a gene product as described herein, referred to herein as peptide mimetics.
  • peptide mimetics can be designed and produced by techniques known to those skilled in the art. See, e.g., U.S. Patent Nos. 5,643,873 and 5,654,276. These mimetics are based on the gene product sequence, and peptide mimetics possess biological or functional activity similar to the biological activity or functional activity of the corresponding peptide compound, but possess a "biological advantage" over the corresponding peptide inhibitor with respect to one, or more, of the following properties: solubility, stability and susceptibility to hydrolysis and proteolysis.
  • Methods for preparing peptide mimetics include modifying the N-terminal amino group, the C-terminal carboxyl group and/or changing one or more of the amino linkages in the peptide to a non-amino linkage. Two or more such modifications can be coupled in one peptide mimetic inhibitor. Examples of modifications of peptides to produce peptide mimetics are described in U.S. Patent Nos. 5,643,873 and 5,654,276.
  • Nucleic acid sequences are defined herein as heteropolymers of nucleic acid molecules.
  • the nucleic acid molecules can be double stranded or single stranded and can be a deoxyribonucleotide (DNA) molecule, such as cDNA or genomic DNA, or a ribonucleotide (RNA) molecule.
  • the nucleic acid sequence can, for example, include one or more exons, with or without, as appropriate, introns, as well as one or more suitable control sequences.
  • the nucleic acid molecule contains a single open reading frame which encodes a desired nucleic acid product.
  • the nucleic acid sequence is "operably linked" to a suitable promoter.
  • a nucleic acid sequence encoding a gene product associated with a feedback loop regulating a hypothalamus-controlled function can be isolated from nature, modified from native sequences or manufactured de novo, as described in, for example, Ausubel et ah, Current Protocols in Molecular Biology, John Wiley & Sons, New York (1998); and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor University Press, New York. (1989). Nucleic acids can be isolated and fused together by methods known in the art, such as exploiting and manufacturing compatible cloning or restriction sites. Typically, the nucleic acid sequence will be a gene which encodes the gene product associated with a feedback loop regulating a hypothalamus-controlled function.
  • operably linked is defined to mean that the gene (or the nucleic acid sequence) is linked to control sequences in a manner which allows expression of the gene (or the nucleic acid sequence). Generally, operably linked means contiguous.
  • Control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable messenger RNA (mRNA) ribosomal binding sites and sequences which control teraiination of transcription and translation.
  • mRNA messenger RNA
  • a recombinant gene (or a nucleic acid sequence) encoding a gene product associated with a feedback loop regulating a hypothalamus-controlled function can be placed under the regulatory control of a promoter which can be induced or repressed, thereby offering a greater degree of control with respect to the level of the product.
  • promoter refers to a sequence of DNA, usually upstream (5') of the coding region of a structural gene, which controls the expression of the coding region by providing recognition and binding sites for RNA polymerase and other factors which may be required for initiation of transcription. Suitable promoters are well known in the art. Exemplary promoters include the SN40 and human elongation factor (EFI).
  • nucleic acid sequence will be incorporated (inserted) in a nucleic acid vector, e.g., a DNA plasmid, virus or other suitable replicon (e.g., viral vector), that can transduce nondividing cells (post-mitotic cells) and express the nucleic acid sequence (e.g., encoded gene product) in the nondividing cells.
  • a nucleic acid vector e.g., a DNA plasmid, virus or other suitable replicon (e.g., viral vector)
  • viral vectors that can transduce nondividing cells include adeno-associated virus, adenovirus, herpesvirus and retroviral vectors based on lentiviruses.
  • lentiviruses examples include human immunodeficiency viruses (e.g., HIN-1, HIV-2, HTV-3), bovine lentiviruses (e.g., bovine immunodeficiency viruses, bovine immunodeficiency-like viruses, Jembrana disease viruses), equine lentiviruses (e.g., equine infectious anemia viruses), feline lentiviruses (e.g., feline immunodeficiency viruses, panther lentiviruses, puma lentiviruses), ovine/caprine lentiviruses (e.g., Brazilian caprine lentiviruses, caprine arthritis-encephalitis viruses, Maedi-Visna viruses, Maedi-Visna-like viruses, Maedi-Visna-related viruses, ovine lentiviruses, Visna lentiviruses), Simian AIDS retroviruses (e.g., human T-cell
  • a nucleic acid sequence encoding a gene product associated with a feedback loop regulating a hypothalamus-controlled function can be inserted into a nucleic acid vector according to methods generally known in the art (see, e.g., Ausubel et al, Eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1998); Sambrook et al, Eds., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor University Press, New York (1989)).
  • Gene products associated with a feedback loop regulating a hypothalamus-controlled function e.g., leptin, ACTH, FSH, LH, prolactin, TRH, somatostatin, PHRH, growth hormone
  • analogs, biologically active fragments and fusion proteins can be directly administered to the hypothalamus via intracranial injection. Administration can also be via transplant of neural tissue, e.g., by injecting neural cells into the brain. Other routes of administration are generally known in the art.
  • Gene products associated with a feedback loop regulating a hypothalamus- controlled function e.g., leptin, ACTH, FSH, LH, prolactin, TRH, somatostatin, PHRH, growth hormone
  • analogs, biologically active fragments and fusion proteins as well as nucleic acid sequences encoding the gene products, analogs, biologically active fragments or fusion proteins, can be administered together with other components of biologically active agents, such as pharmaceutically acceptable surfactants (e.g., glycerides), excipients (e.g., lactose), stabilizers, preservatives, humectants, emollients, antioxidants, carriers, diluents and vehicles.
  • pharmaceutically acceptable surfactants e.g., glycerides
  • excipients e.g., lactose
  • stabilizers e.g., preservatives, humectants, emollients, antioxidants
  • certain sweetening, flavoring and/or coloring agents can also be added.
  • Gene products associated with a feedback loop regulating a hypothalamus- controlled function e.g., leptin, ACTH, FSH, LH, prolactin, TRH, somatostatin, PHRH, growth hormone
  • analogs, biologically active fragments and fusion proteins as well as nucleic acid sequences encoding the gene products, analogs, biologically active fragments or fusion proteins, can be administered prophylactically or therapeutically to a mammal prior to, simultaneously with or sequentially with other therapeutic regimens or agents (e.g., multiple drug regimens), including with other therapeutic regimens used for the treatment of diseases or disorders associated with a specific hypothalamus-controlled function or for regulating a specific hypothalamus- controlled function.
  • Gene products associated with a feedback loop regulating a hypothalamus- controlled function analogs, biologically active fragments and fusion proteins, as well as nucleic acid sequences encoding the gene products, analogs, biologically active fragments or fusion proteins, that are administered simultaneously with other therapeutic agents can be administered in the same or different compositions.
  • Two or more different gene products, analogs, biologically active fragments, fusion proteins and nucleic acid sequences, or combinations thereof, can also be administered.
  • Gene products associated with a feedback loop regulating a hypothalamus- controlled function can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle examples include water, saline, Ringer's solution, isotonic sodium chloride solution, dextrose solution, and 5% human serum albumin.
  • Liposomes and nonaqueous vehicles such as fixed oils can also be used.
  • the vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives).
  • the formulation can be sterilized by commonly used techniques. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences.
  • An effective amount of a gene product associated with a feedback loop regulating a hypothalamus-controlled function, analog, biologically active fragment and fusion protein is defined herein as that amount, or dose, of gene product associated with a feedback loop regulating a hypothalamus-controlled function, analog, biologically active fragment and fusion protein that, when directly delivered to the hypothalamus of a mammal with a disease or disorder associated with a specific hypothalamus-controlled function, is sufficient for therapeutic efficacy (e.g., an amount sufficient for significantly reducing or eliminating signs or symptoms associated with the disease or disorder).
  • An effective amount of a gene product associated with a feedback loop regulating a hypothalamus-controlled function, analog, biologically active fragment and fusion protein is also that amount, or dose, of gene product associated with a feedback loop regulating a hypothalamus-controlled function, analog, biologically active fragment and fusion protein that, when directly delivered to the hypothalamus of a mammal for the purpose of regulating or altering a specific hypothalamus- controlled function is sufficient to result in regulation or alteration of the specific hypothalamus-controlled function.
  • an effective amount of a nucleic acid sequence encoding a gene product associated with a feedback loop regulating a hypothalamus-controlled function, analog, biologically active fragment or fusion protein is defined herein as that amount, or dose, of nucleic acid sequence encoding the gene product, analog, biologically active fragment or fusion protein that, when administered to a mammal with a disease or disorder associated with a specific hypothalamus-controlled function, is sufficient for therapeutic efficacy (e.g., an amount sufficient for significantly reducing or eliminating signs or symptoms associated with the disease or disorder).
  • An effective amount of a nucleic acid sequence encoding a gene product associated with a feedback loop regulating a hypothalamus-controlled function, analog, biologically active fragment or fusion protein is also that amount, or dose, of nucleic acid sequence encoding the gene product, biologically active fragment and fusion protein that, when directly delivered to the hypothalamus of a mammal for the purpose of regulating or altering a specific hypothalamus-controlled function is sufficient to result in regulation or alteration of the specific hypothalamus-controlled function.
  • the effective amount of a gene product associated with a feedback loop regulating a hypothalamus-controlled function, analog, biologically active fragment, fusion protein and nucleic acid sequence encoding the gene product, analog, biologically active fragment or fusion protein is lower than the effective amount for intraventricular or intramuscular delivery to the hypothalamus.
  • the dosage administered to a mammal, including frequency of administration will vary depending upon a variety of factors, including the pharmacodynamic characteristics of the gene product being delivered; size, age, sex, health, body weight and diet of the recipient; nature and extent of symptoms of the disease or disorder being treated or the specific hypothalamic-controlled function being regulated; kind of concurrent treatment, frequency of treatment, and the effect desired.
  • Gene products associated with a feedback loop regulating a hypothalamus- controlled function can be administered in single or divided doses (e.g., a series of doses separated by intervals of days, weeks or months), or in a sustained release form, depending upon factors such as nature and extent of symptoms, kind of concurrent treatment and the effect desired.
  • Other therapeutic regimens or agents can be used in conjunction with the present invention.
  • PCR Polymerase Chain Reaction
  • the 525 bp long product was cloned into Vector-T (Promega, Madison, WI) and sequenced.
  • the hOB cDNA was excised using Agel and Notl and ligated into prAAV.MD (Mandel, R.J. et al, J. Neurosci., 18:4271-4284 (1998)).
  • the prAAV.MD utilizes the human cytomegalovirus (CMV) promoter to drive expression of the transgene.
  • CMV human cytomegalovirus
  • the rAAN.MD.GFP was constructed by inserting the EcoRI-Notl fragment of p ⁇ GFP-1 plasmid (Clontech) into prAAV.MD.
  • the fractions containing rAAV (as assayed by dot-blot analysis using the transgene cD ⁇ A as probe) were pooled and analyzed by replication-center assays to determine the infectious titer, and by plaque- forming assays on 293 cells to determine any adenoviral contamination.
  • the rAAN used in the present study had infectious titers at 3-4 x 10 10 and no contamination of adenovirus could be detected.
  • the viral stocks were desalted using Sephadex-50 columns (Boeringer-Mannheim, Indianapolis, IN) just prior to surgery.
  • mice 70, 10 mice per experimental group
  • Intramuscular injections were performed under light metoxyflurane (Methofane, Abbot Laboratories, Abbott Park, EL) anesthesia, and 50 ⁇ l of vector (IiOB LM and GFP EM groups) or saline (ctrl-Lep ob and normal control) was injected into the right tibialis anterior muscle.
  • the mice were anesthetized using 75 mg/kg pentobarbital.
  • Enzyme-Linked hmnunosorbent Assay ELISA.
  • Human leptin was measured in serum and protein extracts from injections sites using the human leptin immunoassay kit from R&D Systems (Minneapolis, M ⁇ ) according to the supplier's protocol.
  • the GRID software (Interactivision, Copenhagen, Denmark) was used to generate unbiased counting frames.
  • AAV vector encoding green fluorescent protein (GFP) (AAVGFP) (3-4 10 10 cfu/ml) was injected into the lateral ventricle (IN, 5 ⁇ l) or into the hypothalamus (EH, 1 ⁇ l) of mice.
  • GFP green fluorescent protein
  • EH hypothalamus
  • 50 ⁇ l of virus encoding GFP was injected into the right tibialis anterior muscle of mice (EM), a site known to result in robust AAN vector-mediated expression.
  • infected cells were readily detected throughout the ventricular system, including the lateral ventricle on the opposite side to the injection.
  • GFP expressing cells could be detected two to three cell layers into the brain parenchyma at the site of injection, most of the infected cells were found in the ependymal lining of the ventricles.
  • a large number of GFP-expressing cells were detected. Expression of GFP was detected primarily in cells presenting neuronal morphologies. As a consequence, many fibers projecting from the injected site were immunostained against GFP.
  • the pattern of the projecting fibers mainly intrahypothalamic, to the lateral and medial regions, as well as some extrahypothalamic projections (e.g. to the lateral habenula), indicate that the infected cells were neurons of the dorsal and ventral medial hypothalamus (Simerly, R.B., "Anatomical substance of hypothalamic integration", In Ebe rat nervous system, Paxinos, G. ed., Academic Press, San Diego, pages 353-372 (1995)).
  • Stereological quantification of gene transfer revealed that 4,763 ⁇ 2,503 cells expressed GFP after injection of the hypothalamus.
  • Example 2 Levels of Leptin Produced After Intracranial Gene Transfer.
  • AANhOB AAV vector encoding human leptin
  • virus 3-4 x 10 10 cfu/ml
  • EH hypothalamus
  • AANhOB was also injected into muscle (EM, 50 ⁇ l). Twelve weeks after intracranial gene transfer, the site of vector injection was dissected, and tissue extracts were prepared and assayed for leptin, using an ELISA specific for the human gene product.
  • leptin in animals injected with the AAV-leptin construct via the intramuscular route, leptin could be readily detected in the plasma, although the levels were low (up to 250 pg/ml) and variable over a 10-week period. Leptin levels in muscle tissue dissected from the site of virus-injected muscles (median 7; range3-21 pg/ ⁇ g protein) were in the same range as the animals injected intraventricularly.
  • mice injected with leptin virus in the hypothalamus ate less than any other Lep o mice group (3.2 ⁇ 0.4 g/mouse/24 hrs (leptin) versus 5.22 ⁇ 0.2 (GFP), P ⁇ 0.05; Figure IB), an effect accompanied by a mean body weight significantly lower than all other groups of mutant mice (29.9 ⁇ 1.4 g (leptin) versus 37.5 ⁇ 0.6 g (GFP), P ⁇ 0.05; Figure 1 A).
  • the body weight of the EH leptin group was the same as the normal controls (26.7 ⁇ 2.6 g (leptin) versus 21.2 ⁇ 0.4 g (saline normal), P > 0.05; Figure 1A), and the weight of these animals was stable throughout the experiment.
  • the weight loss effect was delayed and partial, not reaching statistical significance until ten weeks post-injection (51 ⁇ 0.8 g (leptin EM) versus 59.5 ⁇ 1.0 (GFP EM), P ⁇ 0.05 and 42.7 ⁇ 2.7 g (leptin EV) versus 57.7 ⁇ 1.0 (GFP EV), P ⁇ 0.05; Figure 1A).
  • intraventricular delivery can allow a nucleic acid product to reach stem cells of the nervous system.
  • intraventricular delivery can be applicable, for example, in degenerative multiple sclerosis therapy.
  • Intraventricular delivery can also be applicable in tumor therapy and stroke therapy.

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Abstract

L'invention concerne des méthodes de régulation d'une fonction spécifique commandée par l'hypothalamus chez un mammifère. L'invention concerne également des méthodes de traitement d'une maladie ou d'un trouble associé à une fonction spécifique commandée par l'hypothalamus.
PCT/US2001/050404 2000-12-21 2001-12-21 Administration directe d'un produit genique a l'hypothalamus WO2002049664A1 (fr)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1996, GEDDES BRAD J ET AL: "Persistent transgene expression in the hypothalamus following stereotaxic delivery of a recombinant adenovirus: Suppression of the immune response with cyclosporin.", XP002198354, Database accession no. PREV199699263534 *
ENDOCRINOLOGY, vol. 137, no. 11, 1996, pages 5166 - 5169, ISSN: 0013-7227 *
LUNDBERG CECILIA ET AL: "Direct delivery of leptin to the hypothalamus using recombinant adeno-associated virus vectors results in increased therapeutic efficacy.", NATURE BIOTECHNOLOGY, vol. 19, no. 2, February 2001 (2001-02-01), pages 169 - 172, XP002198353, ISSN: 1087-0156 *
MIZUNO ET AL: "IN VIVO TRANSFER OF LEPTIN (OBESE) GENE INTO VENTROMEDIAL HYPOTHALAMUS WITH A DEFECTIVE AAV-VECTOR DECREASES BODY WEIGHT AND FOOD INTAKE", SOCIETY FOR NEUROSCIENCE ABSTRACTS, SOCIETY FOR NEUROSCIENCE, US, vol. 22, 1996, pages 324, XP002082895, ISSN: 0190-5295 *
MURPHY ET AL: "Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 94, no. 25, December 1997 (1997-12-01), pages 13921 - 13926, XP002114143, ISSN: 0027-8424 *

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