WO2002049664A1 - Direct delivery of a gene product to the hypothalamus - Google Patents

Abstract

Methods of regulating a specific hypothalamus-controlled function in a mammal are disclosed. Also disclosed are methods of treating a disease or disorder associated with a specific hypothalamus-controlled function.

Description

DIRECT DELIVERY OF A GENE PRODUCT TO THE HYPOTHALAMUS

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 60/257,452, filed on December 21, 2000. The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

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)).

In the normal situation, the central nervous system (CNS) 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 (BBB) 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.

SUMMARY OF THE INVENTION

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.

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.

By "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. By "gene product associated with a feedback loop regulating the hypothalamus-controlled function" is meant a gene product having a receptor in the hypothalamus.

Thus, for example, in the case where body weight is the hypothalamus- controlled function, leptin is a gene product associated with a feedback loop regulating body weight, i the case where parturition is the hypothalamus-controlled function, ACTH, follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin are gene products associated with a feedback loop regulating parturition. In the case where folliculogenesis, ovulation or spermatogenesis is the hypothalamus-controlled function, 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. In the case where cortisol production is the hypothalamus-controlled function, ACTH is a gene product associated with a feedback loop regulating cortisol production. In the case where mammary gland development, lactogenesis or epithelial secretion of mammary epithelium is the hypothalamus-controlled function, 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. In the case where thyroid hormone production is the hypothalamus- controlled function, thyroid hormone-releasing hormone (TRH) and somatostatin are gene products associated with a feedback loop regulating thyroid hormone production. In the case where parathyroid hormone production is the hypothalamus- controlled function, parathyroid hormone releasing hormone (PHRH) is a gene product associated with a feedback loop regulating parathyroid hormone production. In the case where insulin growth factor production is the hypothalamus-controlled function, growth hormone (GH) is a gene product associated with a feedback loop regulating insulin growth factor production. Accordingly, in a particular embodiment, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the discovery that 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. Alternatively, 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. Alternatively, 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.

By "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). By "gene product associated with a feedback loop regulating the hypothalamus-controlled function" is meant a gene product having a receptor in the hypothalamus. As used herein, 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.

Diseases and disorders associated with specific hypothalamus-controlled function(s) 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 entire teachings of which are incorporated herein by reference). 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 feedback loop regulating body weight. Thus, where body weight is the hypothalamus-controlled function, leptin is a gene product associated with a feedback loop regulating body weight.

Leptin is produced by adipocytes in response to increased trigyceride storage, and appears to affect body weight primarily through target cells in the hypothalamus. Although 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, Ν. Engl. J. Med., 334:292-295 (1996); Frederich, R.C. et al., Nat. Med., 1:1311-1314 (1995); Maffei, M. et al., Nat. Med. 1:1155-1161 (1995)), recent studies have shown that obese humans and animals appear to be relatively resistant to the increased plasma levels of leptin (Caro, J.F. et al., Lancet, 348:159- 161(1996); Halaas, J.L. et al. Proc. Natl. Acad. Sci. USA, 94:8878-8883 (1997)). Analysis of the levels of leptin in the cerebrospinal fluid suggests that the uptake of leptin across the blood-brain barrier may be saturable (Caro, J.F. et al., Lancet, 348:159-161 (1996)). Taken together, these results suggest that therapeutic approaches to deliver leptin via the circulation may prove to be problematic.

Although recent clinical trials have suggested that peripherally administered leptin might lead to a reduction in body weight in humans (Heymsfield, S.B. et al., J. Am. Med. Assoc, 282:1568-1575 (1999)), it is likely that the more effective delivery of leptin to cellular targets within the central nervous system (CNS) will be necessary in order to fully reveal the therapeutic potential of the gene product.

In an effort to provide a means for the delivery of leptin which obviates the need for the gene product to transverse the blood-brain-barrier, the use of recombinant adeno-associated vectors to deliver leptin intraventricularly or directly to the hypothalamus was evaluated. As described herein, the delivery of leptin to the hypothalamus through intracranial gene transfer resulted in the efficient normalization of body weights in obese mice, using doses of recombinant virus that yield minimal therapeutic effects when administered intraventricularly or peripherally. These results suggest a new therapeutic avenue to pursue for the treatment of obesity. Thus, in a particular embodiment, 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.

Similarly, where parturition is the hypothalamus-controlled function, ACTH, FSH, LH and prolactin are gene products associated with a feedback loop regulating parturition. Where folliculogenesis, ovulation or spermatogenesis is the hypothalamus-controlled function, 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. Where cortisol production is the hypothalamus-controlled function, ACTH is a gene product associated with a feedback loop regulating cortisol production. Where mammary gland development, lactogenesis or epithelial secretion is the hypothalamus-controlled function, 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. Where thyroid hormone production is the hypothalamus-controlled function, TRH and somatostatin are gene products associated with a feedback loop regulating thyroid hormone production. Where parathyroid hormone production is the hypothalamus-controlled function, PHRH is a gene product associated with a feedback loop regulating parathyroid hormone production. Where insulin growth factor production is the hypothalamus-controlled function, GH is a gene product associated with a feedback loop regulating insulin growth factor production.

Thus, 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,

ACTH fragment, ACTH fusion protein, or a nucleic acid sequence encoding ACTH, ACTH analog, ACTH fragment or ACTH fusion protein. In another embodiment, 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. In a further embodiment, 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. Alternatively, 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. Alternatively, 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.

Alternatively, 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. Alternatively, 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. By "regulating" or "regulation" is meant the ability to control the rate and extent to which a process occurs. For example, regulating body weight refers to the ability to control the rate and extent to which body weight increases or decreases. By "altering" or "alteration" is meant the ability to change, modify or make different relative to a control or baseline.

As used herein, the terms "mammal" and "mammalian" refer 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). Examples of 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 (e.g., leptin, ACTH, FSH, LH, prolactin, TRH, somatostatin, PHRH, growth hormone) can be manufactured according to methods generally known in the art. For example, 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. For example, 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).

Specifically included in the present invention are 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.

Also encompassed by the present invention is the administration of 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. These 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. As such, 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. In one example, 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. Such a gene is typically operably linked to suitable control sequences capable of effecting the expression of the gene product, preferably in the CNS. The term "operably linked", as used herein, 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. In a particular embodiment, 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.

As used herein, the term "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). Other suitable promoters are readily available in the art (see, e.g., Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1998); Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor University Press, New York (1989); and U.S. Patent No. 5,681,735).

Typically, the 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. Viral vectors that can transduce nondividing cells include adeno-associated virus, adenovirus, herpesvirus and retroviral vectors based on lentiviruses. Examples of lentiviruses 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 lymphotropic virus type 4), simian immunodeficiency viruses, simian-human immunodeficiency viruses, human lymphotrophic viruses (e.g., type IE), simian T-cell lymphotrophic viruses. Other suitable viral vectors that can transduce nondividing cells are readily available in the art (see, e.g., Fundamental Virology, 3rd edition, B.N. Fields et al, eds., Lippincott-Raven Publishers, Philadelphia (1996)).

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, as well as nucleic acid sequences encoding the gene products, analogs, biologically active fragments or 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. If desired, 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, 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 formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are 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.

Similarly, 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.

For direct delivery to the hypothalamus, 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, 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 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. The present invention will now be illustrated by the following examples, which are not to be considered limiting in any way.

EXAMPLES

The following materials and methods were used in the work described in the Examples. Polymerase Chain Reaction (PCR). The cDNA encoding the human obese gene (hOB) was amplified from a human fat cell cDNA library (Clontech) using PCR. The PCR amplification was performed using Advantage KlenTaq polymerase (Clontech, USA) and the following primers:

5'-CGGACCGGTATGCATTGGGGAACCCTGTGCGGATTCT-3' (SEQ ID NO.:l) and 5'-AGTCGCGGCCGCTCAGCACCCAGGGCTGAGGTCCAG-3' (SEQ ID NO.:2). 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. The rAAN.MD.GFP was constructed by inserting the EcoRI-Notl fragment of pΕGFP-1 plasmid (Clontech) into prAAV.MD.

rAAV Production. Production of recombinant AAV was performed as previously described

(Snyder, R.O. et al, Hum. Gene Ther., 8:1891-1900 (1997)). Briefly, the recombinant plasmid and the pAD/AVV helper plasmid were co-transfected into 293 cells, which were subsequently infected with El -deleted adeno virus. The cells were harvested 60 hours post-transfection, lysed and the rAAV purified by double- cesium banding. The fractions containing rAAV (as assayed by dot-blot analysis using the transgene cDΝA 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 10¹⁰ 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.

Surgery. All handling of animals was performed according to local regulation and approved by the ethical committee at Boston's Children's Hospital. C57BL/6J-Lep^ob mice (n = 70, 10 mice per experimental group) and control litter mates (n = 10), 5 weeks old at the time of surgery, were obtained from Jackson Laboratory (Bar Harbor, ME).

Intramuscular injections were performed under light metoxyflurane (Methofane, Abbot Laboratories, Abbott Park, EL) anesthesia, and 50 μl 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 intracranial injections were performed using a Kopf stereotaxic frame with mouse adapters at the following coordinates; lateral ventricle (IN): A = 0, L = 1 mm, N = 2.5 mm; hypothalamus (EH) A = 2.1 mm, L = 0.5 mm, N = 5.0 mm. During surgery the mice were anesthetized using 75 mg/kg pentobarbital. Subsequently, 5 μl of vector was injected IN and 1 μl EH. At the time of surgery baseline measurement of body weight and of serum human leptin were obtained. The mice were weighted and bled retro-orbitally biweekly for the duration of the study. At two, four and six weeks, the food intake was monitored over three consecutive days.

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, MΝ) according to the supplier's protocol.

Immunostaining.

At sacrifice, the brains and muscles injected with rAAN.MD.GFP were harvested and processed for immunohistochemistry for GFP. Polyclonal rabbit anti- GFP antibody (1:500, Clontech), and biotinylated swine-anti-rabbit antibody (1:200 DAKO, Glostrup, Denmark) were used. The ABC kit (Vector Laboratories, Birmingham, AL) and 3'3-diaminobenzidine were used to detect the positive signal.

Stereology. The number of GFP -expressing cells in the hypothalamus was estimated using the optical fractionator (Gundersen, H.J. et al, Apmis, 96:857-881 (1988)) and subsequently applying the fractionator formula; N = SQ- x Fl x F2 x F3, where N = total number of GFP-expressing cells, SQ- = sum of cells counted, Fl = fraction of sections used (5 in the present study), F2 = fraction of tissue depth used to collect data (1.33), F3 = fraction of tissue area used to collect data (3.18). The GRID software (Interactivision, Copenhagen, Denmark) was used to generate unbiased counting frames.

Statistics. Data was statistically analyzed using the JMP software (SAS Institute Inc., Gary, NC). Analysis of variance was performed and followed by Tukey-Kramer post-hoc test when appropriate. Data are presented as mean ± SEM or median and range when appropriate.

Example 1 Efficiency of Gene Transfer and Spectrum of Cell Types

Transduced After Intracranial Delivery of Adeno-Associated Viral Vectors. In a first series of experiments aimed at assessing the efficiency of gene transfer and the spectrum of cell types transduced after intracranial delivery of adeno-associated viral (AAV) vectors, an AAV vector encoding green fluorescent protein (GFP) (AAVGFP) (3-4 10¹⁰cfu/ml) was injected into the lateral ventricle (IN, 5 μl) or into the hypothalamus (EH, 1 μl) of mice. As a control, 50 μl 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.

Twelve weeks after injection, either the site of muscle injection or the brains of the injected animals were harvested and examined for GFP expression by visualization of GFP fluoresence (muscle) or by immunohistological staining (brain), using a polyclonal antibody to GFP. Injection of muscle with the GFP virus led to efficient expression of the reporter gene.

In the case of animals in which virus was injected into the lateral ventricle, infected cells were readily detected throughout the ventricular system, including the lateral ventricle on the opposite side to the injection. Although 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. In the animals that received virus directly into the hypothalamus, 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. In a second series of experiments, the levels of leptin produced after intracranial gene transfer, using an AAV vector encoding human leptin (AANhOB) was assessed. As in the case of the studies involving AANGFP, virus (3-4 x 10¹⁰ cfu/ml) was injected into the lateral ventricle (IN, 5 μl) or into the hypothalamus (EH, 1 μl) of mice, hi addition, to provide a basis for comparison of intracranial gene transfer and conventional intramuscular gene transfer. AANhOB was also injected into muscle (EM, 50 μl). 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.

In the case of the intraventricular injections, extracts prepared from the walls of the right lateral ventricle were found to express a maximum of 5 (median 2) pg/μg protein. In the case of the intrahypothalamic injections, tissue levels of leptin were found to be variable, ranging from 7 to 200 (median 133) pg/μg protein. The level of leptin in the samples did not correlate with weight loss, indicating that the variability may result from the center of injection not being included in the dissected tissue, but close enough to the hypothalamic target cells to be effective. Measurement of plasma levels of human leptin after intracranial gene transfer (by either IN or EH routes) revealed no detectable levels of the gene product. In contrast, 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/μg protein) were in the same range as the animals injected intraventricularly.

Example 3 Biological Effects of Administration of Leptin Via Different

Routes of Gene Transfer. To assess the biological effects of the administration of leptin via the different routes of gene transfer (EM, EV, EH), injected animals were periodically analyzed for food intake and body weight over a 12-week period (Figures 1 A and IB).

At the time of surgery (0 weeks), the body weight of all mutant mice were significantly higher than the normal littermate controls (27.9 ± 0.5 g (mutant) versus 16.3 ± 0.5 g (normal), P < 0.05; Figure 1A). At two weeks post-surgery, 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). At four weeks post-surgery, 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. In the two other groups receiving AAV-leptin, 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).

Weight loss consequent to leptin expression was readily evident in photographs of mice at 12 weeks after injection with AANhOB into the lateral ventricle or into hypothalamus. The small weight difference (10 g) between animals receiving intramuscular injections of either GFP- or hOB- encoding vectors was not evident in photographs of mice at 12 weeks after injection.

The results demonstrate both that it is technically possible to deliver leptin directly to the hypothalamus by AAN-mediated gene transfer, and that such a mode of delivery results in increased efficacy relative to transduction of cells in other anatomical locations. In the case of the EH route of vector administration, we found that vector injection of ob/ob mice with AAN-leptin led to normalization of their body weight in four weeks, and this effect was sustained throughout the length of the experiment. Because protein expression from AAN vectors has been reported to reach peak expression levels only after two to four weeks in vivo (Fisher, K.J. et al., Nat. Med., 3:306-312 (1997)), this finding suggests that very low levels of leptin can be effective when present close to its target cells.

Histochemical studies also demonstrated that AAV transduction led to the efficient infection of predominately neurons, consistent with previous reports that have demonstrated a neuronal preference for infection by rAAV (Mandel, R.J. et al., J. Neurosci, 18:4271-4284 (1998); Bartlett, J.S. et al. Hum. Gene Ther, 9:1181- 1186 (1998)). The demonstration that ependymal cells were readily transduced is of considerable interest, in light of the findings that stem cells of the brain have been localized to that anatomical location (Johansson, C.B. et al. Cell, 96:25-34 (1999); Doetsch, F. et al. Cell, 97:703-716 (1999)). hi contrast to the case with EH injections, the effect on body weight had later onset and was only partial in animals receiving injections of rAAV-leptin farther away from the target nuclei (IN and IM). Although the systemic levels of leptin achieved through the EM route would not be expected to result in significant biological effects on weight loss, on the basis of previous studies (Morsy, M.A. et al, Proc. Νatl. Acad. Sci. USA, 95:7866-7871 (1998); Murphy, J.E. et al, Proc. Νatl. Acad. Sci. USA, 94:13921-13926 (1997)), the findings nevertheless indicate that localized delivery of leptin to the hypothalamus is, at a minimum, 50-fold more effective than systemic delivery of leptin by EM gene transfer, and probably much greater. These findings further underscore the importance of achieving high local concentrations of the gene product within the target region.

The finding that intraventricular administration of AAN vectors can lead to transduction of cells throughout the whole ventricular system suggests that this route of infection can be considered for other therapeutic applications in which it is more important to reach many different targets within the CΝS.

For example, intraventricular delivery can allow a nucleic acid product to reach stem cells of the nervous system. As such, intraventricular delivery can be applicable, for example, in degenerative multiple sclerosis therapy. Intraventricular delivery can also be applicable in tumor therapy and stroke therapy.

En conclusion, in conjunction with previous studies in which AAN vectors have been successfully utilized to transduce different cells of the CΝS (Mandel, R.J. et al, J. Neurosci, 18:4271-4284 (1998)); McCown, T.J. et al. Brain Res, 713:99- 107 (1996); Kaplitt, M.G. et al, Nat. Genet, 8:148-154 (1994); Mandel, R.J. et al, Proc. Natl. Acad. Sci. USA, 94: 14083-14088 (1997)), the studies presented here strengthen the notion that AAV vectors may be uniquely suited for a number of different therapeutic applications involving cells of the CNS. The studies herein reinforce the notion that localization of leptin expression to its presumptive site of action in the brain should be an objective of therapeutic strategies involving the gene product, whether delivery is accomplished by protein administration or gene transfer.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAEMSWhat is claimed is:

1. A method of regulating a specific hypothalamus-controlled function in a mammal in need of such regulation comprising direct delivery to the hypothalamus of said mammal: a) an effective amount of a gene product associated with a feedback loop regulating said hypothalamus-controlled function; or b) an effective amount of a nucleic acid sequence encoding a gene product associated with a feedback loop regulating said hypothalamus-controlled function.

2. The method of Claim 1 wherein said feedback loop is a negative feedback loop.

3. The method of Claim 1 wherein said feedback loop is a positive feedback loop.

4. The method of Claim 1 wherein the nucleic acid sequence is incorporated into a viral vector.

5. The method of Claim 1 wherein said specific hypothalamus-controlled function is body weight and said gene product associated with a feedback loop regulating said hypothalamus-controlled function is leptin.

6. The method of Claim 1 wherein said mammal is a rodent.

7. The method of Claim 1 wherein said mammal is a human.

8. A method of treating a disease or disorder associated with a specific hypothalamus-controlled function in a mammal in need of such treatment comprising direct delivery to the hypothalamus of said mammal: a) an effective amount of a gene product associated with a feedback loop regulating said hypothalamus-controlled function; or b) an effective amount of a nucleic acid sequence encoding a gene product associated with a feedback loop regulating said hypothalamus-controlled function.

9. The method of Claim 8 wherein said feedback loop is a negative feedback loop.

10. The method of Claim 8 wherein said feedback loop is a positive feedback loop.

11. The method of Claim 8 wherein the nucleic acid sequence is incorporated into a viral vector.

12. The method of Claim 8 wherein said disease or disorder associated with specific hypothalamus-controlled function in a mammal is obesity, said specific hypothalamus-controlled function is body weight and said gene product associated with a feedback loop regulating said hypothalamus- controlled function is leptin.

13. The method of Claim 8 wherein said mammal is a rodent.

14. The method of Claim 8 wherein said mammal is a human.

15. A method of regulating body weight in a mammal in need of such regulation comprising direct delivery to the hypothalamus of said mammal: a) an effective amount of leptin, leptin analog, leptin fragment or leptin fusion protein; or b) an effective amount of a nucleic acid sequence encoding leptin, leptin analog, leptin fragment or leptin fusion protein.

16. The method of Claim 15 wherein the nucleic acid sequence is incorporated into a viral vector.

17. A method of treating obesity in a mammal in need of such treatment comprising direct delivery to the hypothalamus of said mammal: a) an effective amount of leptin, leptin analog, leptin fragment or leptin fusion protein; or b) an effective amount of a nucleic acid sequence encoding leptin, leptin analog, leptin fragment or leptin fusion protein.

18. The method of Claim 17 wherein the nucleic acid sequence is incorporated into a viral vector.