WO1996039416A1

WO1996039416A1 - Gene therapy for pituitary adenoma and other endocrine disorders

Info

Publication number: WO1996039416A1
Application number: PCT/US1996/008191
Authority: WO
Inventors: Andrew Freese; Eugene S. Flamm; Peter J. Snyder; Thomas Gennarelli; Matthew J. During
Original assignee: The Trustees Of The University Of Pennsylvania; Yale University
Priority date: 1995-06-06
Filing date: 1996-05-31
Publication date: 1996-12-12
Also published as: CA2221604A1; AU6028296A

Abstract

Virus vectors containing selected genes encoding proteins capable of modulating pituitary tumor or gland physiology and methods for using these vectors in the treatment of pituitary adenomas, pituitary hyperfunction and hypofunction, dysfunction of the hypothallamic/pituitary axis, and neuroendocrine diseases mediated by the pituitary gland are provided.

Description

GENE THERAPY FOR PITUITARY ADENOMA AND OTHER ENDOCRINE DISORDERS

FIELD OF THE INVENTION

This invention relates to the field of mammalian gene transfer, tumors of the hypothalamus-pituitary axis, and endocrine disorders. More particularly, methods are provided for delivering of genes that directly or indirectly modulate pituitary tumor and gland physiology. The unique position of the pituitary gland as the key mediator of the hypothalamic-pituitary neuroendocrine axis, located in the brain, but not within the central nervous system, renders genetic intervention in the pituitary gland as clinically relevant and important.

BACKGROUND OF THE INVENTION Significant progress has been made over the past two decades in the medical therapy of pituitary adenomas, and lactotroph adenomas (prolactinomas) in particular. The mainstay of therapy for prolactinomas currently focuses on the use of the ergot derivative, bromocriptine. The primary physiological prolactin inhibitory factor is dopamine. Bromocriptine, a dopamine receptor agonist, apparently acts by suppressing the secretion of prolactin and also retarding the division of prolactin-secreting cells and growth of prolactinomas. M. Ishibashi and T. Yamaji, J. Clin. Endocrinol . Metab. 1995, 60, 599-606.

However, bromocriptine therapy has several problems. Some tumors are resistant to this therapy and continue to grow and secrete abnormal amounts of prolactin. I. Pellegrini et al., Horm. Res. 1989, 31, 19-23. In addition, relatively high doses must be continued on a daily, multiple dose basis as cessation of treatment results in the tumor resuming its previous abnormal secretory and growth pattern. Many patients also find the side effects of bromocriptine unacceptable and cease treatment. New dopaminergic drugs with longer lasting action than bromocriptine, have been reported. However, these new drugs still require continued treatment with attendant side-effects. M.S. Venetikou et al., Acta Endocr±nol (Copenhagen) 1987, 116, 287-92.

An alternative to dopamine agonists consists of co- administration of growth hormone releasing protein (GHRP) and its amino terminal peptide fragment. However, this treatment requires relatively high intravenous daily dosing (0.1 - 100 mg/kg) of poorly bioavailable peptides. B.B. Bercu and R.F. Walker, United States Patent 5,246,920.

Because prolactinomas in men are frequently clinically silent until they reach a macroadenoma size, prolactinomas are more difficult to treat in male patients. Furthermore, once the tumors reach this large size, they can invade relatively inaccessible areas, including the cavernous sinus, rendering surgical removal oftentimes impossible.

Other pituitary adenomas pose even greater challenges for therapy. Those that secrete glycoprotein hormones, including the gonadotropin and thyrotropin secreting tumors, are particularly difficult to treat. Growth hormone secreting tumors are also difficult to treat, although therapy with analogs of the growth hormone inhibitory factor, somatostatin, has yielded encouraging results. However, there are significant systemic side-effects of these analogs as well, and a significant proportion of patients do not respond well clinically.

As the therapies available for most pituitary adenomas are currently suboptimal with regard to patient compliance, efficacy, and required continuous dosing, there is a great need for better therapies. A method for transducing gene sequences encoding proteins which modulate pituitary tumor and gland physiology directly into pituitary cells via the use of adenovirus, adenovirus-associated, and herpes viral vectors has now been developed. Using this method, gene sequences encoding dopamine synthetic enzymes were introduced directly to prolactinoma cells via a viral vector. Subsequent expression of these enzymes increased dopamine synthesis in situ resulting in an inhibition of prolactin secretion. Similarly, a method for transducing gene sequences encoding other hormones, receptors or modulators into the pituitary gland can be used to alter hormone secretion by and growth patterns of other pituitary adenomas, as well as modulate pituitary function in other disease conditions.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method of delivering at least one gene encoding a dopamine synthetic enzyme to pituitary cells which comprises contacting pituitary cells with a viral vector comprising this gene.

Another object of the present invention is to provide a method of modulating pituitary tumor physiology which comprises contacting pituitary tumor cells with a viral vector comprising at least one gene capable of encoding a dopamine synthetic enzyme which modulates pituitary tumor physiology.

Another object of the present invention is to provide viral vectors containing at least one gene encoding a dopamine synthetic enzyme which is capable of modulating pituitary tumor physiology, including, but not limited to, genes encoding tyrosine hydroxylase, amino acid decarboxylase (or dopa decarboxylase) and GTP cyclohydroxylase (responsible for production of the tyrosine hydroxylase cofactor, tetrahydrobiopterin).

Another object of the present invention is to provide viral vectors containing at least one gene encoding other factors that modulate pituitary gland or tumor physiology, including, but not limited to, somatostatin, growth hormone releasing factor (GHRF), activin, follistatin, (luteinizing hormone/follicle stimulating hormone (LH/FSH), corticotropin releasing factor (CRF), corticotropin releasing inhibitory factor (CRIF), adrenocorticotrophic hormone (ACTH), growth hormone (GH), gonadotroph releasing hormone (GNRH), thyroid stimulating hormone (TSH) and other proteins or enzymes endogenous to the hypothalamus-pituitary axis.

Another object of the present invention is to provide viral vectors containing at least one gene encoding receptors for factors that modulate pituitary gland or tumor physiology, including, but not limited to, dopamine receptors (particularly the D2 receptor), somatostatin receptors, activin and follistatin receptors, as well as other receptors for proteins and compounds enumerated above.

Another object of the present invention is to provide viral vectors containing two or more separate genes that each encode a separate protein that may alter pituitary gland or tumor physiology, including, but not limited to, the combination of tyrosine hydroxylase and amino acid decarboxylase, or tyrosine hydroxylase and somatostatin.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 provides schematics of the viral vectors used in the gene transduction experiments. Figure 1A shows a modified human herpes simplex virus-1 vectors, pHSVth, containing a human tyrosine hydroxylase II gene under the control of the herpes immediate early 4/5 promoter (HSV EI-4/5). Figure IB shows an adenovirus vector containing a human tyrosine hydroxylase gene under control of an RSV promoter. Figure 1C shows an adeno-associated virus vector containing a tyrosine hydroxylase gene. Figure ID shows a bicistronic adeno-associated viral vector containing both the tyrosine hydroxylase gene and an amino-acid decarboxylase gene (the gene encoding the next enzyme in the dopamine biosynthetic pathway) in tandem.

Figure 2 is a schematic outline of the biosynthetic pathway of dopamine biosynthesis with the key intermediates and their corresponding enzymes indicated. Figure 3 is the X-gal-based detection (blue cells) of β- galactosidase activity in human pituitary cells following transfection with either an AAV vector with the Lac Z gene as shown in Figure 3A or an Adenovirus vector with the Lac Z gene as shown in Figure 3B, demonstrating the first successful transfer of a transgene (Lac Z) encoding a foreign protein (β-galactosidase) in human pituitary cells. As shown in Figure 3C, negative controls (cells not transfected with the viral vector) do not show blue staining in the cells. Figure 4 is the immunohistochemical detection of TH- transgene expression in the transduced human primary pituitary prolactinoma cells. Adenovirus vector with the TH gene was used in Figure 4A, AAV vector with the TH and AADC genes and the Flag epitope was used in Figure 4B, and control is shown in Figure 4C (absent staining). Controls are mock transductions or transductions with vectors lacking the dopamine synthesis genes of pituitary and prolactinoma cells.

Figure 5 shows the inhibition of prolactin secretion from cells transduced with the adeno-associated virus containing both the TH and AADC genes. The levels of prolactin are assayed in the cell culture media. The percent of prolactin secretion by the adenoma cultures was statistically the same in both lower (depicted as lightly shaded bar) and higher (depicted as darker shaded bar) viral titer-treated wells (2xl0⁵ and 5xl0⁵ particles/ml). In addition, the prolactin secretion (as a percentage of control) was the same in the viral vector-treated cultures as in those cultures exposed to supraphysiological doses of dopamine (depicted as filled bar), confirming that the vectors resulted in the same reduction of prolactin secretion as did supraphysiological doses of dopamine. These results indicate that the vectors will have efficacy at least equal to dopamine receptor analogues, such as bromocriptine.

Figure 6 shows reduction in prolactin secretion by human primary prolactinoma cells in cultures transduced with the adenoviral vector containing the TH transgene, compared to control cells transduced with the non-physiological Lac Z gene (depicted as filled triangle). There was a dose dependent effect with the higher titer of vector (10⁷ particles/ml, depicted as filled circle) resulting in a larger reduction than the lower titer of vector (10⁵ particles/ml, depicted as square) .

Figure 7A shows dopamine secretion induced by an adenovirus viral vector containing the TH gene in human primary prolactinoma cells in culture compared to controls (the prolactinoma cells transduced with the Lac Z gene) . Figure 7B shows L-dopa secretion induced by the same vector. In each situation, a marked increase of both L-dopa and dopamine secretion was noted.

Figure 8 provides cytotoxicity experiments in pituitary cells contacted with these viral vectors. Viral vectors of the present invention are not cytotoxic to human pituitary cells in culture. Pituitary cells were counted in dishes several days following transfeetion with the vectors or control. Control constitutes the buffer, phosphate buffered saline (PBS). Vectors used were Adenovirus with the Lac Z gene and Adenovirus with the TH gene.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that viral vectors containing at least one gene encoding an enzyme involved in dopamine biosynthesis are useful in transducing the gene so that these enzymes are expressed in primary human prolactinoma tumor cells in culture. The resultant in situ increase in dopamine has been shown to inhibit prolactin secretion from these prolactinoma cells. Since the primary prolactin inhibitory factor is dopamine, it is believed that the introduction into the pituitary gland or hypothalamus of genes encoding enzymes involved in dopamine synthesis, which enhances dopamine synthesis and release and inhibits prolactin secretion, will modulate the physiology of prolactinomas.

Several enzymes are very important in the biosynthesis of dopamine. For the purposes of this disclosure, these enzymes are referred to as "dopamine synthetic enzymes". Examples of these enzymes include, but are not limited to, human tyrosine hydroxylase which catalyses a critical hydroxylation from the amino acid precursor tyrosine, and amino acid decarboxylase which catalyses a critical decarboxylation from the amino acid precursor tyrosine. Also included are enzymes which the synthesis of cofactors needed in the synthesis of dopamine. For example, tetrahydrobiopterin, is the cofactor for the rate limiting step in dopamine biosynthesis which is mediated by tyrosine hydroxylase. In primary prolactinoma cultures derived from human patients undergoing pituitary tumor surgery, introduction of viral vectors containing gene sequences encoding human tyrosine hydroxylase and/or amino acid decarboxylase were found to cause a significant reduction in prolactin levels as measured by a radioimmunoassay. In addition, the expression of the transgenes in the pituitary cells following transfection was demonstrated by immunocytochemistry. Viral vectors used in these experiments were either adenovirus or adeno-associated virus vector systems. However, as will be obvious to one of skill in the art upon this disclosure, other viral vector systems can be used. Other hypothalamus-pituitary diseases affecting the neuroendocrine axis including pituitary adenomas and hypo-pituitary states can also be treated in accordance with the methods of the present invention. Viral vectors useful in the present invention can be routinely selected by those of skill in the art upon this disclosure. The vector selected should allow sufficient expression of the gene, while producing minimal viral gene expression. There should be minimal viral DNA replication and ideally no virus replication. In addition, recombination to produce new viral sequences and complementation to allow growth of the defective virus in an animal should be kept to a minimum. Preferred viral vectors which can be used in the present invention include, but are not limited to, adenoviruses, adeno-associated viruses and herpesviruses.

Adenovirus-based vectors are well-suited for^* gene therapy as they appear to be relatively safe and can be manipulated to encode a desired gene product, while at the same time, be inactivated in terms of their ability to replicate in a normally lytic viral life cycle. Adenoviruses are able to infect quiescent cells. Expression of an adenovirus is achieved without integration of the viral DNA into the host cell chromosome thus alleviating concerns about insertional mutagenesis. In addition, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile. Schwartz et al., Am. Rev. Respir. Diε. 1974, 109, 233-238. Extensive studies attempting to establish adenovirus as a causative agent in human cancer have all been negative. Green et al., Proc. Nat 'l Read. Sci. USA 1979, 76, 6606. Adenovirus mediated gene transfer of several different genes to lung tissue has been demonstrated in vivo in animals. Rosenfeld et al., Science 1991, 252, 431-434; Rosenfeld et al., Cell 1992, 68, 143-155.

Pseudo-adenoviruses (PAV) may also be useful as vectors in the present invention. PAV contains no potentially harmful viral genes, has a theoretical capacity for foreign material of nearly 36 kb, may be produced in reasonably high titers, and maintains the tropism of the parent adenovirus for dividing and non-dividing human target cell types. PAVs comprise adenovirus inverted terminal repeats and the minimal sequences of a wild type adenovirus type 2 genome necessary for efficient replication and packaging by a helper virus and genetic material of interest.

Adeno-associated virus (AAV) is a DNA-containing parvovirus that has not been directly linked to any human disease, even though the majority of Americans demonstrate seropositivity to AAV, AAV vectors can be generated which show minimal to no cytotoxicity and minimal or no protein synthesis other than the desired transgene. Samulski et al., J. Virol . 1989, 63, 3822-3828.

Herpes Simplex Virus Type-1 (HSV-1) vectors can be generated through recombinant techniques so that they contain transgenes under the control of selected viral or cellular promoters. By use of vectors with helper viruses or defective HSV-1 mutants, vectors can be produced that show safety and efficacy, with minimal or no cytotoxicity, viral replication and/or recombination. Freese et al., Biochem. Pharmacol . 1990, 40, 2189-2199.

Viral vectors containing at least one gene encoding other factors that modulate pituitary gland or tumor physiology, including dopamine biosynthetic enzymes, dopamine receptors, somatostatin, somatostatin receptors, GHRF, activin, follistatin, activin or follistatin receptors, LH/FSH, CRF,

CRIF, ACTH, GH, GNRH, TSH and other proteins, enzymes or receptors endogenous to the hypothalamus-pituitary axis may also be used in accordance with the teachings of the invention.

In a preferred embodiment, the viral vector of the present invention comprises more than one gene encoding for different enzymes involved in dopamine biosynthesis. For example, the vector construct may contain genes encoding tyrosine hydroxylase and amino-acid decarboxylase. This construct represents an improvement over vectors encoding these two genes separately, as this tandem construct ensures that any transduced cell will receive both required enzymes simultaneously and not be subject to random binomial distribution of each gene delivered separately. Furthermore, this tandem construct is more likely to enhance the production of dopamine, rather than L-dopa.

Viral vectors of the present invention containing at least one gene encoding a dopamine synthetic enzyme are useful in delivering a gene encoding a dopamine synthetic enzyme to pituitary cells comprising contacting pituitary cells with a viral vector a selected gene encoding a dopamine synthetic enzyme. These viral vectors can also be used in the modulation of pituitary tumor physiology, preferably prolactinomas. Pituitary tumor cells are contacted with a viral vector containing a selected gene encoding a dopamine synthetic enzyme capable of modulating pituitary tumor physiology. In a preferred embodiment, the viral vector comprises a gene encoding human tyrosine hydroxylase and a gene encoding amino acid decarboxylase.

The viral vectors of the present invention can also be used to modulate secretion of prolactin by pituitary cells in conditions such as hyperprolactinemia. Hyperprolactinemia is a condition involving increased levels of prolactin in the blood. In women this condition is associated with amenorrhea and galactorrhea. In men, hyperprolactinemia is associated with hypogonadism and impotence. Individuals with hyperprolactinemia many times experience infertility. If associated with enlargement of the pituitary gland resulting from lactotroph hypertrophy, hyperplasia or adenoma formation, these individuals may also suffer from persistent headaches and vision disturbances. Viral vectors of the present invention can be used to decrease the levels of prolactin secreted from the pituitary cells.

In a preferred embodiment the viral vectors of the present invention are administered to a mammal, preferably a human. The vectors can be administered orally or parenterally including intravenously, intramuscularly, intraperitoneally, intranasally, subcutaneously and surgically. When administered parenterally, it is preferred that the vectors be given in a pharmaceutical vehicle suitable for injection such as a sterile aqueous solution or dispersion. Following administration, the mammal is monitored to detect improvement in outward manifestations. Prolactin levels are measured to determine when they approach or reach normal levels. Magnetic resonance imaging can be used to monitor any change in size of pituitary adenomas. Dose and duration of treatment is determined individually depending upon the degree and rate of improvement.

The viral vectors of the present invention provide the first demonstration of gene transduction of human cells of the hypothalamic-pituitary axis and use of gene transduction to modulate hypothalamic-pituitary axis physiology and pituitary tumor physiology, i.e., endocrine secretion. These vectors provide a novel approach to gene therapy and, more specifically, tumor therapy; rather than causing tumor cell death or dysfunction, transducing genetic sequences as described in the present invention results in the production of proteins that either directly or indirectly modulate tumor physiology, hormone secretion and tumor size regression. As will be obvious to those of skill in the art upon this disclosure, in addition to modulating dopamine synthesis, by selecting other appropriate genes, the viral vectors and methods of the present invention are applicable to a variety of therapies of the hypothalamic-pituitary and neuroendocrine axis. In one embodiment, viral vectors can be used to introduce genes encoding proteins into pituitary cells that result in physiological modulation of hormone secretion from these cells. For example, by contacting pituitary cells with a viral vector containing the genetic information encoding somatostatin, growth hormone secretion from somatotroph adenomas can be modulated. In a significant proportion (>20%) of pituitary adenomas, both growth hormone and prolactin are secreted. Thus, a vector containing, for example, both the somatostatin and tyrosine hydroxylase, or other combinations, is especially useful in treating these plurihormonal tumors. The following nonlimiting examples are provided to further illustrate the present invention.

EXAMPLES

Example 1: Vector construction and sequences The human tyrosine hydroxylase II gene is introduced via three viral vectors: a) modified human herpes simplex virus-1 vectors, pHSVth, containing a human tyrosine hydroxylase II gene under the control of the herpes immediate early 4/5 promoter (HSV EI-4/5); b) an adenovirus vector containing a human tyrosine hydroxylase gene; and c) an adeno-associated virus vector containing both a tyrosine hydroxylase and amino acid decarboxylase gene.

Example 2: Prolactinoma cell culture

Human prolactinoma cultures are derived from human prolactinoma tissue obtained in the operating room from patients undergoing transphenoidal surgical resection of their tumors. The tissue is kept in sterile saline on ice and transported immediately to the laboratory. There, the saline is carefully aspirated and the tissue laid out on a Petri Dish. Using two #11 scalpel blades heat sterilized previously, the tissue is minced carefully into small ribbons. Thereafter, a DNAase/trypsin solution in Hanks medium is added and the combined protease solution and prolactinoma sample are transferred using a 2 ml pipette into sterile capped Erlenmeyer flasks. Using a 37°C shaking bath, the tissue is incubated for 1-2 hours until the solution is cloudy, indicating complete digestion and the achievement of a dispersed cell state. This solution is then spun on a table top centrifuge at 5,000 rpm for approximately 10 minutes, and the pellet is resuspended in 40% Dulbecco's modified Eagle's medium, 40% Ham's F12 medium, and 10% fetal calf serum, with 10,000 units of penicillin/streptomycin/100 ml of medium. Cell counts are made using a hemacytometer and 10,000 to 100,000 cells are added to each well of a 48 well Nunclon dish pretreated with poly-1-lysine for 2 hours. The final number of cells per well in each dish is identical, but some variation in the number of cells between tumor samples does occur because of differing quantities of tumor specimen obtained. Each data point represents the mean of at least 3 wells.

Example 3: Gene Transduction

After three days in culture to wash out any residual bromocriptine, the cells were exposed to viral vector (either 2 x 10⁵/ιrtl (low) or 5 x 10⁵/ l (high) particles for AAV vector or 10⁵/ml (low) or 10⁷/ml (high) particles for Adenovirus vector. Every 24 hours thereafter, medium was removed to analyze prolactin secretion; medium was changed every 48 hours. Dopamine secretion was evaluated using buffer with presence of tyrosine and tetrahydrobiopterin. Example 4: Immunohistochemical detection of transgene delivery

Using an antibody directed against either tyrosine hydroxylase or a synthetic epitope placed in the AAV bicistronic construct (the Flag epitope), immunocytochemistry was performed using standard diaminobenzidine secondary antibody techniques. Geller A.I. and Freese A., Proc. Nat 'l Acad Sci USA 1990, 87, 1149-1153. Using the chromogenic substrate for E. coli β-galactosidase, expression of the Lac Z transgene from the viral vectors containing the Lac Z gene in the human pituitary cells was performed, following 15 minute fixation of the cells in 4% paraformaldehyde/phosphate buffered saline. Cells that appear blue are positive for transgene expression, whereas cells that do not turn blue lack β-galactosidase.

Example 5: Reduction of prolactin secretion from transduced prolactinomas

Using an established radioimmunoassay for prolactin available in standardized kits from Nichols Institute, (San

Juan Capistrano), prolactin levels were determined in the media samples from the cultures treated with either controls or experimental viral vectors. The results for the levels of prolactin are demonstrated in Figures 5 and 6.

Claims

What is claimed is:

1. A method of delivering a gene encoding a protein capable of modulating pituitary tumor or gland physiology comprising contacting pituitary cells with a viral vector containing a selected gene encoding a protein capable of modulating pituitary tumor or gland physiology.

2. The method of claim 1 wherein the selected gene encodes a protein endogenous to the hypothalamus-pituitary axis.

3. The method of claim 2 wherein the selected gene encodes a dopamine synthetic enzyme.

4. A method of modulating pituitary tumor or gland physiology comprising contacting pituitary cells with a viral vector containing at least one selected gene encoding a protein capable of modulating pituitary tumor or gland physiology.

5. A method of modulating prolactin secretion in pituitary cells comprising contacting pituitary tumor cells with a viral vector containing at least one selected gene encoding a dopamine synthetic enzyme capable of modulating pituitary prolactinoma physiology.

6. A viral vector comprising at least one selected gene encoding a protein capable of modulating pituitary tumor or gland physiology.

7. The viral vector of claim 6 wherein the selected gene encodes a protein endogenous to the hypothalamus-pituitary axis.

8. The viral vector of claim 7 wherein the selected gene encodes a dopamine synthetic enzyme.