Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-07-24
2002-07-23
Nguyen, Dave T. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S320100, C435S325000, C435S360000, C435S455000
Reexamination Certificate
active
06423693
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the field of recombinant DNA technology. This invention relates to vectors which encode stable messenger RNA (mRNA) and methods of using such vectors. In particular, this invention relates to vectors which establish controlled expression of recombinant genes within a tissue; the expression may be at levels which are useful for gene therapy and other applications. The invention further relates to vectors able to express growth hormone releasing hormone (GHRH) and gene sequences inserted into vectors that control the production of growth hormone releasing hormone in non-human vertebrate animals. The invention is directed further to the use of these vectors in the respective non-human animals to further growth and strengthen their immune systems.
BACKGROUND OF THE INVENTION
None of the information provided herein is admitted to be prior art to the present invention, but is provided only to aid the understanding of the reader.
Growth hormone (GH) secretion by the anterior pituitary is stimulated by growth hormone releasing hormone (GHRH) and inhibited by stomatostatin (SS), both hypothalamic hormones (Scanlong, M. F. et al., 1996
, Hormone Research
149-154). GH enhances protein synthesis, lypolysis, and epiphyseal growth, and is implicated in the regulation of the immune system. GH increases circulating insulin-like growth factor I (IGF-1) levels, which in turn, mediates growth in the liver and peripheral tissues.
The GHRH-GH-IGF-I axis undergoes dramatic changes during the aging process and in the elderly (Iranmanesh et al., 1991
, J. Clin. Endocrin
. &
Metab
. 73:1081-1088; D'Costa A. P. et al., 1993
, J. Reproduction & Fertility—Suppl
. 46:87-98,) with decreased GH production rate and GH half-life, decreased IGF-I response to GH and GHRH stimuli that lead to osteoporosis, increase in fat and decrease in lean body mass and tissue function (Corpas et al., 1993
, Endocrine Rev
. 14:20-39).
In addition, genetic disorders of growth have also been ascribed to defects in the GHRH-GH-IGF-I axis, as those of GHRH receptor (Cao, Wagner, Hindmarsh, Eble, & Mullis, 1995
, Pediatr. Res
. 38:962-966), GH gene (Cogan et al., 1993
, J. Clin. Endocrin
. &
Metab
. 76:1224-1228; Vnencak-Jones et al., 1988
, PNAS
85:5615-5619), GH receptor (Amselem et al., 1993
, Human Molec. Gen
. 2:355-359; Amselem et al., 1991
, Paediatrica Scandinavica—Supplement
377:81-86; Meacham et al., 1993
, J. Clin. Endocrin
. &
Metab
. 77:1379-1383) and pit-1 (Parks et al, 1993
, Hormone Research
40:54-61) a pituitary specific transcription factor. In many cases growth retardation (GR) is a secondary manifestation of an unrelated primary affection (Turner syndrome, chronic renal failure, ovary resistant syndrome) or the exact cause of GR can not be established (Parks et al., in
Molecular Endocrinology: Basic Concepts and Clinical Correlations
(ed. Weintraub, B. D., Raven Press Ltd., New York, 1995) p.473-490).
In these cases of GR where the GHRH-GH-IGF-I axis is unaffected and in elderly, as well as in nonstatural related catabolic conditions (burn, sepsis, trauma associated pathology, chronic obstructive pulmonary disease), GH or GHRH replacement therapy is efficient.
Recombinant GH therapy is currently used in clinics, but a large number of studies have shown that side effects occur frequently, including edema, hypertension, carpal tunnel syndrome, hyperinsulinemia and impaired glucose tolerance (Marcus et al., 1990
, J. Clin. Endocrin
. &
Metab
. 70:519-527; Salomon et al., 1989
, New Engl. J. Med
. 321:1797-1803).
GH and IGF-1 also have beneficial effects on immune function (LeRoith, D. et al.,
Endocrinology
137:1071-1079 (1996)); Kotzmann, H. et al.,
Neuroendocrinology
60:618-625 (1994)). In farm animals, GHRH is galactopoietic (stimulates milk production) with no alteration in milk composition, increases the feed to milk conversion and sustains growth, mostly through increased lean body mass (Enright, W. J. et al.,
Journal of Animal Science
71:2395-2405 (1993); Enright, W. J. et al.,
Journal of Dairy Science
69:344-351 (1986)).
Studies have shown that relatively small amounts of GHRH are required to stimulate the production and secretion of GH in all species. Some benefits of increasing GH in non-human vertebrate animals are improved growth rates, an increase in lean body mass, an increase in feed efficiency in pigs, beef cattle and sheep, increased milk production in dairy cows and goats, and enhanced production of lean meat and egg production in poultry.
GH also enhances the immune system in animals. In animals GHRH will have a great therapeutic utility in the treatment of cachexia in chronic diseases such as cancer, diabetes, due to growth hormone production abnormalities, enhancement of burn and wound healing, bone healing, retardation of the aging process and osteoporosis. However, the greatest use will be in agricultural animals. Intramuscular injection of DNA vector can persist for several months to produce sustained levels of GHRH. The intramuscular delivery of GHRH vector represents a practical method for improving performance in livestock animals.
Current limitations of recombinant GHRH therapy are the high cost of recombinant proteins, the short half-life of the peptides in vivo and the requirement for frequent administration (1-3 times/day) of either subcutaneous or intravenous injections. Using a GHRH injectable DNA plasmid based vector will enhance endogenous GH secretion in vertebrate animals with GH deficiencies in a manner more closely mimicking the natural process and in a less expensive manner than classical therapies.
SUMMARY OF THE INVENTION
The present invention is based in part on the identification of certain nucleic acid sequences which confer advantageous tissue targeting, expression, and secretion properties. Such sequences are utilized in the construction of plasmid vectors encoding GHRH, for delivery and expression of the GHRH coding sequences.
Expression of these vectors can be tissue specific. These vectors are useful in facilitating enhanced expression in tissues as well as in targeting expression with tissue specificity. These vectors can be used to treat diseases by gene therapy by restricting expression of a gene encoded on the vector to targeted tissues. Vectors containing such sequences are able to provide gene delivery and controlled expression of recombinant genes within tissues; such expression can be at levels that are useful for gene therapy and other applications. These vectors can also be used to create transgenic animals for research or livestock improvement.
The ability of the expression vector to provide enhanced product secretion as well as direct expression to specific tissues allows the vector to be used for treating numerous diseases. The above vectors can be used in gene therapy where a vector encoding a therapeutic product is introduced into a tissue so that the tissue will express the therapeutic product. For example, the above vectors may be used for treating muscle atrophy associated with neurological, muscular, or systemic disease or aging by causing tissues to express certain trophic factors.
In addition, the vectors can be used for gene replacement of inherited genetic defects or acquired hormone deficiencies, for vaccination in humans or animals to induce immune responses, or for creating transgenic animals. The transgenic animals can be used as models for studying human diseases, for assessing and exploring novel therapeutic methods, to assess the effects of chemical and physical carcinogens, and to study the effect of various genes and genetic regulatory elements. Furthermore, transgenic animals can be used to develop commercially important livestock species. The above vectors can also be used to transform cells to produce particular proteins and RNA in vitro.
Expression of such vectors having a GHRH encoding sequence in the body of a vertebrate, e.g., a human, can produce both direct and indirect effects. The GHRH produces direct effects by the direct action of the GHRH polypept
Draghia-Akli Ruxandra
Eastman Eric M.
Li Xuyang
Schwartz Robert J.
Baylor College of Medicine
Lyon & Lyon LLP
Nguyen Dave T.
Shukla Ram R.
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