Compositions and methods for modifying the content of polyunsaturated fatty acids in mammalian cells

This application claims priority from U.S. Ser. No. 60/275,222, filed Mar. 12, 2001, the contents of which are incorporated herein by reference in their entirety.

Some of the work presented herein was supported by a grant from the National Institutes of Health (CA79553). The United States government may, therefore, have certain rights in the invention.

This invention relates to compositions and methods for altering the content of polyunsaturated fatty acids in mammalian cells.

Polyunsaturated fatty acids (PUFAs) are fatty acids having 18 or more carbon atoms and two or more double bonds. They can be classified into two groups, n-6 or n-3, depending on the position (n) of the double bond nearest the methyl end of the fatty acid (Gill and Valivety, Trends Biotechnol. 15:401-409, 1997; Broun et al., Annu. Rev. Nutr. 19:197-216, 1999; Napier et al., Curr. Opin. Plant Biol. 2:123-127, 1999). The n-6 and n-3 PUFAs are synthesized through an alternating series of desaturations and elongations beginning with either linoleic acid (LA, 18:2n6) or α-linolenic acid (ALA, 18:3n3), respectively (Gill and Valivety, supra; Broun et al., supra; Napier et al., supra). The major end point of the n-6 pathway in mammals is arachidonic acid (AA, 20:4n6) and major end points of the n-3 pathway are eicosapentaenoic acid (EPA, 20:5n3) and docosahexaenoic acid (DHA, 22:6n3).

An important class of enzymes involved in the synthesis of PUFAs is the class of fatty acid desaturases. These enzymes introduce double bonds into the hydrocarbon chain at positions determined by the enzyme\'s specificity. Although, in most cases, animals contain the enzymatic activity to convert LA (18:2n6) and ALA (18:3n3) to longer-chain PUFA (where the rate of conversion is limiting), they lack the 12- and 15-desaturase activities necessary to synthesize the precursor (parent) PUFA, LA and ALA (Knutzon et al., J. Biol. Chem. 273:29360-29366, 1998). Furthermore, the n-3 and n-6 PUFA are not interconvertible in mammalian cells (Goodnight et al., Blood 58: 880-885, 1981). Thus, both LA and ALA and their elongation, desaturation products are considered essential fatty acids in the human diet. The PUFA composition of mammalian cell membranes is, to a great extent, dependent on dietary intake (Clandinin et al., Can. J. Physiol. Pharmacol. 63:546-556, 1985; McLennan et al., Am. Heart J. 116:709-717, 1988).

To the contrary, some plants and microorganisms are able to synthesize n-3 fatty acids such as ALA (18:3n-3) because they have membrane-bound 12- and 15- (n-3) desaturases that act on glycerolipid substrates in both the plastid and endoplasmic reticulum (Browse and Somerville, Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 467-506, 1991). Genetic techniques have led to the identification of the genes encoding the 12- and 15-desaturases from Arabidopsis thaliana and other higher plant species (Okuley et al., Plant Cell 6:147-158, 1994; Arondel et al., Science 258:1353-1355, 1992). Recently, a fat-1 gene encoding an n-3 fatty acid desaturase was cloned from Caenorhabditis elegans (Spychalla et al., Proc. Natl. Acad. Sci. USA 94:1142-1147, 1997; see also U.S. Pat. No. 6,194,167).

The present invention is based, in part, on the discovery that the C. elegans n-3 desaturase gene, fat-1, can be successfully introduced into other types of animal cells (e.g., mammalian cells), where it quickly and effectively elevates the cellular n-3 PUFA content and dramatically balances the ratio of n-6:n-3 PUFAs. More specifically, heterologous expression of the fat-1 gene in rat cardiac myocytes rendered those cells capable of converting various n-6 PUFAs to the corresponding n-3 PUFA and changed the n-6:n-3 ratio from about 15:1 (an undesirable ratio) to 1:1 (a desirable ratio). In addition, an eicosanoid derived from n-6 PUFA (i.e. arachidonic acid) was significantly reduced in the transgenic cells (as described further below, levels of arachidonic acid can be assessed to determine whether a given nucleic acid encodes a biologically active desaturase; similarly, one can assess the levels of n-6 PUFA; the levels of n-3 PUFA; and the ratio of n-6:n-3 PUFAs). Accordingly, the present invention features compositions (e.g., nucleic acids encoding fat-1, optionally and operably linked to a constitutively active or tissue-specific promoter) and methods that can be used to effectively modify the content of PUFAs in animal cells (i.e., cells other than those of C. elegans, for example, mammalian cells such as myocytes, neurons (whether of the peripheral or central nervous system), adipocytes, endothelial cells, and cancer cells). More generally, a fat-1 sequence or a biologically active variant thereof can be operably linked to a regulatory sequence. Regulatory sequences encompass not only promoters, but also enhancers or other expression control sequence, such as a polyadenylation signal, that facilitates expression of the nucleic acid. The modified cells (whether in vivo or ex vivo (e.g., in tissue culture)), transgenic animals containing them, and food products obtained from those animals (e.g., meat or other edible parts of the animals (e.g., liver, kidney, or sweetbreads)) are also within the scope of the present invention.

In one embodiment, the invention features mammalian cells that contain a nucleic acid sequence encoding the C. elegans n-3 desaturase or biologically active variants (e.g., fragments or other mutants) thereof. Biologically active variants of the n-3 desaturase enzyme are variants that retain enough of the biological activity of a wild-type n-3 desaturase to be therapeutically or clinically effective (i.e., variants that are useful in treating patients, producing transgenic animals, or conducting diagnostic or other laboratory tests). For example, variants of n-3 desaturase can be mutants or fragments of that enzyme that retain at least 25% of the biological activity of wild-type n-3 desaturase. For example, a fragment of an n-3 desaturase enzyme is a biologically active variant of the full-length enzyme when the fragment converts n-6 fatty acids to n-3 fatty acids at least 25% as efficiency as the wild-type enzyme does so under the same conditions (e.g., 30, 40, 50, 75, 80, 90, 95, or 99% as efficient as wild-type n-3 desaturase). Variants may also contain one or more amino acid substitutions (e.g., 1%, 5%, 10%, 20%, 25% or more of the amino acid residues in the wild-type enzyme sequence can be replaced with another amino acid residue). These substitutions can constitute conservative amino acid substitutions, which are well known in the art. Cells that express a fat-1 sequence (optionally, operably linked to a constitutively active or tissue-specific promoter) are valuable aids to research because they provide a convenient system for characterizing the functional properties of the fat-1 gene and its product (cells in tissue culture are particularly convenient, but the invention is not so limited). They also allow one to study any cellular mechanism mediated by n-3 fatty acids without the lengthy feeding procedures of cells or animals that are currently required, and they serve as model systems that can be used, for example, to evaluate existing methods and to design new methods for effectively transferring sequences encoding an n-3 desaturase into cells in vivo. In any of these contexts (e.g., whether the compositions of the invention are being used to treat patients, to generate transgenic animals, or in cell culture assays), nucleic acids encoding fat-1 or a biologically active variant thereof can be co-expressed (by way of the same or a separate vector) with a heterologous gene. The heterologous gene can be, for example, another therapeutic gene (e.g., a receptor for a small molecule or chemotherapeutic agent) or a marker gene (e.g., a sequence encoding a fluorescent protein, such as green fluorescent protein (GFP) or enhanced (EGFP)).

The nucleic acids of the invention can be formulated for administration to a patient. For example, they can be suspended in sterile water or a physiological buffer (e.g., phosphate-buffered saline) for oral or parenteral administration to a patient (e.g., intravenous, intramuscular, intradermal, or subcutaneous injection (in the event the patient has a tumor, the compositions can be injected into the tumor or adminstered to the tissue surrounding the site from which a tumor was removed) or by inhalation).

The invention also features transgenic animals (including any animal kept as livestock or as a food source) that express the C. elegans n-3 desaturase gene or a biologically active variant thereof. Given the discovery that a C. elegans fat-1 gene can be efficiently expressed when delivered to a mammalian cell, this gene can be used to generate transgenic mice or larger transgenic animals (such as cows, pigs, sheep, goats, rabbits or any other livestock or domesticated animal) according to methods well known in the art. Depending on whether the construct used contains a constitutively active promoter or a tissue-specific promoter (e.g., a promoter that is active in skeletal muscle, breast tissue, the colon, neurons, retinal cells, pancreatic cells (e.g., islet cells) etc.) the fat-1 gene can be expressed globally or in a tissue-specific manner. The cells of the transgenic animals will contain an altered PUFA content that, as described further below, is more desirable for consumption. Thus, transgenic livestock (or any animal that is sacrificed for food) that express the desaturase enzyme encoded by the fat-1 gene will be superior (i.e., healthier) sources of food. Food obtained from these animals can be provided to healthy individuals or to those suffering from one or more of the conditions described below.

As noted, the invention features methods of treating patients (including humans and other mammals) who have a condition associated with an insufficiency of n-3 PUFA or an imbalance in the ratio of n-3:n-6 PUFAs by administering a nucleic acid encoding an n-3 desaturase or a biologically active variant thereof (e.g., a fragment or other mutant). Alternatively, one can administer the protein encoded. The methods can be carried out with patients who have an arrhythmia or cardiovascular disease (as evidenced, for example, by high plasma triglyceride levels or hypertension), cancer (e.g., breast cancer or colon cancer), inflammatory or autoimmune diseases (such as rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease (IBD), asthma, chronic obstructive pulmonary disease, lupus, diabetes, Sjogren\'s syndrome transplantation, anlkylosing spondylitis, polyarteritis nodosa, reiter\'s syndrome, and scleroderma), a malformation (or threatened malformaticn, as occurs in premature infants) of the retina and brain, diabetes, obesity, skin disorders, renal disease, ulcerative colitis, Crohn\'s disease, chronic obstructive-pulmonary disease, or who are at risk of rejecting a transplanted organ. Given that fat-1 expression can also inhibit cell death (by apoptosis) in neurons, the methods of the invention can also be used to treat or prevent (e.g., inhibit the likelihood of, or the severity of) neurodegenerative diseases. Accordingly, the invention features methods of treating a patient who has (or who may develop) a neurodegenerative disease such as Parkinson\'s disease, Alzheimer\'s disease, Huntington\'s disease (HD), spinal and bulbar muscular atrophy (SBMA; also known as Kennedy\'s disease), dentatorubral-pallidoluysian atrophy, spinocerebellar ataxia type 1 (SCA1), SCA2, SCA6, SCA7, or Machado-Joseph disease (MJD/SCA3) (Reddy et al. Trends Neurosc. 22:248-255, 1999). As a balanced n-6:n-3 ratio is essential for normal growth and development, and as noted above, the methods of the invention can be advantageously applied to patients who have no discernable disease or condition.

Abbreviations used herein include the following: AA for arachidonic acid (20:4n-6); DHA for docosahexaenoic acid (22:6n-3); EPA for eicosapentaenoic acid (20:5n-3); GFP for green fluorescent protein; Ad.GFP for adenovirus carrying GFP gene; Ad.GFP.fat-1 for adenovirus carrying both fat-1 gene and GFP gene; and PUFAs for polyunsaturated fatty acids.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, useful methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflicting subject matter, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the Examples.