The use of high flash point, low vapor pressure compositions for injection into, and coating of, gas and oil wells and surrounding underground hydrocarbon bearing formations and processing equipment for the purpose of removing scale, paraffins, tars, and other viscous constituents. Treatment results in increased flow of gas and/or oil and decreased adhesion of soil and scale in all aspects of oil and gas recovery, including hydrocarbon bearing formations, casings, lines and pumping equipment. The composition contains about 40 to 99 wt. % of a fatty acid alkyl ester blend and about 1 to 25 wt. % of at least one lower alkyl glycol ether.

This invention relates to the use of high flash point, low vapor pressure compositions for injection into, and coating of, gas and oil wells and surrounding underground formations and processing equipment for the purpose of removing scale, paraffins, tars, and other viscous constituents. Treatment results in increased flow of gas and/or oil and decreased adhesion of soils and scale in all aspects of oil and gas recovery, including formations, casing perforations, casings, lines, tanks, and pumping equipment.
The efficient recovery of oil and gas from wells depends on maintaining clean formations, casing perforations, lines and pumping equipment. Oil wells have a problem with the build up of paraffin, sulfur, scale, heavy oil, and tar by-products. These residues foul the wells and the casing perforations and the lines that carry the oil or gas to the surface, and foul the pumps and metal rods which are used to recover the oil or gas from the well.
Typical prior art methods consist of four major types of products. The first method uses aggressive blends of aromatic and/or aliphatic petroleum hydrocarbons or halogenated hydrocarbons. These products may or may not contain surfactants to allow the products to mix with and emulsify in water to increase cleaning efficiency. The presence of water decreases the efficiency of these compositions significantly. Pure solvent blends are immiscible in water and cannot penetrate to the soils. The addition of emulsifiers allows the solvents to blend with the water, but the resulting solvent/emulsion system is not as efficient a cleaner as the pure solvent.
The second method is the use of bacteria to digest the paraffinic and tar-based soils. This system is very dependent on well temperature and is sensitive to environmental factors such as the composition of the oil. This process is typically slower than the solvent-based process.
The third method is predicated on water-based alkaline, hard-surface cleaners. These cleaners generally incorporate alkaline builders, water-soluble solvents, such as glycol ethers, alcohols, and surfactants. Alkaline builders consist of hydroxide, carbonate, phosphate, and silicate. Water-soluble solvents typically consist of ethylene glycol, diethylene glycol, propylene glycol and dipropylene glycol ethers. Typical surfactants are the categories of alkyl phenol ethoxylates, linear alcohol ethoxylates, or alkyl sulfonates, amphoterics, and fatty acid soaps of alkanolamides. The cleaning efficiency of these alkaline compositions on paraffins and other oil-based soils is typically much lower than that of solvent blends. Further, these compositions are not effective for removing scale.
The fourth method of cleaning involves the use of hot oil, which is injected into the well. The hot oil melts and dissolves the paraffins and other oil byproducts and carries them to the surface. Although this method is efficient, the use of hot oil creates a hazardous condition and can adversely affect the wells.
Typical prior art compositions employ solvents of aromatic and/or aliphatic petroleum hydrocarbons or halogenated hydrocarbons. These solvents produce vapor emissions, commonly known as xe2x80x9cVOCsxe2x80x9d (volatile organic compounds), which typically are toxic. Other cleaning compositions require little or no VOCs, but require high levels of caustic and/or phosphates which cause problems in transportation, use, and disposal.
There are many problems associated with the above-mentioned methods and solvents. For example, halogenated hydrocarbons affect worker health adversely and are ozone-depleting chemicals. Some of the solvents and methods may adversely affect the quality of the oil or gas. Many of the non-halogenated solvents used are either flammable or combustible, resulting in increased fire and explosion risks and higher insurance premiums. Furthermore, the disposal of the spent solvents, in accordance with government regulations, is expensive. In fact, most of the halogenated solvents are subject to high taxes and are in the process of being phased out. In addition to the above-mentioned problems, limits on the amount of VOCs that may be emitted into the atmosphere are set by the Environmental protection Agency, as specified by the xe2x80x9cClean Air Act.xe2x80x9d These limits are determined by the solvent content of the incoming production of raw material versus the solvent content of the waste or finished products.
The bacteria-based cleaning solution also has other disadvantages. The treatment requires that the well be shut down for two weeks to allow the establishment of a bacterial colony. When pumping is resumed, the bacteria are removed with the oil, which results in rapid depletion of the colony and retreatment is required.
An object of the present invention is to provide a method of cleaning gas and oil wells, equipment, well casing perforations, and surrounding formations, pumps, as well as lines, pipes, and the like using an effective cleaning composition which is free of objectionable petroleum-derived or halogenated hydrocarbon and alkaline solvents and useful in removing most of the soils and scale encountered in gas- and oil-well cleaning operations.
Another object is to use a composition which is safe and biodegradable.
A further object is to use a composition capable of functioning at ambient temperatures and at elevated temperatures.
A further object is to use a safe liquid cleaning composition containing fatty acid methyl esters and lower alkyl glycol ethers, which is effective in removing paraffins, tars, related soils, and scale from gas- and oil-wells, casings and equipment.
An additional object is to use a low VOC composition ( less than 50%), which is effective as a cleaner for gas- and oil-wells, casings and equipment.
An additional object of the invention is to use a composition with a flash point above 200xc2x0 F.
A further object is to use a composition which will produce a coating on well casings, lines, pumps, pipes and other equipment, to prevent the adhesion and accumulation of paraffins, other related soils, and scale on these parts, to help retard corrosion, and to allow more efficient operation and consistent production between cleanings.
An additional object is to provide a composition which is low evaporative, widely dispersive, and results in differential wetting of well structures and equipment.
An additional object is to provide enhanced gas and oil recovery from wells.
A further object is a method to separate excess water from recovered crude petroleum oil.
The present invention is directed to a method for removing and preventing the buildup of soils, scale, or both, from gas or oil wells, hydrocarbon bearing formations, and recovery, pumping, storage, and transmission equipment, comprising introducing into said well, formation or equipment a composition comprising about 40 to 99 wt % of a fatty acid alkyl ester blend and about 1 to 25 wt % of at least one lower alkyl glycol ether.
The present invention is also directed to a method for separating excess water, sludge, or both, from recovered crude petroleum oil comprising adding to the crude petroleum oil a composition comprising about 40 to 99 wt % of a fatty acid alkyl ester blend and about 1 to 25 wt % of at least one lower alkyl glycol ether, and then agitating the mixture.
In addition, the present invention provides an improved fracing process of forcing sand and/or another granular material into an oil or gas formation under pressure, whereby the improvement comprises mixing the sand with a composition comprising about 40 to 99 wt % of a fatty acid allyl ester blend and about 1 to 25 wt % of at least one lower alkyl glycol ether.
The most common industrial uses of fatty acid alkyl ester blends are as industrial metal working lubricants, in soap manufacturing, as a plasticizer for waxes and as solvents in the formulation of industrial oils and leather treating compounds. These compounds are also used as textile lubricants and foam depressants, and also in the formulation of low VOC press washes.
It was discovered that combining fatty acid alkyl ester blends with lower alkyl glycol ethers provides cleaning compositions having excellent properties, i.e., high flash point, good soil and paraffin penetration, and excellent protection against resoiling and scale buildup. The compositions used in the methods of the present invention are blended to provide low VOCs (less than 50%) and to provide a composition with a flash point above 200xc2x0 F. TCC (tag closed cup). The compositions are also safe and biodegradable.
The present invention is directed to methods for removing and preventing the buildup of paraffins, other related soils, and scale from gas and oil wells, hydrocarbon bearing formations, and recovery, pumping, storage, and transmission equipment by injecting into the wells and such equipment a cleaning composition comprising about 40 to 99% by weight of a fatty acid alkyl ester blend; and about 1 to 25% by weight of at least one lower alkyl glycol ether, with the remainder being suitable additives. Preferably the composition contains about 60 to 95% by weight, more preferably about 80 to 90% by weight, of the fatty acid alkyl ester blend, and preferably about 1 to 15% by weight of the lower alkyl glycol ether.
There are many types of soils which buildup in gas and oil wells and processing equipment, such as paraffins, tar by-products, and other viscous soils. Further, the buildup of scale is a problem in such wells and equipment. Scale is typically, but not limited to, the accumulation of calcium carbonate deposits and/or iron oxide and other hard residual deposits. The composition of the present invention is effective to remove many types of soils and scale associated with gas and oil recovery and processing and thus enhance the oil recovery process.
The gas and oil processing equipment includes all types and varieties of equipment associated with gas and oil recovery and processing, for example, gas and oil well casings, pumps, pipes, lines, tanks, and the like. It is contemplated that the present composition may be used with all such equipment.
There are several ways that the method of removing or preventing soils and/or scale buildup in gas and oil wells and equipment may be implemented using a composition in accordance with the present invention.
In addition to cleaning the wells and associated equipment, it is often desirable to introduce the composition, through the perforations in the casing, into the surrounding formation. The composition may be forced into the surrounding formation by applied pressure or, if the composition is allowed to set at the bottom of the casing, the composition may seep into the formation without additional pressure. The composition permeates the formation, dissolving blockages in the formation to provide more efficient oil and gas recovery.
A method of cleaning and maintaining a working well, including the surrounding formation, includes the steps of pouring or injecting the composition down the casing side (back lines) of a well and allowing it to mix with the fluid which is already in the well. When enough fluid is present, the composition is then circulated by a pump for 24-72 hours, preferably 48-72 hours. Prior to circulating, the composition may be allowed to set for 8 to 24 hours, for example. The setting time, circulating time and dosage depend on the amount of soil and/or scale anticipated to be present as well as the depth of the well. A basic initial dosage can be, but is not limited to, 20 gallons of composition and for maintaining a clear structure, at least about 5 gallons of composition per well on a periodic basis, e.g. biweekly, monthly, bimonthly.
If there is a great amount of soil and/or scale present in the well or associated equipment, if the pump or rods are frozen, or if the surrounding formation is clogged, alternative methods may be necessary. Thus another method of cleaning and maintaining a working well, including the surrounding formation, initially mixes oil and the composition at ambient temperatures and then introduces the mixture into the casing. The ratio of oil and composition and the amount of the mixture introduced depends on the amount and type of soil and scale present in the well and on associated equipment. The mixture can contain about 10-200 gallons of composition for every 40 barrels of oil. Preferably the mixture contains about 20 gallons of composition for every 40 barrels of oil. The mixture is pumped down the backside or casing side of the well. The mixture is usually allowed to set prior to circulating. The mixture may be used at ambient temperature or may be heated to 150-160xc2x0 F. prior to introduction to the well.
In another embodiment, the composition and oil may be introduced into the well separately, at ambient or elevated temperatures, preferably with the composition being introduced first and forced into the formation by the weight of the oil. The mixture may then be allowed to set, circulated, or both.
In other embodiments water is substituted for the oil. As with the oil, the water and composition may be mixed prior to introduction to the well or introduced separately. The water temperature may be ambient temperature or elevated to about 150-160xc2x0 F. Again, the mixture may be allowed to set, circulated, or both.
After introducing into a well, the composition of the present invention coats the surfaces of the well, formations, and associated equipment to clean and to prevent the future adhesion and accumulation of paraffins, other soils, and scale. The composition may also be applied directly to the equipment. For example, prior to placing rods and casings into gas and/or oil wells, these parts may be sprayed with the composition, or the parts may be dipped into tanks filled with the composition to prevent corrosion and buildup of scale and soils.
The composition may be introduced by means of injection pumps into off-shore gas or oil wells to reduce soils, particularly paraffin, or scale adhesion in well casings and transmission lines. In addition to the problems associated with land oil wells, off shore wells have the further problem of the ocean or sea water behaving as a coolant of the lines and contents between the bottom of the ocean and the platform. Thus off shore wells have a particular problem with paraffin buildup. To treat the lines, 40-50 gallons of the composition, for example, are dropped into the lines. Preferably, the composition is heated prior to being introduced into the lines.
The composition used in the methods of the claimed invention contains ingredients in amounts effective to clean the wells, formations, and equipment and/or to provide an effective coating on their surfaces to prevent future buildup of soils and scale and corrosion. The composition is low evaporative and provides differential wetting of the surfaces; thus, the composition will not evaporate to any significant extent and it will provide a coating that remains on the surface for useful periods of time.
The fatty acid alkyl ester blends useful in compositions of the present invention preferably contain C2 to C8 esters of C4 to C22 fatty acids having the formula:
wherein R1 is C4 to C22 alkyl and R2 is C1 to C8 alkyl. Fatty acid esters are derived from natural products, and thus comprise more than one ester; hence blend. Typically, fatty acid esters are derived from the esterification of fatty acids or the transesterification of animal fats or vegetable oils.
The fatty acid alkyl ester blend more preferably contains methyl, ethyl, n-propyl, isopropyl, or n-butyl esters of C4 to C22 fatty acids. Most preferably, the fatty acid alkyl ester blend contains methyl esters. The fatty acid methyl ester blends are preferably blends with a cloud point of 40xc2x0 F. and a high degree of unsaturation to increase solvency. More preferably, the fatty acid methyl ester blends are derived from soya, canola, and other vegetable oils, with a cloud point of 20xc2x0-32xc2x0 F. and an iodine value of 90-130.
1 to 25 wt. % of a lower alkyl glycol ether may be added as a penetrant, to reduce viscosity of the mixture, as a coupling agent, and/or to increase efficacy on hydrophilic soils. Examples of lower alkyl glycol ethers useful in the present composition include dipropylene glycol monomethyl ether, tripropylene glycol monomethyl ether, ethylene and diethylene glycol ether, methyl, ethyl, propyl and butyl ethers, such as ethylene glycol monobutyl ether, or mixtures thereof.
A polyoxyalkylene glycol ether may be present in the composition in amounts of about 1 to 40 wt. %, preferably 3 to 25 wt. %, and most preferably 3 to 10 wt. %. The actual amount utilized depends on the types of soil present in the equipment to be cleaned and on the water content of the well or equipment being cleaned. Preferably used are polyethylene glycol ethers and polypropylene glycol ethers having the formulas:
Rxe2x80x94Oxe2x80x94(C2H4O)xH and Rxe2x80x94Oxe2x80x94(C3H6O)xH
where, in each formula, R is C1 to C8 alkyl and x is greater than 4. R is preferably methyl, ethyl, propyl, or butyl. More preferably, the polyoxyalkylene glycol ether is an n-butoxy polyalkylene glycol ether. Commercial polyoxyalkylene glycol ether formulations available include Macol 300, Macol 660, WSL-2000, WSL-3520, and WSL-5100 produced by PPG Mazer, Gurnee, Ill. The polyoxyalkylene glycol ether preferably has a molecular weight of between about 200 and 600 and a viscosity of between about 15 and 150 cps when measured at 25xc2x0 C. using a Brookfield LVT Viscometer with a No.2 spindle at 60 r.p.m.
Preferably, at least 1 wt %, more preferably, 1.5 to 3 wt %, of antioxidants are included in the composition. Antioxidants suitable for the present invention include, but are not limited to, (BHT) 2,6-di-tert-butyl-para-cresol, (BHA) 2,6-di-tert-butyl-para-anisole, Eastman inhibitor O A B M-oxalyl bis(benzylidenehydrazide), and Eastman DTBMA 2,5-di-tert-butylhydroquinone. A surfactant may also be added to the composition. Any surfactant suitable for use in cleaning oily soils may be used, such as ethoxylated nonylphenols, linear alcohol ethoxylates, alkanolamine salts of dodecylbenzene sulfonic acid, sulfosuccinates, phosphate esters, alcohol sulfates, quaternary ammonium compounds, amphoteric surfactants, alpha-olefin sulfonates, sorbitan, and fatty acid derivatives. The surfactant is added in an amount effective to perform as a wetting agent and emulsifier, and usually up to 10 wt. %, preferably 1-3 wt %, of the composition.
Commercial surfactants include the EXXATE series of surfactants obtained from EXXON. EXXATE 1000 is an acetic acid ester of C9-C11 branched oxo-alcohol. DBE (DuPont) is a blend of 45-75 wt. % dimethyl glutarate, 10-25 wt. % dimethyl adipate, and 15-30 wt. % dimethyl succinate.
Up to 50 wt. % of other additives may be added, as needed, for particular applications, such as to vary the VOC levels, increase penetration of the mixture, decrease viscosity of the mixture, as couplers for solvents insoluble in the mixture, and to provide solvents for oleophilic and hydrophilic soils. It is within the skill of the art to determine the amount and type of additive needed for a particular application.
Suitable additives include terpenes, terpene alcohols, C8-C14 alcohol ester blends, glycols, acid esters, diacid esters, petroleum hydrocarbons, amino acids, alkanolamines, and amines. Examples of terpenes include d-limonene and xcex1 and xcex2 pinene and terpene alcohols, including a-terpineol. C8-C14 alcohol ester blends include EXXATE 900, 1000, and 1300 from Exxon Chemical; glycols include propylene glycol, dipropylene glycol, and tripropylene glycol. Acid esters include methyl oleate and methyl linoleate, and diacid esters include methyl or butyl diesters of glutaric, adipic, and succinic acids. Petroleum hydrocarbons include AROMATIC 100, AROMATIC 150, ISOPAR M, and ISOPAR K.
Amines such as morpholene, 1,3-dimethyl-2-imidazolidinone, 1,3 propanediamine, 2-amino-1,3-propanediol, and 3-amino propanol, and alkanolamines such as triethanolamine, diethanolamine, 2-aminomethyl propanol, and monoethanolamine act as dispersants for soils and solubilize fatty acids and oils. Amino acids, such as choline and choline hydroxide, provide nontoxic alternatives to monoethanolamine, and act as metal chelators, preferably, methyl or isobutylesters of C4-C6 aliphatic dibasic esters and n-methyl-2 pyrrolidone. Preferably up to 5 wt % of n-methyl-2 pyrrolidone is included.
Other additives typically used in cleaning compositions may be used, including water softening agents, sequesterants, and corrosion inhibitors, which are added in amounts effective to perform their intended function. These additives and amounts thereof are well within the skill of the art. Suitable water softening agents include linear phosphates, styrene-maleic acid co-polymers, and polyacrylates. Suitable sequesterants include 1,3-dimethyl-2-imidazolidinone, 1-phenyl-3-isoheptyl-1,3-propanedione, and 2-hydroxy-5-nonylacetophenoneoxime. Examples of corrosion inhibitors include 2-aminomethyl propanol, diethylethanolamine benzotriazole, and methyl benzotriazole.
All additives preferably have a flash point greater than 190xc2x0 F. TCC in order to achieve a final composition flash point greater than 200xc2x0 F.
Typical treatment of a well requires pumping the cleaning solution into the well casing, and circulating the solution from 24 to 72 hours. A typical well has a 4xc2xdxe2x80x3 casing which extends down through the depth of the well. The 4xc2xdxe2x80x3 casing at the surface is capped by a well head. The well head has a fitting on top through which 2xe2x85x9cxe2x80x3 tubing is inserted. The tubing can extend to the bottom of the well and can be removed in sections for cleaning and maintenance. There are two 2-inch ports on the side of the well head which support the back lines. The normal procedure consists of injecting 5 to 20 gallons of the cleaning solution into the casing side of the well (back lines), and circulating it up the central 2xe2x85x9cxe2x80x3 tubing. The depth of the well, casing diameter, and anticipated amount and nature of the soils, determine the exact dosage needed. The flow of the cleaning solution is maintained for 24 to 72 hours to remove all soils. Depending on buildup, repeated treatments may be provided every 2 to 3 weeks.
Another embodiment of the present invention is directed to a method for separating excess water and sludge from recovered crude petroleum oil. Crude oil contaminated with sludge and/or water is not pure enough for resale. The water or sludge is generally removed by xe2x80x9crollingxe2x80x9d the tank via injecting natural gas under pressure or by adding dry ice to cause agitation of the liquid. The process of oil/water or oil/sludge separation is slow unless an accelerator is added. Thus a further embodiment of the present invention comprises adding to the crude petroleum oil in an amount to accelerate oil/water or oil/sludge separation, a composition comprising about 40 to 99 wt. % of a fatty acid alkyl ester blend and about 1 to 25 wt. % of at least one lower alkyl glycol ether and then agitating to mix.
Typically 1-100 gallons of composition are used to separate water and sludge from 100-400 barrels of oil. The composition is added to the tank and then the tank is xe2x80x9crolledxe2x80x9d by introducing natural gas or carbon dioxide for about 24 hours. The use of the composition in the tank resulted in complete separation of the water, which was removed from the bottom of the holding tank. Standard indicator test strips or indicator reagents for water in oil indicated no detectable level of water.
Further, the composition may be added to crude oil, in amounts of 0.001 to 0.01% by weight, to reduce the formation of sludge and paraffins in oil transported through transmission pipelines, including offshore pipelines.
Other applications using the composition of the present invention include adding the composition during fracing processes, that is the during the process of forcing sand or other material into a hydrocarbon bearing formation under pressure. In accordance with the present invention, the composition is mixed with the sand or other material at a rate of, for example, 1-5 gallons per cubic yard. The sand/composition blend is forced into the a hydrocarbon bearing formation by any one of a variety of methods. Such methods include, but are not limited to, carbon dioxide injection, oil injection, gel injection, and sand/slurry injection.

Cleaning compositions for oil and gas wells, lines, casings, formations and equipment and methods of use


A hydrostatically set packer is disclosed. The actuating piston is locked for run in by a lock sleeve and lock dog arrangement. When the desired depth is reached well pressure is built up to break a rupture disc to allow hydrostatic pressure to act on one side of a lock sleeve. The other side of the lock sleeve is exposed to a chamber under atmospheric or low pressure. Movement of the lock sleeve releases the lock dog allowing piston movement to set the packer. The lock sleeve has a labyrinth seal so that seal leakage below a predetermined level will not prematurely activate the piston lock and prematurely set the packer. A variety of designs for the labyrinth are described.

The field of this invention is labyrinth seal design as well as a particular application to a lock for a hydrostatically set packer.
Hydrostatically set devices for subterranean wells such as packers in the past have relied on an initially locked piston. When the packer was placed at the desired depth, the locking mechanism was released, generally by pressurization of the wellbore, or an electromechanical device or system. The electromechanical devices include, but are not limited to, systems that rely on acoustic, pressure pulse, or vibratory communication methods to enable the setting sequence of the packer or other downhole device. The applied wellbore pressure would break a rupture disc to expose the lock to hydrostatic pressure. In the embodiment of the electromechanical device, the device would expose the lock to hydrostatic pressure. Hydrostatic pressure, acting on one side of the lock against an atmospheric chamber on the other side of the lock, allowed the lock to move. Release of the lock permits piston movement. The piston moves due to the force of hydrostatic pressure across the piston which would set the slips and compress the seal against the borehole wall or tubular, or otherwise actuate the device. A lock ring would hold the set.
A potential problem with this known design was that seal leakage could allow pressure to prematurely communicate to one side of the lock so that the packer would be prematurely unlocked and consequently, hydrostatically set. Thus, an objective of the present invention is to acknowledge that seal leakage is a potential occurrence with drastic and expensive consequences and to deal with that possibility. The objective is met using a wide variety of labyrinth seals on the lock sleeve assembly. Even if leakage of the seals below a predetermined level were to occur, the labyrinth seal would prevent a net force from occurring on the lock sleeve, thus preventing premature hydrostatic setting of the packer.
Labyrinth seals have been used in different tools in downhole applications. They have been used in perforating guns, as shown in U.S. Pat. Nos. 4,886,126 and 5,680,905. They have been used in downhole turbo-machines, U.S. Pat. No. 4,264,285 and in a fluid flow regulator, U.S. Pat. No. 4,858,644. Hydraulically released locks for packers have been used in U.S. Pat. No. 5,320,183.
U.S. Pat. No. 5,720,349 shows the use of a labyrinth seal in an assembly of an anchor, whipstock, and a starter mill. The labyrinth seal compensates for thermal expansion of a fluid filled system to prevent setting of the anchor due to pressure that would have otherwise built up due to thermal effects. This device is focused on compensation for pressure from thermal expansion. On the other hand, U.S. Pat. No. 5,689,905, in the context of a perforating gun, is focused on use of the labyrinth seal to prevent premature actuation of the gun due to seal leakage. Those skilled in the art will appreciate the scope of this invention from the description of the preferred embodiment, which appears below and the claims, which appear thereafter.
A hydrostatically set packer is disclosed. The actuating piston is locked for run in by a lock sleeve and lock dog arrangement. When the desired depth is reached well pressure is built up to break a rupture disc to allow hydrostatic pressure to act on one side of a lock sleeve. The other side of the lock sleeve is exposed to a chamber under atmospheric or low pressure. Movement of the lock sleeve releases the lock dog allowing piston movement to set the packer. The lock sleeve has a labyrinth seal so that seal leakage below a predetermined level will not prematurely activate the piston lock and prematurely set the packer. A variety of designs for the labyrinth are described.

Labyrinth lock seal for hydrostatically set packer


The invention is directed to LERK-6 as a purified and isolated protein, the DNA encoding the LERK-6, host cells transfected with cDNAs encoding LERK-6.

The present invention relates to cytokine polypeptides designated as LERK-6 that bind to the hek or elk receptor, the nucleic acids encoding such polypeptides, processes for production of recombinant LERK-6 polypeptides, and pharmaceutical compositions containing such polypeptides.
Proteins known as the receptor tyrosine kinases have an intrinsic kinase activity that is activated upon ligand binding. This class of proteins is characterized by conserved structural motifs within the catalytic domains (Hanks et al., Science, 242:42, 1988) and can be subdivided into families based on structural features of the regions N-terminal to the catalytic domain.
Boyd et al. (J. Biol. Chem., 267:3262, 1992) purified a cell-surface glycoprotein exhibiting tyrosine kinase activity. The amino acid sequence identified this protein as a member of the eph/elk family, and the protein was thus designated hek (human eph/elk-like kinase). A monoclonal antibody immunoreactive with hek was used to study hek expression on a number of human cell types (Boyd et al., supra). Hek antigen was detected on the human pre-B cell leukemia cell line LK63 (the cell line employed as the immunogen against which the antibody was raised) and the human T-cell leukemia cell line, JM. The Raji B lymphoma cell line showed weak hek antigen expression, and the remaining cell lines tested (both normal and tumor cell lines, among which were hemopoietic cell lines that included pre-B and T-cell lines) were consistently negative. Of the normal and tumor tissue biopsy specimens that were also tested for hek antigen expression, none of the normal tissues was positive and only a very low proportion of hemopoietic tumors was positive.
Expression of hek transcripts in the above-described LK63 and JM cell lines, as well as the human T-cell leukemia cell line HSB-2, has been demonstrated by northern blot analysis (Wicks et al., Proc. Natl. Acad. Sci. USA, 89:1611, 1992). Nucleotide and amino acid sequences for an isolated hek cDNA clone are presented in Wicks et al., supra.
The cell surface protein designated elk is another member of the eph-related tyrosine kinase receptor family of proteins. A partial clone of elk was first discovered in a rat brain cDNA expression library that was screened for proteins expressing tyrosine kinase activity (Letwin et al., Oncogene 3:621, 1988). Later, a composite sequence spanning the entire elk coding region was derived from partial clones isolated from a rat brain cDNA library and a rat cerebellar brain library using the partial clone as a probe (Lhotak et al., Mol. Cell. Biol. 11:2496, 1991).
The hek and elk proteins are closely related to a number of other receptor tyrosine kinases, including the hek homologs mek4 and cek4 (Sajjadi et al. New Biol. 3:769, 1991); eek (Chan et al. Oncogene 6:1057, 1991); erk (Chan et al. supra.), eck (Lindberg et al. Mol. Cell. Biol. 10:6316, 1990); cekS (Pasquale, E. B. Cell Regulation 2:523, 1991); and eph (Hirai et al. Science 238:1717, 1987). The proteins of this subfamily are related not only in their cytoplasmic domains, but also in their extracellular domains, which are 41 to 68% identical. Interestingly, the tissue distributions of these various receptors are diverse. For example, expression of elk mRNA has been reported to be limited to testis and brain (Lhotak et al., supra), whereas eck is found not only in these same two tissues but in lung, intestine, kidney, spleen, ovary, and skin as well. In addition, most eph-related receptors are primarily expressed in the brain. Due to the homology of the receptors in the eph family, a given ligand for one specific receptor may also bind other receptors.
Those ligands that have been identified for the receptor tyrosine kinases are a diverse group of proteins that affect the growth, differentiation, and survival of cells expressing the receptors. Ligands for hek and elk have been isolated, as discussed in more detail below.
Identification of additional ligands for hek and elk that may exist would prove useful in investigating the nature of cellular processes regulated by signaling through these receptors. If enhancement or inhibition of a particular biological signal mediated through these receptors is desired, it is advantageous to identify each of the proteins that may play a role in transduction of such signals. Further, it is known that certain proteins can bind to receptors without initiating signal transduction, including interleukin-1 receptor antagonist protein (Eisenberg et al., Nature 343:341, 1990; Hannum et al., Nature 343:336, 1990; and Carter et al., Nature 344:633, 1990). Identification of additional proteins that bind hek or elk is also desirable in order to determine whether such proteins function as antagonists.
The present invention pertains to mammalian LERK-6 as an isolated or homogeneous protein. In addition, the invention is directed to isolated DNAs encoding mammalian LERK-6 and to expression vectors comprising a cDNA encoding mammalian LERK-6. Within the scope of this invention are host cells that have been transfected or transformed with expression vectors that comprise a cDNA encoding LERK-6, and processes for producing LERK-6 by culturing such host cells under conditions conducive to expression of LERK-6.
In addition, LERK-6 can be bound to a solid phase matrix and used to affinity-purify or separate cells that express hek or elk on their cell surface. The invention encompasses separating cells having the hek or elk receptor on the surface thereof from a mixture of cells in solution, comprising contacting the cells in the mixture with a contacting surface having a LERK-6 molecule thereon, and separating the contacting surface and the solution.
A cDNA encoding murine LERK-6 has been isolated and is disclosed in SEQ ID NO:1. An exon from the human LERK-6 has been isolated and is disclosed in SEQ ID NO:7. DNA encoding a full length human LERK-6 has been isolated and is disclosed in SEQ ID NO:9. This discovery of a cDNA encoding LERK-6 enables construction of expression vectors comprising cDNAs encoding LERK-6; host cells transfected or transformed with the expression vectors; biologically active LERK-6 as homogeneous proteins; and antibodies immunoreactive with LERK-6.
LERK-6 may be useful in the enhancement, stimulation, proliferation or growth of cells expressing the hek or elk receptor. Since the hek or elk receptor is found in the tissue of the brain and testis, treatment of a variety of conditions associated with tissue damage thereof is possible. Moreover, the ligand and receptor complex may be involved in neural growth, development and/or maintenance. While not limited to such, particular uses of the LERK-6 are described infra.
As used herein, the term xe2x80x9cLERK-6xe2x80x9d refers to a genus of polypeptides that bind and complex independently with hek or elk receptor found on T-cells and brain cells. The term xe2x80x9cLERK-6xe2x80x9d encompasses polypeptides having the amino acid sequence 1-184 of SEQ ID NO:2, proteins that are encoded by nucleic acids that contain the nucleic acid sequence of SEQ ID NO:7, and polypeptides having the amino acid sequence 1-184  30 to 213 of SEQ ID NO:10. In addition, LERK-6 encompasses polypeptides that have a high degree of similarity or a high degree of identity with the amino acid sequence 1-184 of SEQ ID NO:2, the amino acid sequence of SEQ ID NO:8, and amino acids 1-184  30 to 213 of SEQ ID NO: 10  NO:10, and which polypeptides are biologically active and bind the hek or elk receptor. In addition, the term xe2x80x9cmurine LERK-6xe2x80x9d refers to biologically active gene products of the DNA of SEQ ID NO: I and the term xe2x80x9chuman LERK-6 refers to biologically active gene products of the DNA of SEQ ID NO:9. Further encompassed by the term xe2x80x9cLERK-6xe2x80x9d are the GPI-linked proteins (which include an extracellular region and a C-terminal hydrophobic region), and soluble or truncated proteins that comprise primarily the receptor-binding portion of the protein, retain biological activity and are capable of being secreted. Specific examples of such soluble proteins are those comprising the sequence of amino acids 1 (Ala)-145 (Asn) of SEQ ID NO:2 and those comprising the sequence of amino acids 1-145  30 to 174 of SEQ ID NO: 10.
The term xe2x80x9chek/elkxe2x80x9d means either hek or elk or both hek and elk. For example, the term xe2x80x9canti-hek/elk antibodiesxe2x80x9d refers to antibodies against either hek or elk. The term xe2x80x9chek/elk-expressing cellsxe2x80x9d refers to cells that express either the hek receptor or the elk receptor, or cells that express both the hek and elk receptors.
The term xe2x80x9cbiologically activexe2x80x9d as it refers to LERK-6, means that the LERK-6 is capable of binding to hek/elk.
xe2x80x9cIsolatedxe2x80x9d means that LERK-6 is free of association with other proteins or polypeptides, for example, as a purification product of recombinant host cell culture or as a purified extract.
A xe2x80x9cLERK-6 variantxe2x80x9d as referred to herein, means a polypeptide substantially homologous to native LERK-6, but which has an amino acid sequence different from that of native LERK-6 (human, murine or other mammalian species) because of one or more deletions, insertions or substitutions. The variant amino acid sequence preferably is at least 80% identical to a native LERK-6 amino acid sequence, most preferably at least 90% identical. The percent identity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Variants may comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known. Naturally occurring LERK-6 variants or alleles are also encompassed by the invention. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the LERK-6 protein, wherein the LERK-6 binding property is retained. Alternate splicing of mRNA may yield a truncated but biologically active LERK-6 protein, such as a naturally occurring soluble form of the protein, for example. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the LERK-6 protein (generally from 1-5 terminal amino acids).
LERK-6 is predicted to be anchored to the cell surface via glycosyl-phosphatidylinositol (GPI) linkage. GPI membrane anchors, including the chemical structure and processing thereof, are described in Ferguson, M. and Williams, A., Ann. Rev. Biochem., 57:285, 1988 (hereby incorporated by reference). When initially expressed, certain proteins comprise a C-terminal hydrophobic domain that contains signals for GPI anchoring. A cleavage site is located upstream, often about 10-12 amino acids upstream of the N-terminus of the hydrophobic domain. Post translational processing includes cleavage of the protein at this cleavage site. A GPI anchor attaches to the newly exposed C-terminal amino acid of the processed, mature protein. Thus, when LERK-6 proteins are expressed in cells that recognize GPI anchoring signals in the hydrophobic domain, the full length amino acid sequence of SEQ ID NO:2 and SEQ ID NO: 10 represents precursor forms of the proteins.
Based on consensus sequences derived from other GPI-anchored proteins, the hydrophobic region in LERK-6 of the present invention likely is between and includes, amino acids 170 (Leu) and 184 (Ser) of SEQ ID NO:2 and amino acids 199 (Leu) and 213(Ser) of SEQ ID NO:10. Between the hydrophobic domain and the receptor-binding domain, is a spacer region of about 22 to 25 amino acids in length. The spacer region extends from approximately amino acid 145 (Asn), 146 (Glu) or 147 (Thr) to about amino acid 169 (His) of SEQ ID NO:2 and from approximately amino acid 174 (Asn), 175(Glu) or 176(Thr) to about amino acid 198(Arg) of SEQ ID NO: 10.
Example 3 describes the construction of a novel hek:Fc fusion protein that may be utilized in the screening for LERK-6. Other antibody Fc regions may be substituted for the human IgG1 Fc region described in the Example. Other suitable Fc regions are those that can bind with high affinity to protein A or protein G, and include the Fc region of human IgG1 or fragments of the human or murine IgG1 Fc region, e.g., fragments comprising at least the hinge region so that interchain disulfide bonds will form. The hek:Fc fusion protein offers the advantage of being easily purified. In addition, disulfide bonds form between the Fc regions of two separate fusion protein chains, creating dimers.
As described supra, an aspect of the invention is soluble LERK-6 polypeptides. Soluble LERK-6 polypeptides comprise all or part of the extracellular domain of a native LERK-6 but lack the GPI signal that would cause retention of the polypeptide on a cell membrane. Soluble LERK-6 polypeptides advantageously comprise the native (or a heterologous) signal peptide when initially synthesized to promote secretion, but the signal peptide is cleaved upon secretion of LERK-6 from the cell. Soluble LERK-6 polypeptides encompassed by the invention retain the ability to bind the hek or elk receptor. Indeed, soluble LERK-6 may also include part of the GPI signal or part of the cytoplasmic domain or other sequences, provided that the soluble LERK-6 protein can be secreted.
Soluble LERK-6 may be identified (and distinguished from its non-soluble membrane-bound counterparts) by separating intact cells which express the desired protein from the culture medium, e.g., by centrifugation, and assaying the medium (supernatant) for the presence of the desired protein. The presence of LERK-6 in the medium indicates that the protein was secreted from the cells and thus is a soluble form of the desired protein.
Soluble forms of LERK-6 possess many advantages over the native bound LERK-6 protein. Purification of the proteins from recombinant host cells is feasible, since the soluble proteins are secreted from the cells. Further, soluble proteins are generally more suitable for intravenous administration.
Examples of soluble LERK-6 polypeptides include those comprising a substantial portion of the extracellular domain of a native LERK-6 protein. An example of a soluble murine LERK-6 protein comprises amino acids 1-145 of SEQ ID NO:2 and amino acids 1-145  30 to 174 of SEQ ID NO:10. In addition, truncated soluble LERK-6 proteins comprising less than the entire extracellular domain are included in the invention. When initially expressed within a host cell, soluble LERK-6 may additionally comprise one of the heterologous signal peptides described below that is functional within the host cells employed. Alternatively, the protein may comprise the native signal peptide. In one embodiment of the invention, soluble LERK-6 can be expressed as a fusion protein comprising (from N- to C-terminus) the yeast xcex1-factor signal peptide, a FLAG(copyright) peptide described below and in U.S. Pat. No. 5,011,912, and soluble LERK-6 consisting of amino acids 1-134 of SEQ ID NO:2 or amino acids 30-163 of SEQ ID NO:10. This recombinant fusion protein is expressed in and secreted from yeast cells. The FLAG(copyright) peptide facilitates purification of the protein, and subsequently may be cleaved from the soluble LERK-6 using bovine mucosal enterokinase. Isolated DNA sequences encoding soluble LERK-6 proteins are encompassed by the invention.
Truncated LERK-6, including soluble polypeptides, may be prepared by any of a number of conventional techniques. A desired DNA sequence may be chemically synthesized using techniques known per se. DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels. Linkers containing restriction endonuclease cleavage site(s) may be employed to insert the desired DNA fragment into an expression vector, or the fragment may be digested at cleavage sites naturally present therein. The well known polymerase chain reaction procedure also may be employed to amplify a DNA sequence encoding a desired protein fragment. As a further alternative, known mutagenesis techniques may be employed to insert a stop codon at a desired point, e.g., immediately downstream of the codon for the last amino acid of the receptor-binding domain.
As stated above, the invention provides isolated or homogeneous LERK-6 polypeptides, both recombinant and non-recombinant. Variants and derivatives of native LERK-6 proteins that retain the desired biological activity (e.g., the ability to bind hek/elk) may be obtained by mutations of nucleotide sequences coding for native LERK-6 polypeptides. Alterations of the native amino acid sequence may be accomplished by any of a number of conventional methods. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene wherein predetermined codons can be altered by substitution, deletion or insertion. Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methods in Enzymol 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462 all of which are incorporated by reference.
LERK-6 may be modified to create LERK-6 derivatives by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives of LERK-6 may be prepared by linking the chemical moieties to functional groups on LERK-6 amino acid side chains or at the N-terminus or C-terminus of a LERK-6 polypeptide or the extracellular domain thereof. Other derivatives of LERK-6 within the scope of this invention include covalent or aggregative conjugates of LERK-6 or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. For example, the conjugate may comprise a signal or leader polypeptide sequence (e.g. the (xcex1-factor leader of Saccharomyces) at the N-terminus of a LERK-6 polypeptide. The signal or leader peptide co-translationally or post-translationally directs transfer of the conjugate from its site of synthesis to a site inside or outside of the cell membrane or cell wall.
LERK-6 polypeptide fusions can comprise peptides added to facilitate purification and identification of LERK-6. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.
The invention further includes LERK-6 polypeptides with or without associated native-pattern glycosylation. LERK-6 expressed in yeast or mammalian expression systems (e.g., COS-7 cells) may be similar to or significantly different from a native LERK-6 polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system. Expression of LERK-6 polypeptides in bacterial expression systems, such as E. coli, provides non-glycosylated molecules.
Equivalent DNA constructs that encode various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences not needed for biological activity or binding are encompassed by the invention. For example, N-glycosylation sites in the LERK-6 extracellular domain can be modified to preclude glycosylation, allowing expression of a reduced carbohydrate analog in mammalian and yeast expression systems. N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. The murine LERK-6 and human LERK-6 proteins comprise three such triplets, at amino acids 13-15, 145-147 and 159-161 of SEQ ID NO:2 and amino acids 42-44, 174-176 and 188-190 of SEQ ID NO:10, respectively. Appropriate substitutions, additions or deletions to the nucleotide sequence encoding these triplets will result in prevention of attachment of carbohydrate residues at the Asn side chain. Alteration of a single nucleotide, chosen so that Asn is replaced by a different amino acid, for example, is sufficient to inactivate an N-glycosylation site. Known procedures for inactivating N-glycosylation sites in proteins include those described in U.S. Pat. No. 5,071,972 and EP 276,846, hereby incorporated by reference.
In another example, sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, preventing formation of incorrect intramolecular disulfide bridges upon renaturation. Other equivalents are prepared by modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present. EP 212,914 discloses the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein. KEX2 protease processing sites are inactivated by deleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivating KEX2 sites. The murine LERK-6 and human LERK-6 contain one KEX2 protease processing site at amino acids 81-82 of SEQ ID NO:2 and amino acids 110-111 SEQ ID NO:10, respectively.
Nucleic acid sequences within the scope of the invention include isolated DNA and RNA sequences that hybridize to the LERK-6 nucleotide sequences disclosed herein under conditions of moderate or severe stringency, and that encode biologically active LERK-6. Conditions of moderate stringency, as defined by Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 101-104, Cold Spring Harbor Laboratory Press, (1989), include use of a prewashing solution of 5xc3x97SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of about 55xc2x0 C., 5xc3x97SSC, overnight. Conditions of severe stringency include higher temperatures of hybridization and washing. The skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as the length of the nucleic acid molecule.
Due to the known degeneracy of the genetic code wherein more than one codon can encode the same amino acid, a DNA sequence may vary from that shown in SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:9 and still encode a LERK-6 protein having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:8, or SEQ ID NO:10, respectively. Such variant DNA sequences may result from silent mutations (e.g., occurring during PCR amplification), or may be the product of deliberate mutagenesis of a native sequence.
The invention provides equivalent isolated DNA sequences encoding biologically active LERK-6, selected from: (a) cDNA comprising the nucleotide sequence presented in SEQ ID NO:1, SEQ ID NO:7, or SEQ ID NO:9; (b) DNA capable of hybridization to a DNA of (a) under moderately stringent conditions and that encodes biologically active LERK-6; and (c) DNA that is degenerate as a result of the genetic code to a DNA defined in (a), or (b) and that encodes biologically active LERK-6. LERK-6 proteins encoded by such DNA equivalent sequences are encompassed by the invention.
DNAs that are equivalents to the DNA sequence of SEQ ID NO:1, and SEQ ID NO:7 or SEQ ID NO:9 will hybridize under moderately and severely stringent conditions to DNA sequences that encode polypeptides comprising amino acids 1-184 of SEQ ID NO:2, SEQ ID NO:8, or amino acids 1-184  30-213 of SEQ ID NO: 10. Examples of LERK-6 proteins encoded by such DNA, include, but are not limited to, LERK-6 fragments (soluble or GPI-linked) and LERK-6 proteins comprising inactivated N-glycosylation site(s), inactivated KEX2 protease processing site(s), or conservative amino acid substitution(s), as described above. LERK-6 proteins encoded by DNA derived from other mammalian species, wherein the DNA will hybridize to the cDNA of SEQ ID NO:1, SEQ ID NO:7 or SEQ ID NO:9 are also encompassed by the present invention.
Variants possessing the requisite ability to bind hek/elk receptor may be identified by any suitable assay. Biological activity of LERK-6 may be determined, for example, by competition for binding to the ligand binding domain of hek/elk receptor (i.e. competitive binding assays).
One type of a competitive binding assay for a LERK-6 polypeptide uses a radiolabeled, soluble LERK-6 and intact hek/elk-expressing cells. Instead of intact cells, one could substitute soluble hek/elk:Fc fusion proteins (such as a hek:Fc or elk:Fc fusion protein) bound to a solid phase through the interaction of a Protein A, Protein G or an antibody to the hek, elk or Fc portions of the molecule, with the Fc region of the fusion protein. Another type of competitive binding assay utilizes radiolabeled soluble hek or elk receptors such as a hek:Fc or elk:Fc fusion protein, and intact cells expressing LERK-6.
Competitive binding assays can be performed following conventional methodology. For example, radiolabeled LERK-6 can be used to compete with a putative LERK-6 homolog to assay for binding activity against surface-bound hek/elk. Qualitative results can be obtained by competitive autoradiographic plate binding assays, or Scatchard plots may be utilized to generate quantitative results.
Alternatively, hek/elk-binding proteins, such as LERK-6 and anti-hek/elk antibodies, can be bound to a solid phase such as a column chromatography matrix or a similar substrate suitable for identifying, separating or purifying cells that express hek/elk on their surface. Binding of a hek/elk-binding protein to a solid phase contacting surface can be accomplished by any means, for example, by constructing a LERK-6:Fc fusion protein and binding such to the solid phase through the interaction of Protein A or Protein G. Various other means for fixing proteins to a solid phase are well known in the art and are suitable for use in the present invention. For example, magnetic microspheres can be coated with LERK-6 and held in the incubation vessel through a magnetic field. Suspensions of cell mixtures containing hek/elk-expressing cells are contacted with the solid phase that has LERK-6 polypeptides thereon. Cells having hek/elk on their surface bind to the fixed LERK-6 and unbound cells then are washed away. This affinity-binding method is useful for purifying, screening or separating such hek/elk-expressing cells from solution. Methods of releasing positively selected cells from the solid phase are known in the art and encompass, for example, the use of enzymes. Such enzymes are preferably non-toxic and non-injurious to the cells and are preferably directed to cleaving the cell-surface binding partner. In the case of hek/elk:LERK-6 interactions, the enzyme preferably would cleave the hek/elk receptor, thereby freeing the resulting cell suspension from the xe2x80x9cforeignxe2x80x9d LERK-6 material. The purified cell population, especially if obtained from fetal tissue, then may be used to repopulate mature (adult) tissues. For example, such purified neural cells can be administered to a patient having a neurodegenerative disorder such as Parkinson""s disease, Alzheiner""s disease, Lou Gehrig""s disease, etc.
Alternatively, mixtures of cells suspected of containing hek/elk+ cells first can be incubated with biotinylated LERK-6. Incubation periods are typically at least one hour in duration to ensure sufficient binding to hek/elk The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides the binding of the cell to the beads. Use of avidin-coated beads is known in the art. See Berenson, et al. J Cell. Biochem., 10D:239 (1986). Wash of unbound material and the release of the bound cells is performed using conventional methods.
As described above, LERK-6 can be used to separate cells expressing hek/elk. In an alternative method, LERK-6 or an extracellular domain or a fragment thereof can be conjugated to a detectable moiety such as 125I to detect hek/elk-expressing cells. Radiolabeling with 125I can be performed by any of several standard methodologies that yield a functional 125I-LERK-6 molecule labeled to high specific activity. Or an iodinated or biotinylated antibody against the hek/elk region or the Fc region of the molecule could be used. Another detectable moiety such as an enzyme that can catalyze a calorimetric or fluorometric reaction, biotin or avidin may be used. Cells to be tested for hek/elk-expression can be contacted with labeled LERK-6. After incubation, unbound labeled LERK-6 is removed and binding is measured using the detectable moiety.
The binding characteristics of LERK-6 (including variants) may also be determined using the conjugated, soluble hek/elk (for example, 125I-hek/elk:Fc) in competition assays similar to those described above. In this case, however, intact cells expressing hek/elk bound to a solid substrate, are used to measure the extent to which a sample containing a putative LERK-6 variant competes for binding with a conjugated a soluble hek/elk to LERK-6.
Other means of assaying for LERK-6 include the use of anti-LERK-6 antibodies, cell lines that proliferate in response to LERK-6, or recombinant cell lines that express hek/elk and proliferate in the presence of LERK-6.
The LERK-6 proteins disclosed herein also may be employed to measure the biological activity of elk or hek proteins in terms of their binding affinity for LERK-6. As one example, LERK-6 may be used in determining whether biological activity is retained after modification of an elk or hek protein (e.g., chemical modification, truncation, mutation, etc.). The biological activity of an elk or hek protein thus can be ascertained before it is used in a research study, or possibly in the clinic, for example.
LERK-6 proteins find use as reagents that may be employed by those conducting xe2x80x9cquality assurancexe2x80x9d studies, e.g., to monitor shelf life and stability of elk protein under different conditions. To illustrate, LERK-6 may be employed in a binding affinity study to measure the biological activity of an elk protein that has been stored at different temperatures, or produced in different cell types. The binding affinity of the modified elk protein for LERK-6 is compared to that of an unmodified elk protein to detect any adverse impact of the modifications on biological activity of elk. Likewise, the biological activity of a hek protein can be assessed using LERK-6.
LERK-6 polypeptides also find use as carriers for delivering agents attached thereto to cells bearing the elk or hek cell surface receptor. Expression of hek antigen has been reported for certain leukemic cell lines, including the human T-cell leukemia cell line designated JM and the human pre-B cell leukemia cell line designated LK63 (Boyd et al., J. Biol. Chem. 267:3262, 1992, and Wicks et al., Proc. Natl. Acad. Sci. USA, 89:1611, 1992). LERK-6 proteins thus can be used to deliver diagnostic or therapeutic agents to these cells (or to other cell types found to express hek on the cell surface) in in vitro or in vivo procedures.
One example of such use is to expose a hek+ or elk+ leukemic cell line to a therapeutic agent/LERK-6 conjugate to assess whether the agent exhibits cytotoxicity toward the leukemic cells. A number of different therapeutic agents attached to LERK-6 may be included in an assay to detect and compare the cytotoxic effect of the agents on the leukemic cells. LERK-6/diagnostic agent conjugates may be employed to detect the presence of hek+ cells in vitro or in vivo.
Diagnostic and therapeutic agents that may be attached to a LERK-6 polypeptide include, but are not limited to, drugs, toxins, radionuclides, chromophores, enzymes that catalyze a colorimetric or fluorometric reaction, and the like, with the particular agent being chosen according to the intended application. Examples of drugs include those used in treating various forms of cancer, e.g., nitrogen mustards such as L-phenylalanine nitrogen mustard or cyclophosphamide, intercalating agents such as cis-diaminodichloroplatinum, antimetabolites such as 5-fluorouracil, vinca alkaloids such as vincristine, and antibiotics such as bleomycin, doxorubicin, daunorubicin, and derivatives thereof. Among the toxins are ricin, abrin, diptheria toxin, Pseudomonas aeruginosa exotoxin A, ribosomal inactivating proteins, mycotoxins such as trichothecenes, and derivatives and fragments (e.g., single chains) thereof. Radionuclides suitable for diagnostic use include, but are not limited to, 123I, 131I, 99mTc, 111In, and 76Br. Radionuclides suitable for therapeutic use include, but are not limited to, 131I, 211At, 77Br, 186Re, 188Re, 212Pb, 212Bi, 109Pd, 64Cu, and 67Cu.
Such agents may be attached to the LERK-6 by any suitable conventional procedure. LERK-6, being a protein, comprises functional groups on amino acid side chains that can be reacted with functional groups on a desired agent to form covalent bonds, for example. Alternatively, the protein or agent may be derivatized to generate or attach a desired reactive functional group. The derivatization may involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to proteins (Pierce Chemical Company, Rockford, Ill.). A number of techniques for radiolabeling proteins are known. Radionuclide metals may be attached to LERK-6 by using a suitable bifunctional chelating agent, for example.
Conjugates comprising LERK-6 and a suitable diagnostic or therapeutic agent (preferably covalently linked) are thus prepared. The conjugates are administered or otherwise employed in an amount appropriate for the particular application.
Another use of the LERK-6 of the present invention is as a research tool for studying the role that LERK-6, in conjunction with elk or hek, may play in growth or differentiation of cells bearing the elk or hek receptor. The LERK-6 polypeptides of the present invention also may be employed in in vitro assays for detection of elk or LERK-6 or the interactions thereof. Likewise, LERK-6 finds use in assays for hek or the interaction of LERK-6 with hek.
As discussed above, when various rat tissues were analyzed for elk mRNA, transcripts were detected only in brain and testis (Lhotak et al., supra). Binding of LERK-6 to eph-related receptors on neural tissue is believed to exert a neuro-protective or neurotrophic effect.
LERK-6 finds use as a tissue culture reagent. A LERK-6 protein can be added to neurons cultured in vitro to enhance the viability or prolong the lifespan of the cultured neurons, thus facilitating research studies, and possible clinical treatment of neural tissue.
One embodiment of the present invention is directed to a method of treating disorders of neural tissue, involving contacting the neural tissue with LERK-6. Such disorders include injury or neurologic diseases, either chronic or acute. A LERK-6 protein may be administered to a mammal to treat such an injury or disease. In one embodiment of the invention, LERK-6 is employed in treating neurodegenerative conditions characterized or mediated, at least in part, by the mechanism of neural death known as excitotoxicity. In addition, LERK-6 may be administered to a mammal to exert a trophic effect on neural tissue. In a patient suffering loss of or damage to neurons due to injury or disease, LERK-6 may enhance the viability of those neurons that have survived.
LERK-6 polypeptides of the invention can be formulated according to known methods used to prepare pharmaceutically useful compositions. LERK-6 can be combined in admixture, either as the sole active material or with other known active materials, with pharmaceutically suitable diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers. Suitable carriers and their formulations are described in Remington""s Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Co. In addition, such compositions can contain LERK-6 complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of LERK-6. LERK-6 can also be conjugated to antibodies against tissue-specific receptors, ligands or antigens, or coupled to ligands of tissue-specific receptors. Where hek/elk is found on neoplastic cells, the LERK-6 may be conjugated to a toxin whereby LERK-6 is used to deliver the toxin to the specific site, or may be used to sensitize such neoplastic cells to subsequently administered anti-neoplastic agents.
LERK-6 can be administered topically, parenterally, or by inhalation. The term xe2x80x9cparenteralxe2x80x9d includes subcutaneous injections, intravenous, intramuscular, intracistemal injection, or infusion techniques. These compositions will typically contain an effective amount of the LERK-6, alone or in combination with an effective amount of any other active material. Such dosages and desired drug concentrations contained in the compositions may vary depending upon many factors, including the intended use, patient""s body weight and age, and route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration can be performed according to art-accepted practices.
LERK-6 polypeptides may exist as oligomers, such as covalently-linked or non-covalently-linked dimers or trimers. Oligomers may be linked by disulfide bonds formed between cysteine residues on different LERK-6 polypeptides. In one embodiment of the invention, a LERK-6 dimer is created by fusing LERK-6 to the Fc region of an antibody (e.g., IgG1) in a manner that does not interfere with binding of LERK-6 to the hek/elk ligand-binding domain. The Fc polypeptide preferably is fused to the C-terminus of a soluble LERK-6 (comprising only the receptor-binding). General preparation of fusion proteins comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991) and Byrn et al. (Nature 344:677, 1990), hereby incorporated by reference. A gene fusion encoding the LERK-6:Fc fusion protein is inserted into an appropriate expression vector. LERK-6:Fc fusion proteins are allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between Fc polypeptides, yielding divalent LERK-6. If fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a LERK-6 oligomer with as many as four LERK-6 extracellular regions. Alternatively, one can link two soluble LERK-6 domains with a peptide linker.
Suitable host cells for expression of LERK-6 polypeptides include prokaryotes, yeast or higher eukaryotic cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-free translation systems could also be employed to produce LERK-6 polypeptides using RNAs derived from DNA constructs disclosed herein.
Prokaryotes include gram negative or gram positive organisms, for example, E. coli or Bacilli. Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E. coli, a LERK-6 polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant LERK-6 polypeptide.
LERK-6 polypeptides may be expressed in yeast host cells, preferably from the Saccharomyces genus (e.g., S. cerevisiae). Other genera of yeast, such as Pichia, K. lactis or Kluyveromyces, may also be employed. Yeast vectors will often contain an origin of replication sequence from a 2xcexc yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657 or in Fleer et. al., Gene, 107:285-195 (1991); and van den Berg et. al., Bio/Technology, 8:135-139 (1990). Another alternative is the glucose-repressible ADH2 promoter described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982). Shuttle vectors replicable in both yeast and E. coli may be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Ampr gene and origin of replication) into the above-described yeast vectors.
The yeast xcex1-factor leader sequence may be employed to direct secretion of the LERK-6 polypeptide. The xcex1-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP 324,274. Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art. A leader sequence may be modified near its 3xe2x80x2 end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
Yeast transformation protocols are known to those of skill in the art. One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp+ transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 xcexcg/ml adenine and 20 xcexcg/ml uracil.
Yeast host cells transformed by vectors containing ADH2 promoter sequence may be grown for inducing expression in a xe2x80x9crichxe2x80x9d medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 xcexcg/ml adenine and 80 xcexcg/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.
Mammalian or insect host cell culture systems could also be employed to express recombinant LERK-6 polypeptides. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988). Established cell lines of mammalian origin also may be employed. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV-1/EBNA-1 cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821, 1991).
Transcriptional and translational control sequences for mammalian host cell expression vectors may be excised from viral genomes. Commonly used promoter sequences and enhancer sequences are derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included.
Exemplary expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986). A useful high expression vector, PMLSV N1/N4, described by Cosman et al., Nature 312:768, 1984 has been deposited as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A-0367566, and in U.S. patent application Ser. No. 07/701,415, filed May 16, 1991, incorporated by reference herein. The vectors may be derived from retroviruses. In place of the native signal sequence, and in addition to an intiator methionine, a heterologous signal sequence may be added, such as the signal sequence for IL-7 described in U.S. Pat. No. 4,965,195; the signal sequence for IL-2 receptor described in Cosman et al., Nature 312:768 (1984); the IL-4 signal peptide described in EP 367,566; the type I IL-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type II IL-1 receptor signal peptide described in EP 460,846.
LERK-6 as an isolated, purified or homogeneous protein according to the invention may be produced by recombinant expression systems as described above or purified from naturally occurring cells. LERK-6 can be purified to substantial homogeneity, as indicated by a single protein band upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
One process for producing LERK-6 comprises culturing a host cell transformed with an expression vector comprising a DNA sequence that encodes LERK-6 under conditions sufficient to promote expression of LERK-6. LERK-6 is then recovered from culture medium or cell extracts, depending upon the expression system employed. As is known to the skilled artisan, procedures for purifying a recombinant protein will vary according to such factors as the type of host cells employed and whether or not the recombinant protein is secreted into the culture medium.
For example, when expression systems that secrete the recombinant protein are employed, the culture medium first may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, (e.g., silica gel having pendant methyl or other aliphatic groups) can be employed to further purify LERK-6. Some or all of the foregoing purification steps, in various combinations, are well known and can be employed to provide a substantially homogeneous recombinant protein.
It is possible to utilize an affinity column comprising the ligand-binding domain of hek/elk receptors to affinity-purify expressed LERK-6 polypeptides. LERK-6 polypeptides can be removed from an affinity column using conventional techniques, e.g., in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized. Alternatively, the affinity column may comprise an antibody that binds LERK-6. Example 5 describes a procedure for employing LERK-6 of the invention to generate monoclonal antibodies directed against LERK-6.
Recombinant protein produced in bacterial culture can be isolated by initial disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or more concentration, salting-out, ion exchange, affinity purification or size exclusion chromatography steps. Finally, RP-HPLC can be employed for final purification steps. Microbial cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
Transformed yeast host cells are preferably employed to express LERK-6 as a secreted polypeptide in order to simplify purification. Secreted recombinant polypeptide from a yeast host cell fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog. 296:171, 1984). Urdal et al. describe two sequential, reversed-phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column.
Useful fragments of the LERK-6 nucleic acids include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target LERK-6 mRNA (sense) or LERK-6 DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the coding region of LERK-6 cDNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniques 6:958, 1988).
Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block expression of LERK-6 proteins. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences. Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10448, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oliginucleotide for the target nucleotide sequence.
Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPO4-mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. Antisense or sense oligonucleotides are preferably introduced into a cell containing the target nucleic acid sequence by insertion of the antisense or sense oligonucleotide into a suitable retroviral vector, then contacting the cell with the retrovirus vector containing the inserted sequence, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or or the double copy vectors designated DCT5A, DCT5B and DCT5C (see PCT Application US 90/02656).
Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
In addition to the above, the following examples are provided to illustrate particular embodiments and not to limit the scope of the invention.


The induction of reducing conditions and stimulating anaerobic process through the addition of species of seaweed (Dulse, Nori, Ascophyllum nodosum, and Kelp) to accomplish accelerated dechlorinization of soil and groundwater contaminated with chlorinated solvents and heavy metals.

This invention aids in the remediation of environmental contaminants in subsurface soils and groundwater via the stimulation of anaerobic processes. Specifically, this invention relates to remediation processes involving the emplacement of solid-phase or aqueous-phase treatment agents. Emplacement of dried seaweed or kelp species as electron donors for microorganisms that carry out reductive dechlorination of chlorinated solvent source areas or plumes is illustrative of the invention.
Various species of Seaweed including Ascophyllum nodosum, Dulse, Nori, and Kelp contain substantial nutrients, beneficial to anaerobic processes. Seaweeds are available in a variety of forms including sheets, meals, flakes and powders that can either be hydrolyzed for solubility or remain insoluble as a slow release remedial product. In addition, dried seaweed can come in various sizes ranging from large granules characteristic of insoluble kelp meal to high mesh sizes of fine powder. The dynamic nature of seaweed has resulted in its wide use in varying commercial fields. Liquid seaweed extract as well as insoluble and (hydrolyzed) soluble dried seaweeds are commonly used as fertilizers to enhance the development and growth rate of plants. Seaweed is also used as a food additive for livestock to promote growth and health. Furthermore, seaweed has been an essential food source for years, used in sushi, chips, seasoning, and even as a dietary supplement for its high nutritional value.
The chemical composition of seaweeds allows for the contribution of stimulating is comprised of fatty acids, carbohydrates, and proteins. Their concentrations of vitamins B2, and B12 in particular make seaweed an excellent alternative for environmental remediation. The species Ascophyllum nodosum contains high levels of enzymes, 17 amino acids, macro and micronutrients, plant hormones (auxins, cytokins, gibberillins), 25% alginic acid, and over 50% of carbohydrates and polysaccharides. Seaweeds also have over 72 minerals, and assorted vitamins (B2, B12, K) that encourage the vigorous and healthy growth of subsurface biological life. Seaweed and Kelp offer the necessary micronutrients and volatile fatty acid precursors that will provide long-term production of organic hydrogen necessary for reductive dechlorination of chlorinated solvents in groundwater and soils. The high concentrations of many of these valuable nutrients provide optimal living conditions for the anaerobic processes responsible for the remediation of contaminated soil and groundwater sites.
Chlorinated solvents are the most common class of ground water contaminants at hazardous waste sites in the U.S. In a list of the top 25 most frequently detected contaminants at such sites, the Agency for Toxic Substances and Disease Registry (ATSDR) found that 10 of the top 20, including two of the top three, were chlorinated solvents or their degradation products. National Research Council, Alternatives for Ground Water Cleanup (National Academy Press, Washington, D.C. 1994). In fact, the same survey found that the most common contaminant, trichloroethylene (TCE), is present in more than 40% of National Priority List sites. The remediation of ground water contaminated by these compounds often presents unique obstacles related to their inherent characteristics, including hydrophobicity and high density. Many commercial process utilize raw vegetable oils and emulsions which co-elute the targeted solvents within the treatment liquid masking the presence of the compound targeted for treatment rather than stimulating the mineralization of said compound.
Natural attenuation of chlorinated solvents by reductive dechlorination often occurs at sites where an electron donor (food source or substrate for microbes) is present along with the chlorinated solvent contamination. As dissolved oxygen and other electron acceptors become depleted some microbes are capable of using the chlorinated solvents as electron acceptors. For selected compounds such as chlorinated ethylenes sequential dechlorination to a harmless byproduct ethylene can be achieved under favorable environmental conditions (EPA/600/R-10 98/128 September 1998).
In recent years efforts have been made to produce this anaerobic treatment effect by injection of electron donor into the subsurface. An overview of these technologies can be reviewed in the EPA document Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents: Fundamentals and Field Applications (EPA 542-R-00-008 July 2000). Other inorganic and organic compounds can be degraded or immobilized under anaerobic conditions including selected toxic metals, nitrate, and MTBE. For sites that do not have sufficient amounts of natural electron donors to drive anaerobic natural attenuation, injection of microbial substrates has proven to be a cost-effective treatment or plume migration control measure. The microbial substrates can be injected into the contaminant source area where residual contamination is adsorbed onto soils or injected in a line across a ground water contaminant plume to form a permeable reactive wall to prevent further contaminant migration.
A wide variety of sugars, alcohols, organic acids, and even molecular hydrogen have been used successfully as electron donors to enhance anaerobic biotransformation processes. Most of these compounds are rapidly consumed after injection and must be replaced by either continuous low concentration delivery systems or with frequent batch additions of additive solution. Contaminant source areas can not be effectively removed or even precisely located for many ground water contaminant plumes. The presence of residual chlorinated solvents adsorbed onto soils or present as dense non-aqueous phase product (DNAPL) serves as an example of persistent ground water plume source areas that can last for many decades. These persistent contaminant source areas continue to contaminate ground water for many years such that continuous operation of recirculation systems or frequent substrate injections can be very costly over the life of a project. Long-term injection of substrates into wells or infiltration galleries often leads to severe bacterial fouling problems adding to project operation and maintenance costs.
Recent interest has developed in the use of materials that slowly biodegrade or slowly release organic matter into ground water over time. A variety of vegetable oils have been demonstrated to be effective electron donors to stimulate anaerobic biodegradation. Although edible oils such as soybean oil have a much lower viscosity than a semisolid product, distribution in saturated soils is difficult. Soybean oil has a viscosity approximately times higher than water, which results in multiphase fluid flow and potential oil blockage of soil porosity. Injection of pure oil or large droplets of emulsified oil blocks soil pores producing treatment zones that are ineffective because they prevent free flow of ground water through the oil treated area. Injection of pure soybean oil into porous soil media has been shown to reduce water permeability by up to 100%.
In addition to slowly biodegradable hydrogen sources, soil and groundwater remediation process that utilize zero-valent metals have been applied with varying success. In the second embodiment of the invention, the addition of zero-valent metals to the micro dried seaweed or kelp allows for maintained reducing conditions resulting in greater longevity of the reactive metal surface. Zero-valent metal particles have been proven to effectively degrade halogenated solvents. For example, the mechanism and reaction rates of which iron reduces chlorinated aliphatics has been studied extensively due to iron's low cost and low toxicity. Additionally, the pathways of the dehalogenation of DNAPL's such as TCE have been proposed. TCE undergoes hydrogenolysis where the replacement of each of the three chlorines occurs sequentially. TCE reduces to cis-1,2-dichloroethene, trans-1,2-dichloroethene, and 1,1-dichloroethene. These intermediates in turn reduce to ethene and ethane.

Method for the treatment of ground water and soils using mixtures of seaweed and kelp

Innovative Environmental Technologies, Inc.