A complex is described for delivery to a cell comprising inserting a nucleic acid into a reverse micelle. The reverse micelle has the property to compact the nucleic acid for easier delivery. Other molecules are used to interact with the nucleic acidxe2x80x94micelle complex to further enhance delivery such as a surfactant having a disulfide bond.

N/A
The invention generally relates to micellar systems for use in biologic systems. More particularly, a process is provided for the use of reverse micelles for the delivery of nucleic acids and genes to cells.
Biologically active compounds such as proteins, enzymes, and nucleic acids have been delivered to the cells using amphipathic compounds that contain both hydrophobic and hydrophilic domains. Typically these amphipathic compounds are organized into vesicular structures such as liposomes, micellar, or inverse micellar structures. Liposomes can contain an aqueous volume that is entirely enclosed by a membrane composed of lipid molecules (usually phospholipids) (R. C. New, p. 1, chapter 1, xe2x80x9cIntroductionxe2x80x9d in Liposomes: A Practical Approach, ed. R. C. New IRL Press at Oxford University Press, Oxford, 1990). Micelles and inverse micelles are microscopic vesicles that contain amphipathic molecules but do not contain an aqueous volume that is entirely enclosed by a membrane. In micelles the hydrophilic part of the amphipathic compound is on the outside (on the surface of the vesicle) whereas in inverse micelles the hydrophobic part of the amphipathic compound is on the outside. The inverse micelles thus contain a polar core that can solubilize both water and macromolecules within the inverse micelle. As the volume of the core aqueous pool increases the aqueous environment begins to match the physical and chemical characteristics of bulk water. The resulting inverse micelle can be referred to as a microemulsion of water in oil (Schelly, Z. A. Current Opinion in Colloid and Interface Science, 37-41, 1997; Castro, M. J. M., Cabral, J. M. S. Biotech. Adv. 6, 151-167, 1988).
Microemulsions are isotropic, thermodynamically stable solutions in which substantial amounts of two immicible liquids (water and oil) are brought into a single phase due to a surfactant or mixture of surfactants. The spontaneously formed colloidal particles are globular droplets of the minor solvent, surrounded by a monolayer of surfactant molecules. The spontaneous curvature, H0 of the surfactant monolayer at the oil/water interface dictates the phase behavior and microstructure of the vesicle. Hydrophilic surfactants produce oil in water (O/W) microemulsions (H0 greater than 0), whereas lipophilic surfactants produce water in oil (W/O) microemulsions. When the hydropbile-lipophile properties of the surfactant monolayer at the water/oil interface are balanced bicontinuous-type microemulsions are formed (H0=0).
Positively-charged, neutral, and negatively-charged liposomes have been used to deliver nucleic acids to cells. For example, plasmid DNA expression in the liver has been achieved via liposomes delivered by tail vein or intraportal routes. Positively-charged micelles have also been used to package nucleic acids into complexes for the delivery of the nucleic acid to cells. Negatively-charged micelles have been used to condense DNA, however they have not been used for the delivery of nucleic acids to cells (Imre, V. E., Luisi, P. L. Biochemical and Biophysical Research Communications, 107, 538-545, 1982). This is because the previous efforts relied upon the positive-charge of the micelles to provide a cross-bridge between the polyanionic nucleic acids and the polyanionic surfaces of the cells. Micelles that are not positively-charged, or that do not form a positively charged complex cannot perform this function. For example, a recent report demonstrated the use of a cationic detergent to compact DNA, resulting in the formation of a stable, negatively-charged particle (Blessing, T., Remy, J. S., Behr, J. P. Proc. Natl. Acad. Sci. USA, 95, 1427-1431, 1998). A cationic detergent containing a free thiol was utilized which allowed for an oxidative dimerization of the surfactant to the disulfide in the presence of DNA. However, as expected, the negatively-charged complex was not effective for transfection. Reverse (water in oil) micelles has also been used to make cell-like compartments for molecular evolution of nucleic acids (Tawfik D. S. and Griffiths, A. D. Nature Biotechnology 16:652, 1998). Cleavable micellar systems was not used in this system.
In addition, Wolff et al. have developed a method for the preparation of DNA/amphipathic complexes including micelles in which at least one amphipathic compound layer that surrounds a non-aqueous core that contains a polyion such as a nucleic acid (Wolff, J., Budker, V., and Gurevich, V. U.S. Pat. No. 5,635,487).
Cleavable Micelles
A new area in micelle technology involves the use of cleavable surfactants to form the micelle. Surfactants containing an acetal linkage, azo-containing surfactants, elimination of an ammonium salt, quaternary hydrazonium surfactants, 2-alkoxy-N,N-dimethylamine N-oxides, and ester containing surfactants such as ester containing quaternary ammonium compounds and esters containing a sugar have been developed.
These cleavable surfactants within micelles are designed to decompose on exposure to strong acid, ultraviolet light, alkali, and heat. These conditions are very harsh and are not compatible with retention of biologic activity of biologic compounds such as proteins or nucleic acids. Thus, biologically active compounds have not been purified using reverse micelles containing cleavable surfactants.
Complexation of Nucleic Acids with Polycations
Polymers are used for drug delivery for a variety of therapeutic purposes. Polymers have also been used for the delivery of nucleic acids (polynucleotides and oligonucleotides) to cells for therapeutic purposes that have been termed gene therapy or anti-sense therapy. One of the several methods of nucleic acid delivery to the cells is the use of DNA-polycation complexes. It was shown that cationic proteins like histones and protamines or synthetic polymers like polylysine, polyarginine, polyornithine, DEAE dextran, polybrene, and polyethylenirine were effective intracellular delivery agents while small polycations like spermine were ineffective. As a result the main mechanism of DNA translocation to the intracellular space might be non-specific adsorptive endocytosis which may be more effective then liquid endocytosis or receptor-mediated endocytosis. Furthermore, polycations are a very convenient linker for attaching specific receptors to DNA and as result, DNA-polycation complexes can be targeted to specific cell types.
There are a variety of molecules (gene transfer enhancing signals) that can be covalently attached to the gene in order to enable or enhance its cellular transport. These include signals that enhance cellular binding to receptors, cytoplasmic transport to the nucleus and nuclear entry or release from endosomes or other intracellular vesicles.
Nuclear localizing signals enhance the entry of the gene into the nucleus or directs the gene into the proxmity of the nucleus. Such nuclear transport signals can be a protein or a peptide such as the SV40 large T ag NLS or the nucleoplasmin NLS.
Other molecules include ligands that bind to cellular receptors on the membrane surface increasing contact of the gene with the cell. These can include targeting group such as agents that target to the asialoglycoprotein receptor by using asiologlycoproteins or galactose residues. Other proteins such as insulin, EGF, or transferrin can be used for targeting. Peptides that include the RGD sequence can be used to target many cells. Chemical groups that react with sulfhydryl or disulfide groups on cells can also be used to target many types of cells. Folate and other vitamins can also be used for targeting. Other targeting groups include molecules that interact with membranes such as fatty acids, cholesterol, dansyl compounds, and amphotericin derivatives.
Size of a DNA complex may be a factor for gene delivery in vivo. Many times, the size of DNA that is of interest is large, and one method of delivery utilizes compaction techniques. The DNA complex needs to cross the endothelial barrier and reach the parenchymal cells of interest. The largest endothelia fenestrae (holes in the endothelial barrier) occur in the liver and have average diameter of 100 nm. The trans-epithelial pores in other organs are much smaller, for example, muscle endothelium can be described as a structure which has a large number of small pores with a radius of 4 nm, and a very low number of large pores with a radius of 20-30 nm.(Rippe, B. Physiological Rev, 1994). The size of the DNA complex is also important for the cellular uptake process. After binding to the target cells the DNA complex should be taken up by endocytosis. Since the endocytic vesicles have a homogenous internal diameter of about 100 nm in hepatocytes, and are of similar size in other cell types, the DNA is compacted to be smaller than 100 nm.
Compaction of DNA
There are two major approaches for compacting DNA:
1. Multivalent cations with a charge of three or higher have been shown to condense DNA. These include spermidine, spermine, Co(NH3)63+,Fe3+, and natural or synthetic polymers such as histone H1, protamine, polylysine, and polyethylenimine. One analysis has shown DNA condensation to be favored when 90% or more of the charges along the sugar-phosphate backbone are neutralized (Wilson, R. W., Bloomfield, V. A. Biochemistry 18, 2192-2196, 1979).
2. Polymers (neutral or anionic) which can increase repulsion between DNA and its surroundings have been shown to compact DNA. Most significantly, spontaneous DNA self-assembly and aggregation process have been shown to result from the confinement of large amounts of DNA, due to excluded volume effect (Strzelecka, T. E., Rill, R. L. Biopolymers 30, 803-14, 1990; Strzelecka, T. E., Rill, R. L. Biopolymers 30, 57-71, 1990). Since self-assembly is associated with locally or macroscopically crowded DNA solutions, it is expected, that DNA insertion into small water cavities with a size comparable to the DNA will tend to form mono or oligomolecular compact structures.
Micelles and Reverse Micelles
Reverse micelles (water in oil microemulsions) are widely used as a host for biomolecules. Examples exist of both recovery of extracelular proteins from a culture broth and recovery of intracellular proteins; Although widely used, recovery of the biomolecules is difficult due to the stability of the formed micelle and due to incomplete recovery during the extraction process. Similarly, purification of DNA or other biomolecules from endotoxin and plasma is difficult to accomplish. One common method employing Triton results in incomplete separation of the DNA or biomolecules from the emulsion.
Reverse micelles have been widely used as a host for enzymatic reactions to take place. In many examples, enzymatic activity has been shown to increase with the used in a micelle, and has allowed enzymatic reactions to be conducted on water insoluble substrates. Additionally, enzymatic activity of whole cells entrapped in reverse micelles has been investigated (Gajar, L., Singh, A., Dubey, R. S., Srivastava, R. C. Applied Biochemistry and Biotechnology, 66, 159-172, 1997). The cationic surfactant cetyl pyridinuim chloride was utilized to entrap Baker""s yeast and Brewer""s yeast inside a reverse micelle.
Micelles have also been used as a reaction media. For example, a micelle has been used to study the kinetic and synthetic applications of the dehydrobromination of 2-(p-nitrophenyl)ethyl bromide. Additionally, micelles have found use as an emulsifier for emulsion polymerizations.
Micelles have been utilized for drug delivery. For example, an AB block copolymer has been investigated for the micellar delivery of hydrophobic drugs. Transport and metabolism of thymidine analogues has been investigated via intestinal absorption utilizing a micellar solution of sodium glycocholate. Additionally, several examples of micelle use in transdermal applications have appeared. For example, sucrose laurate has been utilized for topical preparations of cyclosporin A.
FIG. 1 is a graph illustating the spectra, There are shifts in the position of both the positive and negative bands and in the position of the crossover point for the 20 xcexcL pDNA solution (W0=3.35). Spectra that are similarly shifted are broadly defined asxe2x80x94specta, and are attributed to a condensed form of pDNA. In contrast the spectra of the 60 xcexcL pDNA solution (W0=10.05) resembles the spectra of DNA in buffer alone in respect to crossover point. However is spectra is chbzxurized by an increase in the intensity of the negative band (maximum at 240 nm).xe2x80x9d
The present invention provides for the transfer of polynucleotides, and biologically active compounds into parenchymal cells within tissues in situ and in vivo, utilizing reverse micelles delivered intravasculary, intrarterially, intravenous, orally, intraduodenaly, via the jejunum (or ileum or colon), rectally, transdermally, subcutaneously, intramuscularly, intraperitoneally, intraparenterally, via direct injections into tissues such as the liver, lung, heart, muscle, spleen, pancreas, brain (including intraventricular), spmial cord, ganglion, lymph nodes, lymphatic system adipose tissues, thyroid tissue, adrenal glands, kidneys, prostate, blood cells, bone marrow cells, cancer cells, tumors, eye retina, via the bile duct, or via mucosal membranes such as in the mouth, nose, throat, vagina or rectum or into ducts of the salivary or other exocrine glands.
By xe2x80x9cdeliveredxe2x80x9d we mean that the polynucleotide becomes associated with the cell. The polynucleotide can be on the membrane of the cell or inside the cytoplasm, nucleus, or other organelle of the cell. The process of delivering a polynucleotide to a cell has also been commonly termed xe2x80x9ctransfectionxe2x80x9d or the process of xe2x80x9ctransfectingxe2x80x9d and also it has been termed xe2x80x9ctransformationxe2x80x9d. The polynucleotide could be used to produce a change in a cell that can be therapeutic. The delivery of polynucleotides or genetic material for therapeutic purposes is commonly called xe2x80x9cgene therapyxe2x80x9d. The polynucleotides or genetic material being delivered are generally mixed with transfection reagents prior to delivery. A biologically active compound is a compound having the potential to react with biological components. Pharmaceuticals, proteins, peptides, hormones, cytokines, antigens and nucleic acids are examples of biologically active compounds. The reverse micelle may be negatively-charged, zwitterionic, or neutral. Additionally, the present invention provides for the purification of biomolecules by solubilizing the biomolecule into a cleavable reverse micelle and then cleaving the micelle under conditions that will not destroy the biological activity of the biomolecule. These processes can be used for transferring nucleic acids or biomolecules into cells or an organism such as for drug delivery, or may also be used for analytical methods.
The process of utilizing cleavable reverse micelles for the purification of biomolecules has advantages over current methodology. Isolation of the biomolecule will be enhanced by cleaving the reverse micelle. This will separate the polar group from the non-polar group on the surfactant and therefore eliminate the formation of emulsions and therefore simplify the isolation process. Cleavage conditions will be such that the biological activity of the biomolecule is not destroyed.
Another advantage of the invention is the use of reverse micelles for gene delivery. The reverse micelle can compact the polynucleotide, a critical step for gene delivery, especially in vivo. The micelle containing the compacted polynucleotide can then be utilized as a reaction vesicle in which additional compounds can be added to the DNA. For example, a polycation could be added to the polynucleotide/reverse micelle solution to form a polycation/polynucleotide complex within the reverse micelle. Additionally, the polynucleotide/reverse micelle system is used as a vesicle for template polymerization of the DNA or caging of the DNA in which the polycation is crosslinked. A variety of chemical reactions can take place with in the vesicle preferably without modifying the nucleic acid. The polynucleotide/reverse micelle system also has advantages in that the micelle may be cleaved under physiological conditions involved along the xe2x80x9ctransfection pathway.xe2x80x9d The surfactant can be altered so that micellular cleavage occurs at different point along this pathway. By xe2x80x9ctransfection pathwayxe2x80x9d we mean any point at which the polynucleotide/reverse micelle system is introduced to a solution (i.e., blood, serum) that contains parenchymal cells, or to the cells (for example the skin or mucousal membranes) through the inclusion of the polynucleotide into the nucleus of the parenchymal cell.
In a preferred embodiment, described is a complex for delivery to a cell, comprising: inserting a nucleic acid into a reverse micelle. A compacting agent may be added to the complex as well as a delivery enhancing ligand or compound.
In a preferred embodiment, a process for delivering a complex to a cell is described, comprising inserting a nucleic acid into a reverse micelle.
In a preferred embodiment, the nucleic acid or biomolecule is solubilized into a reverse micelle with an internal water volume for delivery of the biomolecule to parenchymal cells. A compound can be added to the nucleic acid/micelle mixture. Such compounds include polymers such as polyions (polycations such as spermine, polyamines, polylysine, polyethylimine (PEI), and polyanions), proteins, peptides, enzymes, hydrophobic compounds, and amphipathic compounds (to form a second layer around the micelle). Such compounds include compounds that compact the DNA, provide a cell transfer enhancing ligand or provide another layer to the micelle.
In another preferred embodiment, the nucleic acid or biomolecule is complexed with another molecule prior to micelle formation. For example, a polymer such as polylysine is added to the nucleic acid and then crosslinked to cage the nucleic acid. When a nucleic acid is caged a polymer is formed since the polylysine (or any type of polymer) acts as a monomer that is being included in another polymer.
Another preferred embodiment provides a method of making a compound for delivery to a cell, comprising: adding one or more compounds to the nucleic acid or biomolecule/reverse micelle complex prior to delivery to the cell, thereby providing a deliverable complex. For example, another surfactant or a polyion might be added to the complex. The cell can be a prokaryote or eukaryote and can be a plant, animal or mammalian cell.
Another preferred embodiment provides a method of making a compound for delivery to a cell, comprising: modifying a molecule in the presence of the biomolecule/reverse mnicelle complex thereby providing a deliverable complex.
In another preferred embodiment, the parenchymal cell is solubilized within a reverse micelle. A reverse micelle containing a polynucleotide would be added to the parenchymal cell containing reverse micelle. After an appropriate amount of time, the parenchymal cell would be purified, and delivered to a mammal.
In another preferred embodiment the biomolecule is solubilized utilizing one of the following procedures.
Procedure 1.
a) mixing of the biomolecule into an aqueous solution
b) then mixing the aqueous solution containing the biomolecule with a hydrocarbon or halohydrocarbon containing a surfactant with agitation or sonication
Procedure 2.
Mxing of biomolecule into a solution containing a reverse micelle with agitation or sonication.
Procedure 3.
a) mixing the biomolecule into an aqueous solution
b) then extracting the aqueous solution containing the biomolecule with a hydrocarbon or halohydrocarbon containing a reverse micelle and separating the phases.
In another preferred embodiment, the-biomolecule is purified comprising a step in which a reverse micelle is destroyed.
In another preferred embodiment, the biomolecule can be purified by utilizing one of the following procedures.
Procedure 1.
a) mixing of the biomolecule into an aqueous solution
b) then mixing the aqueous solution containing the biomolecule with a hydrocarbon or halohydrocarbon containing a surfactant with agitation or sonication
c) cleaving the reverse micelle
d) extract the biomolecule
Procedure 2.
a) mixing of biomolecule into a solution containing a reverse micelle with agitation or sonication.
b) cleaving the reverse micelle
c) extract the biomolecule
Procedure 3.
a) mixing the biomolecule into an aqueous solution
b) then extracting the aqueous solution containing the biomolecule with a hydrocarbon or halohydrocarbon containing a reverse micelle and separating the phases.
c) cleaving the reverse micelle
d) extract the biomolecule
In another preferred embodiment, the surfactant is a disulfide of general formula A-S-S-B, which contains a hydrophobic group (A) and a hydrophilic group (B).
In another preferred embodiment, the surfactant can be cleaved in the presence of the nucleic acid or biomolecule under conditions that will not destroy the biological activity of the nucleic acid or biomolecule.
In another preferred embodiment, the surfactant could be chemically modified in the presence of the nucleic acid or biomolecule. For example, the surfactant can be polymerized after micelle formation to form a xe2x80x9cshellxe2x80x9d or cage around the nucleic acid. The surfactant could be cleaved separating the bulk of its hydrophilic and hydrophobic parts thus destroying its ability to act as a surfactant.
In yet another preferred embodiment, the possible surfactants can be neutral, negatively charged, or zwitterionic but not positively charged. Neutral surfactants include (but not restricted to) polyoxyethylene alcohol""s, polyoxyethylene isoalcohol, polyoxyethylene p-t-octyl phenol (Triton), polyoxyethylene nonylphenol, polyoxyehtylene esters of fatty acids, polyoxyethylene sorbitol esters (Tween) and lipids. Negatively charged surfactants include (but not restricted to) di-(2-ethyl-hexyl) sodium sulfosuccinate (AOT), sodium dodecylsuphate (SDS), sodium dodecylsuphonate, and sodium dodecyl-N-sarcosinate. The zwitterionic surfactant could contain anionic and cationic groups on the alpha and omega positions of a long aliphatic chain. For zwitterionic surfactants that contain both anionic and cationic groups on the alpha and omega positions of a long aliphatic chain, complex formation should be done under acidic conditions so that the surfactant can have a positive charge that will interact with the nucleic acid. The anionic portion is neutralized by being protonated and therefore interacts with the non-aqueous phase. After formation of the complexes, the complexes are extracted into an aqueous solution containing a higher pH than the pH used to form the complexes.
A biologically active compound is purified is when it is isolated from a mixture of other compounds. It is purified when its purity is increased where purity is defined as the percentage of the mixture containing the desired biologically active compound. Purification can also indicate a process where the purity of the desired compound is not increased but the other compounds within the mixture are changed. Purification also indicates the extraction of a biologic compound from one mixture or solution to another mixture or solution. This can include a process where the desired biologically active compound is moved from one solvent to another (also called extraction) or is solubilized within a solvent. A biologically active compound is a compound having the potential to react with biological components for gene therapy purposes. Pharmaceuticals, proteins, peptides, viruses, antigens, carbohydrates (and congugates), lipids, sacharides, oligonucleotides, and nucleic acids are examples of biologically active compounds.
A surfactant refers to a compound that contains a polar group (hydrophilic) and a non-polar (hydrophobic) group on the same molecule. Cleavable surfactant refers to a surfactant in which the polar group may be separated from the nonpolar group by the removal of a chemical bond located between the two groups, or to a surfactant in which the polar or non-polar group or both may be chemically modified such that the detergent properties of the surfactant are destroyed. Cleavable also means that the surfactant is labile (able to be destroyed or that the detergent properties of the surfactant are able to be destroyed) and that its surfactant properties could be destroyed by other chemical processes than bond cleavage. A disulfide bond that is labile under physiological conditions means the disulfide bond is cleaved more rapidly than oxidized glutathione or any disulfide constructed from thiols in which one of the constituent thiols is more acidic, lower pKa, than glutathione or is activated by intramolecular attack by a free thiol. Constituent in this case means the thiols that are bonded together in the disulfide bond. The surfactant properties of a compound can be destroyed by chemical modification such as converting the polar group into a less polar group. This can be accomplished by a number of chemical modifications including (but not limited to) acylation, alkylation, elimination, reduction or oxidation, of an amine (or its salt), alcohol, diol (di-alcohol) or carboxylic acid groups, or by a multistep process in which several chemical modifications are conducted (for example oxidation of an alcohol to an aldehyde (ketone) followed by nucleophilic addition to the aldehyde (ketone) resulting in an alcohol followed by elimination of the alcohol (or a derivative of it).
Reverse (inverse) micelle refers to a surfactant with an internal aqueous pool. By xe2x80x9caqueousxe2x80x9d we mean containing water, but can include buffers and salts. Non-aqueous solutions are made up of organic solvents such as hydrocarbon or halohydrocarbon. Buffers are made from a weak acid or weak base and their salts. Buffer solutions resist changes in pH when additional acid or base is added to the solution. Salts are ionic compounds that dissociate into cations and anions when dissolved in solution. Salts increase the ionic strength of a solution, and consequently decrease interactions between nucleic acids with other cations.
A reverse micelle is destroyed when the micelle no longer exists and the monophase no longer exists. A reverse micelle is destroyed when the micelle is disrupted. In a preferred embodiment the reverse micelle is destroyed by chemically modifying the surfactant so that water in oil emulsion is destroyed and the phases separate. A destructible reverse micelle is a reverse micelle that can be destroyed such that the water in oil emulsion is destroyed and the phases separate. A destructible reverse micelle can undergo a biological, chemical, or biochemical reaction such that the reverse micelle is destroyed. Biological, chemical, or biochemical reactions involve the formation or cleavage of ionic and/or covalent bonds. In a preferred embodiment the destructible reverse micelle contains a surfactant that is cleavable, destroyable, or chemically modifiable.The surfactant can be a disulfide of the general formula A-S-S-B, in which chemical group A is a hydrophobic group and chemical group B is a hydrophilic group.
The present invention also relates to a method in which a biologically active compound is delivered to a cell comprising a step in which the biologically active compound is mixed with a biologically-labile surfactant. A biologically-labile surfactant is a surfactant in which the hydrophobic moiety is cleaved from the hydrophilic moiety by cellular processes or its surfactant properties are rendered inactive within the cell, tissue or organism. Examples include surfactants that contain disulfide bonds that are labile within the cell, tissue, or organism.
A transfection reagent is a compound or compounds used in the prior art that bind(s) to or complex(es) with polynucleotides and mediates their entry into cells. The transfection reagent also mediates the binding and internalization of polynucleotides into cells. Examples of transfection reagents include cationic liposomes and lipids, calcium phosphate precipitates, and polylysine complexes. At times, the transfection reagent has a net positive charge that binds to the polynucleotide""s negative charge. The transfection reagent mediates binding of polynucleotides to cell via its positive charge (that binds to the cell membrane""s negative charge) or via ligands that bind to receptors in the cell. For example, cationic liposomes or polylysine complexes have net positive charges that enable them to bind to DNA.
Other vehicles are also used, in the prior art, to transfer genes into cells. These include complexing the polynucleotides on particles that are then accelerated into the cell. This is termed biolistic or gun techniques. Other methods include eletroporation in which a device is used to give an electric charge to cells. The charge increases the permeability of the cell.
The term xe2x80x9cnucleic acidxe2x80x9d is a term of art that refers to a polymer containing at least two nucleotides. xe2x80x9cNucleotidesxe2x80x9d contain a sugar deoxyribose (in DNA) or ribose (in RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. xe2x80x9cBasesxe2x80x9d include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and synthetic derivatives of purines and pyrimidines, or natural analogs. Nucleotides are the monomeric units of nucleic acid polymers. A xe2x80x9cpolynucleotidexe2x80x9d is distinguished here from an xe2x80x9coligonucleotidexe2x80x9d by containing more than 80 monomeric units; oligonucleotides contain from 2 to 80 nucleotides. The term nucleic acid includes deoxyribonucleic acid (xe2x80x9cDNAxe2x80x9d) and ribonucleic acid (xe2x80x9cRNAxe2x80x9d). DNA may be in the form of anti-sense, plasmid DNA, parts of a plasmid DNA, vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. RNA may be in the form of oligonucleotide RNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, ribozymes, chimeric sequences, or derivatives of these groups. xe2x80x9cAnti-sensexe2x80x9d is a nucleic acid that interferes with the function of DNA and/or RNA. This may result in suppression of expression. Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, nucleotides, or bases. These include PNAs (peptide nucleic acids), phosphothionates, and other variants of the phosphate backbone of native nucleic acids. In addition, DNA and RNA may be single, double, triple, or quadruple stranded. xe2x80x9cExpression cassettexe2x80x9d refers to a natural or recombinantly produced nucleic acid which is capable of expressing protein(s). A DNA expression cassette typically includes a promoter (allowing transcription initiation), and a sequence encoding one or more proteins. Optionally, the expression cassette may include trancriptilonal enhancers, non-coding sequences, splicing signals, transcription termination signals, and polyadenylation signals. An RNA expression cassette typically includes a translation initiation codon (allowing translation initiation), and a sequence encoding one or more proteins. Optionally, the expression cassette may include translation termination signals, a polyadenosine sequence, internal ribosome entry sites (IRES), and non-coding sequences.
Delivery of a nucleic acid means to transfer a nucleic acid from a container outside a mammal to near or within the outer cell membrane of a cell in the mammal. The term xe2x80x9ctransfectionxe2x80x9d may be used, in general, as a substitute for the term xe2x80x9cdelivery,xe2x80x9d or, more specifically, the transfer of a nucleic acid from directly outside a cell membrane to within the cell membrane. The transferred (or xe2x80x9ctransfectedxe2x80x9d) nucleic acid may contain an expression cassette. If the nucleic acid is a primary RNA transcript that is processed into messenger RNA, a ribosome translates the messenger RNA to produce a protein within the cytoplasm. If the nucleic acid is a DNA, it enters the nucleus where it is transcribed into a messenger RNA that is transported into the cytoplasm where it is translated into a protein. Therefore if a nucleic acid expresses its cognate protein, then it must have entered a cell. A protein may subsequently be degraded into peptides, which may be presented to the immune system.
Polypeptide refers to a linear series of amino acid residues connected to one another by peptide bonds between the alpha-amino group and carboxyl group of contiguous amino acid residues.
xe2x80x9cProteinxe2x80x9d refers herein to a linear series of greater than 2 amino acid residues connected one to another as in a polypeptide. A xe2x80x9ctherapeuticxe2x80x9d effect of the protein in attenuating or preventing the disease state can be accomplished by the protein either staying within the cell, remaining attached to the cell in the membrane, or being secreted and dissociated from the cell where it can enter the general circulation and blood. Secreted proteins that can be therapeutic include hormones, cytokines, growth factors, clotting factors, anti-protease proteins (e.g., alpha1-antitrypsin), angiogenic proteins (e.g., vascular endothelial growth factor, fibroblast growth factors), anti-angiogenic proteins (e.g., endostatin, angiostatin), and other proteins that are present in the blood. Proteins on the membrane can have a therapeutic effect by providing a receptor for the cell to take up a protein or lipoprotein (e.g., low density lipoprotein receptor). Therapeutic proteins that stay within the cell (xe2x80x9cintracellular proteinsxe2x80x9d) can be enzymes that clear a circulating toxic metabolite as in phenylketonuria. They can also cause a cancer cell to be less proliferative or cancerous (e.g., less metastatic), or interfere with the replication of a virus. Intracellular proteins can be part of the cytoskeleton (e.g., actin, dystrophin, myosins, sarcoglycans, dystroglycans) and thus have a therapeutic effect in cardiomyopathies and musculoskeletal diseases (e.g., Duchenne muscular dystrophy, limb-girdle disease). Other therapeutic proteins of particular interest to treating heart disease include polypeptides affecting cardiac contractility (e.g., calcium and sodium channels), inhibitors of restenosis (e.g., nitric oxide synthetase), angiogenic factors, and anti-angiogenic factors.
Biomolecule refers to peptides, polypeptides, proteins, enzymes, polynucleotides, oligonucleotides, viruses, antigens, carbohydrates (and congugates), lipids, and sacharides.
Enzymes are proteins evolved by the cells of living organisms for the specific function of catalyzing chemical reactions.
A chemical reaction can take place within the micelle and nucleic acid complex. A chemical reaction is defined as the formation or cleavage of covalent or ionic bonds. As a result of the chemical reaction a polymer can be formed. A polymer is defined as a compound containing more than two monomers. A monomer is a compound that can be attached to itself or another monomer and thus form a polymer. In one preferred embodiment the surfactant is polymerized by chain or step polymerization and then the surfactant properties are destroyed. This destruction of the surfactant properties could be a accomplished by breaking a chemical bond and separating the hydrophilic and hydrophobic moities.
A chemical reaction can be used to attach a gene transfer enhancing signal to the nucleic acid complex. The gene transfer enhancing signal (or abbreviated as the Signal) is defined in this specification as a molecule that modifies the nucleic acid complex and can direct it or the nucleic acid to a cell location (such as tissue) or location in a cell (such as the nucleus) either in culture or in a whole organism. By modifying the cellular or tissue location of the foreign gene, the expression of the foreign gene can be enhanced.
The gene transfer enhancing signal can be a protein, peptide, lipid, steroid, sugar, carbohydrate, (non-expresssing) nucleic acid or synthetic compound. The gene transfer enhancing signals enhance cellular binding to receptors, cytoplasmic transport to the nucleus and nuclear entry or release from endosomes or other intracellular vesicles.
Nuclear localizing signals enhance the targeting of the gene into proximity of the nucleus and/or its entry into the nucleus. Such nuclear transport signals can be a protein or a peptide such as the SV40 large T ag NLS or the nucleoplasmin NLS. These nuclear localizing signals interact with a variety of nuclear transport factors such as the NLS receptor (karyopherin alpha) which then interacts with karyopherin beta. The nuclear transport proteins themselves could also function as NLS""s since they are targeted to the nuclear pore and nucleus.
Signals that enhance release from intracellular compartments (releasing signals) can cause DNA release from intracellular compartments such as endosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmic reticulum golgi apparatus, trans golgie network (TGN), and sarcoplasmic reticulum. Release includes movement out of an intracellular compartment into cytoplasm or into an organelle such as the nucleus. Releasing signals include chemicals such as chloroquine, bafilomycin or Brefeldin Al and the ER-retaining signal (KDEL sequence), viral components such as influenza virus hemagglutinin subunit HA-2 peptides and other types of amphipathic peptides.
Cellular receptor signals are any signal that enhances the association of the gene with a cell. This can be accomplished by either increasing the binding of the gene to the cell surface and/or its association with an intracellular compartment, for example: ligands that enhance endocytosis by enhancing binding the cell surface. This includes agents that target to the asialoglycoprotein receptor by using asiologlycoproteins or galactose residues. Other proteins such as insulin, EGF, or transferrin can be used for targeting. Peptides that include the RGD sequence can be used to target many cells. Chemical groups that react with sulfhydryl or disulfide groups on cells can also be used to target many types of cells. Folate and other vitamins can also be used for targeting. Other targeting groups include molecules that interact with membranes such as lipids fatty acids, cholesterol, dansyl compounds, and amphotericin derivatives. In addition viral proteins could be used to bind cells.
A polynucleotide can be delivered to a cell in order to produce a cellular change that is therapeutic. The delivery of polynucleotides or other genetic material for therapeutic purposes (the art of improving health in an animal including treatment or prevention of disease) is gene therapy. The polynucleotides are coded to express a whole or partial protein, or may be anti-sense, and can be delivered either directly to the organism in situ or indirectly by transfer to a cell that is then transplanted into the organism. The protein can be missing or defective in an organism as a result of genetic, inherited or acquired defect in its genome. For example, a polynucleotide may be coded to express the protein dystrophin that is missing or defective in Duchenne muscular dystrophy. The coded polynucleotide is delivered to a selected group or groups of cells and incorporated into those cell""s genome or remain apart from the cell""s genome. Subsequently, dystrophin is produced by the formerly deficient cells. Other examples of imperfect protein production that can be treated with gene therapy include the addition of the protein clotting factors that are missing in the hemophilia""s and enzymes that are defective in inborn errors of metabolism such as phenylalanine hydroxylase. A delivered polynucleotide can also be therapeutic in acquired disorders such as neurodegenerative disorders, cancer, heart disease, and infections. The polynucleotide has its therapeutic effect by entering the cell. Entry into the cell is required for the polynucleotide to produce the therapeutic protein, to block the production of a protein, or to decrease the amount of a RNA.
A therapeutic effect of the protein in attenuating or preventing the disease state can be accomplished by the protein either staying within the cell, remaining attached to the cell in the membrane or being secreted and dissociating from the cell where it can enter the general circulation and blood. Secreted proteins that can be therapeutic include hormones, cytokines, growth factors, clotting factors, anti-protease proteins (e.g. alpha-antitrypsin) and other proteins that are present in the blood. Proteins on the membrane can have a therapeutic effect by providing a receptor for the cell to take up a protein or lipoprotein. For example, the low density lipoprotein (LDL) receptor could be expressed in hepatocytes and lower blood cholesterol levels and thereby prevent atherosclerotic lesions that can cause strokes or myocardial infarction. Therapeutic proteins that stay within the cell can be enzymes that clear a circulating toxic metabolite as in phenylketonuria. They can also cause a cancer cell to be less proliferative or cancerous (e.g. less metastatic). A protein within a cell could also interfere with the replication of a virus.
The delivered polynucleotide can stay within the cytoplasm or nucleus apart from the endogenous genetic material. Alternatively, the polynucleotide could recombine (become a part of) the endogenous genetic material. For example, DNA can insert into chromosomal DNA by either homologous or non-homologous recombination.
Parenchymal cells are the distinguishing cells of a gland or organ contained in and supported by the connective tissue framework. The parenchymal cells typically perform a function that is unique to the particular organ. The term xe2x80x9cparenchymalxe2x80x9d often excludes cells that are common to many organs and tissues such as fibroblasts and endothelial cells within the blood vessels. In a liver organ, the parenchymal cells include hepatocytes, Kupffer cells and the epithelial cells that line the biliary tract and bile ductules. The major constituent of the liver parenchyma are polyhedral hepatocytes (also known as hepatic cells) that presents at least one side to an hepatic sinusoid and apposed sides to a bile canaliculus. Liver cells that are not parenchymal cells include cells within the blood vessels such as the endothelial cells or fibroblast cells.
In striated muscle, the parenchymal cells include myoblasts, satellite cells, myotubules, and myofibers. In cardiac muscle, the parenchymal cells include the myocardium also known as cardiac muscle fibers or cardiac muscle cells and the cells of the impulse connecting system such as those that constitute the sinoatrial node, atrioventricular node, and atrioventricular bundle. In a pancreas, the parenchymal cells include cells within the acini such as zymogenic cells, centroacinar cells, and basal or basket cells and cells within the islets of Langerhans such as alpha and beta cells. In spleen, thymus, lymph nodes and bone marrow, the parenchymal cells include reticular cells and blood cells (or precursors to blood cells) such as lymphocytes, monocytes, plasma cells and macrophages.
In the nervous system which includes the central nervous system (the brain and spinal cord) peripheral nerves, and ganglia, the parenchymal cells include neurons, glial cells, microglial cells, oligodendrocytes, Schwann cells, and epithelial cells of the choroid plexus. In the kidney, parenchymal cells include cells of collecting tubules and the proximal and distal tubular cells. In the prostate, the parenchyma includes epithelial cells. In glandular tissues and organs, the parenchymal cells include cells that produce hormones. In the parathyroid glands, the parenchymal cells include the principal cells (chief cells) and oxyphilic cells. In the thyroid gland, the parenchymal cells include follicular epithelial cells and parafolficular cells. In the adrenal glands, the parenchymal cells include the epithelial cells within the adrenal cortex and the polyhedral cells within the adrenal medulla. In the parenchyma of the gastrointestinal tract such as the esophagus, stomach, and intestines, the parenchymal cells include epithelial cells, glandular cells, basal, and goblet cells. In the parenchyma of lung, the parenchymal cells include the epithelial cells, mucus cells, goblet cells, and alveolar cells. In fat tissue, the parenchymal cells include adipose cells or adipocytes. In the skin, the parenchymal cells include the epithelial cells of the epidermis, melanocytes, cells of the sweat glands, and cells of the hair root. In cartilage, the parenchyma includes chondrocytes. In bone, the parenchyma includes osteoblasts, osteocytes, and osteoclasts.
Intravascular refers to an intravascular route of administration that enables a polymer, oligonucleotide, or polynucleotide to be delivered to cells more evenly distributed and more efficiently than direct injections. Intravascular herein means within an internal tubular structure called a vessel that is connected to a tissue or organ within the body of an animal, including mammals. Within the cavity of the tubular structure, a bodily fluid flows to or from the body part. Examples of bodily fluid include blood, lymphatic fluid, or bile. Examples of vessels include arteries, arterioles, capillaries, venules, sinusoids, veins, lymphatics, and bile ducts. The intravascular route includes delivery through the blood vessels such as an artery or a vein. xe2x80x9cIntracoronaryxe2x80x9d refers to an intravascular route for delivery to the heart wherein the blood vessels are the coronary arteries and veins.
Permeability is defined herein as the propensity for macromolecules such as nucleic acids to move through vessel walls and enter the extravascular space. One measure of permeability is the rate at which macromolecules move through the vessel wall and out of the vessel. Another measure of permeability is the lack of force that resists the movement through the vessel wall and out of the vessel. Vessels contain elements that prevent macromolecules from leaving the intravascular space (internal cavity of the vessel). These elements include endothelial cells and connective material (e.g., collagen). High permeability indicates that there are fewer of these elements that can block the egress of macromolecules and that the spaces between these elements are larger and more numerous. In this context, high permeability enables a high percentage of nucleic acids being delivered to leave the intravascular space, while low permeability indicates that a low percentage of the nucleic acids will leave the intravascular space.
The permeability of a blood vessel can be increased by increasing the intravascular hydrostatic pressure. In a preferred embodiment, the intravascular hydrostatic pressure is increased by rapidly (from 1 seconds to 30 minutes) injecting a nucleic acid in solution into the blood vessel, which increases the hydrostatic pressure. In another preferred embodiment, hydrostatic pressure is increased by obstructing the outflow of the injection solution from the tissue for a period of time sufficient to allow delivery of a nucleic acid. Obstructing means to block or impede the outflow of injection fluid, thereby transiently (reversibly) blocking the outflow of the blood. Furthermore, rapid injection may be combined with obstructing the outflow in yet another preferred embodiment. For example, an afferent vessel supplying an organ is rapidly injected while the efferent vessel draining the tissue is blocked transiently (e.g., by ligation, or by an inflated intravascular balloon). The efferent vessel (also called the venous outflow or tract) draining outflow from the tissue is partially or totally clamped for a period of time sufficient to allow delivery of a nucleic acid. In the reverse, an efferent vessel is injected while the corresponding afferent vessel is occluded.
An administration route involving the mucosal membranes is meant to include nasal, bronchial, inhalation into the lungs, or via the eyes.
Transdermal refers to application to mammal skin in which drug delivery occurs by crossing the dermal layer.
Crosslinking refers to the chemical attachment of two or more molecules with a bifunctional reagent. A bifunctional reagent is a molecule with two reactive ends. The reactive ends can be identical as in a homobifunctional molecule, or different as in a heterobifunctional molecule.
Electrostatic interactions are the non-covalent association of two or more substances due to attractive forces between positive and negative charges.
Amphipathic compounds have both hydrophilic (water-soluble) and hydrophobic (water-insoluble) parts.
A polycation is a polymer containing a net positive charge, for example poly-L-lysine hydrobromide. The polycation can contain monomer units that are charge positive, charge neutral, or charge negative, however, the net charge of the polymer must be positive. A polycation also can mean a non-polomeric molecule which contains two or more positive charges. A polyanion is a polymer containing a net negative charge, for example polyglutamic acid. The polyanion can contain monomer units that are charge negative, charge neutral, or charge positive, however, the net charge on the polymer must be negative. A polyanion can also mean a non-polomeric molecule that contains two or more negative charges. The term polyion includes polycation, polyanion, zwiterionic polymers, and neutral polymers. The term zwiterionic refers to the product (salt) of the reaction between an acidic group and a basic group that are part of the same molecule.
Mixing means the method of interdispursing two or more solvents, or solvent(s) and solute(s). Sonication and agitation are forms of mixing. Solvent refers to a material in the liquid phase that can be used to solubilize (dissolve) a compound. Solute refers to a compound dissolved in a solvent.
Hydrocarbon means containing carbon and hydrogen atoms; and halohydrocarbon means containing carbon, halogen (F, Cl, Br, I), and hydrogen atoms.
Alkyl means containing sp3 hybridized carbon atoms; alkenyl means containing two or more sp2 hybridized carbon atoms; aklkynyl means containing two or more sp hybridized carbon atoms; aralkyl means containing one or more aromatic ring(s) in addition containing sp3 hybridized carbon atoms; aralkenyl means containing one or more aromatic ring(s) in addition to containing two or more sp2 hybridized carbon atoms; aralkynyl means containing one or more aromatic ring(s) in addition to containing two or more sp hybridized carbon atoms; steroid includes natural and unnatural steroids and steroid derivatives. A steroid derivative means a sterol, a sterol in which the hydroxyl moity has been modified (for example, acylated), or a steroid hormone, or an analog thereof; carbohydrates include natural and unatural sugars (for example glucose), and surgar derivatives (a surgar derivative means a system in which one or more of the hydroxyl groups on the surgar moitey has been modified (for example acylated), or a system in which one or more of the hydroxyl groups is not present); polyoxyethylene means a polymer having two to six (n=2xe2x88x926) ethylene oxide units (xe2x80x94(CH2CH2O)nxe2x80x94) or a derivative thereof, and R not identified by number is ment to be any compatable group, for example alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, or aralkynyl, and can include heteroatoms (N, O, S), and carbonyl groups.
Hydrophilic groups indicate in qualitative terms that the chemical moiety is water-preferring. Typically, such chemical groups are water soluble, and are hydrogen bond donors or acceptors with water. Examples of hydrophilic groups include compounds with the following chemical moieties carbohydrates; polyoxyethylene, peptides, oligonucleotides and groups containing amites, amides, alkoxy amides, carboxylic acids, sulfurs, or hydroxyls. Hydrophobic groups indicate in qualitative terms that the chemical moiety is water-avoiding. Typically, such chemical groups are not water soluble, and tend not to hydrogen bond. Hydrocarbons are hydrophobic groups.
The terms xe2x80x9ctherapeuticxe2x80x9d and xe2x80x9ctherapeutic resultsxe2x80x9d are defined in this application as levels of gene products, including reporter (marker) gene products, which indicate a reasonable expectation of gene expression using similar compounds (other nucleic acids including other genes), at levels considered sufficient by a person having ordinary skill in the art of gene therapy. For example: Hemophilia A and B are caused by deficiencies of the X-linked clotting factors VIII and IX, respectively. Their clinical course is greatly influenced by the percentage of normal serum levels of factor VIII or IX: less than 2%, severe; 2-5%, moderate; and 5-30% mild. This indicates that in severe patients only 2% of the normal level can be considered therapeutic. Levels greater than 6% prevent spontaneous bleeds but not those secondary to surgery or injury. A person having ordinary skill in the art of gene therapy would reasonably anticipate therapeutic levels of expression of a gene specific for a disease based upon sufficient levels of marker gene results. In the Hemophilia example, if marker genes were expressed to yield a protein at a level comparable in volume to 2% of the normal level of factor VIII, it can be reasonably expected that the gene coding for factor VIII would also be expressed at similar levels.
There are three types of reporter (marker) gene products that are expressed from reporter genes. The reporter gene/protein systems include:
a) Intracellular gene products such as luciferase, xcex2-galactosidase, or chloramphenicol acetyl transferase. Typically, they are enzymes whose enzymatic activity can be easily measured.
b) Intracellular gene products such as xcex2-galactosidase or green fluorescent protein which identify cells expressing the reporter gene. On the basis of the intensity of cellular staining, these reporter gene products also yield qualitative information concerning the amount of foreign protein produced per cell.
Secreted gene products such as growth hormone, factor IX, or alpha1-antitrypgin are useful for determining the amount of a secreted protein that a gene transfer procedure can produce. The reporter gene product can be assayed in a small amount of blood.

Reverse micelles for delivery of nucleic acids


An apparatus for testing the impedance of a medical lead connecting an implantable stimulation device to a nerve or a muscle. The implantable device is of the type comprising a capacitor for stimulating the nerve or the muscle. The system of the invention comprises a current generator for generating a testing current xe2x80x9cIxe2x80x9d during a calibrated testing pulse and a power circuit coupled to the capacitor and to the current generator for determining if the capacitor is charged by the testing current during the calibrated testing pulse.

1. Technical Field
The present invention relates to medical implantable devices and more particularly to the test of medical lead impedance of implantable neurostimulators used for paraplegic people.
2. Background Art
It is a reality that either for natural reasons or consecutively to a malady or a traumatism, nerves or muscles of the human body may be affected and suffer from lack of stimulation or at least from a deficient stimulation. Electronic solutions to replace such deficiencies use Functional Electrical Stimulation devices (FES). State of the art of such devices mainly address the crucial problem of the heart attack with the well-known pacemakers. However, another important concern in which research of implantable solution is very active is the paraplegia domain in which the muscles are inert due to spinal injury.
Contrary to some devices like pacemakers which run continuously and need real time measurements, the implantable devices for paraplegic alternate active and inactive time periods which offer room for different measurements. Furthermore, as the generation of human walking is a complex algorithm, a great number of medical leads are used at the same time to activate the muscles of the inactivated legs (typically 16 or more leads are used as compared to two leads for cardiac pacemaker). In case some of the leads break, the device may be still active, and it is less vital that a real time integrity measurement be made for paraplegia devices than for pacemakers. Therefore, implantable devices for paraplegic do not need to be tested in real time, and checking can be done either at the system setup or during an active stimulation.
Generally, an implantable device performs two main functions: stimulation and telemetry. The stimulation is obtained by the generation of electrical pulses in order to deliver a current through a network of electrodes and medical leads in contact with the part of the body to be stimulated (muscle or nerve). The telemetry function is a feedback operation which allows to get information onto the integrity of the implantable device, such as measuring the level of power supply of the component or testing the value of the lead impedance formed by the association of the stimulated tissue plus the electrode in contact with it plus the wire connecting the electrode to the implantable device.
A first problem arises when the network of electrodes is placed within the body of a patient because several damages may affect the integrity of the leads such as partial or a total fracture in the electrical wires. However, the leads may be tested during the surgical operation which is not the purpose of the present invention.
Moreover, such kind of injury and others such as fibrosis may also appear during the time period the patient is using the stimulation system. It is therefore mandatory that these elements be tested regularly to be sure that stimulation pulses are sent with efficiency. Because of the non-accessibility of the F.E.S. device which is embedded within the patient body, the testing of the components is not easy. However, the testing of lead impedance has been addressed in many patents and only a few are described immediately hereinafter.
U.S. Pat. No. 4,949,720 discloses an apparatus for measuring the lead impedance in a pacemaker. The invention includes a large number of FET transistors operated in parallel to discharge a capacitor though the heart tissue. Pacer lead current is monitored by measuring the current through a small number of these transistors. The current monitoring function is performed by a current-to-voltage converter coupled to an analog-to-digital converter which may make one or more voltage measurements during the output pulse.
U.S. Pat. No. 4,140,131 disclose an apparatus for stimulating body tissue and in particular the heart, as including a device or circuit responsive to the initiation of stimulation and/or to the failure or pending failure of a component of the stimulating apparatus to provide the patient with a perceivable stimulation to a second, different portion of body tissue. There is disclosed an impedance level detector for sensing the impedance presented between the outputs of the stimulation apparatus to provide a warning signal indicating that the output impedance falls outside a predetermined range. In particular, the impedance level detector output is sensed by stimulation control logic to apply a first train of pulses at a first rate to an auxiliary electrode for stimulating the second portion of tissue. Further, there is included a voltage level detector for sensing when the power source voltage depletes below a predetermined level, to actuate the stimulation control logic to provide a second train of warning pulses to the auxiliary electrode, at a second, different rate than that of the first train. In this fashion, the patient not only is warned as to the pending failure or failure of a component of his pacemaker, but also is able to identify the failing component.
With these solutions, either the voltage (V) or the current (I) are measured across the lead impedance (R) and the final value of the impedance is obtained by computing the well-known Ohm""s equation: V=Rxc3x97I.
Another approach to determine the integrity of the leads consists in measuring a stimulation capacitor voltage and to deduce the impedance lead value from a well-known general equation:
V=V0xc3x97e(xe2x88x92T/RC),
wherein V0 is the initial voltage of the stimulation capacitor and (1/RC) is the time constant. Such method is illustrated in the two following patents: In U.S. Pat. No. 5,891,179 from Er et al., a real-time impedance monitoring system is provided for use with an implantable medical device having an implantable electrical lead. The impedance monitoring system includes components for determining the electrical impedance of the lead as a function of time, with the determination being made substantially in real-time, and components for graphically displaying the electrical impedance of the lead as a function of time, with the display also being generated substantially in real-time.
In U.S. Pat. No. 5,201,865 from Kuehn, a method and apparatus for measuring lead impedance during pre selected test mode operation of an implantable body tissue stimulator is presented. Analysis circuitry is periodically triggered into operation, such as on each reprogramming by the physician or periodically as a function of elapsed time or number of stimulation events counted from the preceding measurement. The actual lead impedance, measured from the output circuit from the body tissue-stimulator pulse generator, and taking into account impedance of the interconnection between the lead connector pin and the pulse generator connector block, the lead electrical conductor and its connections with the electrode and the connector pin and the electrode-tissue interface, is calculated as a function of the ratio of the elapsed times that it takes to discharge a capacitor from a first reference voltage to a second reference voltage through a precision resistor and through the lead impedance itself. The calculated lead impedance may be stored in memory with a suitable time tag, employed to automatically effect a change in operating modes or change a lead and electrode selection, if measured lead impedance falls outside normal high and low impedance boundary values. In the pacing context, calculated lead impedance may be employed to adjust sense amplifier sensitivity and pacing output pulse parameters. The method and apparatus may also be employed to calculate cardioversion/defibrillation lead impedance through selective partial discharge of high voltage output capacitors.
The ""179 solution implies that the voltage measurement be picked at the end of the impedance path and thus that an extra-wire from the implant is required. Then, in case of paraplegia where a great number of electrodes are required, such solution is not acceptable due to the number of extra-wires that would be required.
Furthermore, in paraplegic application, the leads are not necessarily proximate to the FES device and the wiring may be long and difficult to access, thereby rejecting a solution as the aforementioned one.
In the ""865 patent, the reader may assume that all the circuitry is powered with a unique common voltage VDD. In the case a high power voltage (i.e. 40 volts) is used for the stimulation capacitor and a lower voltage (5 volts) is used for the rest of the circuitry, this prior art solution is not acceptable as such because the circuitry must be adapted.
An important constraint of paraplegia is the need of interconnection of low powered components (the implant is powered less than 5 v) with high power output stages (high voltage over 40V and high current over 20 mA ) which are needed for the stimulations.
Therefore, even if the cited pacemakers solutions are convenient for the measurement of lead impedance in the cardiology context, none of the aforementioned patents offers a complete solution for testing the functionality of leads impedance in paraplegic implantable device.
Thus, there is still a need for a simple, short time functional testing solution for paraplegic implantable stimulator.
It is therefore a feature of the present invention to provide an apparatus for testing the impedance of a medical lead connecting an implantable stimulation device to a nerve or a muscle. The implantable device is of the type comprising a capacitor for stimulating the nerve or the muscle. The system of the invention comprises a current generator for generating a testing current xe2x80x9cIxe2x80x9d during a calibrated testing pulse and a power circuit coupled to the capacitor and to the current generator for determining if the capacitor is charged by the testing current during the calibrated testing pulse.
Various other objects, features, and attendant advantages of the present invention will become more fully appreciated as the same becomes better understood when considered in conjunction with the accompanying drawings, in which like reference characters designate the same or similar parts throughout the several views.

Method and system for medical lead impedance test

International Business Machines Corporation


A method for performing site-directed mutagenesis characterized in that the method includes the step of carrying out PCR by the use of a double-stranded DNA vector having one or more amber codons, the vector resulting from insertion of a target DNA fragment for site-directed mutagenesis, and at least two kinds of selection primers; and a kit for site-directed mutagenesis for use in the above method, characterized in that the kit includes amber codon reversion primers. According to the present invention, there can be provided a method for performing site-directed mutagenesis and a kit, which is useful for genetic engineering or protein engineering, more simply and rapidly. By using the method and the kit of the present invention, it is possible to efficiently obtain a mutation-introduced gene at the desired position by simply transforming a host with a PCR product obtained by PCR.

This application is the national phase under 35 U.S.C. xc2xa7371 of prior PCT International Application No. PCT/JP97/02355 which has an International filing date of Jul. 7, 1999 which designated the United States of America.
The present invention concerns a method for performing site-directed mutagenesis used in genetic engineering techniques, more easily and efficiently, and a kit for the use in the above method.
In the field of genetic engineering, recently, it is often difficult to obtain a gene product in large amounts while solely relying on techniques of cloning gene to express, and successful cases are few. For this reason, in order to increase a level of expression of a product of a cloned gene, a technique for coinciding a frame and a technique for altering base sequences near the initiation codon without altering amino acid sequences (silent mutation) are minimally required techniques. In addition, in order to carry out cloning of genes and produce more useful protein as a protein to be expressed, a technique is important and essential, which alters the base sequence of a corresponding codon to delete or substitute amino acids and thereby modifies specificities of the protein, such as optimal pH, stability, substrate specificity, Km value, and the like, in the cases where the protein is an enzyme.
As described above, a method for changing a particular base sequence in a cloned gene as desired, i.e. site-directed mutagenesis, is essential to carry out structural and functional analyses of various regulatory regions on genes including RNA, and protein engineering applications using recombinant DNA techniques. In addition, in the research field of protein engineering, a method for performing site-directed mutagenesis is more important, in a view of more rapid and accurate research by introduction of mutations and elimination at the DNA level.
Conventionally, a method for performing site-directed mutagenesis comprises, for instance, the following procedures:
(1) First, a desired DNA to be mutation-introduced is inserted to a vector. Thereafter, the complementary strand of the desired DNA is dissociated by heat-denaturation, in the case of using a double-stranded plasmid DNA, or a M13 phage vector is used to prepare a single-stranded DNA;
(2) An oligonucleotide designed to introduce a desired mutation (mutagenic primer) is annealed with the above single-stranded DNA. Thereafter, a complimentary strand DNA is synthesized in vitro system by a reaction of DNA polymerase and DNA ligase;
(3) Escherichia coli is transformed with the DNA obtained in the above item (2), and then a clone in which a mutation is introduced is selected.
However, since a ratio of a mutant to a parent DNA is extremely low in the case of carrying out only the above procedures, it is necessary to efficiently select a clone resulting from annealing to a mutagenic primer. Therefore, in the step of the above item (3), there is employed a system for selective removal so that a clone harboring the parent DNA does not grow.
Examples of such system are a method utilizing amber mutation (amber codon) [Nucleic Acids Research, 12, No. 24, pp.9441-9456 (1984)]; a method utilizing restriction endonuclease site [Analytical Biochemistry, 200, pp.81-88 (1992); Gene, 102, pp.67-70 (1991)]; and a method utilizing dut (dUTPase) mutation and ung (uracil DNA glycosilase) mutation [Proceedings of the National Academy of Sciences of the USA, 82, pp.488-492 (1985)], etc. However, in these methods of mutagenesis, their procedures are complicated, and much time is consumed. In addition, in these methods, a proportion of obtaining desired clones in which a mutation is introduced is low.
On the other hand, a method of site-directed mutagenesis described in Japanese Patent Laid-Open No. Hei 7-289262 is a method utilizing amber mutation using DNA polymerase and DNA ligase. As compared to the above methods, although in the method described in Japanese Patent Laid-Open No. Hei 7-289262, mutants can be produced at a high efficiency, the two-step transformation into Escherichia coli is necessary, thus hampering simple operation for practically purposes.
In recent years, methods for site-directed mutagenesis based on the wide-spread use of PCR techniques have been developed.
For example, there has been known a method is known, the method comprising the steps of synthesizing a DNA strand to be mutation-introduced using three or more kinds of primers, the primers including a mutagenic primer; thereafter cutting out a DNA strand with restriction endonucleases and then ligating to another vector the resulting DNA strand in which a mutation is expected to be introduced; and transforming a host Escherichia coli with the resulting vector. In addition, Quik Change(trademark) Site-Directed Mutagenesis Kit, manufactured by Stratagene, can be used to obtain a mutation-introduced DNA by means of synthesizing a strand with PCR or DNA polymerase from Pyrococcus furiosus using two kinds of complementary mutagenic oligonucleotides (mutagenic primers), the oligonucleotides being capable of hybridizing to double-stranded DNA to which mutation is to be introduced, thereafter, digesting with restriction endonuclease DpnI the resulting strand without mutation, and then transforming a host Escherichia coli with resultants treated with DpnI. Furthermore, there has been known a method capable of obtaining a mutation-induced DNA comprising adding class IIS restriction endonuclease recognition site at 5xe2x80x2-terminal side of each two kinds of mutagenic oligonucleotides (mutagenic primers), synthesizing strands by PCR, thereafter digesting with class IIS restriction endonuclease a mutation-introduced DNA, ligating and then transforming a host Escherichia coli (U.S. Pat. No. 5,512,463).
As described above, the conventional method using DNA polymerase and DNA ligase necessitates such enzyme reactions and a plurality of steps of transformation for fixing mutation sites, consuming too much time, thereby making it difficult to increase efficiency. Also, in the methods utilizing amber mutation or xe2x80x9cdutxe2x80x9d and xe2x80x9cungxe2x80x9d mutations, single-stranded DNA must be isolated. The method for removing restriction endonuclease site has drawbacks, including limited availability of restriction endonucleases.
In view of the above, a method for performing site-directed mutagenesis using the recently widely spread PCR technique has been developed and brought into actual application. However, its operation is complicated by necessitating three or more kinds of primers, including mutagenic primers, the primers including two or more kinds of mutagenic primers, and by necessitating a restriction endonuclease reaction during the operation, and other aspects. There have been other various problems including extremely reduced mutation efficiency that can result from incomplete restriction endonuclease reaction.
Therefore, an object of the invention is to provide a simple and practical method for performing site-directed mutagenesis using PCR method, and a kit for carrying out the method for performing site-directed mutagenesis.
As a result of intensive investigation to develop an efficient and simple method for performing site-directed mutagenesis, the present inventors have succeeded to obtain a clone in which a desired mutation is introduced, only once transforming Escherichia coli after carrying out PCR. The present invention has been completed, based on such a fact.
Accordingly, the gist of the present invention follows:
[1] a method for performing site-directed mutagenesis characterized in that the method comprises the step of carrying out PCR by the use of a double-stranded DNA vector having one or more amber codons, the vector resulting from insertion of a target DNA fragment for site-directed mutagenesis, and at least two kinds of selection primers;
[2] the method for performing site-directed mutagenesis according to the above item [1], wherein the selection primers are mutagenic primers and amber codon reversion primers;
[3] the method for performing site-directed mutagenesis according to the above item [1] or [2], characterized in that the vector carries a drug resistance gene containing one or more amber codons;
[4] the method for performing site-directed mutagenesis according to any one of the above items [1] to [3], further comprising a step of transforming a suppressor-free (Sup0) host with a PCR product;
[5] the method for performing site-directed mutagenesis according to the above item [4], characterized in that the suppressor-free (Sup0) host is Escherichia coli;
[6] a kit for site-directed mutagenesis for use in the method for performing site-directed mutagenesis according to any one of the above items [1] to [5], characterized in that the kit comprises amber codon reversion primers;
[7] the kit for site-directed mutagenesis according to the above item [6], characterized in that the kit comprises a vector having one or more amber codons;
[8] the kit for site-directed mutagenesis according to the above items [6] or [7], characterized in that the kit comprises a suppressor-free (Sup0) host; and
[9] the kit for site-directed mutagenesis according to the above item [8], characterized in that the suppressor-free (Sup0) host is Escherichia coli.

Method for effecting site-directed mutagenesis


A wireless network communication card usable with a host apparatus is disclosed. The wireless network communication card includes an access point detection circuit that detects a wireless access point through a wireless antenna. A power inlet to which power is supplied in response to an access point detection request from a user when the power of the host apparatus is suspended, the power inlet being connected to the access point detection circuit is included, as is an indicator coupled to said access point detection circuit that indicates a presence of an available wireless access point in response to a detection by said access point detection circuit.

A wireless network communication card usable with a host apparatus is disclosed. The wireless network communication card includes an access point detection circuit that detects a wireless access point through a wireless antenna. A power inlet to which power is supplied in response to an access point detection request from a user when the power of the host apparatus is suspended, the power inlet being connected to the access point detection circuit is included, as is an indicator coupled to said access point detection circuit that indicates a presence of an available wireless access point in response to a detection by said access point detection circuit.

Apparatus and method for detecting a wireless access point for wireless network communication