Disclosed are antisense oligonucleotide, polynucleotide, and peptide nucleic acid compounds that specifically bind to mammalian mRNA encoding a xcex21-adrenoceptor polypeptide and that are useful in the control and/or treatment of cardiac dysfunction, hypertension, hypertrophy, myocardial ischemia, and other cardiovascular diseases in an affected mammal, and preferably, in a human subject. The antisense compounds disclosed herein, and pharmaceutical formulations thereof, provide sustained control of xcex21-adrenoceptor expression over prolonged periods, and achieve therapeutic effects from as little as a single dose. Administration of these antisense compositions to approved animal models resulted in a decrease in blood pressure, but no significant change in heart rate. Use of such antisense compositions in the reduction of xcex21-adrenoceptor polypeptides in a host cell expressing xcex21-adrenoceptor-specific mRNA, and in the preparation of medicaments for treating human and animal diseases, and in particular, hypertension and other cardiac dysfunction is also disclosed.

1. Field of the Invention
The present invention relates generally to the fields of cardiovascular disease and hypertension. More particularly, it concerns antisense oligonucleotide compositions and methods that are useful for reducing hypertension, cardiac hypertrophy, and myocardial ischemia in animals, particularly in mammals such as humans. Specifically, the invention provides antisense oligonucleotide and polynucleotide compositions capable of binding to xcex21-adrenoceptor (xcex21-adrenergic receptor, xcex21-AR)-specific mRNA and inhibiting translation of the xcex21-adrenoceptor mRNA, thereby decreasing the number of xcex21-AR polypeptides in cells capable of expressing this mRNA. Disclosed are antisense oligonucleotide and peptide nucleic acid compositions, pharmaceutical formulations thereof, and vectors encoding antisense oligonucleotides that specifically bind xcex21-AR mRNA and alter expression of the mRNA in a host cell.
2. Description of Related Art
1.2.1 Hypertension
Hypertension is the result of increased arterial resistance to blood flow and left untreated can lead to various pathological consequences. Hypertension affects approximately 40 million people in the United States. Heart attack (Nicholls et al., 1998), kidney damage (Agodoa, 1998), stroke (Chamorro et al., 1998) and loss of vision (Satllworth and Waldron, 1997) are typical conditions that result from high blood pressure. When blood vessels are subjected to high pressure for extended periods of time, they respond by thickening, vasospasm, and internal build-up of lipids and plaques, a condition known as arteriosclerosis. Arteriosclerosis further causes a decreased blood flow to the kidneys, which respond by releasing the protease renin. An overactive renin-angiotensin system is often implicated in the development of hypertension and cardiovascular disease (Nicholls et al., 1998).
Hypertension is often called the xe2x80x9csilent killer,xe2x80x9d since half of the population afflicted with high blood pressure are unaware of the condition. Thus, an initial step in combating hypertension is early detection. Following diagnosis, actions must be taken to control the disorder.
1.2.2 Treatment of Hypertension
Currently, four major categories of hypertensive drugs are administered to treat high blood pressure: (1) Diuretics, typically the drug of choice when the abnormal blood pressure is not very high, increase the rate at which the body eliminates urine and salt, resulting in decreased blood pressure by reducing volume (Moser, 1998); (2) xcex2-adrenergic blockers (xcex2-blockers), typically prescribed in combination with diuretics, lower blood pressure and heart rate (Rodgers, 1998); (3) Calcium channel blockers work by preventing the entry of calcium into cells, which reduces vasoconstriction (Rosenthal, 1993); (4) angiotensin converting enzyme (ACE) inhibitors prevent the narrowing and constriction of blood vessels by blocking the production of the vasoconstrictive peptide angiotensin II (AT-II), a product of ACE (Rosenthal, 1993).
Unfortunately, the short lasting effects of these drugs often require multiple, even daily doses to be therapeutically effective. Poor compliance is a major problem with drug regimens and can lead to a hypertensive crisis if the drug is not taken as scheduled.
The sympathetic nervous system plays a crucial role in the regulation of blood pressure (BP), mainly through activating xcex1- and xcex2-adrenergic receptors xcex21-ARs) in the effector organs, including heart, kidney, and blood vessels. Adrenergic-blocking agents, especially xcex2-blockers, are commonly used in the treatment of hypertension, ischemic heart disease, and arrhythmia (reviewed in Sproat and Lopez, 1991). However, xcex2-blockers cause several side effects, which are usually associated with their central nervous system (CNS) reaction (e.g., sleep disturbance, depression, impotence, dizziness and fatigue) and xcex22-adrenergic antagonistic activity (e.g., increase in peripheral vascular resistance, worsening of asthma symptoms). In addition, because of their short half-life (3 to 10 hrs), xcex2-blockers must be taken daily to be effective. Because a cardiovascular disease such as hypertension is a life-lone disorder, longer-lasting treatment without side effects would be desirable.
Although the precise mechanism underlying the antihypertensive effects of xcex2-blockers remains unclear, it is generally accepted that they antagonize the xcex21-AR activity in heart and kidney, decreasing cardiac output and plasma renin activity (Sproat and Lopez, 1991). A new approach to xcex21-blockade has been designed that reduces the number of receptors. Antisense oligonucleotides (AS-ODN) or antisense DNA designed specifically against B1-ARs might represent a new class of xcex2-blockers. Antisense techniques, through a number of mechanisms (Phillips et al., 1997), can effectively downregulate the expression of target proteins. Clinical trials using antisense in targeting AIDS (Akhtar and Rossi, 1996), cancer (Dachs et al., 1997), and other genetic and acquired diseases (Yla, 1997) indicate their potential clinical usefulness. The antisense approach has several potential advantages over xcex2-blockers. First, the specificity of AS-ODNs is based on DNA sequence. Second, AS-ODNs do not have direct CNS effects, because of the negligible transport of these highly polar molecules through the blood-brain barrier (Agrawal et al., 1991). Third, antisense elements tested in different systems produce long-term effects after single treatment (Gyurko et al., 1997). This prolonged effect can be attributed to 2 features of AS-ODN. One is the extended half-life of chemically modified ODN. The half-life 15-20-mer phosphorothioated ODN is 20 to 50 hrs in rats and mice after intravenous injection (Iversen, 1991; Zhang et al., 1995). The other is associated with the nature of antisense inhibition, which provides a delayed yet prolonged blockade of target proteins distinct from the direct competitive antagonists currently available.
1.2.3 xcex21-Adrenoceptors
xcex21-ARs, which are distributed in the heart, kidney and blood vessels, play a role in the physiological control of blood pressure. For many years xcex2-blocker drugs have been used for the treatment of hypertension through a regimen of daily dosing. The mechanism of control of blood pressure is not precisely known but the value of beta-blockers in hypertension control has been underscored by the reports of the Joint National Committee on High Blood Pressure recommending xcex2-blockers as the first line of defense in the treatment of hypertension. Current xcex2-blocker drugs, however, have certain disadvantages, including: (1) they have to be taken daily, or twice a day and compliance is a problem; (2) they have central effects, leading to psychological changes that contribute to the problem of patient compliance; and (3) many of the xcex2-blockers now available are not specific for xcex21-ARs and, therefore, can have untoward side effects.
As can be understood from the above, there remains a need for an effective xcex21-AR blocker that is highly specific, nontoxic, produces few side effects and does not cross the blood brain barrier to produce psychological changes. Also, a xcex2-blocker that would last several days or weeks would allow patients more flexibility in the regimen of drug dosage by taking drugs infrequently. Thus, the need exists for an effective treatment of cardiac deficiencies (including hypertension, hypertrophy, ischemias, and the like) that circumvents the toxic side effects of existing therapies and provides more specific xcex21-AR inhibition with longer acting effects for improved patient compliance. In addition, methods for delivery of antisense oligonucleotides and polynucleotides to a host cell, and in particular, non-invasive administration of specific antisense constructs to a mammal such as a human subject is particularly desirable.
The present invention overcomes these and other limitations in the prior art by providing antisense nucleic acid compounds and compositions comprising them that specifically inhibit or reduce the expression of an mRNA encoding a xcex21-adrenoceptor (xcex21-AR) polypeptide in a host cell expressing the mRNA. More specifically, the subject invention provides antisense oligonucleotides (AS-ONs) and antisense oligo-peptide nucleic acids (AS-PNAs) that can specifically bind to a mammalian xcex21-AR mRNA, resulting in the reduction or inhibition of translation of the messenger ribonucleic acid (mRNA) into xcex21-AR polypeptide. Such oligonucleotides and PNA may be readily formulated in pharmaceutically-acceptable vehicles and provided directly to host cells, or administered systemically to mammals that express xcex21-AR mRNA to reduce the level of xcex21-AR produced in such cells and affected mammals.
The invention also provides genetic constructs comprising substantially full-length, antisense polynucleotide (AS-PN) sequences operably linked to a suitable promoter that may be used to transform selected cells to endogenously express xe2x80x9cantisensexe2x80x9d mRNA sequences that are complementary to the native xcex21-AR mRNA sequence. When expressed in a suitable host cell, these essentially full length anti-mRNAs specifically bind to the native xcex21-AR mRNA produced in the same host cell, and effectively reduce the availability of native xcex21-AR mRNA that can serve as a template for the translation machinery of the host cell to produce xcex21-AR polypeptide.
The antisense compounds and the genetic constructs of the present invention may be prepared in a variety of compositions, and may also be formulated in appropriate pharmaceutical vehicles for administration to human or animal subjects to inhibit or significantly reduce the expression of xcex21-AR-specific mRNA, and/or to inhibit or significantly reduce the translation of xcex21-AR-specific mRNA into functional xcex21-AR polypeptide. The antisense compounds of the present invention, and compositions comprising them provide new and useful therapeutics for the treatment, control, and amelioration of symptoms of a variety of cardiovascular disorders including hypertension, ischemia, cardiac hypertrophy, myocardial infarction, cardiac dysfunction, and diseases of the heart, that result from, or are exacerbated by, expression of xcex21-AR-specific mRNA in the host cells of the affected mammal. Moreover, pharmaceutical compositions comprising one or more of the nucleic acid compounds disclosed herein, provide significant advantages over existing conventional therapiesxe2x80x94namely, (1) their reduced side effects, (2) their increased efficacy for prolonged periods of time, (3) their ability to increase patient compliance due to their ability to provide therapeutic effects following as little as a single dose of the composition to affected individuals.
Preferred antisense compounds for use in the practice of the present invention are those nucleic acid and peptide nucleic acid sequences that specifically bind to a gene or an mRNA encoding xcex21-AR polypeptide and that inhibit the expression or reduce the level of xcex21-AR polypeptide in a mammalian host cell that expresses the gene and/or the mRNA.
The compounds of the invention, and the pharmaceutical formulations thereof, permit the development of methods for treating hypertension, ischemia, cardiac hypertrophy, cardiac dysfunction, or other cardiovascular diseases through the administration of at least one antisense compound that specifically binds to a mammalian xcex21-AR-specific gene or mRNA, wherein the binding of the antisense compound to the gene or the mRNA alters, decreases, inhibits, and/or prevents transcription of the xcex21-AR-specific mRNA, or, alternatively alters, decreases, inhibits, and/or prevents translation of the xcex21-AR-specific mRNA into functional xcex21-AR polypeptide in a host cell. As described above, the present methods offer significant advantages over traditional pharmacological modalities involving drugs that block or interfere with activity of the xcex21-AR polypeptide (and not the amount of xcex21-AR polypeptide). The present methods also avoid many of the untoward side effects of conventional therapies, avoid invasive surgical procedures, and are effective in lower, less frequent dosing regimens. The ability to dose less frequently represents a key aspect of improving patient compliance in dosing and reduces administration costs associated with more frequent dosage regimens.
In a first embodiment, the invention provides antisense oligonucleotides that specifically bind to a mammalian xcex21-AR-specific gene or a mammalian xcex21-AR-specific mRNA in a host cell, and alter the expression of, or quantity of, xcex21-AR polypeptide produced in the cell.
In a second embodiment, the invention provides antisense peptide nucleic acids that specifically bind to a mammalian xcex21-AR-specific gene or a mammalian xcex21-AR-specific mRNA in a host cell, and alter the expression of, or quantity of, xcex21-AR polypeptide produced in the cell.
In a third embodiment, the invention provides antisense polynucleotides that specifically bind to a mammalian xcex21-AR-specific gene or to a mammalian xcex21-AR-specific mRNA in a host cell, and alter the expression of, or quantity of, xcex21-AR polypeptide produced in the cell. The antisense polynucleotides represent full-length, or substantially full-length sequences that are complementary to a mammalian xcex21-AR-specific mRNA, and that when present in a cell that expresses a mammalian xcex21-AR-specific mRNA, will specifically bind to the xcex21-AR-specific mRNA, thereby reducing the availability of functional mRNA in the cell from which the cellular protein machinery can translate functional xcex21-AR polypeptide. Preferably, these full-length, or substantially full-length polynucleotides are provided to a cell via a genetic construct that comprises a promoter capable of expressing the complementary mRNA in a selected host cell. Such genetic constructs may be provided to suitable host cells using any one of the conventional gene therapy modalities known to those of skill in the art, such as, for example by one or more of the viral, retroviral, adenoviral, or adenoassociated viral constructs commonly exploited for the delivery and expression of heterologous polynucleotides.
In a fourth embodiment, the invention provides compositions that comprise one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compounds and a suitable diluent, carrier, or pharmaceutical vehicle.
In a fifth embodiment, the invention provides pharmaceutical formulations that comprise one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compounds and at least one antihypertensive pharmaceutical compound.
In a sixth embodiment, the invention provides pharmaceutical compositions that comprise at least two or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compounds.
In a seventh embodiment, the invention provides pharmaceutical compositions that comprise at least two or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compounds in combination with at least one antihypertensive pharmaceutical compound.
In an eighth embodiment, the invention provides therapeutic kits and medicaments that comprise at least one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compositions in combination with instructions for using the compositions in the treatment of mammalian cardiac dysfunction or disease. Alternatively, the invention provides kits that comprise at least one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compositions in combination with instructions for using the compositions in the preparation of genetic constructs for the development of suitable gene therapy vectors. Likewise, the invention provides kits that comprise at least one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compositions in combination with instructions for using the compositions in the formulation of multi-drug xe2x80x9ccocktailsxe2x80x9d for the treatment of one or more pathological conditions.
The invention also provides methods for the treatment of one or more cardiac diseases or dysfunctions employing one or more of the antisense compounds or compositions as described herein.
In each of the embodiments described herein, the oligonucleotide, polynucleotide, or peptide nucleic acid compound comprises a sequence region least 9 to about 35 bases in length, wherein the oligonucleotide specifically binds to a portion of mRNA expressed from a gene encoding a mammalian xcex21-AR polypeptide, and further wherein binding of the oligonucleotide to the mRNA is effective in decreasing the activity of xcex21-AR in a host cell expressing the mRNA.
In oligonucleotide and oligo-peptide nucleic acid embodiments, the oligonucleotide or PNA preferably consists of a sequence of from about nine to about 35 nucleotides in length, and preferably comprises a sequence of at least nine, at least ten, at least eleven, at least twelve, at least thirteen at least fourteen, or at least fifteen contiguous bases from any one of the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, as well as the smaller n-mer sequences illustrated in SEQ ID NO:29 through SEQ ID NO:186.
Alternatively, the oligonucleotide or PNA preferably consists of a sequence of from about nine to about 35 nucleotides in length, and preferably comprises a sequence of at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, or at least twenty-five contiguous nucleotides from any one of the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.
Alternatively, the oligonucleotide or PNA preferably consists of a sequence of from about nine to about 35 nucleotides in length, and preferably comprises a sequence of at least twenty-six, at least twenty-seven, at least twenty-eight, at least twenty-nine, at least thirty, at least thirty-one, at least thirty-two, at least thirty-three, or at least thirty-four contiguous nucleotides from any one of the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.
Alternatively, the oligonucleotide or PNA may consist of a sequence of from about nine to about 35 nucleotides in length, and may comprise the entire sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO: 50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.
The oligonucleotide or PNA compound may consist essentially of the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53, or any one or more of the smaller n-mer sequences illustrated in SEQ ID NO:54 through SEQ ID NO:186.
The oligonucleotide or PNA compound may also consist of the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53, or any one or more of the smaller n-mer sequences illustrated in SEQ ID NO:54 through SEQ ID NO:186.
As described above, the length of the preferred oligonucleotide or PNA compounds preferably will be on the order of from about nine to about 35 or so nucleotides. As such, in addition to those compounds that are nine nucleotides in length, and those compounds that are 35 nucleotides in length, all intermediate size compounds are also contemplated to be useful in the practice of the present invention. Thus, oligonucleotide or PNA compounds that are ten nucleotides in length, eleven nucleotides in length, twelve nucleotides in length, thirteen nucleotides in length, fourteen nucleotides in length, fifteen nucleotides in length, sixteen nucleotides in length, seventeen nucleotides in length, eighteen nucleotides in length, nineteen nucleotides in length, twenty nucleotides in length, twenty-one nucleotides in length, twenty-two nucleotides in length, twenty-three nucleotides in length, twenty-four nucleotides in length, twenty-five nucleotides in length, twenty-six nucleotides in length, twenty-seven nucleotides in length, twenty eight nucleotides in length, twenty-nine nucleotides in length, thirty nucleotides in length, thirty-one nucleotides in length, thirty-two nucleotides in length, thirty-three nucleotides in length, and thirty-four nucleotides in length are also contemplated to fall within the scope of the present disclosure. In fact, the preferred oligonucleotide or PNA compounds may also be slightly shorter than the preferred range, that is, they may be about six or about seven or about eight nucleotides in length, or they may even be slightly longer than the preferred range (i.e., they may be about thirty-six or about thirty-seven or even about thirty-eight or so nucleotides in length), and may still function to reduce the level of xcex21-AR polypeptide in a cell, and thus, effective in the treatment of cardiac disorders resulting from an elevated level of xcex21-AR polypeptide.
Likewise, there is no obligate requirement that the antisense compounds be exactly 100% complementary to a given target sequence on the mammalian xcex21-AR mRNA. Nor is there an obligate requirement that the antisense compounds be exactly 100% identical to one of the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53, or any one or more of the smaller n-mer sequences illustrated in SEQ ID NO:54 through SEQ ID NO:186. In fact, the only requirement is that the oligonucleotide or PNA compound have sufficient homology to the gene or the mRNA encoding xcex21-AR polypeptide so that upon specifically binding to the complementary region, a reduction in either the transcription of the xcex21-AR gene and/or a reduction in the translation of xcex21-AR polypeptide from the mRNA is observed. Therefore, the compounds may have 4 or fewer, 3 or fewer, 2 or fewer, or even 1 mismatch from the target sequence to which it specifically binds, and may still be sufficiently active to cause a reduction in xcex21-AR polypeptide within the host cell. Thus, in addition to those compounds disclosed above that comprise sequences that are 100% identical to the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53, or any one or more of the smaller n-mer sequences illustrated in SEQ ID NO:54 through SEQ ID NO:186, the invention also encompasses antisense compounds that comprise sequence regions that are about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, or even about 90% identical to a portion of one of those sequences, so long as the resulting degenerate nucleotide sequence retains sufficient homology so that it specifically binds to the gene or mRNA encoding xcex21-AR polypeptide.
In certain embodiments, the polynucleotide comprises at least 9 contiguous bases from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53. In other aspects of the invention, the polynucleotide comprises DNA, RNA, PNA, or preferably a derivatized or modified polynucleotide, including phosphorothioate derivates and the like.
Preferably the antisense polynucleotide compound comprises a sequence that is substantially the full-length complement of the xcex21-AR mRNA, wherein binding of the substantially full-length complementary sequence to the xcex21-AR mRNA is effective in reducing the translation of the mRNA into the xcex21-AR polypeptide. Preferably the substantially full-length complementary sequence is comprised within a suitable vector that may be delivered to appropriate host cells. The vector preferably comprises a nucleic acid segment that is operably linked to a promoter sequence that expresses the complementary antisense sequence in the host cell. This complementary near full-length sequence is then able to specifically bind to the substantially full length xcex21-AR mRNA and thereby prevent or substantially reduce the translation of the mRNA into xcex21-AR polypeptide. The term substantially full-length is meant to include those sequences that comprise a sequence that is complementary to a region that is at least about 80% or more of the native xcex21-AR-specific mRNA. Thus, for a xcex21-AR mRNA that is 1000 nucleotides in length, a suitable complementary substantially full-length complement is a sequence that is complementary to a region of at least 800 nucleotides of the particular xcex21-AR mRNA. Likewise, all sequences that are greater than 80% of the full-length sequences (i.e. those that are at least 81% full length, at least 82% full length, at least 83% full length, at least 84% full length, at least lo 85% full length, at least 86% full length, at least 87% full length, at least 88% full length, at least 89% full length, at least 90% full length, at least 91% full length, at least 92% full length, at least 93% full length, at least 94% full length, at least 95% full length, at least 96% full length, at least 97% full length, at least 98% full length, and at least 99% full length are also contemplated to fall within the scope of the present disclosure.
When desirable, the clinician may combine two or more of the oligonucleotide, polynucleotide, or PNA compounds disclosed above to provide an antisense xe2x80x9ccocktailxe2x80x9d to effect a more substantial reduction in the levels of xcex21-AR polypeptide. Likewise, the patient or animal to be treated may benefit from a combination therapy involving one or more of the oligonucleotide, polynucleotide, or PNA compounds disclosed herein and at least one anti-hypertensive or other cardiac therapy medicament.
For example, the composition may compris at least a first anti-hypertensive agent. In particular embodiments, the anti-hypertensive agent is selected from the group consisting of captopril (Captopril(copyright)), enalapril (Vasotec(copyright)), ramipril (Altace(copyright)), fosinopril (Monopril(copyright)), lisinopril (Prinivil(copyright), Zestril(copyright)), quinapril (Accupril(copyright)), benazepril (Lotensin(copyright)), trandolapril (Mavik(copyright)), and moexipril (Univasc(copyright)). In other embodiments, the anti-hypertensive agent is selected from the group of angiotensin receptor blockers consisting of candesartan (Atacand(copyright)), losartan (Cozaar(copyright) and Hizaar(copyright)), valsartan (Diovan(copyright)), and irbesartan (Avapro(copyright)).
In still other embodiments, the anti-hypertensive agent is selected from the group of diuretic consisting of dichlorphenamide (Daranide(copyright)), spironolactone (Aldactazide(copyright)), hydrochlorothiazide (Microzide(copyright) and Dyazide(copyright)), triamterene (Maxzide(copyright)), amiloride (Midamor(copyright) and Moduretic(copyright)), torsemide (Demadex(copyright)), ethacrynice acid (Edecrin(copyright)), furosemide (Lasix(copyright)), hydroflumethiazide (Diucardin(copyright)), chlorothiazide (Diuril(copyright)), methylclothiazide (Enduron(copyright)), polythiazide (Renese(copyright)), chlorthalidone (Thalitone(copyright)) and metolazone (Zaroxolyn(copyright)).
In still further embodiments, the anti-hypertensive agent is selected from the group of calcium channel blockers consisting of nifedepine (Adalat(copyright) and Procardia(copyright)), verapamil (Isoptin(copyright), Verelan(copyright), Calan(copyright) and Covera(copyright)), nicardipine (Cardene(copyright)), diltiazem (Tiazac(copyright), Cardizem(copyright) and Dilacor(copyright)), isradipine (DynaCirc(copyright)), nimodipine (Nimotop(copyright)), amlodipine (Norvase(copyright)), felodipine (Plendil(copyright)), misoldipine (Sular(copyright)), and bepridil (Vasocor(copyright)).
In certain preferred embodiments, the composition may further comprise one or more pharmaceutically acceptable vehicles, exemplified by, but not limited to, liposomes, lipid particles, lipid vesicles, nanoparticles, microparticles, nanocapsules, nanospheres, and sphingosomes to facilitate administration of the antisense composition(s) to the affected patient or animal.
In certain aspects of the invention, the antisense composition of the present invention is specific for an mRNA encoding the human xcex21-AR polypeptide. In particular embodiments, the host cell is a mammalian host cell. In certain preferred embodiments of the invention, the host cell is a human cell. In other preferred aspects, the host cell is comprised within a human.
The present invention also provides compositions that comprise at least a first antisense oligonucleotide, PNA, or polynucleotide specific for a mammalian xcex21-AR-specific mRNA as described above, and at least a second antisense compound specific for a mammalian renin, angiotensin, angiotensinogen, angiotensin type 1 (AT-1) receptor mRNA, or an ACE polypeptide.
In certain aspects of the invention, the second antisense compound is specific for a mammalian angiotensinogen mRNA, or an mRNA that encodes one or more RAS pathway-specific enzymes, such as a mammalian ACE polypeptide. In other aspects, the second antisense compound is specific for an mRNA that encodes a transcriptional factor.
Therapeutic combinations of three or more antisense compound are also provided. These cocktail therapies may comprise at least two antisense compounds specific for a mammalian xcex21-AR-encoding gene or mRNA, and at least a third antisense compound specific for the same mRNA, or alternatively, an mRNA encoding another polypeptide in the RAS pathway as described above. The third antisense compound may be specific for renin, AT-1 receptor, ACE, or angiotensinogen, or another gene or mRNA involved in biochemical pathways involved in producing or regulating blood pressure and/or causing or contributing to hypertension, ischemia, cardiac hypertrophy, or other cardiac dysfunction in an affected animal. Alternatively, the constructs may even be specific for one or more particular transcription factor(s) that regulate one or more genes involved in producing hypertension, ischemia, cardiac hypertrophy, or other cardiac dysfunction in a mammal. Such combined therapy approached using two or more antisense oligonucleotides are particularly desirable where enhanced or synergistic activity towards treating hypertension ischemia, cardiac hypertrophy, or other cardiac dysfunction is achieved.
The invention further provides a method for reducing expression of a gene encoding manmmalian xcex21-AR polypeptide in a host cell, the method comprising providing to the host cell an amount of an antisense oligonucleotide, polynucleotide, or peptide nucleic acid that specifically binds to an mRNA encoding the xcex21-AR polypeptide, effective to reduce the amount of xcex21-AR polypeptide in the cell.
Additionally, the invention provides a method for reducing the number of mammalian xcex21-AR polypeptides in a cell, the method comprising introducing into the cell at least a first antisense oligonucleotide, polynucleotide, or peptide nucleic acid that specifically binds to all, substantially-all, or a portion of the mRNA expressed from a gene encoding a mammalian xcex21-adrenoceptor polypeptide, and further wherein binding of the first antisense oligonucleotide, polynucleotide, or peptide nucleic acid to the mRNA is effective in reducing the number of mammalian xcex21-AR polypeptides in the host cell expressing the mRNA.
The invention also provides a method for decreasing or treating hypertension in an animal, the method comprising administering to the animal an effective amount of at least a first antisense oligonucleotide, polynucleotide, or peptide nucleic acid, wherein the compound specifically binds to all, substantially all, or a portion of the mRNA expressed from a gene encoding a mammalian xcex21-AR polypeptide, and further wherein binding of the compound to the mRNA is effective in decreasing the number of such polypeptides in a host cell expressing the mRNA.
The invention additionally provides a method for treating a disease associated with elevated xcex21-AR activity in a mammal, the method comprising administering to the animal an effective amount of at least a first antisense oligonucleotide, polynucleotide, or peptide nucleic acid compound wherein the compound specifically binds to all, substantially all, or a portion of the mRNA expressed from a gene encoding a mammalian xcex21-AR polypeptide, and further wherein binding of the compound to the mRNA is effective in decreasing the receptor activity or receptor number in a host cell expressing the mRNA, such that a decrease in xcex21-adrenoceptor activity is effected, thereby resulting in amelioration of the disease that results from, or is exacerbated by, an elevated level of xcex21-AR activity in the affected animal.
The invention also provides a method for treating a cardiac hypertrophic or ischemic disease that is associated with, that results from, or is exacerbated by, the presence of, or an elevation in, xcex21-adrenoceptor polypeptides in the affected animal. This method generally involves the administration to the animal of one or more compositions that comprise at least a first antisense oligonucleotide, polynucleotide, or peptide nucleic acid compound, wherein the compound specifically binds to all, substantially all, or a portion of the mRNA expressed from a gene encoding a mammalian xcex21-AR polypeptide in an amount, and for a time sufficient to decrease the number, amount, or activity of the xcex21-adrenoceptor polypeptide in a host cell expressing the mRNA.
Further provides are kits for treating hypertension in a human comprising: (a) a pharmaceutically-acceptable formulation comprising at least a first oligonucleotide of at least 9 to about 35 bases in length, wherein the oligonucleotide specifically binds to a portion of mRNA expressed from a gene encoding a mammalian xcex21-AR polypeptide, and further wherein binding of the oligonucleotide to the mRNA is effective in decreasing the activity, amount, or number of receptor polypeptides in a host cell expressing the mRNA; a pharmaceutical excipient; and (b) instructions for using the kit.
In addition to methods involving the delivery of exogenous oligonucleotide compositions to a host cell, or administration of such compositions to an animal in a therapeutic pharmaceutical formulation, the present invention also concerns gene therapy methods for introducing into a host cell a DNA construct that is transcribed by the cell machinery to give rise to an antisense RNA molecule that specifically binds to a portion of an mRNA encoding a mammalian xcex21-AR polypeptide. In a preferred embodiment, such therapies may be accomplished through the use of viral vectors, such as retro-, adeno- or adeno-associated viruses as described hereinbelow.
Regulation of expression of a gene encoding a mammalian xcex21-AR polypeptide in a mammalian cell genomes may also be achieved by integration of a gene under the transcription control of a promoter which is functional in the host and in which the transcribed strand of DNA is complementary to the strand of DNA that is transcribed from the endogenous xcex21-AR-specific polynucleotide sequence(s) one wishes to regulate. The integrated gene, referred to as an antisense gene, provides an RNA sequence capable of binding to naturally existing RNAs, exemplified by mammalian xcex21-AR polypeptide-specific mRNA, and inhibiting its expression, where the anti-sense sequence may bind to the coding, non-coding, or both, portions of the RNA. The antisense construction may be introduced into the animal cell in a variety of ways and be integrated into the animal genome for inducible or constitutive transcription of the antisense sequence.
Another aspect of the invention provides a pharmaceutical composition useful for inhibiting expression of mammalian xcex21-AR-specific mRNA comprising a pharmaceutical carrier and one or more antisense oligonucleotides of the present invention that specifically bind to and reduce expression of the specific mRNA. Another aspect of the invention provides a method for treating hypertension in a human comprising administering to a subject an effective amount of at least one oligonucleotide composition as described herein, in an amount effective to reduce hypertension in the human, or to ameliorate the degree or extent of hypertension in the patient.
The term xe2x80x9cxcex21-ARxe2x80x9d refers to polypeptides having amino acid sequences which are substantially similar to the native mammalian xcex21-AR polypeptide amino acid sequences and which are biologically active and/or which cross-react with xcex21-AR polypeptide-specific antibodies raised against a mammalian xcex21-AR polypeptide or peptide fragment thereof.
The term xe2x80x9cxcex21-ARxe2x80x9d also includes analogs of mammalian xcex21-AR polypeptides that exhibit at least some biological activity in common with native mammalian xcex21-AR polypeptides.
Furthermore, those skilled in the art of mutagenesis will appreciate that other analogs, as yet undisclosed or undiscovered, may be used to construct mammalian xcex21-AR polypeptide analogs or xcex21-AR fusion proteins or identify xcex21-AR-related mRNAs and/or genes using well-known molecular biology techniques, including those described herein. Oligonucleotides complementary to mammalian xcex21-AR polypeptide-encoding mRNAs form the heart of the present invention, especially human xcex21-AR polypeptide-encoding mRNAs.
The oligonucleotides (or xe2x80x9cODNsxe2x80x9d or xe2x80x9cpolynucleotidesxe2x80x9d or xe2x80x9coligosxe2x80x9d or xe2x80x9coligomersxe2x80x9d or xe2x80x9cn-mersxe2x80x9d) of the present invention are preferably deoxyoligonucleotides (ie. DNAs), or derivatives thereof; ribo-oligonucleotides (i.e. RNAs) or derivatives thereof; or peptide nucleic acids (PNAs) or derivatives thereof.
The term xe2x80x9csubstantially complementary,xe2x80x9d when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, an oligonucleotide sequence, is substantially complementary to all or a portion of the selected sequence, and thus will specifically bind to a portion of an Mrna encoding a mammalian xcex21-AR polypeptide. As such, typically the sequences will be highly complementary to the Mrna xe2x80x9ctargetxe2x80x9d sequence, and will have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the complementary portion of the sequence. In many instances, it may be desirable for the sequences to be exact matches, i.e. be completely complementary to the sequence to which the oligonucleotide specifically binds, and therefore have zero mismatches along the complementary stretch. As such, highly complementary sequences will typically bind quite specifically to the target sequence region of the Mrna and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target Mrna sequence into polypeptide product.
Substantially complementary oligonucleotide sequences will be greater than about 80 percent complementary (or xe2x80x98% exact-matchxe2x80x99) to the corresponding Nrna target sequence to which the oligonucleotide specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding Mrna target sequence to which the oligonucleotide specifically binds. In certain aspects, as described above, it will be desirable to have even more substantially complementary oligonucleotide sequences for use in the practice of the invention, and in such instances, the oligonucleotide sequences will be greater than about 90 percent complementary to the corresponding Mrna target sequence to which the oligonucleotide specifically binds, and may in certain embodiments be greater than about 95 percent complementary to the corresponding Mrna target sequence to which the oligonucleotide specifically binds, and even up to and including 96%, 97%, 98%, 99%, and even 100% exact match complementary to all or a portion of the target Mrna to which the designed oligonucleotide specifically binds.
Percent similarity or percent complementary of any of the disclosed sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences. 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 (1986), (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.
Table 1 lists representative oligonucleotide sequences contemplated for use in the practice of the present invention.
N means any nucleotide, e.g., C, A, U (T), or G.
Sequences in bold correspond to portions of the xcex21-AR open reading frame.
In addition to the indicated 35-mers, smaller internal n-mers that comprise from at least 9 bases in length up to the full length 35-mers listed are also contemplated to be useful in the practice of the present invention. For example, in addition to the first indicated full-length oligomer, all internal n-mers are also considered to fall within the scope of this disclosure. Thus for each of the 35-mers (SEQ ID NO:4 to SEQ ID NO:28) all internal 34-mers of each sequence as well as all internal 33-mers, 32-mers, 31-mers, 30-mers, 29-mers, 28-mers, 27-mers, 26-mers, 25-mers, 24-mers, 23-mers, 22-mers, 21-mers, 20-mers, 19-mers, 18-mers, 17-mers, 16-mers, 15-mers, 14-mers, 13-mers, 12-mers, 11-mers, 10-mers, and 9-mers, that are comprised within of each of the disclosed 35-mers are also contemplated to be useful in the practice of the present invention.
For illustrative purposes, all representative n-mers of SEQ ID NO:10 would include the following internal contiguous 9-mer to 34-mer sequences:
CAGCTCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:29)
GCAGCTCGGCATGGGCGCGGGGGTGCTCGTCCTG (SEQ ID NO:30)
AGCTCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:31)
GCAGCTCGGCATGGGCGCGGGGGTGCTCGTCCT (SEQ ID NO:32)
GCTCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:33)
GCAGCTCGGCATGGGCGCGGGGGTGCTCGTCC (SEQ ID NO:34)
CTCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:35)
GCAGCTCGGCATGGGCGCGGGGGTGCTCGTC (SEQ ID NO:36)
TCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:37)
GCAGCTCGGCATGGGCGCGGGGGTGCTCGT (SEQ ID NO:38)
CGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:39)
GCAGCTCGGCATGGGCGCGGGGGTGCTCG (SEQ ID NO:40)
GGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:41)
GCAGCTCGGCATGGGCGCGGGGGTGCTC (SEQ ID NO:42)
GCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:43)
GCAGCTCGGCATGGGCGCGGGGGTGCT (SEQ ID NO:44)
CATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:45)
GCAGCTCGGCATGGGCGCGGGGGTGC (SEQ ID NO:46)
ATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:47)
GCAGCTCGGCATGGGCGCGGGGGTG (SEQ ID NO:48)
TGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:49)
GCAGCTCGGCATGGGCGCGGGGGT (SEQ ID NO:50)
GGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:51)
GCAGCTCGGCATGGGCGCGGGGG (SEQ ID NO:52)
GGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:53)
GCAGCTCGGCATGGGCGCGGGG (SEQ ID NO:54)
GCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:55)
GCAGCTCGGCATGGGCGCGGG (SEQ ID NO:56)
CGCGGGGGTGCTCGTCCTGG (SEQ ID NO:57)
GCAGCTCGGCATGGGCGCGG (SEQ ID NO:58)
GCGGGGGTGCTCGTCCTGG (SEQ ID NO:59)
GCAGCTCGGCATGGGCGCG (SEQ ID NO:60)
CGGGGGTGCTCGTCCTGG (SEQ ID NO:61)
GCAGCTCGGCATGGGCGC (SEQ ID NO:62)
GGGGGTGCTCGTCCTGG (SEQ ID NO:63)
GCAGCTCGGCATGGGCG (SEQ ID NO:64)
GGGGTGCTCGTCCTGG (SEQ ID NO:65)
GCAGCTCGGCATGGGC (SEQ ID NO:66)
GGGTGCTCGTCCTGG (SEQ ID NO:67)
GCAGCTCGGCATGGG (SEQ ID NO:68)
GGTGCTCGTCCTGG (SEQ ID NO:69)
GCAGCTCGGCATGG (SEQ ID NO:70)
GGTGCTCGTCCTGG (SEQ ID NO:71)
GCAGCTCGGCATG (SEQ ID NO:72)
GTGCTCGTCCTGG (SEQ ID NO:73
GCAGCTCGGCAT (SEQ ID NO:74)
TGCTCGTCCTGG (SEQ ID NO:75)
GCAGCTCGGCA (SEQ ID NO:76)
GCTCGTCCTGG (SEQ ID NO:77)
GCAGCTCGGC (SEQ ID NO:78)
CTCGTCCTGG (SEQ ID NO:79)
GCAGCTCGG (SEQ ID NO:80), Etc.
In similar fashion, for illustrative purposes, and in the sake of brevity, representative n-mers of SEQ ID NO:1 would include the following internal contiguous sequences:
CGCGCCCATGCCGA (SEQ ID NO:81);
CCGCGCCCATGCCG (SEQ ID NO:82);
GCGCCCATGCCGA (SEQ ID NO:83);
CCGCGCCCATGCC (SEQ ID NO:84);
CGCCCATGCCGA (SEQ ID NO:85);
CCGCGCCCATGC (SEQ ID NO:86);
GCCCATGCCGA (SEQ ID NO:87);
CCGCGCCCATG (SEQ ID NO:88);
CCCATGCCGA (SEQ ID NO:89);
CCGCGCCCAT (SEQ ID NO:90);
CCATGCCGA (SEQ ID NO:91); and
CCGCGCCCA (SEQ ID NO:92).
Given the benefit of the present disclosure, the skilled artisan would also be able now, in similar fashion, to prepare any and all possible n-mers from each of the disclosed sequences, and from these sequences, identify and select those particular oligonucleotide sequences that specifically inhibit xcex21-AR-specific mRNA expression for therapeutic use by using an acceptable in vitro and/or in vivo assay, such as those described hereinbelow.
Likewise, based upon the sequence of the human xcex21-AR gene, the inventors have identified highly preferred sequences of from about 15 to about 25 nucleotides in length that specifically bind to the mRNA encoding xcex21-AR polypeptide, and that reduce the level of polypeptide in a cell expressing such an mRNA. Illustrative sequences in this size range are identified below:
Length=15 nucleotides:
5xe2x80x2-CACCCCCGCGCCCAT-3xe2x80x2 (SEQ ID NO:93)
5xe2x80x2-ACCCCCGCGCCCATG-3xe2x80x2 (SEQ ID NO:94)
5xe2x80x2-CGCGCCCATGCCGAG-3xe2x80x2 (SEQ ID NO:95)
5xe2x80x2-GCGCCCATGCCGAGC-3xe2x80x2 (SEQ ID NO:96)
5xe2x80x2-CGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:97)
5xe2x80x2-GCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:98)
5xe2x80x2-CCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:99)
5xe2x80x2-CCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:100)
5xe2x80x2-CATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:101)
5xe2x80x2-ATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:102)
Length=16 nucleotides:
5xe2x80x2-GCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:103)
5xe2x80x2-CGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:104)
5xe2x80x2-GCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:105)
5xe2x80x2-CCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:106)
5xe2x80x2-CCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:107)
5xe2x80x2-CATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:108)
5xe2x80x2-ATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:109)
5xe2x80x2-TGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:110)
Length=17 nucleotides:
5xe2x80x2-CGCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:111)
5xe2x80x2-GCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:112)
5xe2x80x2-CGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:113)
5xe2x80x2-GCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:114)
5xe2x80x2-CCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:115)
5xe2x80x2-CCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:116)
5xe2x80x2-CATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:117)
5xe2x80x2-ATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:118)
Length=18 nucleotides:
5xe2x80x2-CCGCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:119)
5xe2x80x2-CGCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:120)
5xe2x80x2-GCGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:121)
5xe2x80x2-CGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:122)
5xe2x80x2-GCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:123)
5xe2x80x2-CCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:124)
5xe2x80x2-CCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:125)
5xe2x80x2-CATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:126)
Length=19 nucleotides:
5xe2x80x2-ACCCCCGCGCCCATGCCGA-3xe2x80x2 (SEQ ID NO:127)
5xe2x80x2-CCCGCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:128)
5xe2x80x2-CCGCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:129)
5xe2x80x2-CGCGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:130)
5xe2x80x2-GCGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:131)
5xe2x80x2-CGCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:132)
5xe2x80x2-GCCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:133)
5xe2x80x2-CCCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:134)
5xe2x80x2-CCATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:135)
Length=20 nucleotides:
5xe2x80x2-CACCCCCGCGCCCATGCCGA-3xe2x80x2 (SEQ ID NO:136)
5xe2x80x2-ACCCCCGCGCCCATGCCGAG-3xe2x80x2 (SEQ ID NO:137)
5xe2x80x2-CCCCCGCGCCCATGCCGAGC-3xe2x80x2 (SEQ ID NO:138)
5xe2x80x2-CCCCGCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:139)
5xe2x80x2-CCCGCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:140)
5xe2x80x2-CCGCGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:141)
5xe2x80x2-CGCGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:142)
5xe2x80x2-GCGCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:143)
5xe2x80x2-CGCCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:144)
5xe2x80x2-GCCCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:145)
5xe2x80x2-CCCATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:146)
Length=21 nucleotides:
5xe2x80x2-CACCCCCGCGCCCATGCCGAG-3xe2x80x2 (SEQ ID NO:147)
5xe2x80x2-ACCCCCGCGCCCATGCCGAGC-3xe2x80x2 (SEQ ID NO:148)
5xe2x80x2-CCCCCGCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:149)
5xe2x80x2-CCCCGCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:150)
5xe2x80x2-CCCGCGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:151)
5xe2x80x2-CCGCGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:152)
5xe2x80x2-CGCGCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:153)
5xe2x80x2-GCGCCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:154)
5xe2x80x2-CGCCCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:155)
5xe2x80x2-GCCCATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:156)
Length=22 nucleotides:
5xe2x80x2-CACCCCCGCGCCCATGCCGAGC-3xe2x80x2 (SEQ ID NO:157)
5xe2x80x2-ACCCCCGCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:158)
5xe2x80x2-CCCCCGCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:159)
5xe2x80x2-CCCCGCGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:160)
5xe2x80x2-CCCGCGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:161)
5xe2x80x2-CCGCGCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:162)
5xe2x80x2-CGCGCCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:163)
5xe2x80x2-GCGCCCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:164)
5xe2x80x2-CGCCCATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:165)
Length=23 nucleotides:
5xe2x80x2-CACCCCCGCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:166)
5xe2x80x2-ACCCCCGCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:167)
5xe2x80x2-CCCCCGCGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:168)
5xe2x80x2-CCCCGCGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:169)
5xe2x80x2-CCCGCGCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:170)
5xe2x80x2-CCGCGCCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:171)
5xe2x80x2-CGCGCCCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:172)
5xe2x80x2-GCGCCCATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:173)
Length=24 nucleotides:
5xe2x80x2-CACCCCCGCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:174)
5xe2x80x2-ACCCCCGCGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:175)
5xe2x80x2-CCCCCGCGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:176)
5xe2x80x2-CCCCGCGCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:177)
5xe2x80x2-CCCGCGCCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:178)
5xe2x80x2-CCGCGCCCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:179)
5xe2x80x2-CGCGCCCATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:180)
Length=25 nucleotides:
5xe2x80x2-CACCCCCCGCGCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:181)
5xe2x80x2-ACCCCCGCGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:182)
5xe2x80x2-CCCCCGCGCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:183)
5xe2x80x2-CCCCGCGCCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:184)
5xe2x80x2-CCCGCGCCCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:185)
5xe2x80x2-CCGCGCCCATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:186)

Antisense compositions targeted to xcex21-adrenoceptor-specific mRNA and methods of use


A motor vehicle has an internal combustion engine and at least one electric motor as a drive motor arranged on a common shaft. The internal combustion engine and/or the electric motor can introduce torque into a transmission downstream in the drivetrain, and a separating clutch is provided between the internal combustion engine and the electric motor. The method increases torque of the electric machine, increasing the rotational speed of the electric machine and setting slip conditions at a clutch of the downstream transmission, engaging the separating clutch to a torque between the reserve torque of the electric motor and the maximum transmissible torque, reducing the transmissible torque of the separating clutch when the rotational speed of the internal combustion engine has reached the ignition rotational speed, ending the slip conditions of the at least one clutch of the downstream transmission, ending the increase of the torque of the electric motor.

1. Field of the Invention
The invention relates to an engine starting method, in particular for an internal combustion engine of a motor vehicle having an internal combustion engine and having at least one electric motor as a drive motor. The invention also relates to a motor vehicle for carrying out an engine starting method.
2. Description of the Related Art
Motor vehicles having an internal combustion engine and an electric motor are known in the prior art and are also referred to as hybrid vehicles. If the internal combustion engine and the electric motor are arranged on one shaft, this arrangement is also referred to as a parallel hybrid.
The motor vehicle may be driven either only by the internal combustion engine, only by the electric motor or by the internal combustion engine and the electric motor simultaneously. The internal combustion engine can be connected to the electric motor by means of a clutch. A transmission can be connected to the electric motor downstream in the torque flow and the drivetrain can be connected to the transmission downstream. The clutch between the internal combustion engine and the electric machine is disengaged when the internal combustion engine is deactivated so that there is no power-transmitting connection between the internal combustion engine and the electric motor. In this operating situation, the electric motor alone drives the vehicle.
The clutch must be engaged if the internal combustion engine is to be started so that the internal combustion engine can be turned over up to the starting rotational speed at which the engine can start of its own accord. Here, however, unpleasant juddering is experienced by the vehicle occupants, because torque that was available for the forward propulsion of the vehicle before the starting of the internal combustion engine suddenly is branched away and no longer available for forward propulsion due to the turning-over of the internal combustion engine.
DE 10 2008 040 662 A1 discloses a system where a clutch torque is raised linearly, then held at a plateau as a function of time, and for thereafter is raised linearly again. The system is intended to determine the breakaway torque of the internal combustion engine, because it changes over the course of the operation of the engine.
This approach, however, entails a method step that fundamentally delays the starting of the engine and also is perceived as being rather unpleasant. Also, the friction power input into the clutch is increased so that the method is not recommended for thermal reasons.

Dr. Ing. h.c. F. Porsche Aktiengesellschaft


A system is provided in order to automatically close a filler inlet compartment lid of a car when the lid is in an unintended open position. When predefined signals which are considered to indicate the termination of a refueling operation occur, the system activates a filler inlet compartment lid closing mechanism so that the filler inlet compartment lid is automatically closed and locked. This can take place when the filler inlet compartment lid is completely open or when the lid is in an only slightly lifted intermediate position. The filler inlet compartment lid may reach such a slightly lifted intermediate position, for example, in a drive-through car wash when triggered by the pressure of the car wash brushes.

This application claims the priority of German application 100 23 382.1, filed May 12, 2000, the disclosure of which is expressly incorporated by reference herein.
The present invention relates to an operating system for a filler inlet compartment lid of a motor vehicle.
Corresponding to design data, the filler inlet compartment lids of current passenger cars are constructed without recessed grips for opening purposes. A mechanism is mounted behind the rearward edge of such a filler inlet compartment lid. After manual pressure from the outside, the mechanism presses the filler inlet compartment lid, during release, approximately 1 cm toward the outside into an intermediate position so that the user can reach behind the edge of the filler inlet compartment lid which will then protrude. After refueling the vehicle, the lid is pressed into the closed position again, against the resistance of the mechanism. When released, the lid will spring only so far to the outside that it is flush with the outer skin. The operation of this system is similar to that of a pressure-operated ball point pen having a cardioid curve or operating according to similar principles.
For reasons of comfort, the triggering force for the above-described pressing mechanism should not exceed a certain amount. There is, therefore, a risk that this mechanism will be triggered in car wash systems. After passing through a car wash system, the filler inlet compartment lid may protrude in the intermediate position. The vehicle user often does not notice this and will drive around with a slightly projecting compartment lid until the projecting lid is noticed. Only after pressure is exercised on the filler inlet compartment lid will this lid be returned into a position flush with the outer skin. Also, if the customer forgets to completely press in the compartment lid after refueling, then the lid will slightly protrude.
It is an object of the invention to provide a system which automatically closes the filler inlet compartment lid if it either has opened in an unintentional manner or was mistakenly not closed.
This object is achieved by using a control unit which responds to signals indicating predefined conditions and which is designed to control the operating system for closing the filler inlet compartment lid. Further features of the invention are also reflected in the claims.
In a system according to the invention, signals are generated under defined conditions such as, for example, after expiration of a predefined time interval following the opening of the filler inlet compartment lid, after a predefined driven distance following a refueling stop, when a predefined speed is exceeded, or under other conditions which can be evaluated as signs that the refueling operation is completed. These signals trigger closing and locking of the filler inlet compartment lid. In this manner, driving around with an accidentally not closed filter inlet compartment lid, in a fully opened or intermediate position, is prevented.
An expedient further development of the invention is that the operating system arranged inside the vehicle body, similar to a central locking system, has a sliding-block part which can be rotated or displaced by a driving element. The sliding-block part has a guide slot which is open on one side and defines two levels. A locking pin can engage into the guide slot. The locking pin is situated on the filler inlet compartment lid and, depending on the position of the sliding-block part, locks the filler inlet compartment lid in the closed position or moves it into a slightly lifted release or intermediate position in which, as mentioned above, the lid can easily be opened manually. Triggering of the sliding-block drive can take place by way of a pressure sensor which, during a slight pressure against the closed filler inlet compartment lid, generates an opening signal which causes a movement of the sliding block into the position in which the filler inlet compartment lid is lifted and is released for opening. After closing the filler piece when the refueling operation is completed, the filler inlet compartment lid, which is normally provided with a dead-center spring, is shut and reaches the slightly opened intermediate position, in which it can again trigger a sensor, which will trigger the sliding-block drive for moving the sliding block into the locking position for the filler inlet compartment lid.
If opening of the filler inlet compartment lid is triggered unintentionally (for example, in a car wash system), or if the filler inlet compartment lid is only leaning, after a closing operation, without triggering the sensor, or remains completely open, then the lid will be automatically closed at a later point in time according to the invention as a function of certain signals, mentioned above by way of example, available in the vehicle. If desired, another signal generator can be provided to indicate the respective position of the sliding block or filler inlet compartment lid on the dashboard. The invention has the advantage that a customer will not be driving around with a projecting filler inlet compartment lid without noticing it after either using a car wash system or refueling. After a defined time or driving distance, or as a function of other conceivable events or conditions, the filler inlet compartment lid will be closed electrically or by another drive (hydraulically, pneumatically, etc.). Advantageously, the entire unit for automatically closing the filler inlet compartment lid can be combined to form a module which can easily be mounted in the vehicle body and connected.
Embodiments of invention illustrated in the drawings will now be explained in detail.

Filler inlet compartment lid operating system

Bayerische Motoren Werke Aktiengesellschaft


A functional electrical stimulation system for generating a data file storing stimulation patterns that can be provided to a stimulator is described. The system includes a host computer system for producing a data structure or data file that describes the patterns and a portable stimulator that using the data structure or data file applies electrical pulses to electrodes carried by a patient. The host computer system and the stimulator system each have a memory storing a table having control and pattern generation information with indexes into a table that separately stores electrode characterization data for each electrode used by the portable stimulator.

This invention relates generally to functional electrical stimulation systems and more particularly to techniques to produce stimulation patterns for functional electrical stimulation systems usable for lower and/or upper extremities.
As is known in the art, research in upper and lower extremity functional electrical systems has been ongoing for a number of years. Functional electrical stimulation uses an electronic system to generate electrical pulses that are delivered to muscles of a patient who has muscle movement impairment. The muscle movement impairment is due to some condition that causes muscle paralysis. This condition can be nerve damage caused by accident or disease. The functional electrical stimulation system produces electrical signals that can be used by the patient to control muscle movement.
Practical electrical stimulation systems require a stimulation system that is small, lightweight and portable. Such a system would to allow a subject patient to operate the system to learn to perform everyday activities. On the other hand a practical system requires sufficient versatility and power to enable practical utilization of generated electrical stimulation patterns to produce a variety of muscle movements.
Several portable systems have been described in the literature. As is known, one of the problems common to these systems is the approach used to generate the electrical signal patterns. The described systems are either built for upper or lower extremity movement but not both or either. In general, the known systems cannot handle different types of sensors. Thus, systems are designed for a particular type of sensor associated with the desired type of stimulation. Further, the approaches used to process inputs to produce stimulation waveform generally are not sufficiently robust to accommodate different sensors.
Different sensors and algorithms are necessary for each type of stimulation because the characteristics of the upper and lower extremity movements are different. Lower extremity movements can be characterized as repetitive, predictable movements, whereas upper extremity movements are more spontaneous in character. A second problem is that for practical, useful movements of muscles generally several muscles must be operated in unison or concert to produce the practical movement. Thus, in addition to providing an algorithm which allows for relatively easy generation of such produced muscle movements, it is also desirable to provide an overall system that can be adapted for upper or lower extremity stimulation or both.

Portable functional electrical stimulation (FES) system for upper or lower extremity applications


A system for detecting substances, the system including a dual-energy radiography (DER) system including a gamma-ray radiation source, including a beam accelerator and a target to which the accelerator sends a beam thereby producing gamma rays, and a plurality of gamma ray detectors positioned to detect gamma rays that pass from the gamma-ray radiation source through an object to be inspected, wherein the DER system is adapted to indicate a presence of a high-Z substance, by detecting a difference in a transmission attenuation characteristic of the high-Z substance as opposed to low-Z and medium-Z substances and differentiating among high-Z substances according to their density, as determined from a plurality of radiographic views.

In recent years, ever-higher priority is being accorded to developing state-of-the-art security-screening methods and devices that will rapidly and reliably detect small quantities (tens of grams) of illicitly-transported SNM. Sensitivity of this order is required to interdict their use in, for example, panic-creating radiological dispersion devices (RDD's) deployed in densely-populated areas, or SNM accretion for the fabrication of improvised nuclear devices (IND's). Indeed, effective countering of these threats is currently considered one of the highest-priority medium-term R&D challenges at the U.S. Dept. of Homeland Security (DHS), among other national and international law-enforcement agencies. For obvious reasons, the severity of the detection problem tends to increase sharply with the mass and volume of the objects to be screened.
The following are examples of the prior art:
a) Passive Radiation-Monitoring Systems:
Such "Portal Monitors" were developed for the purpose of interdicting theft or diversion of SNM from high-security sites. The detection of SNM is based on measuring an increase in radiation intensity above the ambient background. Among others, LANL (Los Alamos National Laboratories) and LLNL (Lawrence Livermore National Laboratories) have been active in developing such systems, which are usually packaged in three distinct categories: 1) Small, Hand-Held Monitors 2) Automatic Pedestrian Monitors 3) Automatic Vehicle Monitors.
Vehicle monitoring systems for detecting SNM are now available from several commercial companies. Among these, Canberra's JPM-12A Vehicle Monitor was developed jointly with LANL. It consists of two large plastic scintillators positioned on either side of the portal, that measure the gamma-rays emitted from SNM. The instrument can detect 10 gr of Pu and 1000 gr of HEU in an unshielded vehicle moving at a speed of 2 m/s. The disadvantage of this method is that gamma detection can be thwarted with relative ease by enveloping the SNM with Pb-sheet, since the minimum detected quantities increase rapidly by an order of magnitude when shielded by several mm of it. In the Pu case, the problem can be countered by using neutron detectors in the portal. However, due to low neutron emission rates, this approach requires large-area neutron detectors. LANL has developed such a system, based on two large-area 3He detectors between which vehicles pass. An equivalent system is currently marketed by TSA-Systems-Ltd., Model NVM-245. NUCSAFE's Vehicle Monitor claims detection of 5 gr of gamma-shielded weapons-grade Pu within 10 s at 1 m distance. Also available are gamma-neutron Vehicle Portal Monitors manufactured by LAURUS-Systems-Inc. Model No. VM-250GN and by POLIMASTER, Model Series PM5000. For the latter, which incorporate sizeable plastic-scintillator detectors as well as large-area 3He proportional counters, detection of 4.3 gr 239Pu and 300 gr 235U at a scan speed of 10 km/hr is claimed. However, in analogy to γ-rays, small Pu quantities might evade detection if some neutron shielding is introduced.
b) Single-Energy Radiography Systems
This method yields information about the contents of a screened object. A number of systems have been developed in recent years by commercial companies. Nearly all of them use high energy bremsstrahlung radiation produced by high-power linacs, although SAIC have built a line of products (VACIS) around a radioactive 60Co source. In several systems, two views at 90° to each other are generated, to obtain more information. Commercial companies such as Smiths-Heimann and Aracor produce linac-based vehicle and marine container inspection systems. They operate at electron energies of ˜10 MeV and employ Cadmium-Tungstate radiation detectors. The spatial resolution permits the detection of 1 mm copper wire. The scan time is ˜3 minutes for a 20 m-long vehicle, but the visual inspection time of the resulting image typically takes 10-15 minutes. These systems penetrate ˜30 cm of steel and the radiation dose to the object is about 150-250 μGy. Their obvious drawback is that they do not automatically identify SNM and in general, the performance of such systems relies heavily on operator skill and judgment.
c) Dual-Energy Radiography (DER) Systems
DER is a well-established technique that has found numerous applications in medical imaging (in particular, in-vivo bone mineral densitometry), environmental studies, material assaying, NDT, NDE, as well as security inspection scenarios. It is based on comparing the transmission attenuation at two energies, a sensitive measure for the atomic number Z of absorbers in the line-of-sight from radiation source to detector.
DER scans are usually performed at energies where the photoelectric component of the transmission attenuation dominates. One particular variant exploits characteristic discontinuities in attenuation when the incident photon energy is varied around inner-atomic-shell (K,L,M, . . . , ) binding energies of a particular element. Typically, the latter vary with Z2. Locating such an energy-discontinuity (by comparing the photon flux transmission above and below it) is a sensitive indication of the presence of the element in question in the field-of-view. However, this variant of the method is unsuitable for inspecting massive cargo such as aviation or marine containers, since the relevant K-binding energies (the highest-energy discontinuities) for Z˜92 are at ˜110 keV, an energy too low to penetrate the inspected items.
In summary, the inadequacy of existing screening methods to effectively interdict the illicit-transport of SNM at the crucial outgoing and incoming control points underscores the need for novel inspection systems that will reliably detect the presence of small quantities of SNM in cargo items ranging in magnitude from small packages, through passenger baggage, palletized cargo and aviation containers, up to a fall-size marine container, loaded land-vehicle or railroad freight-car.

Method and system for detecting substances, such as special nuclear materials


Improved shade mounting brackets. The shade mounting brackets comprise a first bracket and a second bracket. The first and second bracket each have a first arm and a second arm, wherein the first arm has a slot and the second arm has at least one aperture constructed and positioned to receive at least one fastener for securing the bracket to a mounting surface. Both brackets also have a body portion, wherein the body portion has at least one aperture constructed and positioned to receive at least one fastener for securing the bracket to the mounting surface. The first bracket slot constructed substantially in the shape of a cross and having a receiver section adapted to accept a round pin of a shade assembly, and a plurality of retention support sections configured to allow the round pin to be rotatably mounted with respect to said first bracket. The second bracket slot has a plurality of retention support sections; the shape of said retention support sections is adapted to accept the flat spear and sized to prevent the flat spear from rotating with respect to the bracket.

Shade mounting brackets of various types are well known in the art. Such apparatus are used for holding a window shade in the desired position to cover a window. These devices are commonly used in a residential or commercial setting. Window shade mounting brackets are generally sold in pairs, each individual bracket in a pair is usually different from the other bracket of the pair, to some extent at least, since one bracket is designed to receive only the flattened spear end which projects from one end of a window shade roller, and the other bracket is designed to receive only the round pin end which projects from the other end of the window shade roller. When the pin end and the spear end are inserted into their corresponding brackets, the window shade is supported by the pair of brackets. Thus, typically each bracket is dissimilar in construction to the other bracket of the pair.
Depending on the desired mounting position of the bracket on or around the window frame, additional hardware or different bracket configurations may be necessary. As a result, most bracket manufacturers must manufacture several different types of brackets. This generally substantially increases manufacturing costs such as tooling, increases inventory volume, and creates potential manufacturing and packaging difficulties. For example, if several separate manufacturing lines are operating, one for each different bracket configuration, and one line is shut down due to mechanical failure, production eventually stops even though the other lines are in good working order. Additionally, extra care must be taken to ensure that one, and only one, bracket of each design is eventually packaged, and this can increase costs due to the need for special packaging machinery and/or higher inspection standards. Accordingly, the more different bracket designs that need to be supplied, the more likely these problems will occur.
The conventional dissimilar-shaped brackets also often pose a problem for the homeowner who wishes to install a pair of brackets. Additional care must be taken, when mounting a set of brackets, to make sure that the bracket design supplied with the shade will provide the desired mounting position, since brackets are not universal. For example, some designs of mounting brackets are capable of being mounted only to a vertical surface, such as a wall or vertical casement surface, while another, quite dissimilar type of bracket must be separately purchased for ceiling or overhead mounting.
The prior art has attempted to provide universal mounting brackets; however, they have some inherent disadvantages. One of the disadvantages is that the brackets are designed so as to require an open-ended slot for the spear end to be properly inserted into the bracket. An open-ended slot is undesirable because the flat spear end of the shade roller assembly is more likely to fall out of the bracket when someone either pulls the shade down or rolls the shade up. This causes a potentially dangerous situation, because the shade assembly can fall out of the bracket and injure the user.
Included in the prior art are brackets that employ an additional part at the interface of the shade roller and the bracket which can be installed in several different ways to provide the required orientation of the engagement feature while maintaining the alignment of the bracket and roller. An additional piece of hardware is disadvantageous because of both additional manufacturing costs and additional difficulty in handling and installation.
The present invention overcomes these and other problems inherent in existing shade mounting brackets. The present invention provides a pair of universal shade mounting brackets for several types of window shades, which eliminates the manufacture and stocking by distributors and stores of a large number of presently used different bracket configurations. A feature of the pair of brackets is that the brackets may be mounted on the inside of the window frame either at the top of the frame or on the sides of the frame, on the wall outside the frame, or on the ceiling. This universality is distinguished from the common slotted bracket which must be maintained with the slot in an upward orientation, so that the rectangular- or square-shaped spear end of the shade roller does not fall out of the slot in the bracket.


A computer system includes a real-time interrupt that causes the operating system to determine which isochronous tasks are pending. In one embodiment, applications that include isochronous tasks are certified to be well-behaved and the operating system will only initiate applications that are known to be well-behaved by checking a list of certified applications. The operating system will not initiate an application if insufficient resources are available for executing the tasks of the application. Each application informs the operating system of an execution rate and a maximum duration of its isochronous tasks. Prior to initiating an application, the operating system verifies that resources are available to execute the isochronous tasks of the application. The operating system includes a non-maskable interrupt to terminate isochronous tasks. Termination may be necessary if an isochronous task fails to execute within its specified maximum duration. Alternatively, an isochronous task may be preempted to execute a higher priority task. The operating system may include two types of time-slices. Higher priority tasks are allocated to quick slices and lower priority tasks are allocated to standard slices. Standard slices are preemptable and quick slices are not preemptable.

1. Field of the Invention
The present invention relates to operating systems, and more particularly, to processing isochronous data by general-purpose operating systems.
2. Description of the Related Art
Computer systems, such as personal computer systems, were originally developed for business applications such as word processing, databases and spreadsheets, among others. Computer systems, however, are currently being used to handle a number of isochronous tasks, including: multimedia applications having video and audio components, video capture and playback, telephony applications, and speech recognition and synthesis, among others. Generally speaking, isochronous tasks are time-dependent tasks. In other words, the data handled by an isochronous task must be delivered or processed within certain time constraints. Isochronous tasks typically require some degree of precision scheduling and execution.
One problem that has risen is that computer systems originally designed for business applications are not well-suited to the time-dependent requirements of modern multimedia applications. For example, modem personal computer system architectures still presume that the majority of applications executed on the computer system are business applications such as word processing and/or spreadsheet applications, which typically include non-time-dependent tasks. The computer systems typically implement general-purpose operating systems, which may efficiently schedule tasks, but which may not ensure the execution of a task at precise intervals as required for isochronous tasks. For example, a general-purpose operating system may not be able to guarantee the execution of a task within 100 milliseconds. This delay may be unacceptable for isochronous tasks such as audio, video and telephony. The delay may cause perceptible errors such as gaps or altered frequencies in audio signals, blank screens or lines in video signals, and echoes in telephony.
Traditionally, the central processing unit (CPU) of typical computer systems could not efficiently handle isochronous tasks, so isochronous tasks, such as a modem function, typically used dedicated hardware. It is desirable to modify CPUs to handle isochronous tasks so that the dedicated hardware used for isochronous tasks may be eliminated.
Some computer systems implement a real-time operating system (RTOS) to ensure precise execution of time-dependent tasks. Real-time operating systems typically include a clock which starts and stops tasks at regular intervals. The operating system may implement an interrupt which is asserted to start and stop tasks. The tasks typically include a specified rate of execution and a specified maximum duration. The operating system starts a task at the specified rate and expects the task to complete within the specified maximum duration so that a new task may be started. Unfortunately, real-time operating systems depend upon the tasks being well-behaved for correct operation. For example, if a task exceeds its specified maximum duration, the operating system may not be able to execute the next scheduled task. This situation may cause real-time operating systems to crash. In many computer systems, tasks may come from many different sources and it is difficult to guarantee that each task is well-behaved. Accordingly, real-time operating systems are not well-suited to common computing applications.
Several partially effective methods of providing real-time capability to general-purpose operating systems have been used. A general-purpose operating system is an example of a non-real-time operating system. Generally speaking, a non-real-time operating system is an operating system designed to support tasks that do not have well-defined execution durations. One method of providing real-time capability to a general-purpose operating system is to perform real-time tasks during an interrupt service routine. An interrupt causes an interrupt service routine to be executed and real-time work is done within the interrupt service routine. This method creates the problem that the code within the interrupt service routine is uninterruptable. Therefore, the operating system has no way to preempt or terminate a real-time task to initiate another task. Accordingly, if a real-time task exceeds a predetermined duration or gets locked-up, the operating system has no way to take control and may crash.
The Windows 98 operating system attempts to handle real-time and isochronous tasks by implementing a deferred procedure call. When an interrupt initiates a real-time task, the interrupt service routine is limited to gathering critical data, determining the source of the interrupt and requesting a deferred procedure call. The deferred procedure call is put in a FIFO queue. The deferred procedure calls in the FIFO queue are given highest priority by the operating system. Deferred procedure calls are preemptable so that higher priority task may be issued. Unfortunately, this method is not protected against mis-behaving software. For example, if a task initiated by a deferred procedure call disables interrupts, the operating system may not be able to preempt or terminate the task if necessary.
What is desired is a reliable way to execute isochronous tasks in a general-purpose operating system.
The present invention contemplates a computer system that includes a real-time interrupt. The real-time interrupt may cause an operating system to determine whether an isochronous task is pending. If an isochronous task is pending, the operating system executes the isochronous task. In one particular embodiment, the interrupt is derived from a master clock signal that also provides a clock signal to isochronous peripheral devices. In one embodiment, applications that include isochronous tasks are certified to be well-behaved. In one particular embodiment applications are registered with an operating system vendor. The operating system will only initiate an application is known to be well-behaved by checking a list of certified applications.
The operating system may not initiate an application if insufficient resources are available for executing the isochronous tasks of the application. Each isochronous task informs the operating system of an execution interval and a duration. Prior to initiating an application, the operating system verifies that sufficient resources are available to execute the isochronous tasks of the application. In one embodiment, the operating system includes a user interface. In one particular embodiment, if sufficient resources are not available to execute the isochronous tasks of an application, the operating system uses the system interface to query a user whether to suspend an existing application, to increase the percentage of operating system bandwidth allocated to isochronous tasks, or to not initiate the new application.
In one embodiment, the operating system includes a non-maskable interrupt to terminate isochronous tasks. Termination may be necessary if an isochronous task fails to completely execute within its specified duration.
In one embodiment, the operating system includes two types of time-slices. Higher priority tasks are allocated to quick slices and lower priority tasks are allocated to standard slices. Standard slices are preemptable and quick slices are not preemptable. In one embodiment, a standard slice that has been preempted may assert a flag indicating that the task has been preempted and may make the task non-preemptable the next time it is invoked.
Broadly speaking the present invention contemplates a processor, a memory coupled to the processor, interrupt generation circuitry coupled to the processor and configured to output a time-slice interrupt at a known rate, a system clock coupled to the interrupt generation circuitry, and a non-realtime operating system. The non-realtime operating system is configured to operate on the processor and to receive the time-slice interrupt. The operating system is configured to execute a pending isochronous task with a predetermined maximum execution time when the operating system receives the time-slice interrupt. Applications including isochronous tasks are certified to be well-behaved.
The present invention further contemplates a personal computer including: a processor, a main memory coupled the processor, a local bus coupled to the processor, one or more isochronous peripheral devices coupled to the local bus and configured to send or receive isochronous data, interrupt generation circuitry coupled to the processor and configured to output a time-slice interrupt at a known rate, a system clock coupled to the interrupt generation circuitry, and a non-realtime operating system. The non-realtime operating system is configured to operate on the processor and receive the time-slice interrupt. The operating system is configured to execute a pending isochronous task with a predetermined maximum execution time when the operating system receives the time-slice interrupt. Applications including isochronous tasks are certified to be well-behaved.
The present invention still further contemplates a method of reliably executing isochronous tasks in a non-realtime operating system comprising: certifying one or more applications including isochronous tasks to be well-behaved; receiving an interrupt; executing the pending isochronous task; and if no additional isochronous tasks are pending, executing a non-isochronous task.

Isochronous system using certified drivers to ensure system stability


Methods and apparatus are provided for calibrating a voltage controlled oscillator, such as an N-stage voltage controlled ring oscillator. The voltage controlled oscillator comprises a power supply input and at least one gate delay element and has a frequency that is a function of a delay of the gate delay element and a voltage applied to the power supply input. A voltage controlled oscillator is calibrated by varying an output voltage of a programmable voltage source through a range of values; applying the output voltage to the power supply input of the voltage controlled oscillator; comparing an output clock frequency of the voltage controlled oscillator to a reference frequency clock for each of the output voltage values; and selecting a value of the output voltage that provides an approximate minimum frequency difference between the output clock frequency and the reference frequency clock.

The anticipated variation in silicon processing and the range of environmental conditions must be considered when designing an integrated circuit to guarantee that the circuit meets the appropriate performance requirements. The variation in silicon processing and environmental conditions can cause significant circuit performance changes. As a result, compromises must often be made when designing a circuit to guarantee that the circuit is robust even in the presence of all silicon process and environmental effects. For example, the size of a CMOS buffer must be chosen larger than the size required for typical device performance in older to meet performance requirements under a silicon process and environment that produces "slow" devices
When a circuit is designed to meet all requirements in the presence of the full amount of silicon process and environmental conditions, the overall circuit performance is generally compromised. Specifically, a circuit designed under a set of "typical" process conditions must be sized greater than necessary under "typical" processing conditions to meet performance requirements under a set of "slow" process conditions. Supply currents under a "fast" silicon process case are much greater than required and, as a result, signal and power supply trace widths must be increased significantly to reduce the reliability risk of electromigration.
A number of techniques have been proposed or suggested for compensating for process variation and leakage current. For example, the impact of process variation is often reduced by designing a circuit such that the circuit is capable of meeting performance requirements over the entire range of silicon process and environmental conditions. Other techniques employ statistical device models that gauge the probability of a circuit parameter reaching an undesired level. With such statistical models, the "effective" silicon process variation is reduced to a low probability level. The "corner" cases, however are not eliminated
More recently, circuit techniques such as transistor-stacking, the use of multiple threshold devices, applying a body bias to devices subject to high leakage currents or forcing unused circuits in a device to a low leakage state have been employed to minimize device leakage current. Such techniques, however, either require new circuit design topologies not available in many common libraries, add silicon processing steps to allow for multiple CMOS device thresholds or require control techniques to adjust the body voltage of devices. Thus, the design time, circuit area, and cost may all be increased.
A need therefore exists for methods and apparatus for compensating for the impact of silicon process variation. A further need exists for methods and apparatus that compensate for silicon process variation while also reducing device leakage current and maximum device power dissipation

Method and apparatus for calibrating a voltage controlled oscillator by varying voltage applied to power supply input