CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. National Phase application Ser. No. 10/296,849, filed Aug. 27, 2002, which is a National Phase Application under 35 U.S.C. § 371 of Application No. PCT/US2001/17758, filed May 31, 2001, which claims priority to U.S. Provisional Application No. 60/208,944 filed May 31, 2000. All of these applications are incorporated herein by reference
This application is related to U.S. Ser. No. 07/871,973 filed Apr. 22, 1992, now U.S. Pat. No. 5,767,240; U.S. Ser. No. 08/342,297, filed Oct. 17, 1994 (published as WO96/11948), now U.S. Pat. No. 6,174,862; U.S. Ser. No. 60/037,404, filed Feb. 7, 1997 (published as WO98/35042); U.S. Ser. No. 09/187,330, filed Nov. 11, 1998 (published as WO00/27875); U.S. Ser. No. 09/267,511, filed Mar. 12, 1999 (published as WO00/53217); U.S. Ser. No. 60/149,956, filed Aug. 18, 1999 (published as WO01/12654); U.S. Ser. No. 60/208,944, filed May 31, 2000; and U.S. Ser. No. 60/267,805, filed Feb. 8, 2001; herein each incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
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Some experimental results suggest that cognitive enhancers can improve some types of learning and memory. In most cases, cognitive enhancers have been used to treat people with neurological or mental diseases, but there is a growing number of healthy, normal individuals who use these compounds in hopes of getting smarter. In addition, efficacy of these compounds in normal people is uncertain. The identification and isolation of new compounds that would improve cognitive skills would be desirable. The present invention meets this and other needs.
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OF THE INVENTION
Embodiments of the invention provide methods for improving performance (e.g., learning and/or memory) by administering either prenatally or postnatally to a subject an Activity Dependent Neurotrophic Factor (ADNF) polypeptide in an amount sufficient to improve postnatal performance. The ADNF polypeptides include ADNF I and ADNF III (also referred to as ADNP) polypeptides, analogs, subsequences, and D-amino acid versions (either wholly D-amino acid peptides or mixed D- and L-amino acid peptides), and combinations thereof which contain their respective active core sites and provide neuroprotective and growth-promoting functions.
The ADNF I polypeptides have an active core site comprising the following amino acid sequence: Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (“SALLRSIPA” or in short referred to as “SAL” or “ADNF-9”; SEQ ID NO:1). The ADNF III polypeptides also have an active core site comprising a few amino acid residues, namely, the following amino acid sequence: Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (“NAPVSIPQ” or in short referred as “NAP”; SEQ ID NO:2). These ADNF polypeptides have previously been shown, each on their own, to have remarkable potency and activity in animal models related to neurodegeneration.
In one embodiment of the present invention, it is discovered that upon post-natal administration, the ADNF polypeptides also improve performance, such as learning and memory, in animal models that are afflicted with, e.g., neuropathology, Alzheimer's disease, Down's syndrome, age, or mental retardation (e.g., fragile X syndrome), as well as normal animals. The polypeptides of the invention can also be used to improve short term and reference memory.
As such, applications for the ADNF polypeptides of the present invention include improving the performance of subjects with, e.g., neuropathology; sensory-motor problems; improving the performance of subjects impaired in cognitive tasks; improving the performance of subjects with memory deficiencies; improving the performance of normal subjects; and the like. Accordingly, embodiments of the invention in suitable formulations, can be employed for decreasing the amount of time needed to learn a cognitive, motor or perceptual task. Alternatively, invention compounds, in suitable formulations, can be employed for increasing the time for which cognitive, motor or perceptual tasks are retained. As another alternative, embodiments of the invention in suitable formulations, can be employed for decreasing the quantity and/or severity of errors made in recalling a cognitive, motor or perceptual task. Such treatment may prove especially advantageous in individuals who have suffered injury to the nervous system, or who have endured disease of the nervous system. ADNF polypeptides are administered to the affected individual, and thereafter, the individual is presented with a cognitive, motor or perceptual task. Moreover, ADNF polypeptides can be administered to normal subjects to improve their performance (e.g., learning and memory). ADNF polypeptides can be particularly useful for an aged population in which capacity for memory (e.g., short term) has generally declined.
In another embodiment, the present invention is based, in part, on the discovery that when animals in utero are treated with Activity Dependent Neurotrophic Factor (ADNF) polypeptides, the ADNF polypeptides improved the animals' postnatal learning and memory, in particular, spatial learning. Surprisingly, this long term effect of ADNF polypeptides is observed even when a single dose of ADNF polypeptides is prenatally administered in the beginning of pregnancy. Quite surprisingly, this enhanced learning and memory effect of ADNF polypeptides is seen even in animals with normal mental capacity (e.g., normal mice without any mental impairment). Hence, ADNF polypeptides can push normal animals beyond their natural capacity of learning and memory and can improve their cognitive skills.
As described above, these ADNF polypeptides have previously been shown to have remarkable potency and activity in animal models, particularly in those related to neurodegeneration. However, the effects of ADNF polypeptides were observed when they were postnatally administered to the animals. It has now been discovered for the first time that the prenatal treatment with ADNF polypeptides can enhance the animals' postnatal learning and memory, both for normal animals as well as for mentally impaired animals.
The present discovery has significant applications in human subjects in improving their learning, memory, and associated mental processes. Even normal human subjects can benefit from the prenatal treatment with ADNF polypeptides. Moreover, the present discovery has applications in subjects who are mentally compromised. For example, if a fetus is diagnosed as having mental retardation or Down's syndrome, the fetus in utero can be treated with ADNF polypeptides so that its postnatal learning and memory skills can be ameliorated. Even without a specific diagnosis of mental retardation or Down's syndrome, ADNF polypeptides can be prophylactically administered to the fetus in certain circumstances. For example, if there is a family history of mental retardation (e.g., fragile X syndrome), ADNF polypeptides can be prophylactically administered to the fetus in utero. In another example, if the mother is older (e.g., 35 years or older) and thus, has a higher risk of having a baby with Down's syndrome or other genetic defects, ADNF polypeptides can be prophylactically administered to the fetus in utero.
Various parameters can be measured to determine if an ADNF polypeptide or a combination of ADNF polypeptides improves performance (e.g., learning and memory) in vivo. For example, the hidden platform test of the Morris water maize can be used described in the materials and methods section below can be used. Generally, mice that are treated with ADNF polypeptides and control mice (that are not treated with ADNF polypeptides) are trained to escape swimming task by learning the position of a hidden platform and climbing on it. The time it takes them to complete this task is defined as the escape latency. This test can be conducted one or more times daily for a number of days. One parameter that is indicate of improved learning and memory is the reduction in latency in escaping swimming task by climbing onto a hidden platform. See, also, methods described in Gozes et al., Proc. Natl. Acad. Sci. USA 93:427-432 (1996), incorporated herein by reference. Animals treated with suitable ADNF polypeptides would show improvement in their learning and memory capacities compared to the control that are not treated with ADNF Polypeptides. Embodiments of the invention are not limited by examples of test used to measure performance. Any suitable test methods can be used to measure performance, such as learning and memory.
Other methods known in the art can be used in human subjects to determine if an ADNF polypeptide or a combination of ADNF polypeptides improves performance (e.g., learning and memory) in vivo. For example, these methods include assessment of memory or learning over time by the Randt Memory Test (Randt et al., Clin. Neuropsychol. 2:184 (1980), Wechsler Memory Scale (J. Psych. 19:87-95 (1945), Forward Digit Span test (Craik, Age Differences in Human Memory, in: Handbook of the Psychology of Aging, Birren and Schaie (Eds.), New York, Van Nostrand (1977), Mini-Mental State Exam (Folstein et al., J. of Psych. Res. 12:189-192 (1975), or California Verbal Learning Test (CVLT)). See, also, U.S. Pat. No. 6,030,968. In these tests, factors unrelated to effects of ADNF polypeptides (e.g., anxiety, fatigue, anger, depression, confusion, or vigor) are controlled for. See, U.S. Pat. No. 5,063,206. Methods for assessing and controlling for subjective factors is known in the art and determined by such standard clinical tests such as the BECK Depression Scale, Spielberger Trait State Anxiety test, and POMS test (Profile of Mood State).
In one aspect, the present invention provides a method for improving performance (e.g., learning and/or memory), the method comprising administering either postnatally or prenatally to a subject an Activity Dependent Neurotrophic Factor (ADNF) polypeptide in an amount sufficient to improve postnatal performance (e.g., learning and/or memory). Methods of the invention can be applied to any subjects, e.g., subjects who are afflicted with neuropathology, such Alzheimer's disease, Down's syndrome, etc. or normal subjects, either young or old, or subjects in utero. In one embodiment, the ADNF polypeptide is prenatally administered to the subject who has normal mental capacity. In another embodiment, the subject has mental retardation (e.g., fragile x syndrome), a family history of mental retardation, Down's syndrome, or a mother who is at least 35 years of age when pregnant with the subject. Preferably, if the subject has mental retardation, it is not caused by excessive maternal alcohol consumption during pregnancy (i.e., mental retardation is not part of fetal alcohol syndrome).
In one embodiment, the ADNF polypeptide is administered prenatally, e.g., to a pregnant mother, e.g., by intraperitoneal administration or oral administration.
In another embodiment, the ADNF polypeptide is administered postnatally, e.g., by intraperitoneal administration or oral administration. In one embodiment, the ADNF polypeptide is administered at the time of neural tube development and/or closure of the neural tube.
In one embodiment, the method comprises administering an ADNF polypeptide, wherein the ADNF polypeptide is an ADNF I polypeptide comprising an active core site having the amino acid sequence of Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:1)). In another embodiment, the method comprises administering a full length ADNF I polypeptide. In yet another embodiment, the method comprises administering an ADNF I polypeptide which consists of the amino acid sequence of Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:1). In yet another embodiment, the method comprises administering an ADNF I polypeptide, wherein the ADNF I polypeptide is selected from the group consisting of: Val-Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:14); Val-Glu-Glu-Gly-Ile-Val-Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:15); Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:16); Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:17); Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:18); and Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:19). In yet another embodiment, the method comprises administering an ADNF I polypeptide having up to about 20 amino acids at least one of the N-terminus or the C-terminus of the active core site. In certain embodiments, the ADNF I polypeptide has up to 20 amino acids at both the N-terminus and the C-terminus of the ADNF I polypeptide.
In another embodiment, the method comprises administering an ADNF polypeptide, wherein the ADNF polypeptide is an ADNF III polypeptide comprising an active core site having the amino acid sequence of Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2). In yet another embodiment, the method comprises administering a full length ADNF III polypeptide. In yet another embodiment, the method comprises administering an ADNF I polypeptide which consists of the amino acid sequence of Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2). In yet another embodiment, the method comprises administering an ADNF III polypeptide, wherein the ADNF III polypeptide is selected from the group consisting of: Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:20); Leu-Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-Gln-Ser (SEQ ID NO:21); Leu-Gly-Leu-Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-Gln-Ser (SEQ ID NO:22); and Ser-Val-Arg-Leu-Gly-Leu-Gly-Gly-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-Gln-Ser (SEQ ID NO:23). In yet another embodiment, the method comprises administering an ADNF III polypeptide having up to about 20 amino acids at least one of the N-terminus and the C-terminus of the active core site. In certain embodiments, the ADNF III polypeptide has up to 20 amino acids at both the N-terminus and the C-terminus of the ADNF III polypeptide.
In yet another embodiment, the method comprises administering a mixture of an ADNF I polypeptide and an ADNF III polypeptide. Any one or more of the ADNF I polypeptides described herein can be mixed with any one or more of the ADNF III polypeptides described herein in this and other aspects of the invention.
In another embodiment, the active core site of the ADNF polypeptide comprises at least one D-amino acid. In another embodiment, the active core site of the ADNF polypeptide comprises all D-amino acids.
In yet another embodiment, at least one of the ADNF polypeptide is encoded by a nucleic acid which is administered to the subject.
In yet another embodiment, the ADNF polypeptide improves a short term memory. In yet another embodiment, the ADNF polypeptide improves a reference memory. In yet another embodiment, the ADNF polypeptide is administered intranasally or orally.
These and other aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the invention, the accompanying drawings, and the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
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FIG. 1 AF64A-treated rats exhibit an impairment in learning and memory that is ameliorated by intranasal administration of ADNF-9. Two daily water maze tests (A and B, respectively) were performed on adult rats. Groups tested were: 1. control animals treated with vehicle (20 animals, open circles); 2. AF64A-treated animals intranasally administered with vehicle (27 animals, open squares); 3. control animals treated by intranasal administration of ADNF-9 (closed circles, 12 animals); 4. AF64A-treated animals intranasally administered with ADNF-9 (19 animals, closed squares). (A) Latency measured in seconds (mean±standard error of the mean) to reach the hidden platform in its new daily location (indicative of intact reference memory, Gordon et al., Neurosci. Lett. 199:1-4 (1995)) is depicted. Tests were performed over four consecutive days. (B) Latency measured in seconds to reach the hidden platform 0.5 min. after being on it (indicative of intact working memory processes, Gordon et al., Neurosci. Lett. 199:1-4 (1995); Gozes et al., J. Neurobiol. 33:329-342. (1997a)) tested over four consecutive days. There were no differences between animals treated with vehicle and untreated animals (data not shown). (C) On day 5 of testing, the platform was removed and a spatial probe test was performed. The animals were allowed to swim for 120 sec. and the time spent by the animal at the platform quadrant was recorded.
FIG. 2 AF64A-treated rats exhibit impairments in learning and memory that are ameliorated by intranasal administration of NAP. The same experiment reported in FIG. 1 (A, B, C, respectively) was repeated, except that the peptide used was NAP and the number of animals per each of the experimental groups was 10-20 and 27 for the AF64A-treated group.
FIG. 3 Intranasally applied [3H]-NAP reaches the body and the brain. (A) Animals were sacrificed at indicated times after administration and tissue samples were weighed and assayed (in duplicates) for radioactivity in a β-counter, a mean of four animals is depicted. (B) Brains were dissected at indicated time points and radioactivity monitored. (C, D) Intact [3H]-NAP reached the brain after intranasal administration. Radioactive tissue samples (cerebral cortex) were homogenized and subjected to low-speed centrifugation. Supernatants (30 minutes following application, closed circles, C, and 60 minutes following application, closed triangles, D) were analyzed by HPLC fractionation against [3H]-NAP stock (open circles). Samples were monitored for radioactivity (dpm) in a β-counter. All results were calculated to depict radioactivity as fmoles of NAP/g tissue. (E) The experiment was repeated with three additional animals, here the animals were 200 g each instead of 250-300 g in A-D and small visible blood vessels were removed utilizing watchmaker's forceps (no. 5).
FIG. 4 (A) Intranasal application of NAP prevents reduction in choline acetyl transferase activity in AF64A-treated rats. Incorporation of radiolabeled choline into acetyl choline is shown. Results were calibrated against control (100%). Experiments utilizing three animals per group (each in triplicates) were conducted and analyzed as described in the text. (B) AF64A-treated rats exhibit impairments in learning and memory, long-lasting effects of NAP, but not of ADNF-9 treatment. Ten male rats (as described in the methods section) were used per experimental group. Four groups were used, three were treated with AF64A and one group was treated with saline (control). The rats were allowed a week for recovery, and then two AF64A groups were treated (intranasally) with either ADNF-9 or NAP. Following 5 treatment days the animals were allowed to recover for two days and then subjected to daily water-maze tests (as in FIGS. 1 and 2). The difference between this experiment and the experiments in FIGS. 1 and 2 is that the animals did not receive a daily intranasal application of peptides prior to the behavioral test. The figure depicts the second daily test indicative of short-term memory.
FIG. 5 illustrates the effects of prenatal treatment of animals with a mixture of L-NAP and L-SAL (intraperitoneal injection) on learning as assessed by a Morris water maze test.
FIG. 6 illustrates the effects of prenatal treatment of animals with a mixture of D-NAP and D-SAL (oral administration) on learning as assessed by a Morris water maze test.
FIG. 7 illustrates the effects of prenatal treatment of animals with D-SAL (oral administration) on learning as assessed by a Morris water maze test.
FIG. 8 illustrates the effects of prenatal treatment of animals with D-NAP (oral administration) on learning as assessed by a Morris water maze test.
FIG. 9 illustrates the effects of prenatal treatment of animals with a double dose of D-SAL (oral administration) on learning as assessed by a Morris water maze test.
FIG. 10 illustrates the effects of prenatal treatment of animals with a mixture of D-NAP and D-SAL (oral administration) on learning as assessed by a probe test.
The phrase “ADNF polypeptide” refers to one or more activity dependent neurotrophic factors (ADNF) that have an active core site comprising the amino acid sequence of SALLRSIPA (referred to as “SAL”; SEQ ID NO: 1) or NAPVSIPQ (referred to as “NAP”; SEQ ID NO:2), or conservatively modified variants thereof that have neurotrophic/neuroprotective activity as measured with in vitro cortical neuron culture assays described by, e.g., Hill et al., Brain Res. 603, 222-233 (1993); Venner & Gupta, Nucleic Acid Res. 18, 5309 (1990); and Peralta et al., Nucleic Acid Res. 18, 7162 (1990); Brenneman et al., Nature 335, 636 (1988); or Brenneman et al., Dev. Brain Res. 51:63 (1990); Forsythe & Westbrook, J. Physiol. Lond. 396:515 (1988). An ADNF polypeptide can be an ADNF I polypeptide, an ADNF III polypeptide, their alleles, polymorphic variants, analogs, interspecies homolog, or any subsequences thereof (e.g., SALLRSIPA; SEQ ID NO:1 or NAPVSIPQ; SEQ ID NO:2) that exhibit neuroprotective/neurotrophic action on, e.g., neurons originating in the central nervous system either in vitro or in vivo. An “ADNF polypeptide” can also refer to a mixture of an ADNF I polypeptide and an ADNF III polypeptide.
The term “ADNF I” refers to an activity dependent neurotrophic factor polypeptide having a molecular weight of about 14,000 Daltons with a pI of 8.3±0.25. As described above, ADNF I polypeptides have an active site comprising an amino acid sequence of Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (also referred to as “SALLRSIPA” or “SAL” or “ADNF-9”; SEQ ID NO:1). See, Brenneman & Gozes, J. Clin. Invest. 97:2299-2307 (1996), Glazner et al., Anat. Embryol. (In press), Brenneman et al., J. Pharm. Exp. Ther., 285:619-27 (1998), Gozes & Brenneman, J. Mol. Neurosci. 7:235-244 (1996), and Gozes et al., Dev. Brain Res. 99:167-175 (1997), all of which are herein incorporated by reference. Unless indicated as otherwise, “SAL” refers to a peptide having an amino acid sequence of Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:1), not a peptide having an amino acid sequence of Ser-Ala-Leu. A full length amino acid sequence of ADNF I can be found in WO 96/11948, herein incorporated by reference in its entirety.
The terms “ADNF III” and “ADNP” refer to an activity dependent neurotrophic factor polypeptide having a predicted molecular weight of about 95 kDa (about 828 amino acid residues) and a pI of about 5.99. As described above, ADNF III polypeptides have an active site comprising an amino acid sequence of Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (also referred to as “NAPVSIPQ” or “NAP”; SEQ ID NO:2). See, Bassan et al., J. Neurochem. 72:1283-1293 (1999), incorporated herein by reference. Unless indicated as otherwise, “NAP” refers to a peptide having an amino acid sequence of Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2), not a peptide having an amino acid sequence of Asn-Ala-Pro. Full length sequences of ADNF III can be found in WO 98/35042 and WO 00/27875.
The phrase “improving learning and/or memory” refers to an improvement or enhancement of at least one parameter that indicates learning and memory. Improvement or enhancement is change of a parameter by at least 10%, optionally at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, etc. The improvement of learning and memory can be measured by any methods known in the art. For example, ADNF polypeptides that improve learning and memory can be screened using Morris water maze (see, e.g., materials and methods section). See, also, Gozes et al., Proc. Natl. Acad. Sci. USA 93:427-432 (1996). Memory and learning can also be screened using any of the methods described herein or other methods that are well known to those of skill in the art, e.g., the Randt Memory Test, the Wechler Memory Scale, the Forward Digit Span test, or the California Verbal Learning Test.
The term “memory” includes all medical classifications of memory, e.g., sensory, immediate, recent and remote, as well as terms used in psychology, such as reference memory, which refers to information gained from previous experience, either recent or remote (see, e.g., Harrison's Principles of Internal Medicine, volume 1, pp. 142-150 (Fauci et al., eds., 1988).
Pathologies or neuropathologies that would benefit from therapeutic and diagnostic applications of this invention include, for example, the following:
diseases of central motor systems including degenerative conditions affecting the basal ganglia (Huntington's disease, Wilson's disease, striatonigral degeneration, corticobasal ganglionic degeneration), Tourette's syndrome, Parkinson's disease, progressive supranuclear palsy, progressive bulbar palsy, familial spastic paraplegia, spinomuscular atrophy, ALS and variants thereof, dentatorubral atrophy, olivo-pontocerebellar atrophy, paraneoplastic cerebellar degeneration, and dopamine toxicity;
diseases affecting sensory neurons such as Friedreich's ataxia, diabetes, peripheral neuropathy, retinal neuronal degeneration;
diseases of limbic and cortical systems such as cerebral amyloidosis, Pick's atrophy, Retts syndrome;
neurodegenerative pathologies involving multiple neuronal systems and/or brainstem including Alzheimer's disease, AIDS-related dementia, Leigh's disease, diffuse Lewy body disease, epilepsy, multiple system atrophy, Guillain-Barre syndrome, lysosomal storage disorders such as lipofuscinosis, late-degenerative stages of Down's syndrome, Alper's disease, vertigo as result of CNS degeneration;
pathologies associated with developmental retardation and learning impairments, and Down's syndrome, and oxidative stress induced neuronal death;
pathologies arising with aging and chronic alcohol or drug abuse including, for example, with alcoholism the degeneration of neurons in locus coeruleus, cerebellum, cholinergic basal forebrain; with aging degeneration of cerebellar neurons and cortical neurons leading to cognitive and motor impairments; and with chronic amphetamine abuse degeneration of basal ganglia neurons leading to motor impairments;
pathological changes resulting from focal trauma such as stroke, focal ischemia, vascular insufficiency, hypoxic-ischemic encephalopathy, hyperglycemia, hypoglycemia, closed head trauma, or direct trauma;
pathologies arising as a negative side-effect of therapeutic drugs and treatments (e.g., degeneration of cingulate and entorhinal cortex neurons in response to anticonvulsant doses of antagonists of the NMDA class of glutamate receptor).
The term “spatial learning” refers to learning about one\'s environment and requires knowledge of what objects are where. It also relates to learning about and using information about relationships between multiple cues in environment. Spatial learning in animals can be tested by allowing animals to learn locations of rewards and to use spatial cues for remembering the locations. For example, spatial learning can be tested using a radial arm maze (i.e., learning which arm has food) a Morris water maze (i.e., learning where the platform is). To perform these tasks, animals use cues from test room (positions of objects, odors, etc.). In human, spatial learning can also be tested. For example, a subject can be asked to draw a picture, and then the picture is taken away. The subject is then asked to draw the same picture from memory. The latter picture drawn by the subject reflects a degree of spatial learning in the subject.
The term “subject” refers to any mammal, in particular human, at any stage of life. For example, the subject can refer to an embryo, a fetus, a baby, a child, an adolescent or an adult.
A “normal” subject or a subject having “normal mental capacity” refers to a subject whose intellectual functioning level is around or above average (e.g., having an IQ above 75). A “normal” subject can also refer to a subject, such as a fetus, who does not appear to have any mental impairment (e.g., according to an amniocentesis test) and/or has no risk factors (e.g., family history of mental retardation or a mother who consumed alcohol in excessive amount during pregnancy to cause fetal alcohol syndrome in the fetus).
A subject is considered to have “mental retardation” based on the following three criteria: intellectual functioning level (IQ) is below 70-75; significant limitations exist in two or more adaptive skill areas; and the condition is present from childhood (defined as age 18 or less) (AAMR, 1992). Adaptive skill areas are those daily living skills needed to live, work and play in the community. They include communication, self-care, home living, social skills, leisure, health and safety, self-direction, functional academics (reading, writing, basic math), community use and work. See, http://www.thearc.org/faqs/mrqa.html.
The term “Down\'s syndrome” is a chromosome disorder and occurs when, instead of the normal complement of 2 copies of chromosome 21, there is a whole, or sometimes part of an additional chromosome 21.
The term “contacting” is used herein interchangeably with the following: combined with, added to, mixed with, passed over, incubated with, flowed over, etc. Moreover, the ADNF polypeptides or nucleic acids encoding them of the present invention can be “administered” by any conventional method such as, for example, parenteral, oral, topical, and inhalation routes. In presently preferred embodiments, parenteral and nasal inhalation routes are employed.
“An amount sufficient” or “an effective amount” is that amount of a given ADNF polypeptide that improves performance (e.g., learning and/or memory). For example, in the context of improving learning and memory, “an amount sufficient” or “an effective amount” is that amount of a given ADNF polypeptide that reduces the latency in finding a platform in a watermaze test, either in the first daily test (indicative of reference memory) or in the second daily test (indicative of short term memory). The dosing range can vary depending on the ADNF polypeptide used, the route of administration and the potency of the particular ADNF polypeptide, but can readily be determined using the foregoing assays.
The terms “isolated,” “purified,” or “biologically pure” refer to material that is substantially or essentially free from components which normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated ADNF nucleic acid is separated from open reading frames that flank the ADNF gene and encode proteins other than ADNF. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
The term “amino acid” refers to naturally occurring amino acids, amino acid analogs, and amino acid mimetics that function in a manner similar to the naturally occurring and analog amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to synthetic amino acids that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group (e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Both naturally occurring and analog amino acids can be made synthetically. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.
Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
The following groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Serine (S), Threonine (T);
3) Aspartic acid (D), Glutamic acid (E);
4) Asparagine (N), Glutamine (Q);
5) Cysteine (C), Methionine (M);
6) Arginine (R), Lysine (K), Histidine (H);
7) Isoleucine (I), Leucine (L), Valine (V); and
8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
(see, e.g., Creighton, Proteins (1984)).
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides (i.e., 70% identity) that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the complement of a test sequence. Preferably, the percent identity exists over a region of the sequence that is at least about 25 amino acids in length, more preferably over a region that is 50 or 100 amino acids in length.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat\'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat\'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
The phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with a wash in 0.2×SSC, and 0.1% SDS at 65° C.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
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OF THE INVENTION AND PREFERRED EMBODIMENTS
Methods for Improving Performance
The present invention provides a method for improving performance (e.g., learning and/or memory) in a subject. The method comprises administering to the subject, either prenatally or postnatally, an ADNF polypeptide in an amount sufficient to improve post natal performance. In particular, prenatal administration can improve spatial learning in a subject. Candidate subjects who can benefit from such post or prenatal treatment, ADNF polypeptides that can be administered, timing and modes of administration, tests to assess improvement in learning and memory, and methods for producing ADNF polypeptides are described in detail below.
Candidate Subjects for Treatment with ADNF Polypeptides
The prenatal and postnatal treatment with ADNF polypeptides has applications in many types of subjects. For example, normal subjects can benefit from the prenatal treatment of ADNF polypeptides in terms of improving their learning and memory. A normal subject or a subject with normal mental capacity refers to those whose intellectual functioning level, even without the prenatal treatment with ADNF polypeptides, is around or above average (e.g., having an IQ over 75). In the context of a fetus, a normal subject can refer to a subject who does not appear to have any mental impairment (e.g., according to an amniocentesis test) and/or risk factors for mental retardation (e.g., family history of mental retardation or a mother who consumed enough alcohol during pregnancy to cause fetal alcohol syndrome in the subject). A mother who wishes her unborn embryo or fetus to have enhanced capacity for learning and memory can be administered with ADNF polypeptides while the embryo or fetus is in utero.