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  Acetylcholinesterase-derived peptides and uses thereofUSPTO Application #: 20070065882Title: Acetylcholinesterase-derived peptides and uses thereof Abstract: The invention relates to a cell growth and/or differentiation regulatory peptide comprising a sequence of about 9 to about 150 amino acids derived from acetylcholinesterase amino acid sequence, preferably from the C-terminal region of acetylcholinesterase. The invention also relates to pharmaceutical compositions comprising the peptides, particularly for use in promoting survival of stem cells, promoting differentiation of stem cells, promoting growth of stem cells and/or promoting the growth-enhancing effect of a growth factor on stem cells, alone, or in combination with other growth factors. Of particular interest is the use of the peptides in the treatment of thrombocytopenia, post-irradiation conditions, post-chemotherapy conditions, or conditions following massive blood loss and promotion of neural progenitors in use for cell therapies aimed at restoring neural functions in diseased individuals. Further, the invention relates to antibodies against the peptides, inter alia for diagnostic use, for example, the diagnosis of stress-induced male infertility. The invention also relates to in vitro and in vivo methods for screening of drugs that affect the central nervous system, and are potential modulators of interactions between the “readthrough” form of acetylcholinesterase, AChE-R, the intracellular receptor RACK1 and the kinase PKC. (end of abstract)
Agent: Cooper & Dunham, LLP - New York, NY, US Inventors: Hermona Soreq, Amiram Eldor, Sofia Eldor, Varda Deutsch, Dan Grisaru USPTO Applicaton #: 20070065882 - Class: 435007200 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, Involving A Micro-organism Or Cell Membrane Bound Antigen Or Cell Membrane Bound Receptor Or Cell Membrane Bound Antibody Or Microbial Lysate The Patent Description & Claims data below is from USPTO Patent Application 20070065882. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part (CIP) of U.S. patent application Ser. No. 09/998,042, filed Nov. 30, 2001, which is a continuation-in-part of PCT international application No. PCT/IL00/00311, filed May 31, 2000, which claims priority of Israeli Patent Application No. 130224, filed May 31, 1999 and Israeli Patent Application No. 131707, filed Sep. 2, 1999, incorporated herein by reference. FIELD OF THE INVENTION [0003] The invention is directed to the field of stem cell survival and expansion. Specifically, the invention is directed at the stem cell survival and expansion effects of peptides derived from acetylcholinesterase. In addition, the invention relates to a system for screening of nervous system drugs that are directed to central nervous system conditions or disorders. More specifically, the invention relates to the screening of modulators of the AChE-R-PKC.beta.II-RACK1 complex. BACKGROUND OF THE INVENTION [0004] Stress insults evoke a plethora of responses in the organism, affecting the functioning of various systems. [0005] In the hematopoietic system, stress insults are associated with rapid and significant changes in blood cell composition. For example, following massive blood loss, or after surgery, the hematopoietic system responds within hours, by an elevation of the white blood cell and platelet counts. However, the mechanisms responsible for initiating this adjustment are not fully understood. Glucocorticoid hormones, known to be elevated under stress, play a leading role in the adaptive reaction of the bone marrow in response to stress. Glucocorticoid hormones induce absolute increases in all hematopoietic lineages, especially myeloid cells. This involves a cascade of events culminating in changes in the proliferation, differentiation and apoptotic events characteristic of each of the hematopoietic cell lineages [Lansdorp (1995) Exp. Hematol. 23, 187-91]. Also, significant changes occur under glucocorticoid hormones in the levels of hematopoietic growth factors controlling the proliferation of stem cells from which blood cells develop. [0006] Hematopoietic stem cells (HSCs) are pluripotent, in that they give rise to all blood cell lineages. These cells migrate during ontogeny to settle in the bone marrow as a permanent self-renewing source of blood cells. Under normal conditions the vast majority of HSCs are non-dividing, but under conditions of development or stress they can undergo clonal expansion and self-renewal [Keller and Snodgrass (1990) J. Exp. Med. 171, 1407-18]. A large number of cytokines and growth factors, such as stem cell factor (SCF), thrombopoietin (TPO), and FLT-3 ligand, are thought to mediate the proliferative capacity of HSCs, through specific receptors, c-kit, c-mpl and flt3/flk-2, respectively. Alone, their capacity to stimulate proliferation is limited. For example, SCF can maintain survival for a few days in vitro, but not the self-renewal of HSCs (Li and Johnson, Blood 84, 408-14, 1994). However, when used in combination, these growth factors acquire a potent co-stimulatory effect. The early phase of adaptation of the hematopoietic system to stress (first 24 hr), requires coordinator(s), such as leu-enkephalin, which modulate the effects of growth factors on stem cells. However, leu-enkephalin is present in the circulation only immediately following the stress insult, whereas the modulation of hematopoiesis continues long after that phase. Therefore, additional long-acting modulators remain to be identified. [0007] The enzyme acetylcholinesterase (AChE) is expressed in brain tissue, but also in most, if not all, of the mammalian hematopoietic cell lineages. AChE is expressed in many parts of the vertebrate embryo, with a developmentally regulated pattern in specific cell types and tissues during the embryonic and adult stages. AChE diversity is noted in several pathological states, such as Alzheimer's disease, where AChE activity was shown to decrease, not only in the primary site of the disease, the brain, but also in the hematopoietic system. [0008] It has now surprisingly been found that the C-terminal peptides of AChE-S and AChE-R have independent biological activities. Specifically, it has been found that these peptides promote stem cell survival. It has also been found that these peptides promote stem cell expansion, when used in combination with growth factors. Further, it has been found that such peptides are capable of augmenting hematopoiesis in vivo. [0009] In the central nervous system (CNS), physiological stress induces rapid and robust signaling processes in mammalian brain neurons. These processes are known to suppress long term potentiation (LTP) [Vereker, E. et al. (2000) J Neurosci, 20, 6811-9], augment long term depression (LTD) [Xu, L. et al. (1997) Nature, 387, 497-500], and induce release of synaptic vesicles, potentiating neurotransmission [Stevens, C. F. and Sullivan, J. M. (1998) Neuron, 21, 885-93]. At the long term, stress-induced signaling attenuates the stress response, enabling the organism to be less excessively affected by a stressful event. This induces neuronal dendrite branching [Sousa, N. et al. (2000) Neuroscience, 97, 253-66] and synapse re-organization [McEwen, B. S. (1999) Ann Rev Neurosci, 22, 105-22]. However, the molecular pathway(s) leading from short to long term processes and which enable the adjustment to stressful stimuli, are not yet known. [0010] Ample information suggests the involvement of specific protein kinases in at least some of these stress-induced processes. The enzymatic activity of certain subtypes of protein kinase C (PKC) [Coussens, L. et al. (1986) Science, 233, 859-66] was shown to be subject to changes (i.e. biochemical activation, membrane translocation) under physiological [Hu, G. Y. et al. (1987) Nature, 328, 426-9], biochemical [Macek, T. A. et al. (1998) J Neurosci, 18, 6138-46] and cytoarchitectural [Tint, I. S. et al. (1992) Proc Natl Acad Sci USA, 89, 8160-4] responses at the cellular and organismal levels. A relevant mediator of the stress-related changes in PKC activities is likely to be largely absent from brain neurons under normal conditions, but should be induced rapidly and for long periods following stress insults. [0011] A relevant putative mediator of the stress-related changes in PKC activities should be intracellular in its location and capable of activating or translocating active PKC within neuronal perikarya. The "readthrough" acetylcholinesterase variant AChE-R is a promising candidate for this role [Soreq, H. and Seidman, S. (2001) Nat Rev Neurosci, 2, 294-302]. Brain AChE-R is exceedingly rare in the adult, non-stressed brain. Various stress insults induce AChE-R overproduction through alternative splicing, creating a different C-terminal domain from that of synaptic AChE (AChE-S). AChE-R levels rise rapidly under acute psychological stress [Kaufer, D. et al. (1998) Nature, 393, 373-7] or chemical neurotoxication [Shapira, M. et al. (2000) Hum Mol Genet, 9, 1273-81] and stay elevated for over two weeks following head injury [Shohami, E. et al. (2000) J Mol Med, 78, 228-36]. Being a secretory protein, AChE-R fulfills the extracellular function of reducing the stress-induced acetylcholine levels. In parallel, it accumulates in neuronal cell bodies [Sternfeld, M. et al. (2000) Proc Natl Acad Sci USA, 97, 8647-52], where acetylcholine hydrolysis is unlikely. Transgenic mice overexpressing neuronal AChE-R, but not the normally abundant synaptic variant AChE-S, display reduced levels of stress-associated neuropathologies [Sternfeld et al. (2000) id ibid.]. This suggests distinct stress-related function(s) for the AChE-R protein. Intriguingly, the unique C-terminal domain of AChE-R does not participate in acetylcholine hydrolysis for which the core domain, common to all of the AChE variants is sufficient [Duval, N. et al. (1992) J Cell Biol, 118, 641-53]. [0012] Using a yeast two-hybrid screen, the inventors discovered that the C-terminal domain of AChE-R forms a tight complex with RACK1 [PCT/IL00/00311]. Interestingly, the inventors have shown that PKC.beta.II is also part of this complex (FIG. 1), and all three proteins can be co-immunoprecipitated. [0013] In search for the marker of the transition between short and long term processes following stress stimuli, the inventors have demonstrated that interaction with AChE-R activates PKC.beta.II and facilitates its translocation into densely packed neuronal clusters (FIGS. 4 and 5), which may be causally involved with the stress-protection capacity of overexpressed AChE-R. [0014] In view of these unprecedented results, it this an object of this invention to provide a method for screening of nervous system drugs that modulate the trimeric complex AChE-R/PKC/RACK1 interactions. SUMMARY OF THE INVENTION [0015] This invention is directed at a cell growth and/or differentiation regulatory peptide comprising a sequence of about 9 to about 150 amino acids derived from Acetylcholinesterase amino acid sequence. The said sequence preferably contains a region predicted to be rich in beta-pleated sheet structure and turns. Also preferably, the said sequence contains a predicted amphipathic helix structure. In another embodiment of the peptide of the invention, the said sequence is derived from the C-terminal region of acetylcholinesterase. [0016] The said sequence is preferably derived from the readthrough or synaptic variant of acetylcholinesterase, preferably from the mature form thereof. The said sequence is preferably about 20 to about 70 amino acids in length. More preferably, the said sequence is SEQ ID: No. 1, SEQ ID: No. 2, SEQ ID: No. 3, SEQ ID: No.7 or SEQ ID: No.8. Still more preferably, the said peptide is SEQ ID: No. 1, SEQ ID: No. 2, SEQ ID: No. 3, SEQ ID: No.7 or SEQ ID: No.8. [0017] A peptide of the invention which is a cyclic peptide is also considered to be within the scope of the invention. [0018] The peptide of the invention is preferably synthetic and preferably comprises the amino acid sequence of SEQ ID: No. 1, SEQ ID: No. 2, SEQ ID: No. 3, SEQ ID: No.7 or SEQ ID: No.8. More preferably, the peptide has the amino acid sequence denoted by SEQ ID: No. 1, 2, 3, 7 or 8. The peptide is preferably linear and synthetic. [0019] In one embodiment, the invention provides a peptide capable of promoting cell survival and/or differentiation and comprising the amino acid sequence denoted by SEQ ID: No. 1, SEQ ID: No. 2 or SEQ ID: No. 3, and functional analogues and derivatives thereof. [0020] In another embodiment, the invention provides a peptide of the invention which is a hematopoietic stem cell growth and/or differentiation regulatory peptide. Preferably, said peptide is capable of promoting stem cell survival and/or myeloid and megakaryocytic differentiation and comprises the amino acid sequence denoted by SEQ ID: No. 1, SEQ ID: No. 2 or SEQ ID: No. 3, or functional analogues and derivatives thereof. Said peptide may be either linear or cyclic, and is preferably synthetic. [0021] In a still further embodiment, the invention provides a peptide thereof for use in ex vivo or in vivo expansion of hematopoietic stem cells and of neural progenitors. Continue reading... Full patent description for Acetylcholinesterase-derived peptides and uses thereof Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Acetylcholinesterase-derived peptides and uses thereof patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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