Patent Publication Number: US-2011078804-A1

Title: Angiogenin and Amyotrophic Lateral Sclerosis

Description:
RELATED APPLICATIONS 
     This application is a continuation of PCT application number PCT/US2009/040616 designating the United States and filed Apr. 15, 2009 which claims the benefit of U.S. provisional patent application No. 61/045,046, filed Apr. 15, 2008, both of which are hereby incorporated by reference in their entireties. 
    
    
     STATEMENT OF GOVERNMENT INTERESTS 
     This invention was made with government support under the National Institutes of Health grant number CA105241. The Government has certain rights in the invention. 
    
    
     FIELD 
     The present invention relates to methods and compositions for treating neurodegenerative diseases and disorders such as, for example, amyotrophic lateral sclerosis. 
     BACKGROUND 
     Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with specific loss of motor neurons in the brain, brain stem, and spinal cord (Pasinelli and Brown (2006)  Nat. Rev. Neurosci.  7:710). The average age of onset is 55 years with upper and lower motor neuron signs, including distal muscle weakness and wasting, increased muscle tone with hyperreflexia, and at times diaphragmatic and/or bulbar weakness. Atypical forms can include symptoms of dementia and/or Parkinsonism. Both familial and sporadic forms of the disease have been reported and described as having no distinguishable differences in symptoms, progression, and histological abnormalities. All forms of ALS progress to generalized amyotrophy, culminating in respiratory failure and death after an average duration of four years (Rowland and Shneider (2001)  N. Engl. J. Med.  344:1688). 
     ALS was first referred to as Charcot&#39;s sclerosis in honor of the French neurobiologist Jean-Martin Charcot who originally described this disease in 1869 (Charcot and Joffory (1869)  Arch. Physiol. Neurol. Pathol.  2:744). It is now more commonly known as Lou Gehrig&#39;s disease in the United States, in memory of the great baseball player who developed the disease in the 1930s. It is a devastating disease as there is almost no cognitive impairment at a time when nearly complete paralysis of arms, legs, and the muscles necessary for speech has developed (Rippon et al. (2006)  Arch. Neurol.  63:345). The only recognized treatment for ALS is riluzole, which extends survival by only about 3 months with no improvement in motor muscular functions (Bensimon et al. (1994)  N. Engl. J. Med.  330:585). 
     The incidence of ALS is estimated at 0.4-1.8 per 100,000 population (Pasinelli and Brown (2006)  Nat. Rev. Neurosci.  7:710; Gros-Louis et al. (2006)  Biochim. Biophys. Acta.  1762:956). Approximately 90% of ALS cases occur in individuals with no known family history, while the remaining cases are attributable to familial inheritance in either an autosomal dominant or recessive manner (Pasinelli and Brown (2006)  Nat. Rev. Neurosci.  7:710; Boillee et al. (2006)  Neuron  52:39). Mutations in the Cu/Zn superoxide dismutase gene 1 (ALS1; SOD1), have been identified in approximately 20% of familial (Andersen et al. (1997)  Brain  120:1723; Bruijn and Cudkowicz (2006)  Expert Rev. Neurother.  6:417; Niemann et al. (2004)  J. Neurol. Neurosurg. Psychiatry  75:1186) and in approximately 3% of sporadic (Corrado et al. (2007)  J. Neurol. Sci.  258:123; Jones et al. (1995)  J. Med. Genet.  32:290) ALS patients. Currently, SOD1 is the only known autosomal dominant gene in which mutations have been functionally associated with ALS, although three other loci have been identified for typical autosomal dominant ALS by linkage analysis (ALS3, ALS6, and ALS7) (Pasinelli and Brown (2006)  Nat. Rev. Neurosci.  7:710; Gros-Louis et al. (2006)  Biochim. Biophys. Acta.  1762:956). Other dominantly inherited genetic loci, associated with an atypical ALS phenotype, have also been identified (ALS with dementia/parkinsonism, MAPT; progressive lower motor neuron disease, DCTN1; and ALS8, vesicle-associated membrane protein (VAPB)). In autosomal dominant ALS with frontotemporal dementia (ALSFTD), genetic linkage has been reported to 9q21-q22 (Chen et al. (2006)  Neurology  67:508). Mutations in the SETX gene have been identified in juvenile onset autosomal dominant ALS. Lastly, genetic loci identified for juvenile onset autosomal recessive disease include Alsin (ALS2) and linkage for ALS5 to 15q15.1-q21 (Pasinelli and Brown (2006)  Nat. Rev. Neurosci.  7:710; Gros-Louis et al. (2006)  Biochim. Biophys. Acta.  1762:956). It is notable that besides SOD1, the other genes and loci described above have only been found in very few ALS patients, and often in atypical ALS with slow progression. 
     Genetic association studies have also identified several risk factors in ALS, including deletions or insertions in the neurofilament (NF) heavy chain gene (Al-Chalabi et al. (1999)  Hum. Mol. Genet.  8:157; Figlewicz et al. (1994)  Hum. Mol. Genet.  3:1757; Tomkins et al. (1998)  Neuroreport.  9:3967), polymorphisms in vascular endothelial growth factor (VEGF) (Lambrechts et al. (2003)  Nat. Genet.  34:383) and in the hemochromatosis gene HFE (Goodall et al (2005)  Neurology  65:934). Moreover, 5 base pair deletions in mitochondrial cyclooxygenase I (COX1) (Comi et al. (1998)  Ann. Neurol.  43:110) and a T4272C mutation in isoleucine tRNA synthesase (IARS2) (Borthwick et al. (2006)  Ann. Neurol.  59:570) have been linked to ALS, although only in a single case. A common mitochondrial DNA deletion mutation (mt DNA4977) is increased in the brain of ALS patients (Dhaliwal and Grewal (2000)  Neuroreport  11:2507). The apolipoprotein E (ApoE) epsilon4 allele has been associated with decreased survival of ALS patients (Drory et al. (2001)  J. Neurol. Sci.  190:17). Copy number variation in survival motor neuron (SMN) has also been shown to be a susceptibility factor (Veldink et al (2005)  Neurology  65:820). More recently, whole genome association (WGA) studies have identified genetic variations in dipeptidyl-peptidase 6 (DPP6) (van Es et al. (2008)  Nat. Genet.  40:29), and inositol 1,4,5-triphosphate receptor 2 (ITPR2) (van Es et al. (2007)  Lancet Neurol.  6:869) genes in ALS patients. WGA has also identified a minor association of a SNP near the FIJI 0986 gene to ALS Dunckley et al. (2007)  N. Engl. J. Med.  357:775). Most recently, missense mutations in the coding region of TARDBP encoding the Tar DNA binding protein TDP-43 were found in both familial and sporadic ALS patients (Gitcho et al. (2008)  Ann. Neurol.  63:535; Kabashi et al. (2008)  Nat. Genet.  40:572; Sreedharan et al. (2008)  Science  319:1668; Van Deerlin (2008)  Lancet Neurol.  7:409; Yokoseki et al. (2008)  Ann. Neurol.  63:538; Rutherford et al. (20080)  PloS Genet.  4:e1000193). following the discovery that TDP-43 is a major constituent of the neuronal cytoplasmic inclusions (Neumann et al. (2006)  Science  314:130). So far, a total of 26 different mutations have been identified in 39 of 2395 ALS patients. The overall frequency of TARDBP mutation in ALS is 1.6% (3.6% in FALS and 1.0% in SALS), which places TARDBP as the second most frequently mutated gene so far identified in ALS (after SOD1). 
     Since 2004, angiogenin (ANG), a 14 kDa angiogenic ribonuclease, has emerged as an important gene and/or genetic factor in ALS (Greenway et al. (2006)  Nat. Genet.  38:411). A total of 15 different missense mutations in the coding region of ANG have been identified in 33 of 3001 patients from Irish, Scottish, Swedish, North American, French, and Italian ALS populations (Greenway et al. (2006)  Nat. Genet.  38:411; Conforti et al. (2007)  Neuromuscul. Disord .; Gellera et al. (2007)  Neurogenetics ; Wu et al. (2007)  Ann. Neurol.  62:609; Paube et al. (2008) 65:1333), which places ANG as the third most frequently mutated gene in ALS (after SOD1 and TARDBP). Importantly, while wild type (WT) ANG induces angiogenesis, stimulates neurite outgrowth of motor neurons and protects them from hypoxia-induced death, mutant ANG proteins lack these activities (Wu et al. (2007)  Ann. Neurol.  62:609; Subramanian et al. (2008)  Hum. Mol. Genet.  17:130). Therefore, ANG appears to be the first loss-of-function gene so far identified in ALS (Wu et al. (2007)  Ann. Neurol.  62:609). The genes and genetic factors whose alterations predispose to ALS are listed below. 
     Genes and genetic factors associated with ALS are as follows (disease type/locus/gene/inheritance/clinical features/reference): ALS1/21q22.21/SOD1/autosomal dominant (AD)/adult onset/typical ALS/(Rosen et al. (1993)  Nature  362:59); ALS2/2q33/Alsin/autosomal recessive (AR)/juvenile onset, atypical ALS and primary lateral sclerosis (PLS), slow progression/(Yang et al. (2001)  Nat. Genet.  29:160); ALS3/18q21/unknown/AD/adult onset, typical ALS/(Hand et al. (2002)  Am. J. Hum. Genet.  70:251); ALS4/9q34/SETX/AD/juvenile onset, atypical ALS, slow progression/(Chen et al. (2004)  Am. J. Hum. Genet.  74:1128); ALS5/15q15.1-21.1/unknown/AR/juvenile onset, atypical ALS, slow progression, no pseudobulbar signs/(Hentati et al. (1998)  Neurogenetics  2:55); ALS6/16q12/unknown/AD/adult onset, typical ALS, short duration/(Abalkhail et al. (2003)  Am. J. Hum. Genet.  73:383); ALS7/20pte1-13/unknown AD/adult onset, typical ALS, short duration/(Sapp et al. (2003)  Am. J. Hum. Genet.  73:397); ALS8/20q13.33/VAPB (vesicle-associated membrane protein, a 27.2 kDa homodimer belonging to a family of intracellular vesicle-associated/membrane-bound proteins that are presumed to regulate vesicle transport)/AD/adult onset, atypical ALS, slow progression/(Nishimura et al. (2004)  Am. J. Hum. Genet.  75:822); ALS-frontotemporal dementia (FTD)/9q21-22/unknown/AD/adult onset, ALS with FTD/(Hosler et al. (2000)  JAMA  284:1664); ALS-PD/17q21/MAPT (microtubule-associated protein Tau)/AD/adult onset, ALS with Parkinsonism and dementia/(Hutton et al. (1998)  Nature  393:702); progressive lower MND/2p13/DCTN1 (dynactin p150 subunit, a major component of the dynein complex that comprises the major zonal retrograde motor (a mutation in the p150 subunit appears to affect the binding of the dynactin-dynein motor to microtubules))/AD/adult onset, lower motor neuron disorder/(Puls et al. (2003)  Nat. Genet.  33:455); ALS, sporadic/6q12, 22q12.1-q13.1, 6q21.3/VEGF (vascular endothelial growth factor, an essential angiogenic factor that also regulates neurogenesis); NFHC (neurofilament heavy chain); HFE (a hemochromatosis gene involved in iron metabolism)/risk factors/adult onset, typical ALS/(Lambrechts et al. (2003)  Nat. Genet.  34:383, Al-Chalabi et al. (1999)  Hum. Mol. Genet.  8:157, Figlewicz et al. (1994)  Hum. Mol. Genet.  3:1757, Tomkins et al. (1998)  Neuroreport.  9:3967, Goodall et al (2005)  Neurology  65:934); ALS, familiar and sporadic/14q11.2/ANG/AD/adult onset, typical ALS (Greenway et al. (2006)  Nat. Genet.  38:411, Gellera et al. (2007)  Neurogenetics , Wu et al. (2007)  Ann. Neurol.  62:609). ALS, familiar and sporadic/1p36/TARDBP/AD/adult onset, typical ALS (Gitcho et al. (2008)  Ann. Neurol.  63:535; Kabashi et al. (2008)  Nat. Genet.  40:572; Sreedharan et al. (2008)  Science  319:1668; Van Deerlin (2008)  Lancet Neurol.  7:409; Yokoseki et al. (2008)  Ann. Neurol.  63:538; Rutherford et al. (20080)  PloS Genet.  4:e1000193). 
     The present understanding of the pathobiology of ALS is largely from examinations of post-mortem samples of patients and from studies of SOD1 mutations. Many theories of ALS pathogenesis have been proposed including oxidative stress, excitotoxicity, mitochondrial dysfunction, defective axonal transport, abnormal protein aggregation, and loss of trophic and angiogenic factor support (Pasinelli and Brown (2006)  Nat. Rev. Neurosci.  7:710; Cleveland and Rothstein (2001)  Nat. Rev. Neurosci.  2:806; Goodall and Morrison (2006)  Expert Rev. Mol. Med.  8:1; Lambrechts et al. (2006)  Trends Mol. Med.  12:345). Each of these hypotheses is supported by some experimental evidence, but at the same time is undermined by contradictory data. For example, motor neuron damage as a result of oxidative stress is a key hypothesis in ALS. Oxidative damage increases with age so it fits with the middle-life onset of the disease. Several studies have confirmed the presence of elevated oxidative metabolism in ALS, such as the detection of increased biochemical markers of oxidative injury in post mortem examinations of ALS patients (Simpson et al. (2003)  Curr. Opin. Rheumatol.  15:730). It is also supported by the acquired capacity of some forms of mutant SOD1 to catalyze the production of reactive oxygen species (ROS) such as superoxide anions (O 2   − ) and peroxynitrite ( − ONOO) through either copper catalysis or improper copper and zinc binding (Beckman et al. (1993)  Nature  364:584; Estevez et al. (1999)  Science  286:2498; Wiedau-Pazos et al. (1996)  Science  271:515). However, the concept that mutant SOD1 provokes aberrant oxy-radical reactions has been controversial. For example, oxidative markers are detected in SODG93A mice but not in SODG37A mice, although both developed ALS symptoms (Bruijn et al. (1997)  Proc. Natl. Acad. Sci. USA  94:7606). Deletion of copper chaperone for SOD1 diminished the copper load but did not affect the development of ALS (Wong et al. (2000)  Proc. Natl. Acad. Sci. USA  97:2886). Moreover, copper-binding-site-null SOD1 causes ALS in transgenic mice (Wang et al. (2003)  Hum. Mol. Genet.  12:2753). 
     Therefore, the etiology of ALS is likely to be multi-factorial, involving the interplay of several mechanisms to initiate disease and propagate the spread of motor neuron death. A generally accepted hypothesis at present is that multiple factors, both genetic and environmental, cause mitochondrial dysfunction and excitotoxicity, lead to abnormal protein precipitation, and finally cell apoptosis (Goodall and Morrison (2006)  Expert Rev. Mol. Med.  8:1). 
     There is presently no effective pharmacologic treatment for ALS to halt neuronal death or even slow it appreciably. Riluzole, the only drug approved for ALS since 1995, only extends survival by 2-3 months if it is taken for 18 months. Riluzole is thought to act in part by limiting glutamate release. It preferentially blocks tetrodotoxin sensitive sodium channels, which are associated with damaged neurons (Song et al. (1997)  J. Pharmacol. Exp. Ther.  282:707). This reduces influx of calcium ions and indirectly prevents stimulation of glutamate receptors. Together with direct glutamate receptor blockade, the effect of the neurotransmitter glutamate on motor neurons is greatly reduced. Riluzole was approved for use in ALS after two independent clinical trials showed a marginal increase in the survival time of ALS patients (Bensimon et al. (1994)  N. Engl. J. Med.  330:585; Lacomblez et al. (1996)  Lancet  347:1425). Unfortunately, patients taking riluzole do not experience any slowing in disease progression or improvement in muscle function. Therefore, riluzole does not present a cure, or even an effective treatment, and the search for better therapeutic agents continues. 
     In the last two decades, over twenty double-blind, placebo-controlled trials have been reported, involving several thousands ALS patients. Most of them were based on efficacy in early animal models including the wobble mouse, the neuromuscular degeneration mouse and the progressive motor neuropathy mouse. However, each of them failed in the human trials. The growing consensus is that these three early mouse models have little in common with ALS and should not be used as models of ALS (Cleveland and Rothstein (2001)  Nat. Rev. Neurosci.  2:806). 
     Mutant SOD1 transgenic mice are a better model for ALS as SOD1 mutations are the cause of approximately 20% of the familiar ALS and approximately 3% of the sporadic ALS, and, therefore, approximately 4% of all ALS cases. More than 100 mutations in SOD1, distributed throughout the gene, have been found in ALS patients (Goodall and Morrison (2006)  Expert Rev. Mol. Med.  8:1). Although it is still unknown why the mutant form of this abundant and ubiquitously expressed enzyme is specifically toxic to motor neurons and causes ALS, it is clear that mice over-expressing the mutant SOD1 genes develop symptoms mimicking that of human ALS patients. Many of the agents undergoing clinical trials in ALS have shown good effects in the SOD1 G93A  mice, both in reducing the rate of disease progression and in prolonging survival. However, the benefits in the mouse have not been translated into clinical efficacy except in the case of riluzole. All the others have failed, including brain-derived neurotrophic factor (BDNF) (Kasarkis (1999)  Neurology  52:1427), ciliary neurotrophic factor (CNTF) (Akbar et al. (1997)  Neuroscience  78:351), insulin-like growth factor 1 (IGF1) (Borasio et al. (1998)  Neurology  51:583; Lai et al. (1997)  Neurology  49:1621), and glial-derived neurotrophic factor (GDNF) (Manabe et al. (2003)  Neurol. Res.  25:195). Additional animal models based on a better understanding of ALS pathobiology are needed for screening and testing of candidate agents for clinical trials. One of the purposes of this application is to develop such an animal model based on ANG mutations. The following compounds have been tested or are currently under testing in clinical trials of ALS (Cleveland and Rothstein (2001)  Nat. Rev. Neurosci.  2:806; Goodall and Morrison (2006)  Expert Rev. Mol. Med.  8:1; McGeer and McGeer (2005)  BioDrugs  19:31). 
     Antioxidants tested in SOD1 G93A  mice include: SOD1 (Jung et al. (2001)  Neurosci. Lett.  304:157), catalase (Reinholz et al. (1999)  Exp. Neurol.  159:204), desmethylsegeline, lipoic acid (Andreassen et al. (2001)  Exp. Neurol.  167:189), ginseng root (Jiang et al. (2000)  J. Neurol. Sci.  180:52), ginko biloba extract (Ferrante et al. (2001)  J. Mol. Neurosci.  17:89), N-acetylcycteine (Andreassen et al. (2000)  Neuroreport  11:2491), MnSOD (Andreassen et al. (2001)  Exp. Neurol.  167:189), nNOS null (Facchinetti et al. (1999)  Neuroscience  90:1483), general NOS inhibitor (Trieu et al. (2000)  Biochem. Biophys. Res. Commun.  267:22), ascorbate (Nagano et al. (1999)  Neurosci. Lett.  265:159), vitamin E (Gurney et al. (1996)  Ann. Neurol.  39:147), carboxyfullerenes (Dugan et al. (1997)  Proc. Natl. Acad. Sci. USA  94:9434), and Glutathione peroxidase (Cudkowicz et al. (2002)  Neurology  59:729). Antioxidants tested in ALS clinical trials include: vitamin E (Desnuelle et al. (2001)  Amyotroph. Lateral Scler. Other Motor Neuron Disord.  2:9), N-acetylcycteine, selegeline, coenzyme Q10 (currently undergoing clinical trials) and AEOL 10150 (currently undergoing clinical trials). 
     Anti-inflammatory and immunomodulatory agents tested in SOD1 G93A  mice include: cycloporin (Keep et al. (2001)  Brain Res.  894:327), aspirin (Barneoud and Curet (1999)  Exp. Neurol.  155:243), indomethecin, FK506 (Anneser et al. (2001)  Neuroreport  12:2663), Celecoxib (Drachman et al. (2002)  Ann. Neurol.  52:771), and glatiramer acetate (Habisch et al. (2007)  Exp. Neurol.  206:288). Anti-inflammatory and immunomodulatory agents tested in ALS clinical trials include: ganglioside (Harrington et al. (1984)  Neurology  34:1083), Celecoxib (Cudkowicz et al. (2006)  Ann. Neurol.  60:22), Cyclosporine (Appel et al. (1988)  Arch. Neurol.  45:381), Azathioprine, Cyclophosphamide, Plasmaphoresis (Patten (1986)  Crit. Rev. Clin. Lab. Sci.  23:147), intravenous immunoglobulin (Meucci et al. (1996)  J. Neurol.  243:117), total lymphoid irradiation (Haverkamp et al. (1994)  Ann. Neurol.  36:253), glatiramer acetate (currently undergoing clinical trials) (Gordon et al. (2006)  Neurology  66:1117), and thalidomide (currently undergoing clinical trials). 
     Anti-apoptosis agents tested in SOD1 G93A  mice include: zVAD-fmk (Li et al. (2000)  Science  288:335), p53 (Prudlo et al. (2000)  Brain Res.  879:183), p35, Bc12 (Kostic et al. (1997)  Science  277:559), Caspase 1 (Friedlander et al. (1997)  Nature  388:31), Bax, PARP (Andreassen et al. (2001)  Exp. Neurol.  167:189), Minocycline (Kriz et al. (2002)  Neurobiol. Dis.  10:268), Sodium phenylbutyrate (Ryu et al. (2005)  J. Neurochem.  93:1087), and Arimoclomol (Kieran et al. (2004)  Nat. Med.  10:402). Anti-apoptosis agents tested in ALS clinical trials include: minocycline (Gordon et al. (2007)  Lancet Neurol.  6:1045), sodium phenylbutyrate (currently undergoing clinical trials), and arimoclomol (currently undergoing clinical trials). 
     Antiglutamatergic agents tested in SOD1 G93A  mice include: Riluzole (Gurney et al. (1996)  Ann. Neurol.  39:147), Topiramate (Maragakis et al. (2003)  Neurosci. Lett.  338:107), EAAT2 (Guo et al. (2003)  Hum. Mol. Genet.  12:2519), and AMPA antagonist (Canton et al. (2001)  J. Pharmacol. Exp. Ther.  299:314). Antiglutamatergic agents tested in ALS clinical trials include: Riluzole (Bensimon et al. (1994)  N. Engl. J. Med.  330:585; Lacomblez et al. (1996)  Lancet  347:1425), Lamotrigine (Ryberg et al. (2003)  Acta. Neurol. Scand.  108:1), Topiramate (Cudkowicz et al. (2003)  Neurology  61:456); Dextromethorphan (Gredal et al. (1997)  Acta. Neurol. Scand.  96:8), Gabapentin (Miller et al. (1996)  Neurology  47:1383), Talampanel (currently undergoing clinical trials), Tamoxifen (currently undergoing clinical trials). 
     Antiviral agents tested in ALS clinical trials include: guanidine (Norris Jr. (1973)  N. Engl. J. Med.  288:690), amantidine (Munsat et al. (1981)  Neurology  31:1054), interferon-α (Mora et al. (1986)  Neurology  36:1137), and isoprinosine (Fareed and Tyler (1971) Neurology 21:937). 
     Trophic factors tested in SOD1 G93A  mice include: BDNF (Kasarkis (1999)  Neurology  52:1427), GDNF (Manabe et al. (2003)  Neurol. Res.  25:195), VEGF (Azzouz et al. (2004)  Nature  429:413), IGF (Borasio et al. (1998)  Neurology  51:583; Lai et al. (1997)  Neurology  49:1621), HGF (Kadoyama et al. (2007)  Neurosci. Res.  59:446), BMP7 (Dreibelbis et al. (2002)  Muscle Nerve  25:122), Cardiotropin 1 (Bordet et al. (2001)  Hum. Mol. Genet.  10:1925), Leukemia inhibitory factor (Azari et al. (2001)  Brain Res.  922:144). Trophic factors tested in ALS clinical trials include: BDNF (Kasarkis (1999)  Neurology  52:1427), GDNF (Manabe et al. (2003)  Neurol. Res.  25:195), IGF (Borasio et al. (1998)  Neurology  51:583; Lai et al. (1997)  Neurology  49:1621), CNF (Akbar et al. (1997)  Neuroscience  78:351), Thyrotrophin-releasing hormone (Munsat et al. (1992)  Neurology  42:1049), Xaliproden (Meininger et al. (2004)  Amyotroph. Lateral Scler. Other Motor Neuron Disord.  5:107). 
     A calcium regulator tested in SOD1 G93A  mice includes: Calbindin (Piper et al. (1996)  J. Neurosci. Methods  69:171). Clacium regulators tested in ALS clinical trials include: Verapamil (Miller et al. (1996)  Muscle Nerve  19:511) and Nimodipine (Miller et al. (1996)  Neuromuscular Disorder  6:101). 
     A compound involved with energy metabolism tested in SOD1 G93A  mice includes: creatine (Klivenyi et al. (1999)  Nat. Med.  5:347). Compounds involved with energy metabolism tested in ALS clinical trials include: creatine (Groeneveld et al. (2003)  Ann. Neurol.  53:437) and branched chain amino acids (Plaitakis et al. (1988)  Lancet  1:1015). 
     The proteasome inhibitor ritonavir is currently undergoing ALS clinical trials. 
     Structural proteins NF-H (Couillard-Despres et al. (1998)  Proc. Natl. Acad. Sci. USA  95:9626), NF-L (Kong and Xu (2000)  Neurosci. Lett.  281:72-4) and NF-L deletion (Williamson et al. (1998)  Proc. Natl. Acad. Sci. USA  95:9631) were tested in SOD1 G93A  mice. 
     Compounds involved with metal ion regulation tested in SOD1 G93A  (superoxide dismutase 1 having an alanine substitution at glycine residue 93) mice include: Trientine (Nagano et al. (1999)  Neurosci. Lett.  265:159), D-penicillamine (Hottinger et al. (1997)  Eur. J. Neurosci.  9:1548) and copper chaperone for superoxide dismutase 1 (SOD1) deletion (Silahtaroglu et al. (2002)  BMC Genet.  3:5). 
     SUMMARY 
     Hundreds of compounds have been tested in mouse models and many of them have been tested in human clinical trials. Among the various categories of inhibitors, neurotrophic factors are particularly relevant to the data presented herein on ANG. CNTF, GDNF, and IGF1 all showed good effects in the SOD1 G93A  mouse model, but trials in human ALS have been disappointing. VEGF is a prominent angiogenic factor and a new trophic factor linked to ALS. Since the demonstration that deletion of the hypoxia-response element in the promoter of VEGF causes motor neuron degeneration in mice (Oosthuyse et al. (2001)  Nat. Genet.  28:131) and that polymorphisms in the VEGF promoter that reduce VEGF expression are associated with ALS in the populations of Sweden, Belgium, UK (Lambrechts et al. (2003)  Nat. Genet.  34:383), and New England (Terry et al. (2004)  J. Neurogenet.  18:429), various attempts have been made to target VEGF as a therapeutic approach for ALS. VEGF delivered into SOD1 G93A  rats intracerebroventricularly (Storkebaum et al. (2005)  Nat. Neurosci.  8:85) or into SOD1 G93A  mice with a retrogradely transported lentiviral vector (Azzouz et al. (2004)  Nature  429:413) has been shown to improve motor neuron function and prolong survival of the SOD1 G93A  transgenic animals. VEGF overexpression also improves motor muscular function and increases the survival in SOD1 G93A  transgenic mice (Wang et al. (2007)  J. Neurosci.  27:304). However, intraperitoneal injection of VEGF in SOD1 G93A  mice had only a modest effect in delaying disease onset and in prolonging survival (Zheng et al. (2004)  Ann. Neurol.  56:564). 
     The present invention is based in part on the surprising discovery that injected ANG protein can improve one or more physical characteristics of a neurodegenerative disorder such as, e.g., ALS. For example, it has been surprisingly discovered that i.p. injection of ANG protein improved motor muscular function by 15-fold and increased life span of SOD1 G93A  mice by 26%. Mutant ANG proteins did not exhibit this activity. 
     The beneficial effect of ANG both in survival and in the improvement of the motor muscular function of SOD G93A  mice is the most significant among all the compounds and neurotrophic factors that have been tested so far including IGF, VEGF, BDNF, CNTF, and GDNF. Remarkably, ANG is effective when it is given after the disease onset, thus making it a good drug candidate for neuromuscular disorder, e.g., ALS, therapy. In certain exemplary embodiments, the dosing regimen will be optimized and technology will be developed to allow sustained and localized delivery of ANG in order to both maximize the benefits of ANG while minimizing potential side-effects. Thus, ANG protein holds promise as a therapeutic agent for neuromuscular disorders such as, e.g., ALS. 
     Accordingly, a method of therapeutically treating a neurodegenerative disorder (e.g., ALS and/or spinal muscular atrophy) in a subject in need thereof is provided. In certain exemplary embodiments, the method includes administering (e.g., intravenously administering, intramuscularly administering, subcutaneously administering, intraperitoneally administering, intrathecally administrating and/or intraventricularly administrating) to the subject a therapeutically effective amount of a composition including an isolated angiogenin polypeptide, allowing the isolated angiogenin polypeptide to pass through one or both of the blood brain barrier and the blood spinal cord barrier, and reducing one or more symptoms of the neurodegenerative disorder in the subject such that the neurodegenerative disorder is therapeutically treated. In other exemplary embodiments, the method includes administering (e.g., intravenously administering, intramuscularly administering, subcutaneously administering, intraperitoneally administering, intrathecally administrating and/or intraventricularly administrating) to the subject a therapeutically effective amount of a composition including an isolated nucleic acid sequence encoding an angiogenin polypeptide, expressing the angiogenin polypeptide in the subject, allowing the angiogenin polypeptide to pass through one or both of the blood brain barrier and the blood spinal cord barrier, and reducing one or more symptoms of the neurodegenerative disorder in the subject such that the neurodegenerative disorder is therapeutically treated. In still other exemplary embodiments, the method includes administering (e.g., intraventricularly administering and/or intrathecally administering) directly to the central nervous system (CNS) of a subject (using, e.g., an infusion pump and/or a delivery scaffold) a therapeutically effective amount of a composition including an isolated angiogenin polypeptide or a therapeutically effective amount of a composition including an isolated nucleic acid sequence encoding an angiogenin polypeptide, expressing the angiogenin polypeptide in the subject, and reducing one or more symptoms of the neurodegenerative disorder in the subject such that the neurodegenerative disorder is therapeutically treated. 
     In certain aspects, a method includes one or any combination of the following steps: allowing nuclear translocation of the isolated angiogenin polypeptide; allowing the isolated angiogenin polypeptide to stimulate ribosomal RNA transcription; allowing the isolated angiogenin polypeptide to stimulate ribosomal biogenesis; allowing the isolated angiogenin polypeptide to stimulate cell (e.g., a spinal cord cell and/or one or both of a neural cell (e.g., a motor neuron) and an endothelial cell) proliferation; allowing the isolated angiogenin polypeptide to stimulate cell differentiation (e.g., an undifferentiated cell is stimulated to differentiate into a neural cell); and/or allowing the isolated angiogenin polypeptide to stimulate angiogenesis. In certain aspects, a method includes allowing nuclear translocation of the isolated angiogenin polypeptide, allowing the isolated angiogenin polypeptide to stimulate ribosomal RNA transcription, allowing ribosomal biogenesis, allowing cell proliferation and allowing angiogenesis. In certain aspects, a method includes allowing the isolated angiogenin polypeptide to stimulate angiogenesis in the CNS of a subject. 
     In certain aspects, one or more symptoms of ALS, e.g., motor neuron degeneration, muscle weakness, muscle atrophy, fasciculation development, frontotemporal dementia and/or premature death are improved in the subject. In certain aspects, the angiogenin polypeptide enters one or both of the brain and the spinal cord. In other aspects, one or both of muscle coordination and muscle function are improved. In other aspects, the survival of the subject is prolonged. In still other aspects, a nucleic acid sequence is administered using a gene therapy vector. 
     In certain exemplary embodiments, a transgenic animal (e.g., a transgenic mouse and/or transgenic rat) model of ALS including a human ANG gene including a mutated human ANG gene encoding an angiogenin polypeptide with one or more of the following amino acid substitutions: M(-24)I, F(-13)S, P(-4)S, Q12L, K17I, K17E, D22G, S28N, R31K, C39W, K40I, I46V, K60E, P112L, V113I, H114R, R121H and exhibiting one or more symptoms of ALS is provided. In certain aspects, the one or more symptoms of ALS include one or more of motor neuron degeneration, muscle weakness, muscle atrophy, fasciculation development, frontotemporal dementia and/or decreased lifespan. 
     In certain exemplary embodiments, a knockout animal (e.g., a knockout mouse and/or a knockout rat) model of ALS, including an ANG1 gene knockout and exhibiting one or more symptoms of ALS is provided. In certain aspects, the one or more symptoms of ALS include one or more of motor neuron degeneration, muscle weakness, muscle atrophy, fasciculation development frontotemporal dementia and/or premature death. 
     In certain exemplary embodiments, ANG mutant polypeptides are provided. In certain aspects, a mutant ANG polypeptide is administered to a subject or a cell to regain and/or enhance one or more ANG functions in the subject or cell. 
     In certain exemplary embodiments, a method of increasing one or more ANG activities in a subject including administering to the subject a composition comprising an isolated angiogenin polypeptide having at least one mutation, and allowing isolated angiogenin polypeptide having at least one mutation to increase one or more ANG activities in the subject is provided. In certain aspects, the one or more ANG activities are selected from one or more of angiogenesis, ribonucleolytic activity, binding ANG receptor, activating tissue plasminogen activator, enhancing motor muscular function, enhancing neurite outgrowth, enhancing neurogenesis, enhancing survival of motor neurons, crossing the blood brain barrier, crossing the blood spinal cord barrier, enhancing survival of a subject having ALS and any combination thereof. In certain aspects, the isolated angiogenin polypeptide having at least one mutation has a D116H substitution. In certain aspects, the subject lacks endogenous ANG or has one or more ANG mutations. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which: 
         FIG. 1  schematically depicts the conceptual framework of the interaction between ANG and its target cells. ANG, shown in yellow, can bind to both the receptor and the binding protein, shown in white and orange, respectively. The majority of the ANG and its binding protein complex will dissociate from the cell surface and activate tissue plasminogen activator (tPA) to produce plasmin, and induce cell invasion into the extracellular matrix. Binding to the 170 kDa receptor induces second messengers and triggers signal transduction. Upon binding, ANG is also internalized and translocated to the nucleus where it accumulates in the nucleolus. Each of these individual steps are necessary for angiogenesis. 
         FIG. 2  schematically depicts that ANG-stimulated rRNA transcription is a permissive factor for cell proliferation induced by other angiogenic factors. Without intending to be bound by scientific theory, the inventors&#39; propose that ANG is a permissive factor for other angiogenic proteins to induce cell proliferation. Growth factors such as VEGF activate PI3K-AKT-mTOR pathway to enhance ribosomal protein production, but it is unclear how rRNA is proportionally increased. The experimentation described herein shows that ANG is translocated to the nucleus where it enhances rRNA transcription so that ribosome biogenesis can occur. Since ribosomes are essential for protein translation and cell proliferation, it has been demonstrated by the data presented herein that inhibiting ANG abolishes cell proliferation stimulated by other angiogenic factors including, but not limited to, bFGF and VEGF. 
         FIGS. 3A-3E  depict the detection of ANG protein in the ventral horn area of spinal cord of ALS patients. Spinal cord sections of non-ALS subjects were from anonymous autopsy materials of Brigham and Women&#39;s Hospital. ALS-1 and -2 were from the National Disease Research Interchange. ALS-3 to −6 were from Dr. Bob Brown. ANG protein was stained by IHC with 26-2F (10 μg/ml at 4° C. for 16 h). (A) Images taken at 200× using Olympus camera DP70 attached to Olympus IX81 microscope. (B)-(E) Quantitative analyses by ImageJ software. Pixel-counting algorithm was used to obtain the chromogen specific areas that was then converted to μm 2 . To obtain photon counts, the images were transformed in gray scale and the cumulative grayscale values of each of the three primary colors (red, green, and blue) was determined. (B) average motor neuron size. (C) total area covered by ANG-positive motor neuron in a ventral horn. (D) average photon counts in each motor neuron. E, total photon counts in an entire ventral horn. 
         FIGS. 4A-4F  depict immunofluorescence (IF) detection of ANG protein in the ventral horn of human spinal cord. Human spinal cords from non-ALS controls and ALS patients were stained with anti-ANG mAb and anti-vWF pAb, respectively. Alexa 488-labeled goat anti-mouse IgG (green) and Alexa 555-labeled goat anti-rabbit IgG (red) were used to visualize ANG and vWF, respectively. Motor neurons and blood vessels were indicated by arrows and arrow heads, respectively. 
         FIGS. 5A-5B  depict in situ hybridization (ISH) detection of ANG mRNA in spinal cord. Human spinal cords from non-ALS and ALS patients were stained with human ANG-specific riboprobe labeled with digoxiginin. An alkaline phosphatase-conjugated anti-digoxiginin antibody was used to visualize the signal. (A) images. (B) total photon counts of ANG mRNA in the ventral horn motor neurons determined with ImageJ. 
         FIG. 6A-6J  depict the level of ANG protein and mRNA in mouse spinal cords. Spinal cords from wild-type (WT) and SOD1 G93A  mice were stained with an anti-R165 and detected by IHC (A, B) or by IF (C, D). (E) quantitative analysis of IHC images from A and B. (F) Western blotting analysis of mouse ANG protein from spinal cord extracts. (G)-(J) A mouse ANG1-specific riboprobe was labeled with digoxiginin and used to detect the mRNA level of mouse ANG1. (K) Quantitative analysis of ISH images from (G) and (I). 
         FIG. 7A-7F  depict decreased blood vessel size in the ALS spinal cord. Non-ALS and ALS human spinal cords (A)-(C), and WT and SOD1 G93A  mice (D)-(F) were stained with an anti-vWF antibody. Blood vessels (brown staining) in a total of 10 microscopic areas of the ventral horns were counted (B) and (E), and the diameter of each counted vessels was measured (C) and (F), and the mean±SD values were shown. 
         FIG. 8  depicts that i.p. injected ANG reaches the CNS. PBS or human ANG protein, 10 μg/mouse, was injected i.p. into 11-week-old WT mice and SOD1 G93A  mice. The animals were sacrificed 2.5 h post-injection after cardiac perfusion with PBS. The spinal cords were processed for IHC detection of human ANG with the mAb 26-2F. 
         FIGS. 9A-9D  depict the effect of i.p. injection of ANG protein on SOD1 G93A  mice. (A) qPCR detection of SOD1 G93A  DNA. Data shown are the relative value to mouse GAPDH DNA. (B) ANG treatment enhances the muscle strength of the hind legs. (C) ANG treatment enhances motor function. Starting from 11 weeks of age, mice were treated with a weekly i.p. injection of WT or P112L ANG protein at 10 mg per mouse. PBS- (green), ANG- (red) and P112L mutant ANG- (black) treated mice were tested on a rotarod (20 rpm). (D) Survival curve of the three groups of mice. 
         FIGS. 10A-10B  graphically depict the efficacy of three routes of administration. Wild-type ANG was administered into the SOD G93A  mice by subcutaneous (s.c.), i.p., or intravenous (i.v.) injection starting at week 10 and continued weekly. Rota-rod test was performed at week 11 and weekly afterward. Each group had six mice. 
         FIG. 11  graphically depicts the efficacy of three doses of ANG. Wild-type ANG was administered into the SOD G93A  mice by i.p. injection starting at week 10 at a dose of 0.1, 1, and 10 μg/mouse and continued weekly. Rotarod tests were performed at week 11 and weekly thereafter. Each group had five mice. 
         FIG. 12A-12D  depicts the effect of ANG on motor neuron and blood vessels of SOD1 G93A  mice. WT and PBS or ANG-treated SOD1 G93A  mice were sacrificed at week 15, at which time the ANG-treated mouse was at its peak performance on rotarod test. The lumbar region of the spinal cord was removed, fixed, embedded, and sectioned. (A) IHC with a human SOD-specific antibody (top panels); Niss1 staining showing the motor neurons in the ventral horn (middle panels); and IHC with an anti-vWF antibody to show blood vessels (bottom panels). (B) Number of large motor neuron (&gt;250 μm 2 ) per section. (C) blood vessel density. (D) blood vessel size. A total of 500 vessels were measured in each sample. P values were calculated from Student t-test. 
         FIG. 13A-13D  depict that ANG stimulates proliferation and differentiation of P19 cells. (A) IF detection of endogenous mouse ANG by R165. (B) human ANG was incubated with P19 cells for 2 hours and detected by IF with 26-2F. C, P19 cells were treated with or without ANG (50 ng/ml). On day 4, newly formed embryoid bodies were collected and counted. (D) P19 embryoid bodies were resuspended and cultured in the absence or presence of 50 ng/ml ANG for another 10 days. Neuronal processes are indicated by arrows. 
         FIG. 14A-14C  depict the effect of angiogenin on NSC-34 motor neuron-like cells. (A) Nuclear translocation of exogenous angiogenin in NSC-34 cells. The cells were incubated with 0.5 μg/ml WT or P112L mutant human angiogenin for 2 hours, fixed and stained with mAb 26-2F and Alexa488-labeled 2 nd  antibody and with DAPI. (B) Angiogenin stimulated NSC-34 cell proliferation. Cells were seeded in 48-well plates at 5×10 3  cells per well and stimulated with angiogenin at 0.5, 1, and 10 μg/ml for 7 days. Cell numbers were determined by a Coulter counter. (C) Angiogenin stimulated neurite outgrowth. NSC-34 cells were cultured in the absence or presence of 0.5 μg/ml angiogenin for 7 days, and stained with 5,6-carboxyfluorescein diacetate. 
         FIG. 15A-15D  depict the effect of ANG on P19 cell apoptosis. Cells were serum-starved in the absence or presence of ANG for 24 hours. Apoptotic cells were stained by EB (A), counted and the percentage calculated (B). (C) Caspase activities were measured using Caspase-Glo 3/7 Assay kit (Promega). (D) P19 cells were cultured on PA6 cells in the presence of 0.5 μM retinoic acid for 10 days. The cells were then put at 20° C. for 40 minutes with or without ANG (0.5 μg/ml), and subject to IF detection of neurofilaments. 
         FIG. 16  depicts the detection of ANG in the neuromuscular junction (NMJ). Mouse tibialis anterior muscle was removed and frozen section of 40 μm cut. ANG was stained with pAb R165 and Alexa 488-goat anti rabbit IgG. NF were stained with anti-NF medium subunit mAb and Alex a 555-goat anti mouse IgG. 
         FIGS. 17A-17C  depict the generation of ANG1 floxed mice. (A) mRNA levels of 6 mouse ANG isoforms in the spinal cord by qRT-PCR. (B) construction of targeting vector. (C) RT-PCR (left) and Western blotting (right) analyses of ANG1 mRNA and ANG protein, respectively, in the spinal cord of WT and KO mice. 
         FIGS. 18A-18D  depict the generation of ANG transgenic mice. (A) Schematic view of the expression vector. A DNA fragment containing human ANG cDNA an IRES-controlled AcGFP expression cassette was flanked by chicken β-actin promoter and the rabbit β-globin PolyA signal. (B) Genotyping of the 17 pups for human ANG DNA. Mice 83, 86, 89 and 95 have been confirmed to be founders. (C) Establishment of two transgenic lines. Founders 89 and 94 were backcrossed with WT mice twice. (D) IHC detection of human angiogenin on the spinal cord sections from WT and ANG transgenic lines 89 and 94 with the human angiogenin-specific mAb 26-2F. 
         FIG. 19  depicts the amino acid sequences of mammalian ANGs. By convention, the first amino acid in the mature protein is designated as 1. The signal peptide is underlined. Mutations are bracketed in red. Human is set forth as SEQ ID NO:1; chimpanzee is set forth as SEQ ID NO:2; gorilla is set forth as SEQ ID NO:3; mouse is set forth as SEQ ID NO:4; rat is set forth as SEQ ID NO:5; bovine is set forth as SEQ ID NO:6; porcine is set forth as SEQ ID NO:7. 
     
    
    
     DETAILED DESCRIPTION 
     In certain exemplary embodiments, methods and compositions including one or more ANG polypeptides and/or one or more nucleic acid sequences encoding one or more ANG polypeptides are provided for treating a neurological disorder (e.g., ALS) in a subject. As used herein, the term “ANG polypeptide” refers to an angiogenic ribonuclease (e.g., ANG in humans) or portion thereof having one or more ANG properties: 1) promoting angiogenesis; 2) having ribonucleolytic activity; 3) binding the ANG receptor; 4) activating tissue plasminogen activator; 5) enhancing motor muscular function (e.g., in an individual having ALS); 6) enhancing neurite outgrowth (e.g., in an individual having ALS); 7) enhancing neurogenesis (e.g., in an individual having ALS); 8) enhancing survival of motor neurons (e.g., in an individual having ALS); 9) crossing the BBB; 10) crossing the BSCB; 11) enhancing survival of in an individual having ALS; and/or 11) enhancing and/or restoring one or more functions described above in an individual (e.g., an individual having an ANG mutation or deletion). In certain exemplary embodiments, an ANG polypeptide can eliminate, ameliorate and/or decrease one or more symptoms associated with ALS: 1) degeneration of motor neurons; 2) muscle weakness; 3) muscle atrophy; 4) motor neuron degeneration (e.g., upper- and/or lower motor neurons); 5) fasciculation development; 6) frontotemporal dementia and/or 7) decreased lifespan. 
     As used herein, the terms “subject,” “individual” and “host” are intended to include living organisms such as mammals. Examples of subjects and hosts include, but are not limited to, horses, cows, sheep, pigs, goats, dogs, cats, rabbits, guinea pigs, rats, mice, gerbils, non-human primates (e.g., macaques), humans and the like, non-mammals, including, e.g., non-mammalian vertebrates, such as birds (e.g., chickens or ducks) fish or frogs (e.g.,  Xenopus ), and non-mammalian invertebrates, as well as transgenic species thereof. 
     As used herein, the terms “neurological disorder” and “neurological disease” include, but are not limited to, neuromuscular disorders, Alzheimer&#39;s disease, aphasia, Bell&#39;s palsy, Creutzfeldt-Jacob disease, cerebrovascular disease, encephalitis, epilepsy, Huntington&#39;s disease, pain, phobia, movement disorders (e.g., Parkinson&#39;s disease), sleep disorders, Tourette syndrome, multiple sclerosis, neural tumors, neural autoimmune disorders (e.g., multiple sclerosis) pediatric neural disorders (e.g., autism, dyslexia, cerebral palsy and the like) and the like. As used herein, the terms “neuromuscular disorder” and “motor neuron disorder” include, but are not limited to, disorders such as ALS, Guillain-Barre syndrome, Charcot-Marie-Tooth disease, spinal muscular atrophy (SMA), muscular dystrophy, spastic paraplegia and the like. 
     In certain exemplary embodiments, a therapeutic amount of one or more ANG polypeptides and/or one or more nucleic acid sequences encoding one or more ANG polypeptides is administered to an individual in need thereof, e.g., for the treatment of a neurological disorder such as, e.g., ALS. The one or more ANG polypeptides and/or one or more nucleic acid sequences encoding one or more ANG polypeptides described herein can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the one or more nucleic acid molecules or polypeptides and a pharmaceutically acceptable carrier. 
     As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutically acceptable carriers and their formulations are known to those skilled in the art and described, for example, in Remington&#39;s Pharmaceutical Sciences, (19th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa. 
     In certain exemplary embodiments, pharmaceutical formulations of a therapeutically effective amount of one or more ANG polypeptides or one or more nucleic acid sequences encoding one or more ANG polypeptides one or more test compounds, or pharmaceutically acceptable salts thereof, are administered by intravenous injection, intraperitoneal injection, oral administration or by other parenteral routes (e.g. intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration), or by intrathecal and intraventricular injections into the CNS, in an admixture with a pharmaceutically acceptable carrier adapted for the route of administration. 
     In certain exemplary embodiments, pharmaceutical formulations of a therapeutically effective amount of one or more ANG polypeptides or one or more nucleic acid sequences encoding one or more ANG polypeptides, or pharmaceutically acceptable salts thereof, are administered directly to the central nervous system (CNS), e.g., intrathecally, intracerebrally, via the olfactory nerve, via the olfactory epithelium, via the spinal cord and the like. Direct administration to the CNS can be performed using an implantable infusion pump or a transplanted delivery scaffold, for example. Implantable infusion pumps are commercially available (Medtronic, Inc., Minneapolis, Minn.) and are described in U.S. Pat. Nos. 5,711,316, 5,814,014 and 7,232,435. Delivery scaffolds for use in the CNS are known in the art and described in, e.g., Lee et al. (2006)  Toxicol. Appl. Pharm.  215:64; Proceedings of the IEEE 31 st  Annual Northeast Bioengineering Conference (2005) 1-3, ISBN: 0-7803-9105-5 INSPEC Accession Number: 8487652, DOI:10.1109/NEBC.2005.1431898, Posted online: 2005-05-23 09:06:59.0; DeLaporte and Shea (2007)  Adv. Drug Deliv. Rev.  59:292; and Nomura et al. (2006)  J. Neurotrauma  23:496. 
     Solutions or suspensions used for parenteral, intradermal, subcutaneous or central nervous system application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. 
     Methods well known in the art for making formulations are found, for example, in Remington&#39;s Pharmaceutical Sciences (19th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa. Compositions intended for oral use may be prepared in solid or liquid forms according to any method known to the art for the manufacture of pharmaceutical compositions. The compositions may optionally contain sweetening, flavoring, coloring, perfuming, and/or preserving agents in order to provide a more palatable preparation. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier or excipient. These may include, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, sucrose, starch, calcium phosphate, sodium phosphate, or kaolin. Binding agents, buffering agents, and/or lubricating agents (e.g., magnesium stearate) may also be used. Tablets and pills can additionally be prepared with enteric coatings. 
     Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In certain exemplary embodiments, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and/or sodium chloride, will be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. 
     Sterile injectable solutions can be prepared by incorporating one or more ANG polypeptides or one or more nucleic acid sequences encoding one or more ANG polypeptides described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. 
     Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: A binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic, acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant: such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. 
     In one embodiment, the one or more ANG polypeptides or one or more nucleic acid sequences encoding one or more ANG polypeptides described herein are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. 
     Nasal compositions generally include nasal sprays and inhalants. Nasal sprays and inhalants can contain one or more active components and excipients such as preservatives, viscosity modifiers, emulsifiers, buffering agents and the like. Nasal sprays may be applied to the nasal cavity for local and/or systemic use. Nasal sprays may be dispensed by a non-pressurized dispenser suitable for delivery of a metered dose of the active component. Nasal inhalants are intended for delivery to the lungs by oral inhalation for local and/or systemic use. Nasal inhalants may be dispensed by a closed container system for delivery of a metered dose of one or more active components. 
     In one embodiment, nasal inhalants are used with an aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A non-aqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers may be used to minimize exposing the agent to shear, which can result in degradation of the compound. 
     Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions. 
     Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. 
     The one or more ANG polypeptides or one or more nucleic acid sequences encoding one or more ANG polypeptides described herein can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. 
     In one embodiment, the one or more ANG polypeptides or one or more nucleic acid sequences encoding one or more ANG polypeptides described herein are prepared with carriers that will protect them against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. 
     It is especially advantageous to formulate oral, parenteral or CNS direct delivery compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. 
     Toxicity and therapeutic efficacy of one or more ANG polypeptides and/or nucleic acid sequences encoding one or more ANG polypeptides described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. 
     Data obtained from cell culture assays and/or animal studies can be used in formulating a range of dosage for use in humans. The dosage typically will lie within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. 
     In certain exemplary embodiments, a method for treatment of a neurological disorder (e.g., ALS), includes the step of administering a therapeutically effective amount of an agent (e.g., one or more ANG polypeptides or one or more nucleic acid sequences encoding one or more ANG polypeptides) to a subject. As defined herein, a therapeutically effective amount of agent (i.e., an effective dosage) ranges from about 0.0001 to 30 mg/kg body weight, from about 0.001 to 25 mg/kg body weight, from about 0.01 to 20 mg/kg body weight, from about 0.1 to 15 mg/kg body weight, or from about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of one or more ANG polypeptides or one or more nucleic acid sequences encoding one or more ANG polypeptides can include a single treatment or, in certain exemplary embodiments, can include a series of treatments. It will also be appreciated that the effective dosage of agent used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result from the results of diagnostic assays as described herein. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. 
     In certain exemplary embodiments, vectors such as, for example, expression vectors, containing a nucleic acid encoding one or more ANG polypeptides described herein are provided. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. 
     In certain exemplary embodiments, the recombinant expression vectors comprise a nucleic acid sequence (e.g., a nucleic acid sequence encoding one or more ANG polypeptides described herein) in a form suitable for expression of the nucleic acid sequence in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence encoding one or more ANG polypeptides is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel;  Gene Expression Technology: Methods in Enzymology  185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors described herein can be introduced into host cells to thereby produce proteins or portions thereof, including fusion proteins or portions thereof, encoded by nucleic acids as described herein (e.g., one or more ANG polypeptides). 
     In certain exemplary embodiments, nucleic acid molecules described herein can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat. No. 5,328,470), or by stereotactic injection (see, e.g., Chen et al. (1994)  Proc. Natl. Acad. Sci. U.S.A.  91:3054). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, adeno-associated virus vectors, and the like, the pharmaceutical preparation can include one or more cells which produce the gene delivery system (See Gardlik et al. (2005)  Med. Sci. Mon.  11:110; Salmons and Gunsberg (1993)  Hu. Gene Ther.  4:129; and Wang et al. (2005)  J. Virol.  79:10999 for reviews of gene therapy vectors). 
     Herpesvirus Vectors 
     Herpesvirus is trophic for cells of the nervous system (e.g., neural cells). Various defective (i.e., non-replicating, non-infectious) herpesvirus vectors have been described, such as a defective herpes virus 1 (HSV-1) vector (WO 94/21807, WO 92/05263). 
     Adenovirus Vectors 
     Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types in vivo, and have been used extensively in gene therapy protocols, including for targeting genes to neural cells. Various serotypes of adenovirus exist. Of these serotypes, preference is given to using type 2 or type 5 human adenoviruses (Ad-2 or Ad-5) or adenoviruses of animal origin (see WO 94/26914). Adenoviruses of animal origin include, but are not limited to, adenoviruses of canine, bovine, murine (e.g., May 1), ovine, porcine, avian, and simian (e.g., SAV) origin. In certain exemplary embodiments, the adenovirus of animal origin is a canine adenovirus, such as a CAV2 adenovirus (e.g., Manhattan or A26/61 strain (ATCC VR-800)). Various replication defective adenovirus and minimum adenovirus vectors have been described for gene therapy (See, e.g., WO 94/26914, WO 95/02697, WO 94/28938, WO 94/28152, WO 94/12649, WO 95/02697 WO 96/22378). 
     Adeno-Associated Viruses 
     The adeno-associated viruses (AAV) are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation. The use of vectors derived from AAVs for transferring genes in vitro and in vivo has been described (See, e.g., WO 91/18088; WO 93/09239; U.S. Pat. No. 4,797,368, U.S. Pat. No. 5,139,941, EP 488528). Replication defective recombinant AAVs can be prepared by co-transfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes), into a cell line which is infected with a human helper virus (for example an adenovirus). The AAV recombinants which are produced can then be purified by standard techniques. 
     Retrovirus Vectors 
     In certain exemplary embodiments, a gene expressing ANG or a portion or mutant form thereof can be introduced in a retroviral vector (See, e.g., U.S. Pat. No. 5,399,346; U.S. Pat. No. 4,650,764; U.S. Pat. No. 4,980,289; U.S. Pat. No. 5,124,263; EP 453242, EP178220; WO 95/07358). Retroviruses are integrating viruses which infect dividing cells. The retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). In recombinant retroviral vectors, the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest. These vectors can be constructed from different types of retrovirus, such as, for example, murine Moloney leukemia virus, murine Moloney sarcoma virus, Harvey sarcoma virus, spleen necrosis virus, Rous sarcoma virus, Friend virus and the like. Suitable packaging cell lines have been described, such as, for example, the cell line PA317 (U.S. Pat. No. 4,861,719); the PsiCRIP cell line (WO 90/02806) and the GP+envAm-12 cell line (WO 89/07150). In addition, the recombinant retroviral vectors can contain modifications within the LTRs for suppressing transcriptional activity as well as extensive encapsidation sequences which may include a part of the gag gene. Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art. Retrovirus vectors can also be introduced by recombinant DNA viruses, which permits one cycle of retroviral replication and amplifies transfection efficiency (See, e.g., WO 95/22617, WO 95/26411, WO 96/39036, WO 97/19182). 
     In certain exemplary embodiments, lentiviral vectors can be used to provide highly effective expression of a gene of interest as lentiviruses can change the expression of their target cell&#39;s gene for up to six months. They can be used, for example, in non-dividing or terminally differentiated cells such as neurons, macrophages, hematopoietic stem cells, retinal photoreceptors and muscle and liver cells, cell types for which previous gene therapy methods could not be used. The vectors can efficiently transduce dividing and non-dividing cells in these tissues, and maintain long-term expression of the gene of interest. Lentiviral packaging cell lines are available and known generally in the art. They facilitate the production of high-titer lentivirus vectors for gene therapy. An example is a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line which can generate virus particles at titers greater than 10 6  IU/ml for at least 3 to 4 days. The vector produced by the inducible cell line can be concentrated as needed for efficiently transducing non-dividing cells in vitro and in vivo. 
     Non-Viral Vectors 
     A vector can be introduced in vivo in a non-viral vector, e.g., by lipofection, with other transfection facilitating agents (peptides, polymers and the like), or as naked DNA. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection, with targeting in some instances (Felgner, et. al., 1987; Felgner and Ringold, 1989; see Mackey, et al., 1988; Ulmer et al., 1993). Useful lipid compounds and compositions for transfer of nucleic acids are described in WO 95/18863, WO 96/17823 and in U.S. Pat. No. 5,459,127. Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as, e.g., a cationic oligopeptide (See, e.g., WO 95/21931), peptides derived from DNA binding proteins (See, e.g., WO 96/25508) a cationic polymer (See, e.g., WO 95/21931) and the like. A relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has been described (See, e.g., WO 99/01157; WO 99/01158; WO 99/01175). DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun (ballistic transfection), or use of a DNA vector transporter (See, e.g., Canadian Patent Application No. 2,012,311). Receptor-mediated DNA delivery approaches can also be used. U.S. Pat. Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal. 
     Expression vectors described herein can be designed for expression of one or more ANG polypeptides in prokaryotic or eukaryotic cells. For example, one or more vectors encoding one or more ANG polypeptides can be expressed in bacterial cells such as  E. coli , insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel,  Gene Expression Technology: Methods in Enzymology  185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. 
     Expression of proteins in prokaryotes is most often carried out in  E. coli  with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988)  Gene  67:31-40); pMAL (New England Biolabs, Beverly, Mass.); and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. 
     In certain exemplary embodiments, the expression vector encoding one or more ANG polypeptides is a yeast expression vector. Examples of vectors for expression in yeast  S. cerevisiae  include pYepSec1 (Baldari, et. al., (1987)  EMBO J.  6:229-234); pMFa (Kurjan and Herskowitz, (1982)  Cell  30:933-943); pJRY88 (Schultz et al., (1987)  Gene  54:113-123); pYES2 (Invitrogen Corporation, San Diego, Calif.); and picZ (Invitrogen Corporation). 
     Alternatively, one or more ANG polypeptides can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983)  Mol. Cell. Biol.  3:2156-2165) and the pVL series (Lucklow and Summers (1989)  Virology  170:31-39). 
     In certain exemplary embodiments, a nucleic acid described herein is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987)  Nature  329:840) and pMT2PC (Kaufman et al. (1987)  EMBO J.  6:187-195). When used in mammalian cells, the expression vector&#39;s control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T.  Molecular Cloning: A Laboratory Manual.  2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. 
     In certain exemplary embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include lymphoid-specific promoters (Calame and Eaton (1988)  Adv. Immunol.  43:235), in particular promoters of T cell receptors (Winoto and Baltimore (1989)  EMBO J.  8:729) and immunoglobulins (Banerji et al. (1983)  Cell  33:729; Queen and Baltimore (1983)  Cell  33:741), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989)  Proc. Natl. Acad. Sci. U.S.A.  86:5473), pancreas-specific promoters (Edlund et al. (1985)  Science  230:912), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990)  Science  249:374) and the α-fetoprotein promoter (Campes and Tilghman (1989)  Genes Dev.  3:537). 
     In certain exemplary embodiments, host cells into which a recombinant expression vector of the invention has been introduced are provided. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. 
     A host cell can be any prokaryotic or eukaryotic cell. For example, one or more ANG polypeptides can be expressed in bacterial cells such as  E. coli , viral cells such as retroviral cells, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. 
     Delivery of nucleic acids described herein (e.g., vector DNA) can be by any suitable method in the art. For example, delivery may be by injection, gene gun, by application of the nucleic acid in a gel, oil, or cream, by electroporation, using lipid-based transfection reagents, or by any other suitable transfection method. 
     As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection (e.g., using commercially available reagents such as, for example, LIPOFECTIN® (Invitrogen Corp., San Diego, Calif.), LIPOFECTAMINE® (Invitrogen), FUGENE® (Roche Applied Science, Basel, Switzerland), JETPEI™ (Polyplus-transfection Inc., New York, N.Y.), EFFECTENE® (Qiagen, Valencia, Calif.), DREAMFECT™ (OZ Biosciences, France) and the like), or electroporation (e.g., in vivo electroporation). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. ( Molecular Cloning: A Laboratory Manual.  2nd, ed., Cold Spring harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals. 
     Embodiments of the invention are directed to a first nucleic acid (e.g., a nucleic acid sequence encoding one or more ANG polypeptides) or polypeptide sequence (e.g., one or more ANG polypeptides) having a certain sequence identity or percent homology to a second nucleic acid or polypeptide sequence, respectively. 
     Techniques for determining nucleic acid and amino acid “sequence identity” are known in the art. Typically, such techniques include determining the nucleotide sequence of genomic DNA, mRNA or cDNA made from an mRNA for a gene and/or determining the amino acid sequence that it encodes, and comparing one or both of these sequences to a second nucleotide or amino acid sequence, as appropriate. In general, “identity” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov (1986)  Nucl.    
     Acids Res. 14:6745. An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the “BestFit” utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). 
     One method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages, the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “match” value reflects “sequence identity.” Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found at the NCBI/NLM web site. 
     Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. Two DNA sequences, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 80%-85%, at least about 85%-90%, at least about 90%-95%, or at least about 95%-98% sequence identity over a defined length of the molecules, as determined using the methods above. As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.; Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press. 
     Two nucleic acid fragments are considered to “selectively hybridize” as described herein. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit a completely identical sequence from hybridizing to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern blot, Northern blot, solution hybridization, or the like, see Sambrook, et al., supra). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target. 
     When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence “selectively hybridize,” or bind, to each other to form a hybrid molecule. A nucleic acid molecule that is capable of hybridizing selectively to a target sequence under “moderately stringent” conditions typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe. Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe. Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization, supra). 
     With respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as varying wash conditions. The selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook et al., supra). 
     As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% identical to each other typically remain hybridized to each other. In one aspect, the conditions are such that sequences at least about 70%, at least about 80%, at least about 85% or 90% or more identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley &amp; Sons, NY (1989), 6.3.1-6.3.6. A non-limiting example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50° C., at 55° C., or at 60° C. or 65° C. 
     In certain exemplary embodiments screening assays for identifying modulators, i.e., candidate or test compounds or agents (e.g., antibodies, peptides, cyclic peptides, peptidomimetics, small molecules, small organic molecules, or other drugs) which have a stimulatory effect on one or more ANG polypeptides and an inhibitory effect on one or more neurodegenerative diseases (e.g., ALS) are provided. 
     As used herein, the term “small molecule” refers to a molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 25 daltons and less than about 3000 daltons, usually less than about 2500 daltons, more usually less than about 2000 daltons, usually between about 100 to about 1000 daltons, more usually between about 200 to about 500 daltons. 
     In certain exemplary embodiments, assays for screening candidate or test compounds which bind to or modulate (e.g., stimulate) one or more ANG polypeptides and modulate (e.g., inhibit) one or more neurodegenerative diseases (e.g., ALS) are provided. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S. (1997)  Anticancer Drug Des.  12:145). 
     It is to be understood that the embodiments of the present invention which have been described are merely illustrative of some of the applications of the principles of the present invention. Numerous modifications may be made by those skilled in the art based upon the teachings presented herein without departing from the true spirit and scope of the invention. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference in their entirety for all purposes. 
     The following examples are set forth as being representative of the present invention. These examples are not to be construed as limiting the scope of the invention as these and other equivalent embodiments will be apparent in view of the present disclosure, figures, tables, and accompanying claims. 
     Example 1 
     Angiogenin 
     ANG was originally isolated from the conditioned medium of HT-29 human colon adenocarcinoma cells based solely on its angiogenic activity in the chicken embryo chorioallantoic membrane (CAM) angiogenesis assay (Fett et al. (1985)  Biochemistry  24:5480). Subsequently, ANG has been found to have a wide tissue distribution with the liver being the major source for circulating ANG (Weiner et al. (1987)  Science  237:280). ANG is a member of the pancreatic ribonuclease A (RNase A) superfamily with a 33% amino acid identity and an overall homology of 56% to that of RNase A (Strydom et al. (1985)  Biochemistry  24:5486). ANG has a unique ribonucleolytic activity that is several orders of magnitude lower than that of RNase A but is important for its biological activity (Shapiro et al. (1986)  Biochemistry  25:3527). The amino acid residues important for catalysis are conserved in all vertebrate ANG from fish to human (Riordan (2001)  Methods Enzymol.  341:263). Extensive work on site-directed mutagenesis has shown that ANG variants with reduced enzymatic activity invariably have reduced angiogenic activity (Shapiro et al. (1986)  Biochemistry  25:3527; Curran et al. (1993)  Biochim. Biophys. Acta.  1202:281; Hallahan et al. (1992)  Biochemistry  31:8022; Hallahan et al. (1991)  Proc. Natl. Acad. Sci. USA  88:2222; Harper et al. (1990)  Biochemistry  29:7297; Shapiro et al. (1989)  Biochemistry  28:1726; Shapiro and Vallee (1989)  Biochemistry  28:7401; Shapiro and Vallee (1992)  Biochemistry  31:12477; Shapiro et al. (1987)  Proc. Natl. Acad. Sci. USA  84:8783). Structural work indicated one of the reasons for ANG to have a reduced ribonucleolytic activity is that the side chain of Gln 117 occupies part of the enzymatic active site so that substrate binding is compromised (Acharya et al. (1994)  Proc. Natl. Acad. Sci. USA  91:2915; Russo et al. (1994)  Proc. Natl. Acad. Sci. USA  91:2920). 
     ANG is angiogenic, whereas the prototype family member RNase A is not. Two important structural differences between ANG and RNase A are responsible for this discrepancy. The first is the segment from amino acid residues 59 to 68 that forms the receptor binding site (Hallahan et al. (1991)  Proc. Natl. Acad. Sci. USA  88:2222; Hu et al. (1991)  Proc. Natl. Acad. Sci. USA  88:2227) in ANG and is very different in RNase A (Acharya et al. (1994)  Proc. Natl. Acad. Sci. USA  91:2915; Acharya et al. (1995)  Proc. Natl. Acad. Sci. USA  92:2949). Therefore ANG binds to its target cells (including endothelial cells, cancer cells and motor neurons) but RNase A does not. ANG binds to endothelial cells specifically (Badet et al. (1989)  Proc. Natl. Acad. Sci. USA  86:8427) and induces second messenger responses including diacylglycerol and prostacyclin (Bicknell and Vallee (1988)  Proc. Natl. Acad. Sci. USA  85:5961; Bicknell and Vallee (1989)  Proc. Natl. Acad. Sci. USA  86:1573), and activates MAP kinase (Liu et al. (2001)  Biochem. Biophys. Res. Commun.  287:305) and AKT (Kim et al. (2007)  Biochem. Biophys. Res. Commun.  352:509). 
     Another structural difference between ANG and RNase A is that ANG has a nuclear localization signal (NLS) consisting of 291MRRRGL35 (SEQ ID NO:8), whereas RNase A does not (Moroianu and Riordan (1994)  Biochem. Biophys. Res. Commun.  203:1765). Therefore ANG undergoes nuclear translocation in endothelial cells where it accumulates in the nucleolus (Moroianu and Riordan, J. F. (1994)  Proc. Natl. Acad. Sci. USA  91:1677; Hu and Riordan (2000)  J. Cell. Biochem.  76:452), binds to the promoter region of ribosomal DNA (rDNA) and stimulates ribosomal RNA (rRNA) transcription (Xu et al. (2002)  Biochem. Biophys. Res. Commun.  294:287, Xu et al. (2003)  Biochemistry  42:121), an essential step for ribosome biogenesis and therefore for protein translation and cell proliferation. 
     An ANG binding protein has been identified from the surface of endothelial cells (Hu et al. (1991)  Proc. Natl. Acad. Sci. USA  88:2227) and has been characterized to be a type of smooth muscle actin (Hu et al. (1993)  Proc. Natl. Acad. Sci. USA  90:1217; Moroianu et al. (1993)  Proc. Natl. Acad. Sci. USA  90:3815). An approximately 170 kDa ANG receptor has also been identified from the endothelial cell surface to mediate nuclear translocation of ANG and cell proliferation (Hu et al. (1997)  Proc. Natl. Acad. Sci. USA  94:2204). Expression of the binding protein and the receptor on endothelial cells seems to be mutually exclusive. The binding protein is expressed on the surface of confluent cells. Binding of ANG to the binding protein activates tissue plasminogen activator (tPA) (Hu and Riordan (1993)  Biochem. Biophys. Res. Commun.  197:682) thereby inducing cell invasion and migration (Hu et al. (1994)  Proc. Natl. Acad. Sci. USA  91:12096). After the leading cells migrate away, the local cell density decreases which triggers the expression of an ANG receptor. Binding of ANG to the receptor stimulates cell proliferation so that the gap created by the migrating cells is filled. Therefore, ANG is a multifunctional angiogenic molecule that plays a role in several steps in the angiogenesis process including cell invasion, proliferation, and tube formation.  FIG. 1  summarizes the findings presented herein regarding the mechanism of ANG-induced angiogenesis. 
     Example 2 
     Role of ANG in rRNA Transcription 
     ANG has been shown to undergo nuclear translocation in endothelial cells (Moroianu and Riordan, J. F. (1994)  Proc. Natl. Acad. Sci. USA  91:1677; Hu and Riordan (2000)  J. Cell. Biochem.  76:452; Li et al. (1997)  Biochem. Biophys. Res. Commun.  238:305) and in various types of human cancer cells (Tsuji et al. (2005)  Cancer Res.  65:1352; Yoshioka et al. (2006)  Proc. Natl. Acad. Sci. USA  103:14519). Nuclear translocation of ANG in endothelial cells is under tight regulation and is cell density-dependent. It decreases as cell density increases and ceases when cells are confluent (Hu and Riordan (2000)  J. Cell. Biochem.  76:452; Hu et al. (1997)  Proc. Natl. Acad. Sci. USA  94:2204). Nuclear translocation of ANG occurs through receptor-mediated endocytosis (Moroianu and Riordan, J. F. (1994)  Proc. Natl. Acad. Sci. USA  91:1677) and is independent of microtubules systems and lysosomal processing (Li et al. (1997)  Biochem. Biophys. Res. Commun.  238:305) and ANG seems to enter the nuclear pore by the classic nuclear pore input route (Moroianu and Riordan (1994)  Biochem. Biophys. Res. Commun.  203:1765). Nuclear translocation of exogenous ANG was very fast. When exogenous ANG is added to the cell culture, nuclear ANG is detectable within 2 minutes and is saturated in 30 minutes (Hu and Riordan (2000)  J. Cell. Biochem.  76:452). Upon arriving at the nucleus, ANG accumulates in the nucleolus (Moroianu and Riordan, J. F. (1994)  Proc. Natl. Acad. Sci. USA  91:1677) where ribosome biogenesis takes place. Nuclear ANG has been shown to bind to the promoter region of rDNA (Xu et al. (2003)  Biochemistry  42:121) and stimulates rRNA transcription (Xu et al. (2002)  Biochem. Biophys. Res. Commun.  294:287; Kishimoto et al. (2005)  Oncogene  24:445). 
     Cell growth requires the production of new ribosomes. Ribosomal biogenesis is a process involving rRNA transcription, processing of the pre-rRNA precursor and assembly of the mature rRNA with ribosomal proteins (Comai (1999)  Braz. J. Med. Biol. Res.  32:1473; Melese and Xue (1995)  Curr. Opin. Cell. Biol.  7:319; Stoykova et al. (1985)  J. Neurochem.  45:1667). The rate-limiting step in ribosome biogenesis is the synthesis of rRNA. Therefore, rRNA transcription is an important aspect of growth control. When cells are quiescent, the overall rate of protein accumulation is reduced. On mitogenic stimulation the synthesis of rRNA is accelerated, and the production of ribosomal proteins and translation factors increases before cells reach S phase (Rosenwald (1996)  Cancer Letters  102:113; Clarke et al. (1996)  J. Biol. Chem.  271:22189; Rosenwald (1996)  Bioessays  18:243). The rate of growth is directly proportional to the rate of protein accumulation and this is related to ribosome content (Baxter and Stanners (1978)  J. Cell Physiol.  96:139). As ribosome biogenesis is a limiting factor for cell duplication, the rate of cell proliferation could be controlled by modulating the expression of nucleolar proteins involved in rDNA gene transcription, rRNA processing, and transport of transcripts to the cytoplasm. Nuclear localization of those nucleolar proteins from the cytoplasm or from outside of the cells would then be an important factor to control ribosome biogenesis. 
     ANG appears to be one of the proteins that is translocated to the nucleus where it regulates rRNA transcription in its targeting cells. Therefore, ANG-stimulated rRNA transcription has been proposed as a general requirement for angiogenesis and is a common crossroad that all the angiogenic factors need to go through (Kishimoto et al. (2005)  Oncogene  24:445). In other words, ANG is a permissive factor for other angiogenic factors. Experimental evidence for this contention includes: (1) nuclear translocation of endogenous ANG in endothelial cells is stimulated by other angiogenic factors including acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) (Kishimoto et al. (2005)  Oncogene  24:445); (2) knocking-down ANG expression in endothelial cells inhibits bFGF- and VEGF-induced cell proliferation accompanied with a decrease in rRNA transcription. Addition of exogenous ANG can completely restore the proliferative activity of these angiogenic factors (Kishimoto et al. (2005)  Oncogene  24:445); (3) ANG-specific inhibitors had no effect on binding of VEGF and bFGF to their receptors but inhibit angiogenesis induced by them (Hirukawa et al. (2005)  Clin. Cancer Res.  11:8745).  FIG. 2  summarizes the function of ANG-stimulated rRNA transcription in cell proliferation. 
     Example 3 
     ANG Mutation is ALS Patients 
     Recently, linkage analysis in Irish and Scottish ALS populations identified an association of the G allele of the single nucleotide polymorphism (SNP) rs11701 in the coding region of ANG (representing the amino acid residue G86 in the mature protein) (Greenway et al. (2004)  Neurology  63:1936). Subsequently, seven heterozygous missense mutations in ANG were identified in 15 patients by sequence screening of 1,629 individuals with ALS (Greenway et al. (2006)  Nat. Genet.  38:411), an overall frequency of approximately 1% with an over representation of familial ALS (FALS) (4/259, 1.5%) over sporadic (SALS) (11/1370, 0.8%). From sequencing an additional 298 ALS patients of a Northern American cohort, an additional four mutations in the ANG gene were identified (1.3% frequency) (Wu et al. (2007)  Ann. Neurol.  62:609). More recently, 7 more mutations were identified in 9 of the 737 Italian ALS patients (Gellera et al. (2007)  Neurogenetics ). ANG mutations in Italian population also seem to segregate FALS (3/132, 2.3%) from SALS (6/605, 1%) with an overall frequency of 1.2%. Id.  FIG. 19  lists the amino acid sequences of primate and mammalian ANG with the mutations bracketed. 
     So far, a total of 15 missense mutations (at 14 positions) in the coding region of ANG have been identified in 37 of the 4193 ALS patients of the Irish, Scottish, Swedish (Greenway et al. (2006)  Nat. Genet.  38:411), North American (Wu et al. (2007)  Ann. Neurol.  62:609), Italian (Gellera et al. (2007)  Neurogenetics ), and French (Paube et al. (2008)  Arch. Neurol.  65:1333) populations. Among these mutations, three occurred in the signal peptide regions and 10 in the mature protein. In the six sequencing efforts carried out so far, a total of 3341 healthy controls have been included and two mutations in the ANG gene were found in healthy controls (Greenway et al. (2006)  Nat. Genet.  38:411; Gellera et al. (2007)  Neurogenetics ). The first is a K17I mutation that was found in an apparent healthy 65-year-old male of European descent. The second is the 146V mutation that was found in 11 of the 1568 Italian healthy control patients (Corrado et al. (2007)  J. Neurol. Sci.  258:123; Conforti et al. (2007)  Neuromuscul. Disord .; Gellera et al. (2007)  Neurogenetics ; Del Bo et al. (2006)  Neurobiol Aging ). Therefore, I46V mutation does not seem to be associated with Italian ALS patients but does seem to be associated with the Scottish ALS patients in which 3 of the 398 ALS patients but none of the 299 controls harbor the 146V mutation (Greenway et al. (2006)  Nat. Genet.  38:411). Table 1 lists the frequencies of ANG mutations occurred in 3001 ALS patients. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                 Number  
                   
               
               
                   
                 Mutation 
                 of Cases 
                 References 
               
               
                   
               
             
            
               
                   
                 M(−24)I 
                 2 
                 Conforti et al. (2007) 
               
               
                   
                   
                   
                 Neuromuscul. Disord.; 
               
               
                   
                   
                   
                 Gellera et al. (2007) 
               
               
                   
                   
                   
                 Neurogenetics 
               
               
                   
                 F(−13)S 
                 1 
                 Conforti et al. 
               
               
                   
                 P(−4)L 
                 2 
                 Gellera et al.; 
               
               
                   
                   
                   
                 Wu et al. (2007) Ann. 
               
               
                   
                   
                   
                 Neurol. 62:609 
               
               
                   
                 Q12L 
                 2 
                 Greenway et al. (2006) Nat. 
               
               
                   
                   
                   
                 Genet. 38:411 
               
               
                   
                 K17I 
                 4 
                 Greenway et al.; 
               
               
                   
                   
                   
                 Wu et al. 
               
               
                   
                   
                   
                 Van Es et al (2009) 
               
               
                   
                   
                   
                 Neurology 71:287) 
               
               
                   
                 K17E 
                 2 
                 Greenway et al. 
               
               
                   
                 S28N 
                 1 
                 Wu et al. 
               
               
                   
                 R31K 
                 1 
                 Greenway et al. 
               
               
                   
                 C39K 
                 2 
                 Greenway et al. 
               
               
                   
                 K401 
                 3 
                 Greenway et al. 
               
               
                   
                 I46V 
                 10   
                 Corrado et al. (2007) J. 
               
               
                   
                   
                   
                 Neurol. Sci. 258:123; 
               
               
                   
                   
                   
                 Greenway et al.; 
               
               
                   
                   
                   
                 Gellera et al. 
               
               
                   
                 P112L 
                 1 
                 Wu et al. 
               
               
                   
                 V113I 
                 2 
                 Gellera et al. 
               
               
                   
                 H114R 
                 1 
                 Gellera et al. 
               
               
                   
                 R121H 
                 1 
                 Paube et al. (2008) Arch. 
               
               
                   
                   
                   
                 Neurol. 65:1333 
               
               
                   
               
            
           
         
       
     
     Two new mutations, D22G and K60E, have been discovered in our ongoing effort to screen more ALS patients. 
     Example 4 
     Properties of Mutant ANG Proteins 
     Except for the three mutations in the signal peptide regions (M-24I, F-13S, P-4S) and the two in the coding region (V113I, H114R) that were just reported in the most recent publication (Gellera et al. (2007)  Neurogenetics ), all the mutant ANG proteins have been prepared and characterized by ribonuclease assay (Wu et al. (2007)  Ann. Neurol.  62:609; Crabtree et al. (2007)  Biochemistry  46:11810), nuclear translocation assay (Wu et al. (2007)  Ann. Neurol.  62:609), and angiogenesis assay (Id.). Except for R31K, all of these mutant proteins have severely impaired ribonucleolytic activity ranging from &lt;1% (K40I) to 19% (K17E) of the wild-type ANG. R31K has 69% of the wild-type enzymatic activity (Crabtree et al. (2007)  Biochemistry  46:11810). Because the RNase activity of ANG has been proven to be essential for angiogenesis (Shapiro et al. (1986)  Biochemistry  25:3527; Shapiro and Vallee (1989)  Biochemistry  28:7401), these mutant ANG proteins are likely to be inactive in inducing angiogenesis. Some of them also seem to have a reduced thermal stability (Crabtree et al. (2007)  Biochemistry  46:11810). Among the three mutant ANG proteins (K17I, S28N, P112L) tested in the nuclear translocation assay, S28N and P112L do not undergo nuclear translocation and K17I has a reduced capacity (Wu et al. (2007)  Ann. Neurol.  62:609). Two types of angiogenesis assays have been used to test the angiogenic activity of the mutant ANG proteins. The endothelial cell tube formation assay on fibrin gel was used to examine the mutants identified from the Northern American ALS cohort and the results showed that all three mutants (K17I, S28N, P112L) are inactive. Id. The aorta ring assay was used to test three of the seven mutants identified from the Irish and Scottish ALS populations. All three mutants tested (Q12L, C39W, K40I) were inactive in the aorta ring angiogenesis assay (Crabtree et al. (2007)  Biochemistry  46:11810). Taken together, these results demonstrate that ANG mutations identified in ALS patients are associated with a functional loss of the angiogenic activity of the ANG protein. 
     Wild-type ANG has been shown to induce neurite outgrowth and pathfinding of motor neurons derived from P19 embryonal carcinoma cells (Subramanian and Feng (2007)  Hum. Mol. Genet.  16:1445). ANG also protects P19-derived motor neuron from hypoxia-induced cell death, but the ALS-associated mutant ANG proteins (Q12L, C39W, K40I) lack this neuroprotective activity (Subramanian et al. (2008)  Hum. Mol. Genet.  17:130). Moreover, these mutant ANG proteins are cytotoxic to the P19-derived motor neurons and induce their degeneration, suggesting that ANG mutations may even be causative to ALS. Id. 
     Example 5 
     Expression of ANG in the CNS 
     Mouse ANG is strongly expressed in the developing mouse nervous system both in the brain and in the spinal cord (Subramanian and Feng (2007)  Hum. Mol. Genet.  16:1445). Immunohistochemistry (IHC) and immunofluorescence (IF) have been used to show that ANG expression is the strongest in the presumptive forebrain, midbrain, hindbrain and spinal cord at 9.5 day pc. Id. At 11.5 day pc, ANG expression remains high in the telencephalon, mesen and mylencephalon as well as in the spinal cord, spinal ganglia and choroids plexus. Id. Until mid-gestation, ANG expression is stronger in the nervous system than in any other tissues. Co-staining with Peripherin and Isletl showed that ANG is expressed in mouse motor neurons. 
     IHC was also used to detect the expression of human ANG in normal spinal cords obtained from fetal (ranging from 15 to 30 weeks gestation) and adult human autopsies. Strong ANG staining was observed in the spinal cord ventral horn motor neurons of both fetal and adult cases (Wu et al. (2007)  Ann. Neurol.  62:609). ANG was also detected in the extracellular matrix and interstitial tissues in all cases, consistent with ANG being a secreted protein. It appears that ANG expression in the spinal cord is down-regulated as development proceeds but is still strongly expressed in the adulthood. Strong cytoplasmic and nuclear accumulation of ANG in motor neurons of both prenatal and adult spinal cords suggest a physiological role of ANG, both early in development and later in adulthood, and supports the hypothesis that ANG mutations are likely relevant to ALS pathology. 
     In the developing human brain, we have detected strong ANG expression in other types of neural cells such as ependymal cells, purkinje cells and glial cells in the cerebellum ( FIG. 15A ) and the motor neurons of cranial nerves and substantia nigra ( FIG. 15B ). It is of interest to note that neurons that express ANG strongly are all related to motion. 
     Double IF with an anti-ANG monoclonal antibody (mAb) and anti-von Willebrand factor (vWF) polyclonal antibody (pAb) showed that ANG is localized in both endothelial cells and motor neurons of spinal cord tissues, suggesting that ANG may also mediate angiogenesis in the spinal cord, and may play a role in maintaining the physiological health of motor neurons. Id. Thus, ANG abnormalities may have a dual role in ALS—directly through motor neuron function and indirectly through endothelial cells and aberrant angiogenesis. 
     Example 6 
     ANG Protein Level is Decreased in Spinal Cord Motor Neurons of ALS Patients 
     This example demonstrates that ANG expression was decreased in the spinal cord of ALS patients that do not harbor ANG mutations. IHC with the human ANG-specific mAb 26-2F showed that ANG protein level was markedly decreased in both motor neurons (indicated by arrows) and stroma (indicated by stars) of the spinal cord of human ALS patients as compared to that of the non-ALS controls ( FIG. 3A ). A total of six ALS cases and six non-ALS control spinal cords were examined. 
     The mAb 26-2F is known to be specific for human ANG. It does not recognize any other human proteins. An isotype-matched, non-immune IgG was used as a negative control and no signals were observed in both ALS and non-ALS specimens under the same concentration. Moreover, an affinity-purified anti-human ANG polyclonal antibody (pAb) R113 was also and the results were the same to that obtained with mAb 26-2F. These data confirm specificity of the IHC and the subsequent IF results. 
     Imaging quantification was performed with the ImageJ software. The entire ventral horn was analyzed. The average size of the ANG-positive motor neuron was 1245±170 and 385±63 μm 2 , respectively, in normal and ALS spinal cord ( FIG. 3B ), representing a 69% decrease in ALS (p&lt;3.3×10 −5 ). The total area covered by ANG-positive motor neuron in the ventral horn of normal and ALS patients are 15.5±2.6×10 3  and 2.8±0.8×10 3  μm 2 , respectively ( FIG. 3C ), representing a 72% decrease in ALS (p&lt;4.9×10 −5 ). To obtain the ANG staining intensity in motor neurons, the photon counts of the adjacent non-motor neuron area were used as the controls. The average photon counts per motor neuron of normal and ALS spinal cord were 6.3±0.7×10 5  and 7.8±1.2×10 4 , respectively ( FIG. 3D ), representing a 78% decrease in ALS (p&lt;4.1×10 −6 ). The total photon counts ( FIG. 3E ) in the motor neuron in normal and ALS spinal cord were 6.7±1.2×10 6  and 5.3±0.9×10 5 , respectively, representing a 92% decrease in ALS (p&lt;1.6×10 −5 ). These results indicate that ANG protein level in the spinal cord of ALS patients was dramatically decreased. 
     Example 7 
     ANG Protein Level was Decreased in Both Motor Neurons and Endothelial Cells of ALS Spinal Cord 
     Double IF staining with 26-2F ( FIGS. 4A-B ) and anti-von Willebrand factor (vWF) ( FIGS. 4C-D ) was carried out to detect ANG and blood vessels, respectively. The merged images of ANG and vWF staining shows that ANG is located both in motor neurons (indicated by arrows) and in blood vessels (indicated by arrow heads) and its level in both cell types is decreased in the spinal cord of ALS patients ( FIGS. 4E-F ). 
     Example 8 
     ANG mRNA Levels Decreased in ALS Spinal Cord 
     In situ hybridization (ISH) was used to check the mRNA level of ANG in the ALS and control spinal cords. Strong staining of ANG mRNA in the motor neurons was observed in non-ALS spinal cord, which was dramatically decreased in ALS spinal cords ( FIG. 5A ). Quantitative analysis of the ISH images indicate that the total photon counts of ANG mRNA staining in the motor neurons of the entire ventral horn of ALS and non-ALS spinal cord were 5.3±0.8×10 5  and 6.4±0.8×10 5 , respectively ( FIG. 3E ), representing an 88% decrease in ALS samples. 
     The results shown in  FIGS. 3-5  indicate that ANG is strongly expressed in normal human spinal cords and that its expression is decreased in ALS patients. At present, it is unknown whether decreased ANG level in the spinal cord of ALS patients is a cause or a consequence of motor neuron degeneration. However, these findings indicate that a deficiency in ANG activity might be related to ALS pathogenesis. These results also indicate that local production of ANG may be more relevant to ALS. Without intending to be bound by scientific theory, transcription from the distal promoter (i.e., non-liver) may be affected in ALS patients differently from transcription from the proximal promoter (i.e., liver-specific). 
     Example 9 
     Protein and mRNA Levels of Mouse Angiogenin (ANG) is Decreased in the Spinal Cord of SOD1 G93A  Mice 
     Transgenic mice over-expressing the G93A mutant SOD1 gene develop symptoms mimicking that of human ALS patients and are an established model for ALS research (Gurney (1997)  J. Neurol Sci.  152 Suppl. 1:S67). IHC, IF and Western blotting were used to determine the protein level and use ISH for the mRNA level of mouse ANG in the spinal cord of WT and SOD1 G93A  mice. Animals were sacrificed at 14 weeks of age after transcardiac perfusion and the spinal cords were processed for IHC ( FIG. 6A-B ) and IF ( FIG. 6C-D ) with an anti-mouse ANG IgG R165. 
     Mouse ANG protein levels in the ventral horn motor neurons of SOD1 G93A  spinal cord were much lower than that of WT mice at the same age. The antibody used in these experiments was prepared using recombinant mouse ANG1 and was affinity-purified. While humans have only one ANG gene, mice have 6 ANG isoforms. It is not known at present whether R165 recognizes the other ANG isoforms, but ANG1 was the predominant isoform expressed in the spinal cord. The signals in  FIGS. 6A-D  were most likely from ANG1. No IHC or IH signals were detected when a non-immune rabbit IgG was used under the same conditions (30 μg/ml IgG). Quantitative image analysis indicated that the total photon counts of mouse ANG in SOD1 G93A  and WT mice were 1.1±0.2×10 6  and 5.4±0.7×10 6 , respectively, representing an 80% decrease (p=1.1×10 −7 ) in SOD1 G93A  mice ( FIG. 6E ). 
     Next, ISH was performed using a riboprobe specific for ANG1 to detect the mRNA level of ANG1 ( FIGS. 6F-I ). ANG1 mRNA levels in the spinal cord of SOD1 G93A  mice ( FIGS. 6H-I ) was significantly lower than that of the WT ( FIGS. 6F-G ). Quantitative image analyses indicated that the photon counts in SOD1 G93A  and WT mice were 5.9±0.4×10 4  and 2.2±0.2×10 4 , respectively ( FIG. 6J ), representing a decrease of 63% (p=4.4×10 −6 ). Therefore, both ANG1 protein and mRNA levels were decreased in the motor neurons in SOD1 G93A  mice. It is currently unknown how SOD1 G93A  expression results in the decrease of ANG expression in the spinal cord motor neurons. 
     Example 10 
     Decreased Blood Vessel Size in Spinal Cords of Human ALS Patients and SOD1 G93A  Mice 
     The density and size of the blood vessels in control and ALS spinal cords were investigated. IHC with an anti-vWF antibody was used to stain the blood vessels ( FIG. 7A ). The numbers of the vessels in five randomly selected areas from each of the two ventral horns (n=5) were counted and their sizes measured.  FIG. 7B  shows that the density of blood vessels in the ALS spinal cord was marginally but statistically significantly increased (p=0.0054). However, the average size of the vessel was dramatically decreased (p=1×10 −9 ) ( FIG. 7C ). Similarly, the vessel density in the SOD1 G93A  and WT mice was not much different (p=0.23) ( FIG. 7E ), but the vessel size was significantly decreased ( FIG. 7F , p=0.0001). These results indicate that motor neuron degeneration may be associated with an irregular vasculature in the spinal cord. 
     Example 11 
     Intraperitoneally Injected ANG Protein Reaches at Spinal Cord 
     Without intending to be bound by scientific theory, one of the reasons for a relative minimal effect of exogenous trophic factors and other types of therapeutic proteins could be their failure to cross the blood brain barrier (BBB) and blood spinal cord barrier (BSCB). Gene therapy is therefore an alternative approach for ALS therapy. Many strategies are under investigation, including the delivery of genes encoding neurotrophic factors, anti-apoptotic drugs and antioxidants using viral vectors administered directly into the affected areas of the central nervous system (CNS), or through retrograde transport to motor neurons from intramuscular injection, or through ex vivo gene transfer (Federici and Boulis (2006)  Muscle Nerve  33:302). Besides lentiviral vector-mediated delivery of VEGF (Azzouz et al. (2004)  Nature  429:413), AAV-mediated delivery of IGF-1 (Kaspar et al. (2003)  Science  301:839),  GDNF  (Wang et al. (2002)  J. Neurosci.  22:6920), and Bcl-2 (Azzouz et al. (2000)  Hum. Mol. Genet.  9:803) genes have also been shown to be effective in the SOD1 G93A  transgenic mice. 
     To ascertain the feasibility of systemic administration of ANG protein in potential ALS treatment, it was determined whether i.p.-injected ANG could reach the spinal cord. For this purpose, WT and SOD1 G93A  mice, 11 weeks of age, were injected with 10 μl of PBS or 10 μg of human WT ANG. IHC with the human ANG-specific mAb 26-2F showed that ANG reached the spinal cord ( FIG. 8 ) of both WT and SOD1 G93A  mice. As the mice were transcardiac perfused with 30 ml PBS before sacrifice, it was unlikely that the spinal cord samples were contaminated with the human ANG that entered circulation after i.p.-injection. Moreover, the same amount of EGF, bFGF, and RNase A were injected in the same manner, and no significant amount of these proteins was detected in the spinal cord (data not shown). RNase A has a 56% homology to ANG at the amino acid level (Strydom et al (1986)  Biochemistry  25:3527), has the same molecular weight, similar pI, and a very similar 3-D structure (Acharya et al et al. (1991)  PNAS  92:2049), but is not angiogenic. The unique property of .p.-injected ANG protein crosses the BBB or BSCB allowed the systemic administration of ANG protein to assess its therapeutic activity toward ALS in SOD1 G93A  mice. 
     Example 12 
     ANG Treatment Improved Motor Muscular Function and Survival of SOD1 G93A  Mice 
     The effect of ANG on motor muscular function of SOD1 G93A  mice was investigated. A total of 36 SOD1 G93A  mice were separated into three groups. Each group had 12 litter- and gender-matched mice. Quantitative PCR was performed on every SOD1 G93A  mouse to ensure that a relatively equal copy number of the transgene was present in each mouse ( FIG. 9A ). 
     Eleven week old mice were treated with weekly i.p. injections of PBS, WT or the P112L mutant ANG protein at 10 μg per mouse, and their motor muscular function was tested on a rotarod in a blinded fashion.  FIG. 9B  shows that ANG-treated mice were able to extend their hind legs when lifted by the tail, but the PBS-treated mice failed to do so, indicating that the muscle strength was enhanced by ANG treatment. To compare the effect of P112L mutant with WT ANG, IHC was performed with the mAb 26-2F to ensure that i.p.-injected P112L mutant protein indeed reached spinal cord as did WT protein. Rotarod performance showed that ANG treatment dramatically increased the time the animals could stay on the rotarod as compared to the PBS and P112 treatments ( FIG. 9C ), indicating that i.p. ANG treatment enhanced the motor coordination and muscular function of the SOD1 G93A  mice. Student&#39;s t-test indicated that the difference in the rotarod time was highly significant from week 12 to 18 (p&lt;0.0001 in all these 7 weeks, indicated by *). At the peak performance (at week 14), ANG-treated mice were able to stay on the rotarod for an average of 1726±778 sec, a 32-fold increase over that of the PBS-treated control mice (53±3 sec) ( FIG. 9C ). At the same week, P112L ANG-treated mice were able to stay on the rotarod for an average time of 51±21 sec. 
     ANG treatment increased the survival of these animals by four weeks ( FIG. 9D ), representing a 23% increase in the life span of these mice. These results indicate that ANG treatment may prolong the life of ALS patients as well as improving quality of life. In certain exemplary embodiments, an increased life expectancy of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more is obtained. It is important to note that the P112L mutant protein had no beneficial effect on SOD1 G93A  mice on either the rotarod test or the tail suspension test, and did not prolong their survival. These results demonstrate that the beneficial effect of ANG on SOD1 G93A  mice is dependent on the angiogenic activity. They also support the hypothesis that ANG mutations are associated with ALS pathogenesis. ANG had no detectable effects on WT mice at the same dose on rotarod test. No side effects in animal grooming behavior were observed. 
     The efficacy of different routes of ANG administration including intravenous (i.v.), subcutaneous (s.c.) and i.p. at the same dose (10 μg per mouse) and same interval (every week) were compared. All three routes of administration were effective in increasing the performance on rotarod with the descending order of i.v.&gt;i.p.&gt;s.c. ( FIG. 6 ). For example, at week 13 (after 2 injections), the time the mice were able to stay on a rotating rotarod were 161±15, 259±27, and 1770±310 sec, respectively, for the s.c., i.p., and i.v. ANG treatment, respectively. All three routes of ANG administration show significantly better performance on the rotarod as compared to the untreated group (19±16 sec). Intravenous injection provides the fastest improvement in motor muscular function ( FIG. 10 ). The maximal effect of i.v. and i.p. injection appeared at week 13 and 14, respectively. Please note the different scales used in the two panels in order to reveal the difference in the beneficial effect of 3 routes of administration. Among the three routes of administrations, s.c. injection was the least effective. However, the beneficial effect of s.c. injection of angiogenin in improving motor muscular function of SOD G93A  is still better than that mediated by IGF and VEGF reported in prior art (Gage et al. (2003), Science 301, 839; Azzouz et al. (2004) Nature 429, 413). 
     In order to determine the optimal dose of angiogenin in improving the motor muscular function of SOD G93A  mice, we compared the effect of i.p injection of ANG protein at 0.1, 1, and 10 μg per mouse per week. As shown in  FIG. 11 , 1 μg per mouse per week was the most effective dose, while all three doses significantly improved the motor muscular function of SOD G93A  mice as determined by their performance on a rotarod. 
     Example 13 
     ANG Treatment Improved Motor Neuron Survival and Normalized Spinal Cord Vasculature 
     WT mice and SOD1 G93A  mice treated with PBS or ANG protein were sacrificed at week 15, and the lumbar regions of the spinal cord was removed and processed for histology and IHC. First, an anti-human SOD1 IgG was used to ensure that the transgene expression was not interfered with ANG treatment ( FIG. 12A , top panels). Nissl staining showed that there was a clear loss of motor neurons in the spinal cord of PBS-treated SOD1 G93A  mice compared to those of WT mice ( FIG. 12A , middle panels). ANG treatment clearly increased the number of motor neurons ( FIG. 12B ). The number of the motor neurons with a soma size larger than 250 μm 2  was 21.9±2.1, 12.3±1.3, and 18.7±2.3, respectively, in WT, PBS- and ANG-treated SOD1 G93A  mice. ANG treatment thus significantly enhanced motor neuron survival (p=0.024). 
     IHC with an anti-vWF antibody was performed ( FIG. 12A , bottom panel) and the density and size of the blood vessels determined. There were no significant differences in the density of the blood vessels between WT (82±23/mm 2 ), PBS— (68±13/mm 2 ) and ANG-treated SOD1 G93A  (73±14/mm 2 ) mice ( FIG. 12C ). However, ANG treatment significantly increased the size of the blood vessels in SOD1 G93A  mouse spinal cords. The average diameter of the WT mouse, PBS- and ANG-treated SOD1 G93A  mice were 3.3±1.5, 1.9±0.7, and 2.8±1.6 μm, respectively ( FIG. 12D ). Without intending to be bound by scientific theory, it is possible that ANG treatment improves the motor muscular function through enhancement of motor neuron survival as well as normalization of spinal cord vasculature. 
     Example 14 
     ANG Stimulates P19 Cell Proliferation and Differentiation 
     P19 cells are mouse pluripotent embryonal carcinoma cells that have the stem cell-like property of having the ability to both self-renew and differentiate into various types of neural cells (Bain et al. (1994)  Bioessays  16:343; McBurney and Rogers (1982)  Dev. Biol.  89:503).  FIG. 13A  shows that undifferentiated P19 cells were stained positive with mouse ANG-specific pAb R165. The role of endogenous ANG in P19 cells is currently unknown. However, human ANG, when added exogenously, underwent nuclear translocation in P19 cells ( FIG. 13B ) and stimulated the formation of embryoid bodies, the undifferentiated suspended aggregates ( FIG. 13C ), indicating that ANG stimulates P19 cell proliferation. If these embryoid bodies were resuspended and continued to culture, long neuronal processes formed in the presence of ANG but not in its absence ( FIG. 13D ), indicating that ANG also induces cell differentiation. These results support a role of ANG in neurogenesis. 
     Example 15 
     ANG Undergoes Nuclear Translocation in NSC-34 Motor Neuron Cells and Enhances Neurite Outgrowth 
     NSC-34 cells are neuroblastoma-spinal cord hybrid cells derived from somatic cell fusion between neuroblastoma N18TG2 cells and motor neuron-enriched embryonic day 12-14 spinal cord cells (Cashman et al. (1992)  Dev. Dyn.  194:209). NSC-34 cells display motor neuron like characteristics including generation of action potentials, expression of NF triplet proteins, and acetylcholine synthesis, storage, and release (Matusica et al. (2007)  J. Neurosci. Res .; Durham et al. (1993)  Neurotoxicology  14:387). Exogenously added human angiogenin was found to undergo nuclear translocation in NSC-34 cells, whereas the P112L mutant angiogenin failed to do so ( FIG. 14A ). This is consistent with a role of angiogenin in stimulation of rRNA transcription. Angiogenin induced NSC-34 cell proliferation in a dose-dependent manner ( FIG. 14B ). Moreover, it also promoted neurite outgrowth of NSC-34 cells ( FIG. 14C ). 
     Example 16 
     ANG Prevented Stress-Induced Apoptosis of P19 Cells and Embryonic Mouse Cortical Neurons 
     Serum withdrawal induced P19 cells apoptosis ( FIG. 15A , left) that could be prevented by addition of ANG protein ( FIG. 15A , middle). In this experiment, apoptotic cells were stained red by ethidium bromide (indicated by arrows), whereas non-apoptotic cells were stained green by acridine orange.  FIG. 15B  shows that the protective activity of ANG was dose-dependent. Caspase-3 and -7 activities were measured using a luminescent assay, and the results shown in  FIG. 15C  indicate that ANG prevented caspase 3/7 activation in a dose-dependent manner. In order to know whether ANG protect stress-induced neuron degeneration, mouse embryonic cortical neurons were isolated and cultured for 10 days, and then subjected to hypothermia treatment at 20° C. for 40 min in the absence or presence of 0.5 μg/ml ANG. Neurofilament staining showed that significant fragmentation of the neuronal processing occurred in the absence of ANG but not in the presence of ANG ( FIG. 15D ). These results indicate that ANG is able to protect stress-induced neuron degenerations. 
     Example 17 
     Detection of ANG at the Neuromuscular Junction (NMJ) 
     Double IF was performed to detect whether ANG was located at the NMJ.  FIG. 16  demonstrates that in mouse tibialis muscle, ANG (green) and neurofilament (red) was colocalized at the NMJ. 
     Example 18 
     Generation of Conditional ANG1 Knockout Mice 
     To elucidate the role of ANG in motor neuron physiology and the effect of ANG deficiency on motor neuron degeneration, we are in the process of creating ANG knockout mice. Although humans have only a single ANG gene, mice have six (Cho and Zhang (2006)  Gene  373:116). It is not possible to knockout all of them simultaneously because they are spread out over ˜8 million base pairs, with many intervening genes. Fortunately, one of the mouse angiogenins, ANG1, is clearly the prominent form and the homologue of the human protein in terms of sequence, functional properties, gene organization, and expression.  FIG. 17A  shows real time RT-PCR analysis of the relative mRNA levels of the 6 mouse ANG isoforms in the spinal cord. In these experiments, the efficiency of the primers to amplify each ANG isoform was determined by performing a dilution curve. A plot of Ct values for each of the dilution against the fold of dilution was obtained and the efficiency of the primers was calculated from the slop of this linear graph. GAPDH mRNA was used as the internal control. The efficiency of the GAPDH primers was determined in the same way. The relative mRNA level of a particular ANG isoform to GAPDH was determined from the primer efficiency and the numbers of cycles needed to reach a preset threshold. The relative mRNA levels of the different ANG isoforms were then calculated. ANG6 mRNA was not detectable in all the samples prepared from 5 different mice. The relative mRNA level for ANG1, 2, 3, 4, and 5 was 98.2±3.9, 0.063±0.044, 1.66±0.22, 0.09±0.022, and 0.99±0.64, respectively ( FIG. 13A ). Therefore, ANG1 is clearly the major isoform in the spinal cord, and, without intending to be bound by scientific theory, knocking out ANG1 will probably suffice for investigating the function of human ANG. 
     In order to distinguish and differentiate the role of ANG in development and in ALS pathology, a conditional knockout strategy to delete the coding exon of the gene was used. ANG1-positive clones were obtained from screening a C57BL/6 BAC library and confirmed by PCR. An approximately 11.7 kb region of the ANG1 gene was then subcloned for construction of the targeting vector using a homologous recombination-based technique. This region contains the single coding exon of ANG-1, ˜2.3 kb of the 5′ flanking region (short homology arm (SHR)) and approximately 8.9 kb of the 3′ flanking region (long homology arm, LHR). The gene segment was inserted into the backbone vector pSP72 (Promega) containing an Amp selection cassette. A pGK-gb2 LoxP/FRT-flanked Neomycin (Neo) cassette was inserted 161 nt upstream from the coding exon, and an additional LoxP site was inserted 80 nt downstream from the coding exon ( FIG. 17B ). In addition, the Neo cassette was flanked by FRT sites to allow selective removal by Flp recombinase ( FIG. 17B ). The FRT sites were inserted for the purpose to generate only ANG1 loxed mice without Neo so that tissue-specific and inducible KO mice can be generated. 
     F1 heterozygotes with ANG1 gene floxed were obtained from the above construct after the standard ES cell electroporation, blastocyst injection and chimera backcrossing. After sequencing confirmation of the LoxP sites, homozygous ANG1 LoxP/LoxP  mice were obtained. They were then crossed with EIIa-Cre mice to generate mosaic mice. The mosaic mice were backcrossed with WT mice to obtain conventional ANG1 KO mice.  FIG. 17C  shows that no ANG1 mRNA was detectable from the spinal cord of the KO mice, confirming the successful knockout of ANG1 gene. However, a week but recognizable ANG protein band was still present in ANG1 KO mice. Without intending to be bound by scientific theory, this band was one or more of the other ANG isoforms that could still be recognized by the R165 anti-mouse ANG antibody. This raised the possibility that the various ANG isoforms may be redundant. However, since the sum of the mRNA of ANG2-6 was less than 3% of that of ANG1 ( FIG. 17A ) and there was no evidence of upregulation of other ANG isoforms in ANG1 KO mice, it is expected that these mice will be suitable for use to evaluate the role of ANG in ALS pathology. It is notable that all the ANG mutations so far identified in ALS patients are heterozygous and that ANG protein levels in human ALS and SOD1 G93A  mouse spinal cord are still detectable. 
     So far, 84 heterozygotes and 75 homozygotes ANG1 KO mice that have both ANG1 gene and the Neo cassette removed were obtained. These mice were various ages ranging from 3 weeks (the youngest homozygotes) to 3 months (the oldest heterozygote). Body weights of the ANG1 null mice are indistinguishable from that of WT littermates thus far. Motor coordination and muscular function were being tested on a rotarod, and no difference has been detected yet. Homozygous KO mice exhibited no fertility problems and are not embryonic lethal. However, 6 ANG1 KO mice have developed hydrocephalus before reaching the age of 8 weeks, suggesting a role of ANG1 in development. In order to distinguish the role of ANG during development from that in adult onset motor neuron degeneration, conditional KO mice are being generated by breeding ANG1 LoxP/−,Neo  mice with Flp mice that carry a transgene for a variant of Flp recombinase under the control of the human β-actin promoter. Mice that carry two floxed ANG1 alleles and no Neo were successfully produced. So far, one male (#1659) and two female (#1661 and 1664) mice have been obtained. This line is currently being amplified and will be used to cross with various Cre mice for conditional knockout as described below. 
     Example 19 
     Generation of Human ANG Transgenic Mice 
     To understand whether ANG over-expression will prevent SOD G93A -induced ALS development or delay the disease onset, and improve the motor muscular function of SOD G93A  mice, we plan to generate ANG: SOD G93A  double transgenic mice. For this purpose, we decided to first make wild-type human ANG transgenic mice. Human ANG cDNA including the segment encoding the signal peptide was ligated into pCAGGS between the chick β-actin promoter and the IRES-controlled GFP gene that is followed by the SV40 early polyadenylation signal ( FIG. 18 ). The sequence of the vector has been confirmed and a linearized fragment with SalI and PstI was transfected into LNCaP human prostate cancer cells to test the ANG and GFP expression levels. Transfected cells were sorted by GFP expression and showed a 25-fold increase in ANG secretion as determined by ELISA. The linearized fragment (2 ng/μl) was than injected into 240 embryos, 210 of them were transferred into 7 recipient mothers and 17 pups have been obtained and have been genotyped ( FIG. 18B ). 
     The primer set used for genotyping is specific for human ANG cDNA and will amplify a fragment of 101 nt. Four founders have been obtained (Mouse numbers 83, 86, 89 and 94). Two of the founders (89 and 94) were backcrossed with WT mice and two transgenic lines were established ( FIG. 18C ). ELISA analysis showed that line 89 and 95 have circulating human angiogenin level of 90 and 35 ng/ml in the plasma. 16 and 8 transgenic mice from line 89 and 95 were obtained, respectively. IHC examination of human angiogenin expression in the spinal cord showed that human angiogenin, the transgene product, was detected in the motor neurons (indicated by arrows) as well as in other type of cells in the spinal cord. 
     Example 20 
     Assess the Therapeutic Activity of Systemic Administration of ANG Protein in SOD G93A  Mice 
     Preliminary studies have shown that i.p. injection of ANG protein reaches the CNS and dramatically improves motor neuron function in SOD G93A  mice ( FIG. 9 ). We will optimize the dosing, duration, frequency and routes of administration to obtain the most effective treatment regimen. We will also determine distribution and stability of systemically administered ANG protein. If ANG knockout and mutant ANG transgenic animals, to be generated described herein, develop ALS-like phenotype, the effect of systemic administration of ANG protein in these mice will also be studied. 
     Generate ANG:SOD G93A  Double Transgenic Mice and Examine the Effect of ANG Overexpression on SOD G93A -Induced ALS Phenotype 
     Founders of transgenic mice expressing wild-type human ANG have been obtained ( FIG. 18 ). ANG:SOD G93A  double transgenic mice will be created by breeding ANG and SOD G93A  transgenic mice. Motor muscular function and development of an ALS-like phenotype will be examined and compared to that of SOD G93A  mice. The effect of ANG overexpression will be compared to that of systemically delivered ANG protein in alleviating ALS symptoms of SOD G93A  mice. 
     Create ANG1 Knockout Mice and Characterize the Effect of ANG Deficiency on Motor Neuron Function 
     Conditional ANG knockout mice are being generated and we have obtained heterozygous mice whose ANG1 gene has been flanked with loxP sites (ANG1 LoxP/loxP  mice). Initially, the ANG gene will be deleted by mating with Ella-Cre mice strain to generate the conventional knockout. If this knockout is embryonic lethal, the mating will be performed with tamoxifen-inducible Cre mice (β-actin-Cre/ESR or ubiquitin-Cre/ESR) to delete the ANG1 gene at various stage during development and in adulthood. Endothelial cell- and motor neuron-specific deletion of the ANG1 gene will be obtained by mating ANG1 loxP/loxP  mice with Tie2-Cre and Mnx1-Cre mice, respectively. 
     Generate Mutant ANG Transgenic Mice and Characterize the Effect of ANG Mutant Proteins on Motor Neuron Function 
     The mutant ANG proteins found in ALS patients are not only inactive in inducing angiogenesis and neurite outgrowth but are also toxic to motor neurons in culture. Therefore, in addition to the haploinsufficiency caused by the loss-of-function mutation in one allele, a toxic gain-of-function of mutant ANG may also be an underlying mechanism of ALS pathogenesis. To test this hypothesis, transgenic mice expressing the mutated human ANG genes (P112L, K17I, and P(-4)S) will be generated and examined. 
     Example 21 
     ANG Expression Level, Distribution and Stability 
     All the necessary reagents and methods have been developed for detection of both ANG proteins and mRNA. A mAb, 26-2F, that is specific for human ANG will be used for IHC and IF detection of wild-type and mutant human ANG, and for double antibody ELISA assay together with an anti-human ANG pAb (R113) for the measurement of ANG content in tissues and in the circulation. R113 will also be used for Western blotting analyses of human ANG because it is known that the mAb 26-2F does not work very well in Westerns. We also have in hand an anti-mouse ANG pAb (R165) that has ˜100-fold higher sensitivity to mouse ANG than human ANG. R165 will then be used to detect mouse ANG in IHC and Western. Quantitative real-time RT-PCR for both human ANG and mouse ANG1 mRNA are also currently in the lab. All these reagents and methods were developed in our lab for our ongoing studies on the role of ANG in angiogenesis and in cancer progression. They will be applied to this application. 
     Functional Evaluations of Mice 
     Performance on a rotarod will be tested on a weekly basis for each animal to evaluate motor disability and the effect of ANG knockout or overexpression of mutant ANG proteins on motor muscular impairment, or the effect of wild-type ANG overexpression or systemic administration in improving motor function. The animals will be placed on a rotating rod that is set at a fixed speed or, when necessary (such as for super-performing mice), increases in both speed and the number of revolution over time. The duration for which the animals are able to stay on the rod is the indication of the severity of motor impairment. Each test will have 3 trials and the longest time will be recorded. For ALS therapy experiments with ANG proteins, one week prior to injection, the mice will receive two sessions of training to practice staying on the rotating rod. 
     Mice will be evaluated weekly for motor muscular functions using 6 indices: left and right forelimb flexion during suspension by the tail, left and right hindlimb flexion when the forelimbs remain on a hard surface and the hindlimbs are lifted up and back by the tail, and the ability to resist lateral pulsion toward the left and right. In addition, mice are tested for their ability to stand on an inclined plane (angle board) while facing left, right, and upward. A composite motor neuron score (McIntosh et al. (1989)  Neuroscience  28:233), on a scale of 0 (complete loss of function) to 4 (normal function), is generated by combining the scores for each test. 
     Assessment of Muscle Function and Number of Motor Units 
     The number of functional motor units in each extensor digitorum longus (EDL) muscle will be determined. Isometric contractions will be elicited by stimulating the EDL motor nerve using square-wave pulses of 0.02 ms duration and supramaximal intensity via silver-wire electrodes. Contractions will be elicited by trains of stimuli at frequencies of 20, 40 and 80 Hz. The half-relaxation time values will be measured. The number of motor units in the EDL muscles will be assessed by stimulating the motor nerve with stimuli of increasing intensity, resulting in stepwise increments in twitch tension because of successive recruitment of motor axons (Kieran et al. (2004)  Nat. Med.  10:402). 
     Fatigue Test 
     EDL is a fast muscle that fatigues quickly when repeatedly stimulated, producing a characteristic fatigue pattern from which a “fatigue index” can be calculated (Dick et al. (1995)  Neuromuscul. Disord.  5:371). For this purpose, EDL muscles will be stimulated at 40 Hz for 250 ms every second, and contractions will be recorded. The decrease in tension after 3 minutes of stimulation is measured, and the fatigue index is calculated as (initial tetanic tension−tetanic tension after 3 minutes of stimulation)÷initial tetanic tension. 
     Animal Survival 
     Animals will be monitored daily. Survival time is measured by recording the date that each animal reaches the end stage. When mice are unable to right themselves in 30 seconds after being placed on their sides, they are defined as end stage and scored “moribund”. 
     Motor Neuron Survival 
     After transcardiac perfusion with 4% paraformaldehyde, the lumbar region of the spinal cord will be removed and 20 μm transverse sections will be cut and Nissl staining will be performed with gallocyanin. Gallocyanin-stained motoneurons located within the sciatic motor pool, in which a nucleolus is clearly visible (as shown in  FIG. 8 ), will be counted in each ventral horn on every third section between the L2 and L5 levels of the spinal cord. This method avoids the possibility of counting the same cell twice. The soma size of the surviving motor neurons will be measured with a micro cell caliper under the microscope. 
     Immunohistochemistry 
     Spinal cords are removed, fixed, and sectioned at 7 μm for IHC examinations. Antibodies against Nestin, NeuN, GFAP, myelin basic protein, NF, and MAP2 will be utilized to visualize neural stem cells, neurons, astrocytes, oligodendrocytes, axonal filaments, and dendrites, respectively. Anti-vWF antibody will be used to assess vascularization. Anti-ANG mAb 26-2F will be used to determine distribution and localization of wild-type and mutant human ANG. Motor neurons will be visualized with choline acetyltransferase staining. For this purpose, the spinal cord will be sectioned in 40 μm thickness. 
     Statistics 
     Cumulative survival statistics of paralysis, rotarod and survival will be calculated by using Kaplan-Meier statistics. Log-rank P values will be calculated to compare the survival curves. Rotarod performance, grip strength and body weight data will be analyzed by using repeated measures ANOVA. To analyze rotarod performance and grip strength, a zero value will be assigned to deceased mice. Cox regression analysis and ANOVA-paired t-test will be used to analyze the therapeutic effect of ANG proteins. All data will be reported as the mean±SE. 
     Example 22 
     Specific Designs 
     Assess the Therapeutic Activity of Systemic Administration of ANG Protein in SOD G93A  Mice 
     Rationale 
     The hypothesis that ANG has therapeutic value in SOD G93A  mice has been proven by the data presented in Examples 1-16. It has been shown that i.p. administration of wild-type human ANG protein but not the P112L mutant form improves the motor muscular function and prolongs survival of SOD G93A  mice ( FIG. 9 ), accompanied by an increase of spinal cord motor neuron survival and function ( FIG. 12 ). It has also been shown that i.v., i.p., and s.c. routes all significantly improve motor muscular function of the SOD G93A  mice. Since ANG treatment was started after the disease onset (at 10 weeks of age), the results are particularly relevant to human disease. The objective of this experiment is therefore to obtain the optimal regimen of ANG protein administration in the SOD G93A  mouse model for the ultimate purpose to develop ANG for ALS treatment in human patients. 
     Experimental Design 
     It has been determined that intravenous (i.v.) route provided the fastest enhancement of motor muscular function. This experiment will be repeated with an additional control group to test P112L mutant ANG protein that has been shown to be ineffective when injected i.p. ( FIG. 9 ). In this repeat, s.c. will not be included because it is clear that s.c. is less effective than i.p. ( FIG. 10 ). Therefore, there will be 4 groups of mice that will be treated by wild-type ANG i.p., wild-type ANG i.v., P112L ANG i.v., and PBS, respectively. Another purpose of this experiment is to check whether P112L mutant ANG protein, when injected i.v., will exacerbate the disease in SODG93A mice. This is related to the experiments described herein where it is planned to generate transgenic mice expressing the mutant form of ANG. If P112L mutant ANG protein exacerbates the disease symptoms and progression, its effect on wild-type mice will be examined. 
     Mouse Versus Human ANG 
     It was determined from the results of i.p.-injected ANG ( FIGS. 9-11 ) that the beneficial effect waned after its initial peak at week 15. It could be due to down-regulation of ANG receptor resulting in desensitization of the mice to ANG treatment. It could also be due to an immune reaction of the mice to human ANG. The reasons that human ANG was used in the initial therapy experiment were two-fold. First, ANG distribution and stability could be detected by human ANG-specific mAb 26-2F. Secondly, wild-type could be compared with the mutant form of ANG. These two objectives have now been met. It has been determined that i.p.-injected wild-type and mutant ANG reaches the CNS ( FIG. 8 ) but only wild-type ANG has therapeutic activity ( FIG. 9 ). The effect of mouse ANG will be examined in a similar fashion. Both i.p. and i.v. routes will be used and human ANG at the same dosing will be used as control. Recombinant mouse ANG will be prepared with the same pET11 system (Holloway et al. (2001)  Protein Expr. Purif.  22:307) that is routine in our lab. If mouse ANG shows better efficacy, it will be used in the subsequent experiments to determine the optimal dosing regimen. 
     Dosing and Frequency of Treatment 
     It has been determined that 1 μg/mouse is superior to 0.1 and to 1 μg ( FIG. 11 ). The optimal dose will then be in the range between 0.1 and 10 μg. Accordingly. the efficacies of weekly injections of 0.1, 0.2, 0.5, 1, 2, 5, and 10 μg ANG per mouse will be compared. A 5-fold increase in the time the mice can stay on a rotarod and a 2 week increase in survival (equivalent to 8 months in human) will be considered effective. Since 0.1 μg per mouse was still effective, the dosage will be lowered further until the minimum effective dose is determined. 
     Next, the optimal interval of drug administration will be determined. For this purpose, ANG will be injected at a given dose (the minimum effective dose determined above by weekly injection) daily, every other day and every 3 days and compare with the results of weekly injection. This experiment will be repeated at the maximum effective dose (when it reaches the plateau) to see whether a further increase in efficacy is possible by giving the ANG protein more frequently. 
     Distribution and stability of systemically administered ANG protein: It was determined in the preliminary studies that i.p.-injected ANG cross the BBB and BSCB, and reaches at the CNS ( FIG. 8 ). The mechanism ANG employs to cross the BBB and BSCB is unknown. Although it will be an important research project, especially in connection with an ANG receptor study, it is beyond the scope of this application and the program relevance. However, it is important to know the distribution and stability of systemically injected ANG in the spinal cord, in the brain stem and in the motor cortex of the brain for our effort to develop ANG protein as an ALS therapeutic. For this purpose, IHC with the human ANG-specific mAb 26-2F and double IF with vWF (for blood vessels) and choline acetyltransferase (for motor neurons) will be used to examine the localization of ANG. Without intending to be bound by scientific theory, ANG is expected to be observed in both motor neurons and in endothelial cells. The concentration of ANG in these motion related nervous systems will be determined by ELSA analysis from snap-frozen tissues. Western blotting with pAb R113 that has a 100-fold higher sensitivity for human ANG than for mouse ANG will be used to determine the stability of ANG in these tissues. 
     Without intending to be bound by scientific theory, it is expected that the therapeutic effect of systemically injected ANG protein in SOD G93A  mice will be confirmed. Without intending to be bound by scientific theory, it is expected that the optimal regimen including the dose, frequency of injection, and the route of administration will be determined. By functional evaluation of the mice, combined with morphological determination of motor neuron number and size, and pathological examination of the motor neuron processing, as well as the localization of injected ANG, mechanistic insight into how ANG prevents motor neuron degeneration may be obtained. One potential problem in this set of experiments is that the efficiency of ANG to cross BBB and BSCB may be low so a relatively high ANG amount may be needed to reach a therapeutically effective concentration in the spinal cord. The best dose determined so far was 1 μg per mouse, which will be translated to 3 mg per 75 kg human patient. To reduce possible side-effects of injected angiogenin protein, the enzymatically and angiogenically more active ANG variant D116H, that is two orders of magnitude more potent in inducing angiogenesis (Harper et al. (1990)  Biochemistry  29:7297) will be tested. 
     Assess the Therapeutic Activity of ANG Variants. 
     3 ANG variants with an altered nuclear localization sequence (NLS) of  30 KRRRG 34  (SEQ ID NO:10),  30 MRRRK 34  (SEQ ID NO:11),  30 KRRRK 34  (SEQ ID NO:12), respectively, will be generated and tested in the in vitro enzymatic assay and motor neuron culture assay described herein. The variant that shows the highest in vitro activity will be further tested in ALS mice. In case that the NLS variants do not have an enhanced activity, the therapeutic activity of ANG variant D116H, that is known to be 2 orders of magnitude more potent in inducing angiogenesis, will be assessed. 
     Determination of Maximum Tolerated Dose (MTD) 
     MTD will be determined in a 28-day, repeat-dose tolerance study in WT mice and in accordance with Society of Toxicologic Pathology—proposed guidelines for repeat-dosing toxicity studies. Twelve-week old mice will be randomly assigned to experimental groups representing different doses of WT or G34K ANG, administered at 0, 10, 20, 40, 80, 160, and 320 μg per mouse (6 mice per group). This dose range encompasses the demonstrated effective dose of ANG (10 μg) and the proposed maximum effective dose (200 μg). Body weights, to be measured twice per week, and direct observations of general health and behavior, to be recorded daily, will provide the primary indicators of tolerance to the drug. MTD will be the maximum dose tested that does not cause limiting toxicity as defined by a loss of ≧10% of starting body weight, inactivity/lethargy (≧2 days), inability or unwillingness to eat and/or drink (≧2 days), hunched posture, or other signs indicating moribundity. In addition, complete necropsies will be performed and tissues with grossly visible lesions will be fixed in formalin, paraffin-embedded and stained with H&amp;E for microscopic evaluation 
     Toxicological Screening of ANG 
     Besides the gross health indices described above, the potential toxicologic effects of ANG will be assessed by hematologic and histopathologic evaluations. The weights of epididymal fat pads will be measured as an assessment of body fat and as an additional indicator of general health. Blood samples will be taken and blood cell counts and serum chemistry will be determined. Because ANG is an angiogenic molecule, special attention will be paid to the toxicity related to excessive angiogenesis. Histology and organ weight data will be used to determine whether extramedullary hematopoiesis in the spleen or thymic hypertrophy occur after treatment. Weights of the testes and epididymides will be measured to determine whether any abnormal growth occurs. Whether or not the treated animals are able to achieve litters during the treatment and whether there difference in the sizes of these litters will be ascertained. The angiogenesis status will be examined by IHC with an anti-CD31 antibody and the neovessel density in the liver, kidney, skin, brain and spinal cord will be determined. If the vessel density increases by 20% after ANG treatment, the effect of ANG administration on the animals in responding to tumor inducing agents will be examined. A two stage protocol consisting of a single application of 9,10-dimethyl-1,2-benzanthracene (DMBA) followed by repeated irradiation with UVB (280-320 nm) will be used. DMBA will be applied in a single injection of 50 μl of a 0.5% solution in acetone to the dorsal surface on postnatal day 5. UVB treatment will consist of 3 exposures per week beginning with a dose of 100 mj/cm 2 . Mice will be monitored daily and tumor volume will be measured 3 times a week with a microcaliper. 
     Pharmacokinetics, Biodistribution, and Stability of i.p. Administered ANG Protein 
     The pharmacokinetics (PK) of ANG (WT or G34K) in SOD1 G93A  mice will be measured following an i.p. injection at the optimal dosing regimen, followed by the collection of blood, liver, kidney, brain, and spinal cord tissues at different time (1, 2, 4, 8, 16, 24, 36, 48, 72, and 96 h, or until the second injection starts). Three mice will be used in each data point. The concentration of ANG in tissue samples and in blood stream will be determined by ELISA. IHC with the human ANG-specific mAb 26-2F and double IF with vWF and choline acetyltransferase will be used to examine the localization of ANG. It is expected that ANG will be observed in both motor neurons and endothelial cells. Western blotting with pAb R113 will be used to determine ANG stability. 
     Effect of Retrograde AAV Delivery of ANG Gene on SOD1 G93A  Mice 
     The strong early protection of ANG protein to the SOD1 G93A  mice ( FIG. 9 ) indicates a possible function of ANG in NMJ function. This is supported by the preliminary finding that ANG is detectable at the NMJ ( FIG. 16 ). Therefore, the effect of AAV-mediated retrograde ANG gene delivery on SOD1 G93A  mice will be assessed. AAV vector containing human ANG cDNA was prepared and shown to express human ANG in NSC34 cells. They will be injected bilaterally into the hindlimb quadriceps and intercostal muscle of the animals, with an initial dosage of 1×10 10  particles per injection. AAV-GFP will be used as control vector. Transductiuon of human ANG in the lumbar and thoracic spinal cord motor neurons will be examined by IHC with mAb 26-2F. Motor neurons and glial cells in these sections will be labeled with CHAT and GFAP staining, respectively, so that motor neuron-specific expression of human ANG will be confirmed. Levels of ANG in the blood, muscle biopsy, and lumbar spinal cord will be determined by ELISA. Functional and pathological examination of treated animals will be carried out as described in the above sections, and the results will be compared with that of i.p.-delivered ANG protein. It is expected that better efficacy will be achieved with the AAV-delivery method. It has been reported that up to 1.1% of the AAV-IGF-1 virus injected was transported to the lumbar region of the spinal cord (Kasper et al. (2003)  Science  301:839). IGF-1 levels in the lumbar spinal cord and in the quadriceps muscle can reach to 175 and 111 ng/ml, respectively. If ANG transduction is similar to that IGF-1, AAV-mediated delivery will reach much higher protein level in the spinal cord and muscle. 
     To understand whether retrograde transport of AAV-ANG to the spinal cord is necessary or whether muscle transduction of ANG plays a more important role to achieve therapeutic effects, vesicular stomatitis virus glycoprotein-pseudotyped lentiviral (LV) vector will be used to deliver ANG cDNA and compare it with AAV vector. This vector does not retrogradely transport to the spinal cord and maintains long-term expression in the muscle only (Kafri et al. (1997)  Nat. Genet.  17:314). This lentiviral vector has been used to direct muscle specific expression of IGF-1 (Kasper et al. (2003)  Science  301:839). It has also been used to encode ANG siRNA in prostate cancer (Ibaragi et al. (2009) Mol. Cancer. Res. 7:415). A comparison between AAV- and LV-mediated ANG deliveries will likely determine whether ANG plays a direct role in NMJ. 
     Generation of ANG:SODG93A Double Transgenic Mice and Examination of the Effect of ANG Over-Expression on the SODG93A-Induced ALS Phenotype 
     Rationale 
     The hypothesis to be tested in this experiment is that over-expression of wild-type ANG may prevent or delay SOD G93A -induced motor neuron degeneration. This hypothesis is formulated from the finding that ANG expression is dramatically decreased in spinal cord of human ALS patients and in that of SOD G93A  mice as compared to their normal counterparts ( FIG. 3 ). It is also supported by our finding that systemic administration of ANG protein improves motor muscular function and prolongs survival of SOD G93A  mice ( FIGS. 5-8 ). 
     Experimental Design 
     Characterization of wild-type ANG transgenic mouse line: Transgenic mice over-expressing wild-type ANG have just been generated ( FIG. 18 ). These founder mice will be backcrossed to the wild-type to establish transgenic lines. The expression level of human ANG transgene in the spinal cord will be determined by ELISA and its localization will be visualized by IHC. The effect of ANG over-expression on motor neuron physiology during development will be assessed by Nissl staining Motor neurons, microglia and astrocytes can be isolated in parallel from the same embryonic spinal cord samples and cultured in vitro (Gingras et al. (2007)  J. Neurosci. Methods  163:111). The effect of ANG over-expression on hypoxia-induced degeneration and N-methyl-D-asparate (NMDA)-induced excitotoxicity will be examined with the control cells from wild-type littermates. 
     Generation and Characterization of Double Transgenic Mice 
     Male SOD G93A  mice will be crossed with ANG female mice to obtain double transgenic mice that express both G93A SOD and wild-type ANG. Genotyping of the double transgenic mice should be straightforward. Both male and female double transgenic mice will be compared with the corresponding single transgenic and wild-type controls. Functional evaluation, morphological and pathological examinations will be carried out as described above. The efficacy of ANG over-expression in protecting SOD G93A  mice from developing ALS-like symptom will be compared to that obtained from i.v.- or i.p.-injection of exogenous ANG protein as described herein. 
     Cell Culture Study 
     Motor neurons, microglia and astrocytes will be isolated (Gingras et al. (2007)  J. Neurosci. Methods  163:111) from the double transgenic (ANG:SOD G93A ), single transgenic (ANG or SOD G93A ) and wild-type mice. The growth and differentiation pattern of these cells from the 4 different types of mice will be compared and the effect of ANG transgene over-expression can be determined. Its activity will be compared to exogenously added ANG to see whether ANG acts in a cell autonomous manner. If the double transgenic mice show improved motor muscular function and prolonged survival, but motor neurons isolated from these mice lack this beneficial effect upon ANG treatment, motor neurons will be co-cultured with astrocytes or microglia or both to check whether ANG produced in one type of cells acts on the other (the paracrine mechanism). If the beneficial effect of ANG over-expression seen in mice can not be mimicked in cell culture, it may suggest that ANG mainly acts on promoting angiogenesis that indirectly helps the function of motor neurons. In this case, spinal cord endothelial cells will be isolated (Ge and Pachter (2006)  J. Neuroimmunol.  177:209) and examined for their proliferation rate, hypoxia and peroxide-induced apoptosis, and responsiveness to other angiogenic stimulators including bFGF and VEGF. In combination with the experiments described herein, it is planned to knockout ANG1 gene in motor neurons and in endothelial cells separately, to get a better understanding of how ANG plays a role in motor neuron physiology. 
     ANG is known to stimulate Erk (Liu et al. (2001)  Biochem. Biophys. Res. Commun.  287:305) and AKT (Kim et al. (2007)  Biochem. Biophys. Res. Commun.  352:509) phosphorylation in endothelial cells. Therefore, the effect of ANG over-expression on Erk and AKT phosphorylation will be examined. It has been determined previously that the primary function of ANG in its target cells including proliferating endothelial cells and various cancer cells is to stimulate rRNA transcription (Xu et al. (2002)  Biochem. Biophys. Res. Commun.  294:287; Tsuji et al. (2005)  Cancer Res.  65:1352; Yoshioka et al. (2006)  Proc. Natl. Acad. Sci. USA  103:14519; Kishimoto et al. (2005)  Oncogene  24:445) through binding to the promoter region of rDNA (Xu et al. (2003)  Biochemistry  42:121). Thus, the status of nuclear translocation of ANG in these cells will be ascertained by IF. The steady-state level of rRNA in these cells will also be determined by Northern blotting and de novo synthesis of rRNA by metabolic labeling (Kishimoto et al. (2005)  Oncogene  24:445). 
     Without intending to be bound by scientific theory, it is expected that wild-type ANG over-expression will improve the motor muscular function of SOD G93A  mice and prolong their survival at least as effectively as the systemic administration of ANG protein. It is likely that wild-type ANG over-expression is much more effective. It is also possible that it may have a preventive function or may delay the disease onset due to constitutive expression of the ANG transgene. If ANG over-expression is much more effective than ANG protein administration, in addition to systemic administration of ANG protein, a gene therapy method using AAV-mediated retrograde delivery of ANG gene through intramuscular injection at hindlimb quadriceps. This method has been successfully used to deliver IGF gene into the spinal cord of SOD mice (Kaspar et al. (2003)  Science  301:839). AAV vectors containing ANG cDNA under the control of CMV promoter have already been prepared and are in use for our other project on the mechanism of ANG-induced angiogenesis, so it can be readily applied to this project if necessary. 
     In our effort to generate human ANG transgenic mice, the construct (2 ng/μl) was injected into 240 embryos and transferred 210 embryos into 7 recipient mothers but only 17 pups were born and only 4 of them carry the transgene. While having 4 founder mice to work with is good, without intending to be bound by scientific theory, it is kept in mind that this is a somewhat low rate for transgenic mice production and may indicate that universal over-expression of ANG could be problematic. If it is unfortunately true, a different promoter such as the Mnx1 and Tie2 promoter will be chosen to obtain relatively motor neuron and endothelial cell specific over-expression of ANG. In any event, this initial experiment will guide subsequent steps. 
     Create ANG Knockout Mice and Characterize the Effect of ANG Deficiency on Motor neuron function 
     Rationale 
     The hypothesis to be tested in this experiment is that ANG deficiency will impair motor neuron development and cause motor neuron degeneration. This hypothesis is supported by the findings that ANG mutations identified in ALS patients are loss-of-function mutations (Wu et al. (2007)  Ann. Neurol.  62:609; Crabtree et al. (2007)  Biochemistry  46:11810). It is also supported by the report that human ANG is strongly expressed in the motor neurons of both fetal and adult spinal cords (Wu et al. (2007)  Ann. Neurol.  62:609) and that mouse ANG is strongly and selectively expressed in the CNS during development (Subramanian and Feng (2007)  Hum. Mol. Genet.  16:1445). Further supporting evidence is from the data presented in Preliminary Studies that ANG expression is dramatically decreased in ALS spinal cord ( FIG. 3 ). 
     Design 
     ANG1 gene floxed (ANG1 loxP/+ ) mice have been generated ( FIG. 17 ) and are currently being backcrossed to obtain more heterozygous (ANG1 loxP/+ ) and homozygous (ANG1 loxP/loxP ) mice that will be bred with various Cre mice to generate either constitutional or tissue specific ANG knockout either constitutively or to be induced at various stages during the development and in adulthood. 
     Breeding Strategy 
     ANG1 gene floxed mice have been created and have been crossed with EIIa-Cre mice to generate conventional ANG1 KO mice ( FIG. 17 ). While these conventional KO mice await further phenotype development and characterization, preliminary studies suggest that inducible and conditional knockout is advisable as the role in adult onset diseases could be distinguished and differentiated from that during development. ANG1 LoxP/−,Neo  mice have been crossed with Flp mice and founders that carry two floxed Ang1 alleles and no Neo cassette have been generated. These Neo-deleted, ANG1 floxed homozygotes will be used for tissue-specific knockout and for deleting ANG1 at certain time during development or in the adulthood. 
     Two types of inducible Cre mice can be used to make inducible ANG1 knockout mice. The first is the tamoxifen-inducible Cre and the second is interferon-inducible Cre. Tamoxifen-inducible Cre is favored because interferon may complicate the disease course as it has been shown to modulate ALS (Mora et al. (1986)  Neurology  36:1137; Holmoy et al. (2006)  Amyotroph. Lateral Scler.  7:183; Linda et al. (1998)  Exp. Neurol.  150:282). B6.Cg-Tg(Cre/esr1)5Amc/J (β-Actin-Cre/ESR, Jackson Lab Stock Number: 004682) mice that have a tamoxifen-inducible Cre-mediated recombination system driven by the chicken β-actin promoter coupled with CMV immediate-early enhancer (Hayashi and McMahon (2002)  Dev. Biol.  244:305) were selected. ANG1 deletion after tamoxifen induction in various tissues will be checked by PCR and the protein level will be examined by IHC and Western blotting analyses. Tamoxifen-inducible Cre mice under the control of ubiquitin promoter are also available (Ubiquitin-Cre/ESR, Jackson Lab Stock Number: 007001). If, for some reason, 13-Actin-Cre/ESR does not result in an efficient recombination in the spinal cord, ubiquitin-Cre/ESR mice will be used as an alternative. 
     Tie2-Cre (Jackson Lab Stock Number: 004128) (Koni et al. (2001)  J. Exp. Med.  193:741) and Mnx1-Cre (Jackson Lab Stock Number: 006600) (Arber et al. (1999)  Neuron  23:659) mice direct Cre recombinase expression in endothelial cells and motor neurons, respectively, and will be crossed with ANG1 LoxP/loxP  mice to obtain endothelial cell- and motor neuron-specific deletion of ANG1 gene. Characterization of these cell-specific ANG knockout mice will help understanding whether ANG deficiency in endothelial cells or in motor neurons, or in both, is the likely cause of motor neuron degeneration. 
     If ANG1 deletion in embryonic endothelial cells and motor neurons are lethal, inducible cell specific Cre mice will be used to knockout ANG1 in endothelial and neurons in adult mice (motor neuron-specific inducible Cre mice are not available, so only mice that have inducible Cre in all neural cells will be used). For this purpose, tamoxifen-inducible endothelial cells-specific Cre mice, L43T1e2ERT2Cre (Forde et al. (2002)  Genesis  33:191), available from TaconicArtemis and tamoxifen-inducible neuron-specific Cre mice, Thy1-Cre/ESR1-EYGP (Arenkiel et al. (2007)  Neuron  54:205), available for Jackson Laboratory (Stock Number: 007606), will be used. 
     Characterization of ANG Knockout Mice and Cells 
     The general methods described above will be used to evaluate the motor muscular function and to characterize the morphological and pathological changes of the various ANG1 knockout mice. Cell biology studies will be carried out to confirm and to supplement the in vivo findings and to gain insight into the mechanism of ANG deficiency-induced motor neuron degeneration. The spinal cord endothelial cells will be isolated according to the published method (Ge and Pachter (2006)  J. Neuroimmunol.  177:209). The main function of ANG in cultured endothelial cells has been shown to be related to rRNA transcription (Xu et al. (2003)  Biochemistry  42:121; Kishimoto et al. (2005)  Oncogene  24:445), accordingly, the status of rRNA in these ANG1 knockout endothelial cells will be determined by means of Northern blotting and metabolic labeling analyses as described (Kishimoto et al. (2005)  Oncogene  24:445). Spinal cord motor neurons will be isolated (Gingras et al. (2007)  J. Neurosci. Methods  163:111). The effect of ANG1 knockout on a panel of cellular activities including the oxidative status, excitotoxicity, abnormal protein precipitation and aggregation, mitochondrial abnormalities, cytoskeletal defects, axonal transport, and neuroinflammation; all of these have been proposed to play a role in ALS (reviewed Goodall and Morrison (2006)  Expert Rev. Mol. Med.  8:1) and can be studied by established methods. Id. In the ongoing work on the role of ANG in prostate cancer, it was determined that that knocking-down ANG1 in prostate cancer cells inhibits rRNA transcription thereby inducing cell apoptosis. The level of rRNA transcription in these cells will be determined and the effect of ANG1 knockout on cell apoptosis will be determined by TUNEL staining and the level and activity of caspase 1 and caspase 3. Caspase 1 and 3 have been shown to be the prominent caspases involved in apoptosis in ALS (Friedlander (2003)  N. Engl. J. Med.  348:1365). Another rationale to initially focus on cell apoptosis is the common pathway of ALS pathogenesis regardless of the causative factors. Id. 
     ANG1 floxed mice have been obtained and all the Cre mice proposed are available either from Jackson Lab or from Taconic. Technical difficulties are not foreseen in generating these various knockout mice. Without intending to be bound by scientific theory, it is expected that ANG1 knockout will impair motor neuron functions and the mice will show motor muscular defects so that the role of ANG in motor neuron physiology can be illuminated and whether ANG deficiency is an underlying pathogenesis of ALS can be determined. The outcomes from functional evaluation, morphological and pathological examinations, as well as cell culture studies will allow us to determine the likely mechanism by which ANG insufficiency causes ALS. 
     One potential problem in these experiments is the possibility that the ANG1 knockout may have no consequences because of the compensations by the other 5 ANG isoforms. This probability is relatively small because ANG1 is the prominent isoform and because it has been shown in prostate cells that knocking-down ANG1 does not induce over-expression of other mouse ANG isoforms. However, it is a real possibility as the mouse does have 6 isoforms and the function of these isoforms are not well characterized (Cho and Zhang (2006)  Gene  373:116). If the ANG1 knockout mice are completely normal, the expression level of the other ANG isoforms will be determined by qRT-PCR, and the total protein levels by the polyclonal antibody R165 that does not distinguish mouse ANG isoforms. If the other isoforms are upregulated and the total ANG protein levels are relatively stable before and after ANG1 knockout, it suggests that mouse ANG is redundant. In this case, on the one hand, it implies the importance of ANG and the prediction that human ANG gene mutation would be pathogenic because humans have only one ANG gene. On the other hand, if the role of ANG on motor neuron physiology and pathology can not be studied in ANG1 knockout mice, the transgenic mice that over-express the dominant negative forms of mutant ANG described herein will need to be relied upon. 
     Generate Mutant ANG Transgenic Mice and Characterize the Effect of ANG Mutant proteins on motor neuron function 
     Rationale 
     The hypothesis to be tested in this experiment is that overexpression of the mutant forms of ANG might cause motor neuron degeneration. This hypothesis is supported by the findings that all the 14 ANG mutations so far found in ALS patients ( FIG. 14 ) are heterozygous. This could suggest an essential role of ANG in development and that homozygous mutations will be lethal so people with homozygous ANG mutation will not be born or live. If this is true, the reason for heterozygous ANG mutation to cause motor neuron degeneration could be due to haploinsufficiency and this will be studied by ANG1 knockout mice as described herein. On the other hand, this phenomenon could also be explained if the mutant ANG proteins have gain-of-function toxicity to motor neurons while they have lost functions in inducing angiogenesis. In this case, mice over-expressing the mutant ANG proteins may develop ALS-like symptoms. This hypothesis is also supported by the observation that the mutant ANG proteins are not only inactive in protecting cultured motor neurons from hypoxia-induced death but also are toxic (Subramanian et al. (2008)  Hum. Mol. Genet.  17:130). 
     Design 
     It is planned to generate 3 transgenic lines that over-express the mutant ANG P112L, K17I and P(-4)S respectively. These three mutants were among the four mutations found in the Northern American ALS patients (Wu et al. (2007)  Ann. Neurol.  62:609). The fourth mutation is S28N. We have shown that the three mutations occurred in the mature peptide (K17I, S28N, and P112L) all result in a complete loss of angiogenic activity as a result of reduced ribonucleolytic activity, impaired nuclear translocation, or both (Wu et al. (2007)  Ann. Neurol.  62:609). P(-4) mutation is in the signal peptide so it may affect protein secretion. We have made an ANG-GFP fusion construct, in which GFP is fused to the C-terminal of ANG, and transfected it into 293 cells that were co-cultured with HeLa cells. Preliminary studies indicate that the wild-type ANG-GFP is secreted by 293 cells and is taken up by HeLa cells whereas the P(-4) ANG-GFP is not secreted by 293 cells. In another preliminary experiment with NSC34 cells, we found that K17I and P112L mutant ANG inhibit wild-type ANG-induced neuronal processing. In a recent publication, ANG mutant proteins Q12L, C39W, and K40I, found in the Irish and Scottish ALS populations, have been shown cause degeneration of P19-derived motor neurons. Therefore, generation and characterization of transgenic mice expressing mutant ANG proteins is warranted. 
     Constructs encoding the mutant ANG forms have already been prepared in pGEX-4T-2 vector where ANG gene is flanked by EcoRI and BamHI sites. Id. It will be excised by EcoRI and BamHI digestion and the fragment will be ligated into the pCAGGS vector ( FIG. 11 ) to replace the DNA encoding the wild-type ANG. The same protocol will be used to generate these transgenic lines as has been done with the wild-type ANG transgenic mice ( FIG. 12 ). Functional, morphological and pathological evaluations will be carried out as described above (section 4.1.1-4.1.8). If these transgenic mice develop ALS-like symptom, motor neurons, microglia and astrocytes will be isolated (Gingras et al. (2007)  J. Neurosci. Methods  163:111) and characterized in detail for their sensitivity to hypoxia-induced degeneration and to NMDA-induced excitoxicity. Apoptosis will be determined by TUNEL staining and by measuring the levels and activities of caspase 1 and caspase 3. Changes in NF bundling and axonal transportation as a result of over-expression of the mutant ANG form will also be examined. In all these experiments, corresponding cells isolated from wild-type mice and from transgenic mice over-expressing wild-type ANG will be used as controls. 
     It is hard to predict whether over-expressing the mutant form of ANG will cause ALS in mice. However, it is expected that the motor neurons and other types of cells isolated from these mice will have some significant difference from that isolated from wild-type mice in their growth properties. Especially, it is expected that motor neurons over-expressing mutant ANG will have greater sensitivity to hypoxia and to inducers of excitotoxity. Combined with the results from ANG1 knockout experiments described herein, it is expected that an in-depth understanding of the role of ANG in motor neuron physiology and the effect of its deficiency or mutations in motor neuron degeneration will be obtained. 
     If the mutant ANG transgenic mice develop ALS-like symptoms, they will be valuable assets for the ALS research community. These mice will be made available to ALS researchers through the “Shared Model Organisms Plan” of Harvard Medical School. They can be used together with the SOD G93A  transgenic mice for screening of lead compounds for ALS therapy. The first experiment to be performed with the mice would be to inject wild-type ANG and to see whether this treatment will alleviate the ALS symptom as it does in SOD G93A  mice. 
     Develop Biocompatible CNS Delivery Vehicle for ANG 
     It has been shown that i.p. injection of ANG protein reaches the CNS and dramatically improves motor neuron function in SOD G93A  mice. The experiments proposed in Example 16 will optimize the dosing, duration, frequency and routes of administration to obtain the most effective treatment regimen. The stability, distribution and effective concentration of ANG in the spinal cord will be determined. These data will be used to guide us to develop biopolymer-encapsulated ANG gels that will be delivered into CNS directly to improve the therapeutic activity of ANG. The ANG biogels will be customized to control the ANG release rate so that an effective concentration of ANG in the CNS can be sustained locally to avoid repeated injection and distant side-effects. 
     Although the effect of i.p.-injected ANG in improving the neuromuscular function and survival of SOD G93A  mice is the best among all the compounds and factors that have been tested so far ( FIGS. 9C &amp; 9D ), the therapeutic activity of ANG wanes after 7 weeks and animals die after 5 weeks prolonged life. Bolus doses and repeated injections of ANG may also result in unwanted side-effects as ANG is a known angiogenic factor. To achieve a long-term beneficial and survival effect and to minimize side-effects to the other tissues and organs, the therapeutic activity of ANG will be assessed when is delivered into the spinal cord directly by slow-releasing biopolymer gels that are known are well tolerated when injected into the CNS. ANG biogels will be prepared with different release rates such that the effective ANG concentration in the spinal cord as described herein will be achieved. The therapeutic activity of these ANG biogels will be determined and whether sustained local delivery of ANG is more effective, longer acting, and leads to fewer distant side-effects will be tested. Enzymatically and angiogenically more active ANG variant(s) for delivery, including M30K, G34K, M30KG34K, and D116H will be generated.