LEADING TO PROGNOSIS AND THERAPEUTIC TREATMENT FOR ALS DISEASE

FiEtD

The field includes modulation of AIS biomarkers to homeostasis by (SM604, the correlation with prognosis of ALS disease progression, and the correlation with therapeutic effect on ALS disease progression.

RELATED APPLICATIONS

This Application takes priority from U.S.S.N. 62/185,278, filed June 25, 2015, entitled

'Methods of using GM604 in modu ating ALS disease biomarkers leading to prognosis and therapeutic treatment for ALS disease', by Pui-Yuk Dorothy Ko, incorporated by reference in its' entirety.

BACKGROUND

The following includes information that may be useful in understanding the present inventions, it is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced Is prior art.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. It affects the neurons in the brain and the spina! cord. Motor neurons reach from the brain to the spinal cord and from the spinal cord to the muscles throughout the body. When the motor neuron dies, the ability of the brain to initiate and control muscle movement is lost. The name Amyotrophic lateral sclerosis means when the muscle ("Myo") does not ("A") receive nourishment {'Trophic"), the muscles "atrophies" or wastes away. This leads to the local area {"lateral"} of muscles where portion of the nerve ceils signal and control the muscles degenerates and leads to scarring or hardening ("sclerosis"} in the local area. As ALS disease progresses, people lose the ability to speak, eat, move and breathe. The motor neurons that provide voluntary movements and muscle control dies, in the ALS patient's own words, they are watching themselves dying off a little each day, trapped in a body that cannot move.

Amyotrophic Lateral Sclerosis (ALS, also known since the late 1930s as tou Gehrig's Disease, and also known as motor neuron disease In other countries) is a uniformly-fatal neurodegenerative disease in which the neurons controlling the voluntary skeletal muscles wither and die. This renders the patient progressively paralyzed until respiratory faiiure finally ends life. The average survival from diagnosis is between 2-5 years. The disease was first described in medical literature by Jean-Martin Charcot in 1859 and 145 years later there is still no truly-effective treatment for this savage disease (X!ong ZQ et at 2002, Fas(t) balls and Lou Gehrig disease. A clue to selective vulnerability of motor neurons? Neuron, 12: 35 (6); 1011- 1013),

ALS was for approximately 100 years thought to be a cell-autonomous disease in which some intrinsic failing of the motor neurons alone caused their death. Since the turn of last century, after discovery of the fi rst genetic cause and subsequent development of the transgenic rodent model, the disease has been realized to be non-ce l-autonomous. In fact, it Is now understood that multiple cellular and support systems are involved in the development and progression of ALS. llieva H, et aS, 2009, Non-cell autonomous toxicity in neurodegenerative disorders; ALS and Beyond. J Cell Biol, 187:761-772.). Addressing only a single pathway is therefore unlikely to produce enough benefit to successfully treat a multi-factor disease.

Most drug actions are single targeted by either increasing the desired target (agonist) or decreasing the undesirable target (antagonist). For example, if the over expression of X causes certain disease, the drug can be an antibody binding to X to decrease its presence.

Central Nervous system diseases are very complex and involve more than a single disease pathway or a single target. What is needed is a high level upstream master regulator which is active and ^' functional in the nervous system development at the embryonic stage, one that may provide mu!ti- targeted therapeutic effect. The invention satisfies this need. Art MNTF analog G 604 was shown to have therapeutic effect through modulating multi-targets is an aspect of the invention. GM604 is the name assigned for ALS therapeutic treatment. 6M602 is assigned for ischemic stroke treatment . G 608 is assigned for Parkinsonigned for treatment.

B8J€F SUMMARY

The inventions described and claimed herein have many attributes and embodiments including, but not limited to, those set forth or described or referenced in this Summary, The inventions described and claimed herein are not limited to or by the features or embodiments identified in this Summary, which ^' is ^' included for purposes of illustration only and not restriction. These and other aspects and embodiments of the inventions described and claimed herein will be apparent from and throughout the application and claims, all of which shall be considered to be a part of the written description thereof. in one aspect, the invention is directed toward methods of monitoring, prognosing, and treating ALS.

Accordingly, in one embodiment a method of monitoring ALS is provided where GiV¾604 is administered to a subject to regulate at least one of the following biomarkers; TDP-43, SODl, and Tau, The regulation of these biomarkers is monitored after administration of GIV1604, and a known parameter associated with ALS disease progression is measured to make an evaluation of ALS disease progression. Then, a determination is made as to whether the regulation of the blornarker correlates with an ALS disease progression determination.

In another embodiment, a method of prognosis for ALS is provided where GM604 is administered to a subject to regulate at least one of the following biomarkers: TDP-43, 5QD1, and Tau, The regulation of these btomarkers is monitored after administration of G 6G4, and a known parameter associated with ALS disease progression is measured to make an evaluation of ALS disease progression. Then, a determination is made as to whether the biomarker predicts the ALS disease progression determination, in another embodiment _, , a method of regulating biomarkers in a subject is provided. In this embodiment, a candidate subject for administration of 6 8G4 is selected. The GM6G4 is administered to the subject in a preselected amount to the subject where the administration regulates at least one of the following biomarkers: TDP-43, SODI, and Tau.

A method of treating ALS is also provided. In one embodiment, the method is carried out by administering GM60 to a subject to regulate at least one of the following biomarkers: TDP-43, SGDi, and Tau. The regulation of the biomarkers {activity and/or expression) is monitored and observed, and then an observation is made to determine whether the regulation of the biomarker correlates with ALS disease progression. In addition to the three biomarkers TDP-43, SODI, and Tau, additional biomarkers such as Cystatin-C, I S1, IRS2, AKT1, PI 3, and C9orf72 can be monitored. In certain preferred embodiments, at least one biomarker is regulated, in other embodiments, two or more biomarkers are regulated. In another aspect, administration of G 604 results in a homeostasis of one or more biomarker. in another aspect, administration of 6fvi60 results in a down regulation of one or more biomarker, in another aspect, administration of GM6Q4 results in an up regulation of one or more biomarker.

BR!EF DESCRSPTiON OF THE DRAWINGS

In Fig. 1 to Fig. 4, The Y-axis represents the percentage change from baseline, where the mean of percentage change in a group is the mean of the percentage changes in each subject of that group.

F G. 1. shows Plasma TOP 43 vs. Tim through Week 12. The Y-axis represents the percentage change from baseline, where the mean of percentage change in a group is the mean of the percentage changes in each subject of that group. Shown on the X-axis are time points in weeks, including at 0, 2, 4, 6, 8, 10, 12 weeks. The triangles in the p fs represent the placebo and the circles represent subjects with GM604 treatment. The dotted fine represents the slope of subjects treated with placebo whil the solid line represents the slope of subjects treated with GIVI604. As shown, piasma TOP-43 was reduced 30% below baseline at week 12.

Fig. 2 shows plasma TDP-43 percent change versus baseline level. The Y-axis represents the percentage change from baseline, where the mean of percentage change in a group is the mean of the percentag changes in each subject of that group. The X-axis represents individual visits at separate time points, that are not to scale however, such that V4 is 1 week after treatment at the beginning of the second week, V6 is the end of th second week when the sixth and final dosage administration of GM6G4 was administered, V7 is 6 weeks after baseiine, and VS is 12 weeks after baseline.

Fig. 3 shows plasma totai Tau percent change versus baseiine level. The Y-axis represents the percentage change from baseline, where the mean of percentage change in a grou is the mean of the percentage changes in each subject of that group. The X-axis represents individual visits at separate time points, that are not to scale however, such that V4 is 1 week after treatment at the beginning of the second week, V6 is the end of the second week when the sixth and final dosage administration of GIV1604 was administered, V7 is 6 weeks after baseiine, and V8 Is 12 weeks after baseiine.

Fig, 4 shows plasma total SOD1 percent change versus baseiine level. The Y-axis represents the percentage change from baseline, where the mean of percentage change in a group is the mean of the percentage changes in each subject of that group. The X-axis represents individual visits at separate time points, that are not to scale however, such that V4 is 1 week after treatment at the beginning of the second week, V6 is the end of the second week when the sixth and final dosage administration of G 604 was administered, V? is 6 weeks after baseline, and V8 is 12 weeks after baseline.

DETAILED DescRtpno

The isolation and characterization of two motoneuronotrophic factors (MNTFi and MNTF2) from rat muscle tissues, as well as the subsequent cloning of a recombinant NTFI-F6 gene derived from a human retinobiastoma cDNA library, is previously described in Appiicanf s prior U.S. Patent No. 6,309,877. The NTF1-F6 gene sequence encodes a 33 amino acid sequence referred to herein as SEQ ID NO:l having the following amino acid sequence: LffT F GDTtNCWMLSAFSft¥¾KCLA£G H DG PTQ [SEQ IO MO;l],

The naturally occurring and recombinant MNTFI polypeptides were shown to selectively enhance the survival in vitro of anterior horn motor neurons isolated from rat lumbar spinal cord expiants. Photomicrographs of treated cultures exhibited neurits outgrowth of myelinated nerve fibers and a marked reduction in the growth of non-neuronal cells, e.g. glial cells and fibroblasts. Similarly, in vivo administration of MNTFi to surgically axotomized rat peripheral nerves resulted in a markedly higher percentage of surviving motor neurons than untreated controls, which could be blocked by co- administratio of anti-M NTFi monoclonal antibody.

Further beneficial effects of MNTFI were demonstrated n rats subjected to spinal cord hemf-section, repaired by a peripheral 'ner e autograft and implanted with MNTFl-containing gel sections in close proximity to the nerve graft junctions with spinal cord, MNTFi treated animals exhibited greater numbers of surviving motor neurons, improved recovery of motor and sensory function, reduced inflammatory response (fewer infiltrating macrophages and lymphocytes) and reduced collagen- containing scar tissue formation at the site of the graft, normal Schwann ceil morphology and normal myelinated and non-myelinated nerve fiber formation.

The proposed approach of using neurotrophic growth factors for the treatment of amyotrophic lateral scierosis has been studied a lot in the 1990s. Recent review bring back interest to the use of neurotrophic growth factors, despite possible reasons for clinical faiiure in the past and reasons for a renewal of hope in this powerful class of drugs for the treatment of ALS, {Henriques A ef al. 2010, Neurotrophic Growt Factors for the Treatment of Amyotrophic Lateral Sclerosis: Where do we stand? Frontiers in Neuroscience. Vol 4 _; Article 32, 1-14).

Motoneurono Trophic Factor (MNTF) peaks in expression during week 9 in human fetus gestation period. {Di„ X. Et al. 1998. Localization and roorphometrie study on Motoneuronotrophic factor 1 and its receptor in developing chorionic villi of human placenta. Acta Anatoroica Sinica 29: 86-89). This expression of MNTF in the developing human embryo prompted us to reason that MNTF may promote the differentiation and/or survival of motor neurons. Indeed, in US Patent No. 8,986,676, it vvas shown that M TF and its peptide analog with retinoic acid have induced differentiation of embryonic stem cell into motor neuron and induced proliferation of the neurons. MNTF or its analog also demonstrated neuroprotective effect via a protein kinase pathway which is modulated by a sonic hedgehog independent pathway to ameliorate or inhibit the progression of neuronal disorder. The inventors have also shown that MNTF is involved in modulating the expression or activity of one or more proteins including a tyrosine kinase, a growth factor, a insulin receptor, S6F-1 receptor, SGF-2 receptor, Shh, Akt, Bad (Scl-2 antagonist of ceii death), PI (3,4,5) P3-dependent kinase {PD 1J, Bax, p53 gene product, ppSO-Src, JAK 2, nitric oxide synthases {NOS}, glycogen synthase kinase 3 (GS ), caspase, P!3 kinase {Phosphatidylinositol 3-kinase), and as.

Two previously unrecognized overlapping domains within the NTF1-F6 molecule that appear to be sufficient for the known biobgicai activities of MNTFl have now been identified. Each of these domains, designated herein as the "W LSAFS" and "FSRVA * domains, are sufficient to stimulate the proliferation of motor neuron derived eel! lines in a manner similar to the MNTFi-FS 33-mer, Similarly, the "FSRYAR" domain is sufficient to direct selective reinnervation of muscle targets by motor neurons in vivo in a manner similarto the MNTF1-F6 33-mer. in addition, the "FSRYAR" domain provides an antigenic epitope sufficient to raise antibody that recognizes any MNTF peptide containing the "FSRYAR" sequence, including the MNTF1-F6 33-mer,

Novel peptides and composition from active fragments of MNTF that are capable of modulating viability and growth in neuronal ceils, and to methods of modulating neuronal cell viability and growth employing the novel peptides and compositions, containing either a "WMLSAF5 domain" or "FSRYAR domain", which is sufficient for neurotrophic or neurotropic function is described in US Patent 7,183,373. The polypeptide domain demonstrated therein were sufficient for the selective maintenance and axonai regeneration of neuronal ceils, and to peptides and/or mo!ecuies capable of mimicking their structure and/or function. Preferred embodiments of that invention comprise a peptide having the amino acid sequence: FSRYAR [SEQ !D NO:2], the sequence of 6M6, also known as 6 604 for ALS indication, as welt as analogues thereof. Preferably such analogues are functional equivalents of the G 604 [SEQ ID NO: 2] .

GM604 is the active domain of fVl TF, an endogenous master neural growth regulator present during the feta! development phase when neurons are being created and reaching terminal synaptic targets. The fetal phase is the most intense and rapid period of human growth and development, especially within the CUS. In preclinical studies, treatment of GM6Q4 in rodents demonstrated that it promotes neural regeneration and exhibit both the trophic and tropic effects (Chau Rfvl et al. 1990 Neuronotrophsc Factor, Chin J. Neuroanat. 6:129-138; Chau RMW et al. 1992. Muscle neurotrophic factors specific for anterior horn motoneurons of rat spinal cord. Recent Adv. Cell Moi Biol. 5:89-94; Yu J. et al. 2008. !Viotoneuronotrophic Factor analog Gfv 604 reduces infarct volume and behavioral deficits .following. transient ischemia in the mouse. Brain Res. 1238:143-153; US 7,183,373).

From the inventors in vitro and in vivo studies, we now understand that the !MNTF 6 mer described herein and referred to as GM6G4 involves multiple mechanisms of action. GM604 binds to the insulin receptor and causes autophosphorylation of Tyr 1162/1163 of insulin receptor and SGF-1 (US 8,986,676). G 604 also activates and modulates pathways through PI3K, as shown in the in vitro study with SH-SY5Y cells Parkinson Disease mode!, treatment with wortmannin (PI3K inhibitor) abrogated the effects of M NTF. implying effect through PBK pathway {US 8,673,852). The inventors have also shown th role of 6M60 in anti-apoptosis, neurogenesis and anti-inflammation. in U.S. Patent Mo. 8 _f 673, 852, the inventors showed that GM604 was able to penetrate the b!ood brain barrier.

A biomarker is defined as any characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacogenomics processes to a therapeutic intervention. Both the FDA and E A recognize the increasingly important role of biomarkers in the drug-development process, For devastating diseases, a search of disease-related biomarker can expedite the identification of a drug target. The use of disease-reiated biomarker as surrogate end points in clinical trials has expedited drug approval in oncology. The potential ciinica! benefits for disease-specific biomarkers include a more rapid and accurate disease diagnosis, and potential reduction in size and duration of clinical drug trials, which would speed up drug development. The application of biomarkers into drug development of ALS disease should both determine if a drug hits its proposed target ("target biomarkers") and whether the drug alters the course of disease ("efficacy biomarke s''},

A surrogate end point is defined as a biomarker that is intended to substitute for a known clinical end point, such as in the case of AIS, A surrogate endpoint is expected to predict benefit (or harm, or lack of benefit) based on epidemiologic, therapeutic, pathophysiologic o other scientific evidence. (Biomarkers Definitions Working Group (2001), Biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clin. Pharmacol. Ther. ;69{3);89-95) Such biomarkers are also frequently used to monitor disease progression in response to therapy.

The diagnosis of ALS is currently limited to evaluations based upon a clinical examination, electrophysiological f indings _, , medical history, and exclusion of ^■ confounding disorders. ALS can be difficult to diagnose during the early stages, and the diagnostic process takes as long as between 13 to 18 months. (A, Radunovic et a!. 2007. Clinical care of patients with amyotrophic lateral sclerosis. The Lancet Neurology, vol. 6, no. 10, pp. 913-925). Reliable biomarkers can serve as a surrogate of disease progression, which would allow the objective measurement of secondary endpoint for drug efficacy rather than relying solely on survival or the revised ALS functional rating scale (ALSFRS-R). ALSFRS-R is a quaiity-of-iife scale but with low sensitivity. Other commonly used clinical measures include muscle strength testing by grip strength machine or Hand Held Dynamometer), respiratory function testing reported as forced vital capacity (FVC). None of these measurements provides any insight into the biological mechanisms of disease or disease progression. Therefore, many investigators for ALS have searched for biomarkers of ALS for provide disease progression to provide insight into the mechanisms of disease.

Despite intensive research that has been conducted over the past 20 years, we also do not currently have practical diagnostic biomarkers. Currently there are no reliable and robust biomarkers capable of indicating the progression of ALS in the clinic or for therapeutic triais. A reliable progression marke should enable shorte trials on a smaller number of patients to be conducted. (H, Ryberg et ai. 2008, Protein biomarkers for amyotrophic lateral sclerosis. Expert Review of Proteomics. vol. 5, no. 2, pp. 249-262; , R, Turner et ai, 2009. Biomarkers in amyotrophic lateral sclerosis. The Lancet Neurology, vol. S, no. 1, pp. 94-109.}. An absence of reliable prognostic biomarkers combined with a lack of understanding of the mechanisms underlying ALS is a substantial problem for furthering the understanding and treatment of this devastating disease. Over the last two decades the search for biomarker has been relentless, and yet none of the proposed biomarkers has been translated into effective tools in the clinical setting. Two obstacles that have been neglected but which are vital toward achieving a better understanding and potential treatments for ALS are developing reliable prognostic biomarkers and a multi-target approach that is not dependent on one single mechanism. Both of these vita! components are provided by the instant invention. in this subject invention, we have identified a combination of biomarkers that are prognostic of ALS and we have shown that G 60 is capable of regulating these biomarkers. With data collected from a Phase 2A double blinded randomized placebo controlled clinical trial with GM604 in ALS patients, 6 604 demonstrated its ability to bring homeostasis in several ALS disease related genes, especially TDP-43, SODl and Tau with statistical signif icance when the plasma biomarkers levels in G 604 treated patients were compared with placebo treated patients. The data from human clinical trial confirmed in vitro data in DNA microarray and PCR arrays in neuronal cells.

Aspects of the invention may also be described as follows;

1. A method of monitoring ALS, the method comprising the steps of: i) administering G 604 to a subject to regulate at least one biomarker in the subject selected from the group consisting of TDP-43,, SODI, and Tau, ii) monitoring the regulation of the at least one biomarker after administration of IMTF, iii) measuring a known parameter associated with ALS disease progression to make an evaluation of ALS disease progression, and iv} making a determination if the regulation of said biomarker correlates with an ALS disease progression determination.

2. A method of monitoring ALS, the method comprising the steps of: i) administering Grv 604 to a subject to regulate the a biomarker, wherein the biomarker comprises TDP-43, it) monitoring the regulation of TDP-43 after administration of MNTF, iii) measuring a known parameter associated with ALS disease progression to make an evaluation of ALS disease progression, and iv) making a determination if the regulation of said biomarker correlates with an ALS disease progression determination.

3. A method of monitoring ALS, the method comprising the steps of: I) administering GIV1604 to a subject to regulate the a biomarker, wherein the biomarker comprises SODl, »} monitoring the regulation of SODl after administration of SvlNTF, iii) measuring a known parameter associated with ALS disease progression to make an evaluation of ALS disease progression, and iv) making a determination if the regulation of said biomarker correlates with an ALS disease progression determination.

4. A method of monitoring ALS, the method comprising the steps of; i) administering GM604 to a subject to regulate the a biomarker, wherein the biomarker comprises Tau, it ^' i monitoring the regulation of Tau after administration of NTF, iii} measuring a known parameter associated with ALS disease progression to make an evaluation of ALS disease progression, and iv) making a determinatio if the regulation of said biomarker correlates with an ALS disease progression determination.

5. A method for monitoring and determining the efficacy of treatment for ALS, the method comprising the steps of: i) selecting a patient, ii J quantifying a biomarker for ALS in said patient, wherein th biomarker is selected from the group consisting of TDP-43, 5QD1, and Tau, iii) classifying the patient as in need of treatment for ALS if the quantity of said biomarker is determined to be above a predetermined leve! for a selected subject, iv} administering G!v1604 to a subject classified as in need of treatment to regulate the biomarker, and v} correlating the regulation of the biomarker with an improvement in ALS disease progression,

6. A method for monitoring and determining the efficacy of treatment for ALS, the method comprising the steps of : i) selecting a patient, it) quantifying a biomarker for ALS in said patient, wherein the biomarker comprises TDP-43, iii) classifying the patient as in need of treatment for ALS if the quantity of said biomarker is determined to be above a predetermined level for a selected subject, Iv) administering GM604 to a subject classified as in need of treatment to regulate TDP-43, and v) correlating the regulation of the biomarker with an improvement in ALS disease progression.

7. A method for monitoring and determining the efficacy of treatment for ALS, the method comprising the steps of: i) seiecting a patient, ii) quantifyirig a biomarker for ALS in said patient, wherein the biomarker comprises SOD1, iii) classifying the patient as in need of treatment for ALS if the quantity of said biomarker is determined to be above a predetermined level for a selected subject, iv) administering G S04 to a subject classified as in need of treatment to regulate SOD1, and v) correlating the regulation of the biomarker with an improvement in ALS disease progression.

8. A method for monitoring and determining the efficacy of treatment for ALS, the method comprising the steps of: i) selecting a patient, ii) quantifying a biomarker for ALS in said paiient, wherein the biomarker comprises Tau, Hi) classifying the patient as in need of treatment for ALS if the quantity of said biomarker is determined to be above a predetermined level for a selected subject, iv) administering GIV56G4 to a subject classified as in need of treatment to regulate Tau, and v) correlating the regulation of the biomarker with an improvement in ALS disease progression.

9. A method of prognosis for ALS, the method comprising the steps of. i) administering GIVS604 to a subject to regulate at least one biomarker in the subject selected from the group consisting of TDP-43, SOD1, and Tau, ii) monitoring the regulation of the at least one biomarker after administration of GM604, Hi) measuring a known parameter associated with ALS disease progression to make an evaluation of ALS disease progression, and iv) making a determination if the regulation of said biomarker predicts the ALS disease progression determination.

10. A method of regulating biomarkers in a subject, the method comprising the steps of: i) selecting a subject for administration of GM8G4, ii) administering the 6 604 in an preselected amount to the subject, iii) wherein the administration regulates at least one biomarkers se!ected from TDP-43, SODi, and Tau,

11. A method of regulating biomarkers in a subject, the method comprising the steps of: i) selecting a subject fo administration of G 804, ii) administering the G 604 in an preselected amount to the subject, iii) wherein the administration regulates the biomarkef TDP-43.

12. A. method of regulating biomarkers in a subject, the method comprising the steps of: i) selecting a subject for administration of GIV1604, ii) administering the GM604 n an preselected amount to the subject, lit) wherein th administration regulates the biomarker SOD1.

13. A method of regulating biomarkers in a subject, the method comprising the steps of: i) selecting a subject for administration of G 604, ii) administering the G 8D4 in an preselected amount to the subject, iii) wherein the administration regulates the biomarker Tau.

14. A method of treating AIS, the method comprising the steps of: i) administering GM604 t a subject to regulate at least one biomarker in the subject selected from the group consisting of TDP-43, SODl, and Tau, ») observing the regulation of the at least one biomarker after administration of !NTF, ii:} correlating the regulation of the biomarker with an improvement in AIS disease progression,

15. A method of treating AIS, the method comprising the steps of: i) administering 6M604 to a subject to regulate a biomarker comprising TDP-43, ii) observing the reguiation of TDP-43 after administration of MNTF, iii) correlating the regulation of TDP-43 with an improvement in ALS disease progression,

16. method of treating ALS, the method comprising the steps of: i) administering GIV16G4 to a subject to regulate a biomarker comprising SODl, ii) observing the reguiation of SODl after administration of NTF, lis} correlating the reguiation of SODl with an improvement in ALS disease progression.

17. method of treating ALS, the method comprising the steps of: i) administering 6M604 to a subject to regulate a biomarker comprising Tau, ii) observing the reguiation of Tau after administration of MNTF, iii) correlating the reguiation of Tau with an improvement in ALS disease progression.

18. A method of monitoring ALS, the method comprising the steps of: i) administering GM604 to a subject to regulate at least one biomarker in the subject seiected from the group consisting of TOP-43, SODl, Tau, Cystatin-C, !RSl, IRS2, AKTl, PI 3, and C9orf72, ii) monitoring the reguiation of the at least one biomarker after administration of MNTF, iii) measuring a known parameter associated with ALS disease progression to make an evaluation of ALS disease progression, and iv) making a determination if the reguiation of said biomarker correlates with an ALS disease progression determination.

19. A method according to any one of claims 1-17, where the administration of GMS04 regulates at least one of the following Cystatin-C, IRS1, IR52, AKTl, PIK3, and C9orf72,

20. A method according to any one of claims 1-18, wherein two or more of the biomarkers are regulated.

21. A method according to any one of claims 1-18,. wherein said regulation results In a homeostasis of one or more biomarker.

22. A method according to any one of claims 1-13, wherein said reguiation resufts in a down regulation of one or more biomarker. 23, A method according to claim 22, wherein said down regulation results in a down regulation of the mRNA expression levels one or more biomarker.

24, A method according to ciaim 22, wherein said down regulation results in a down regulation of the protein expression levels one or more biomarker.

25, A method according to ciaim 22, wherein said down regulation results in a down regulation of the activity of one or more biomarker.

26, A method according to any one of claims 1-18, wherein said regulation results in an up regulation of one or more biomarker.

27, A method according to claim 26, wherein said up regulation resu!ts in an up reg latio of the mRNA expression levels one or more biomarker.

28, A method according to claim 22, wherein said up regulation results in an up regulation of the protein expression levels one or more biomarker.

29, A method according to claim 22, wherein said up regulation results in an up regulatio of the activity of one or m re biomarker.

30, A method according to any one of ciaims 1, 2, S, 9, 10, 11, 14, or 15 wherein TDP-43 mRNA amount or expression level was reduced by about 10%, 20%, or about 30% below baseline levels.

31, A method according to any one of ciaims 1, 2, 5, 9, 10, 11, 14, or 15 wherein plasma TDP-43 protein amount was reduced by about 10%, 20%, or abou 30% below baseSine levels.

32, A method according to any one of claims 1, 2, 5, 9, 10, 11, 14, or 15 wherein plasma TDP-43 protein amount was reduced by about 30% below baseline levels at week 12.

The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLE i Selection of TDP-43, Tau and SOOl as AIS biomarkers TDP-43 is a pathologic hallmark of ALS and prior studies have shown increased feveSs of TDP-43 in ALS biofiuid sampfes.f ^' asai, T et af, 2009. increase TDP-43 protein in cerebrospinaS fluid of patients with amyotrophic lateral sclerosis. Acta Neuropathol 117:55-62; Noto Y et a!. 2011. Elevated CSF TDP-43 !eveis in amyotrophic lateral sclerosis: specificity, sensitivity, and a possible prognostic value. Amyotrophic Lateral Scier, 12(2): p.140-143; Steinacker, P. et al. 2008. TDP-43 in cerebrospinal fluid of patients with frontotemporai lobar degeneration and amyotrophic !abera! sclerosis. Arch Neurol., 64(11): p.1481-1487.) A primary feature of ALS is an accumulation of the protein TDP-43, too much of which is toxic to ceiis. Over 90% of ALS cases exhibit TDP-43 based pathology, developing a treatment that keeps protein levels just right is imperative. Scientists are looking to develop a drug that can target a protective mechanism nonsense-mediated mRNA decay (NMD) triggered by overexpression of wild type (WT) ΪΩΡ- 43,( Barmada S et at 2015. Ameliora tion of toxicity in neuronal models of amyotrophic lateral sclerosis by hUPFl, PNAS 112 (25) 7821-7826.} An in vitro study using patient derived motor neurons generated from induced Piuripotent Stem cells f ^' iPS cells) to screen for drugs that reduce expression levels of TDP-43 showed higher levels of TDP-43 can be neurotoxic and generate cytoplasmic aggregates that impede cellular functions. Compounds that reduce TDP-43 level improve the neurite health. {Egawa N et af. 2012. Drug screening for ALS using patient-specific induced piuripotent stem ceils. Sci Trans! Med 4. 145ral04^, GM604 targeted over expression of TDP-43 and lowered TDP-43 levels. The slope in plasma TDP-43 through week 12 in GM604 treated patients {-3.513 pg mL/wk) is tower than that in placebo treated patients (0,493 pg/ml/wk) with statistical significance, p=0.0078. in this trial TDP-43 plasma was reduced significantly by 30% at week 12 below baseline.

Tau has been a biomarker for neurodegeneration for many years and is extensively marked for Alzheimer's disease, Tau was used as a biomarker to monitor effects of memantine treatment in ALS patients in a phase 2 clinical tria . That trial showed that reduction of Tau !evels in ALS patients due to drug treatment correlated to reductions of clinical parameters of ALS disease progression. {Levine TD et al. 2010. A piiot trial of memantin and riluzole in ALS: correlation to CSF biomarkers. Amyotrophic Lateral Sclerosis, 11:514-519}, in our ALS Phase 2A trial, p!asma Total TAU {evidence of broken axon and continue neuronal degeneration} reduction achieved statistical significance in percentage change between the treated and piacebo patients at 6 weeks, p=0.0369. The toxicity of 50D1 is the result of a gain of toxic function rather than a loss of enzymatic function; thus reducing concentration of the mutant protein is predicted to slow progression of SODl- linked amyotrophic lateral sclerosis. fRothstein JD. 2009, Current hypotheses for the underlying biology of amyotrophic lateral sclerosis. Ann Neurol. 65 Supp! 1:53-9). SODl levels tested in animal models by anti-sense oligonucleotide treatment showed SODI is a good pharmacodynamics marker and lowering SODl prolonged survival. There is a lack of correlation between CSF SODl levels and ALS disease severity and progression, suggesting that once patients become sick, SODl remains stable throughout the disease course unless there is a treatment that can lower SODl level. fWiner L et ai. 2013, SODl in cerebral spinal fluid as a pharmacodynamics marker for antisense oligonucleotide therapy. JAMA Neurol,, 70(2); 201- 207). Lowering SODl was hypothesized to be a therapeutic strategy and phase 1 clinical trial with intrathecal ants-sense oligonucleotide administration was performed and biof!uid SODl level was monitored. {Miller TM et at 2013. An antisense oligonucleotide against SODl delivered intratheca!iy for patient with SODl familial amyotrophic Iaterai sclerosis: a phase 1, randomized, first-in-man study. Lancet Neurol. 12:435-442). The Over expression of wild-type human SODl can also be neurotoxic and may more generally be involved in the pathogenesis of ALS. (Graffmo K et ai, 2012. Expression of wild-type human superoxide dssmutase-1 in mice causes amyotrophic iaterai sclerosis. Human Molecular 6enetics.l-10). All these studies suggested that bwering SODl may be a therapeutic strategy to treat ALS. In this trial SODl plasma in 6M604 treated group showed significant reduction trend when compared with placebo group at 2 weeks, p=O.Q55G.

Biomarke Data Analysis in Phase 2A clinical trial Genervon Bsopharmaceuticafs engaged iron Horse Diagnostic, inc. and its director Robert

Bowser, PhD to test and analyze the CSF and Plasma biomarkers data from GALS-OGl Phase 2A clinical trial. The same biomarker assays are used to test CSF and plasma biomarkers. The piasma btomarkers show more robust results than CSF biomarkers. Biomarker data is basically not able to be influenced by placebo effect. The biomarker study was designed to illuminate whether GM604 might modulate certain genes (e.g. target biomarkers), a process that may take hours to change the protein expression and days or weeks to improve th neurological system.

In the trial summarized in Table 1, five biomarkers were tested and data collected. As shown in the Table 6M604 modulated one target (SODl Plasma and CSF}, one efficacy (Total TAU Plasma,), two target/efficacy biomarkers {TDP-43 Piasma and Cystatin C CSF) and one prognostic biomarker (pNFH CSF). Table i

The observed effects of GM604 on TDP-43, SODl and Cystatin C are consistent with the Neuroprotective properties of GMS04 reported in previous Genervon's PCR studies of Gfv1504 with SHSYSY cells as presented in Example 2,

Biomarkers data may have confirmed the hypotheses and show a trend that 6 604 (1) is a master regulator that cure ALS diseases by responding to distress signals from multiple affected genes (targets), {2} modulates multiple genes by up and down regulate each gene closer to a normal range, and (3) brings homeostasis of affected biological systems. Single target drug molecules such as antibody, inhibitor or blockers usually are unidirectional, e.g. stopping the effect of certain undesirable gene expressions or increase certain gene expression to compensate for the lack thereof. Master regulator peptide drug is not only modulating multiple targets but in each gene the master regulator modulation should be bi-directional depending on the distress signals from the targeted gene. This behavior is consistent with our DNA micro-array and PCR array studies results.

Results of the Disease Progression Analyses for the CSF Biornarkers

There we e no statistically significant differences In the slopes between G1V3604 and placebo for the CSF biornarkers, although there was one non-significant sizable difference at week 6 as shown on Table i. The slope fo GM604 for SOD1 was -1.874 through Week 6 and 15,225 for placebo. The baseline SOOl level in the CSF of GM604 treated patient 0201 was four fold that of other ALS patients in this trial. His SOD1 CSF level was reduced by 50% at end of drug treatment and remained 40% below basal level at the end of study. His ALSF S-R rate of progression was reduced when compared to historic controls, Cystatin C is often reduced i the CSF of ALS patients versus controls, increasing CSF

Cystatin C could have a neuroprotective effect. In the clinical trial biomarker testing CSF Cystatin C level had modest increase in treated group {slope =0.786 @ week 6} but decrease in placebo group (slope 2,284 @ week ,6), although no statistical significance. The clinical biomarker test results seem to confirm that Cystattn C is both a target and efficacy marker of 6M604. ALS patients pNFH are >400pg/ml in CSF and >50 g/m! in plasma. The higher the pNFH, the higher disease Progression Rate. 6M604 treated group had decrease in the CSF pNFH levels while the placebo had less decrease, although no statistical significance.

Results from three plasma n½i«srkers; TDP-43, Toi si Tau and SOD.1

Figure 1 is a plot s owin percentage change in lasma TDP 43 from baseline vs. Time through Week 12, Shown on the X-axis are time points in weeks, including at. Q, 2, 4„ 6„ 8, ί 0, 12 weeks. Plasma TDP-43 was reduced 30% below baseline at week 12. The slope from baseline to week 12 were -3.513 and 0.493 for GM604 treated group and placebo group respectively, p-0.0078. Significan differences, in percentage change between the GM604 treated and placebo patients were also observed in Plasma Total Tau at 6 weeks (p=0.036 ) and SGDiplasma at 2 weeks (p=0.055).

Fig, 2 is a bar chart showing plasma TDP-43 percent change versus baseline level. The X-axis represents .individual visits at separate time points. TDP-43 is a gene .modulated (decreased transcription) by GM604 and thus is also a target of the drug. When TDP-43 data is graphed as percent, change over time versus baseline, the GM604 treated group exhibited decreases at each, visit when compared to baseline, with, more significant decreases by visit 8. Subject 0 _. 01 is removed from this analysis due to hemolysis of the baseline plasma, sample. The placebo group displayed small decreases at visit 4, and modest increases at visits ? and 8. The slope in plasma TDP- 43 through week 12 in GM604 treated (-3.513 pg/mL wk) is lower than placebo (0.493 pg/mL/wk) with statistical significance, p ^::: 0,OO78, It appears that GM604 may have targeted TDP-43 and lowered TDP- 43 levels. Lowering TDP-43 could be a good indication for modulating neurodegeueration.

For Plasma total Tau _> when the results are graphed, as a percent change over time versus baseline (Figure 3) at individual visits at separate time polities, we noted reductions in plasm total Tau at visits 7 and 8 for the GM604 group, but no consistency was observed in the trend of the placebo group. At visit 7 (6 weeks) the GM604 treatment group exhibited a reduction in plasma total Tau and the placebo group an increase in plasms total Tau that is statistically significant (p - 0,0369), Subject 0202 (1,527 pg/mi-out!ier) is removed, from the placebo group in this fignre

The SODi gene is modulated (decreased transcription) b GM604 (prior Genervon s v i t o date results) and therefore is a target of the drug. As shown in Figure 4, the percent change of SODI levels in plasma over time verses baseline at individual visits at separate time points, exhibited a trend with near statistically significant reduced levels in the GM604 treated patients versus the piacebo group at the end of the treatment time (v6, /?=0.055). Also, GM604 treated patient 0101 exhibited a shar reduction in SODi levels in the plasma between visits 4 and 6 dining drag treatment (drop of 60% from 360.52 ng ml to 143.93 ng/ml), suggesting a drug effect. This drop in SO I levels suggests that GM604 may modulate gene expression towards & more homeostatie level typlcai of healthy adults. The baseline plasma sample for patient 1 1 was hemoiyzed and therefore this subject was not included i the data analysis of percent change versus the baseline. .The trends observed in this study would suggest that GM604 may be modulating abnormal SODi protein levels in the CSF and blood of ALS patients.

©M604 regulates homeostasis In addition to testing the biomarker changes in Phase 2A trial patient population,, biomarkers changes were also tested and analyzed with samples from the single, very advanced ALS patient who was treated with GM604 with the approval of the FDA through compassionate use. He has biomarker values below the norma! range at baseline before treatment as we have predicted, 6M60 increased the expression of SOD1. Tau and Cystatln C from the baseline data. The importance of this data is that it showed that GM604 can modulate the same faiomarkers in both directions. Most ALS patients in the Phase 2A trial within 2-years of disease onset have those biomarker data above the normal range and they were lowered by GM604. This advance patient whose biomarker levels were below normal had the same biomarker levels increased. This is the hallmark of homeostatlc process for the health of living organism.

Exam le 2

Biomarker Data and pra-Clinical mRNA PC Array _. Data

Another aspect of the invention is the regulation of various targets of 6IV1604 at the mRNA level, as well as the quantification and correlation of thi reguiation of GM604 targets by in vitro techniques such as the use of microarrays and PCR, Prior ts the 6ALS-001 clinical trial, Geoervon expiored what genes are modulated by GM604 by in vitro methods in DNA microarray and PCR array. The DNA microarray quantified the effect of Gfv1604 on the gene expression profile in the SH-SY5Y {neuroblastoma) ceils, using DNA microarray technique. The resulting data were then analyzed with Genesifter software. A study with PCR array was carried out with a number of ALS genes that were modulated in DNA microarray. SHSY5Y cells were incubated in the presence and absence of G 604. 6 604 was co-cultured with SH5Y5Y cells at multiple concentrations,

RNAs were extracted at 2 hours, 4 hours, 12 hours, 24 hours and 48 hours from the culture incubated with 6M604, and compared with RNA extracted at 0 hour and 48 hour from the control samples. The extracted RNAs were purified and assessed the fold reguiation in comparison with control sampie at 0 hour. The expression of genes of interest was measured by real-time quantitative PCR with PCR with SioRad Detection System.

In the PCR array study, there is a dose dependent effect on modulation by GM604. The modulation effect was also observed longitudinally at 0, 2, 4, 12, 24, 48, hours, with control observed at 0 h and control at 48 hr. PCR array confirmed that 6M604 modulates the mRNA ievels corresponding to the same way G 604 modulates the genes in the ONA microarray.

The results of Foid changes of purified tota! RNA in real-time quantitative PCR of SHSY5Y cells incubated with G 604 and extracted RNA at various time points are summarized below. Table 2

There are a large number of genes related to ALS disease that were modulated in both DNA microarray and PCR array of GM604, indicating that these genes may be targeted by GM604. However the biomarkers for ALS are at an early stage of development and very few good assays were developed and none of them is vaiidated. Therefore Genervon selected only SODl, TDP-43 (TARDBP) and Cystatin C (CST3) from the PCR array tested list and tested for protein expressions in the GALS-G01 ciinica! trial patient CSF and plasma samples and found correiations between the PCR array results and the ciinica! trial biomarker test results.

One of he therapeuticstrategies to treat ALS is by lowering SODl, For SODl, in the clinical trial biomarker test results, SODl ievels in both CSF and piasma are reduced at the end of drug treatment (when samples are collected 2-6 hours after dosing at visit 6), but does not continue to go down and gradually return to a leve! between baseiine and end of drug treatment. As seen in the PCR array, the effect of G 604 at 4 hours lowers SODl level to 0.48, but does not tower the expression of SODl at 48 hours anymore. This may explain why the effects of GM604 on SOD1 levels are more prominent and imminent at the end of drug treatment but not as prominent after cessation of drug treatment. The biomarker results seem to confirm SOD! Is a target of 6 604.

The effect of GM604 on TDP-43 (TARDBP) seems to be longer lasting as shown on the PCR array results. At 48 hours, TDP-43 level still remained low at 0.48. We did not test beyond 48 hours to see how long the effect wiiS last. This may explain why GM604 treatment has a significant effect on t e plasma biomarker TDP-43 across the time interval of 12 weeks, with p=0.0078 when comparing slope from baseline to week 12 between GIV1604 treated and placebo. The biomarker test results seem to confirm that TDP-43 is a target of GMS04.

Cystatin C is often reduced in the CSf of ALS patients versus controls, increasing CSF Cystatin C could have a neuroprotective effect, Cystatin C (CSTS) was tested in PCR array and showed fast increase at 2 hours, peaked at 12 hours, then the modulation effect gradually decreases but stii! has some effect at 48 hours. GEV1604 effect on Cystatin C may be longer lasting than SODl but not as long lasting as in TDP-43. That may explain why in the clinical trial biomarker testing that CSF Cystatin C level had modest increase in treated group. The clinical biomarker test resu!ts seem to confirm that Cystatin C is both a target and efficacy marker of 6MS04.

EXAMPLE 3 HUMAN DATA: Biomarke and Clinical Evaluation of GIV1604 fo Detection and Prognosis of

ALS

* * *

A trial with the title "6M604 Phase 2A randomized double-blind placebo cont olled PiSot TtiaS in Amyotrophic Lateral Sclerosis Disease {ALS}* was completed in 2014. {Protocol no, GALS-001, ! D #118420; c|irticaltriafe,gOV NCT01S54294)The Phase 2A trial was designed to test for safety and to determine whether a six-dose treatment will initiate the disease modification process in ALS patients, such modification to be observed by clinical outcome and measured by biological effects as evidenced by biomarker changes. Both biomarker and clinical data in this small trial demonstrated positive effect in disease modification. Molecular {biomarker} measures in particular are usually immune to change as a result of the psychology of the patient commonly known as placebo effect. The bio markers data showed that most treated patients are good responders to GM604 treatment in ALS disease modification.

A total of twelve patients were enrolled in the ALS Phase 2A study. Six subjects enrolled at Site I and six subjects enrolled at Site 2. Dosing was by slow bolus intravenous administration, once a day for 3 times a week for 2 weeks. Eight patients were randomized to receive 320 mg 6M604 per dose and four patients to receive placebo. Our inclusion/exclusion criteria required the ALS patients in the Phase 2A trial to be "definite" ^" ' ALS sufferers according to El Escorta! criteria (i.e., the disease progression must involve upper and Sower motoneuron and the bulbar region) who had experienced disease onset within 24 months prior to the beginning of the trial. The goal is to enroll fast progressing patients. Our dosing regimen was delivered via intravenous bolus, dosing three times: a week {Mo day, Wednesday, Friday) for two consecutive weeks, for a total of 6 doses. There was no dosing after the two weeks' treatment, and the patients were followed and evaluated for the next ten weeks. The clinical data for the ALSFRS-R, FVC, TUG, and the Grip Strength and HHD were measured at Baseline, Week 2, Week 6, and Week 12. The primary endpolnt was the percentage change at week 12 from baseline of each biomarker in the CSF of each subject. Secondary ertdpoints include percentage change of CSF or plasma biomarkers from baseline to different time points, progressive change in clinical outcomes from baseline to different time points for ALSFRS-R, FVC, grip strength, muscle strength and Time Up and Go test (TUG). Secondary analyses also include comparison of slopes (change in the rate of deciine) for any hint of disease modification. Additionally., placebo patients from a large database of recent clinical trials by the Northesaf ALS Consortium (NEALS) were matched for baserine features and showing stabie rates of decitne as historical controls. The biomarker data and the clinical data are presented in the following pages.

GALS001 trial data showed that 6M604 can start the healing process of ALS by six doses treatment and showed how long the healing process can sustain. Our clinical data showed that the most robust drug effect is at visit 6 (right after the last dosing at Week 2} and visit 7 at Week 6 (4 weeks after end of dosing) but iess robust by visit 8 at Week 12 {10 weeks after dosing).

Because this Phase 2A trial is a pilot study with a very small sample size, it is not reasonabie to expect useful levels of statistical power. The goal was to confirm safety and to find significant efficacy trends in not only clinical data but to correlate with biomarkers data. An unexpected surprise is that both clinical and biomarker data in this small trial have big treatment effect and are well correlated. Summary of Clinical Data

The ci.in.icai eridpoints for the ALSFRS-R _S FVC, TUG, and the Grip Strength and

HHD were measured at Baseline, Week 2, Week 6, aad Week 12. This summary will focus on the. FVC and ALSF S. The Grip Strength and HHD assessment had great variability doe to the different handedness of the patients along with the disease potentially affecting one side of the body in a slightly different manner than the other side. The Grip Strength and HHD results will not be presented here. TUG is also not a good clinical measurement for ALS trial because as ALS disease progressed, many ALS patients will not be able to perform TUG _S such as in this trial, 50% of placebo patient, were not able to perform TUG at week 12. TUG results will not be reported in here.

FVC

The Forced Vital Capacity (FVC) measures the maximal volume of gas that can be expired as forcefully and rapidly as possible after a maximal inspiration to total lung capacity. This measurement will help determine the patient's breathing ability- The operator of the spirometry machine will reset the machine, start and stop the FVC data collection. The testing procedure requires a lot of coaching from the evaiuator: instruct the patient to have an abrupt and unhesitating start, smooth and continuous exhalation until complete, no coughing, during first second, instruct the patient to inspire fully as rapidly as possible, instruct the patient to exhal as rapidly, forcefully, and completely as possible (a minimum of six-seconds of exhalation is recommended), minimum of three acceptable efforts, repeatabfe, acceptable efforts to breathe normally through the pneumotach. Because the accuracy of FVC measurement depends a lot on the operator/evaluator's coaching, the same operator/evaluator should perform FVC testing on the same patient at different time points to avoid inter-rator error.

At Site i, same operator/evaluator performed all the FVC measurements. At Site 2, several different operators/evaiuators performed FVC measurements even for the same patient and is more probable to have inter-rater variability. Also, out of only 8 patients enrolled, one of the patient enrolled at Site 2 was from a foreign country and d d not come back for visit 12 FVC data collection and ^' only left with. FVG data from 7 patients. That renders the Site 2 FVC data not as reliable as Site 1 data, in the pre-specified analysis of FVC from baseline to visit 12 for both Site I and Site 2 combined, there were trends for improvements, (Table 3, - 11.5 vs -4,7}. in light of Site 2 FVC data not being reliable,. FVC data from Site 1 alone is analyzed. Statistically significant differences were observed in change from baseline to week 12 when comparing treatment with placebo using data from Site 1 (Table 4, -28 vs 4.8, p=0.0268). Statistical analysis of the FVC data are presented in the following tables:

Table 3 Change from Saseline to Week 12 in FVC

Table 4 Change from Baseline to Week 12 in FVC at Site 001

Time Point Placebo 6M60

Baseline

N 2 4

Mean 73.5 89.5

Week 12

N 2 4

Mean 45.5 84.8

Change from Baseline

N 2 4

Mean -28.0 -4.8

P-va!ues

Two-sam le t Test 0,0268 Wiicoxon Rank Sum Test 0,1052

The fact that these p-values are significant at one site, or approaching significance with the nonparametric Wiicoxon Rank Sum Test, with so few patients is remarkable. The six doses over two weeks seemed to have started the disease modification process.

ALSFRS-

AISFRS-R is a disease specific subjective functional rating scaie for the AIS patient to answer 12 questions, with 4 being fu!iy functional and 0 being totally not functional, it is assumed that a healthy person has ALSFRS-R score of 4S, As AIS disease progresses, the score decreases. ALSFRS-R is a commonly used clinical outcome endpoint, but it is susceptible to the placebo effect and lowers the power of the study. All the ALS patients enrolled in Phase 2A trial are diagnosed as definite per E! Escoriai criteria and their disease progression as faster than average. Their ALSFRS-R should decrease faster than average ALS patient. Even though there were no significant differences between GM604 and placebo in the decline of ALSFRS-R score at week 12, the smaller decrease in ALSFRS-R showed a trend of disease modification when compare GM6CJ4 treated with placebo patients, -2.7 vs -3.5 respectively {-4.7% versus -9% respectively} in ALSFRS-R score. in a comparison between the slope of ALSFRS-R decline before and after treatment, the slope for the placebo group changes minimally before {-0.037/day} and after (-0.034/day) treatment, while the slope for the GM604 group changes noticeably before and after treatment from -0.046/day before treatment to -0,032/day after treatment. It was found that the slope for the active treatment changed had a 30% decrease in slope compared to ^' before the treatment. SSVIS04 in Phase 2A ALS trial achieved a positive trend of slo wing down disease progression as measured by ALSFRS-R slope. Th ALSFRS-R slope per month was also compared to a historical placebo control of the Ceftriaxone trial showing a statistical power {-0,99 vs -1,97, p=0,0047j. This further analysis was specified in the FDA approved protocol,.

Summary of Ms and SAEs of Phase 2A in ALS tria?

1. Safety and to!erability were evaluated based on the results of adverse events, vital signs, electrocardiography {ECG) measurements, physical and neurological examinations, safety laboratory monitoring, and hypersensitivity and injection site reactions. 2. Of all patients enrolled in the study, 9 patients reported 1 adverse event over the course of the study. Overall, in the 6M604 treatment group, 5 out of S patients experienced at least 1 Treatment-emergent adverse events (TEAE) and 4 out of 4 patients in the placebo treatment group experienced at least 1 TEAE. No unexpected findings in the context of the known safety profile (clinical or pre-clinical) of the GM604 were observed in this study. Consistent with the protocol-defined expected adverse reactions, the most frequently reported AEs by GM604-treated patients in the present study were fails (4 patients, 50%), puncture site pain (3 patients, 37.5%), rash (2 patients) and headache {2 patients, 25%), Of these most commonly reported TEAEs in GM6Q4-treated patients, fails (1 patient, 25%), puncture site pain {1 patient., 25%) and headache (2 patients, 50%) were reported in piacetao-f reated patients,

3. Adverse events in the general disorders and administration site conditions system organ class were the most frequently experienced AE (7 patients and 61 total events in both the GM604 and place bo-treated groups),

4. There was 1 serious adverse event SAE) experienced by a patient in the GM6Q4 treatment group. Patient 0203 who flew back to Germany experienced shortness of breath 24 days after baseline visit that required inpatient hospitalization. The investigator determined the SAE was unrelated to the investigational product,

5. No deaths or withdrawals due to adverse events occurred.

6. There were no clinically meaningful differences noted between patients who received GSV16Q4 and those who received placebo for changes over time in clinical laboratory tests, hematology parameters, or urinalysis results. There were no clinically meaningful differences noted between patients who received G 604 and those who received placebo for changes over time in ECGs, vital signs, physical findings, neurological examination, or other observations related to safety. 7. Grade 1 hypersensitivity reactions were reported by 1 patient receiving placebo treatment (Visit 2) and by I patient receiving GM604 treatment (Visit 5). Ail other patients reported an absence of hypersensitivity (Grade 0) reactions. There were no concerns regarding QT prolongation as no patient receiving treatment with G 604 had OJT or QTcB result above 450 msec. EXAMPLE 4

Clinical Evaluation of 6SVS804 for Treatment of End Stage A IS Patient

This proposed individual patient trial (GALS-C) is for compassionate use for an end stage ALS patient. The rationale is "tim is brain/neurons". We hope to re-innervate the compromised motor neurons in time befbre their rapid progression towards death. We also want to compare how GM604 works in patients with disease onset within 2 years (Phase 2A clinical trial patients) and in an end stage ALS patient.

Case Report

GALS Com assionate Use (GALS-C)

A 46 year old male ALS patient was first diagnosed with the disease in Q1 2005 and by Q3 2008 was quadriplegic and on a ventilator. The FDA approved a compassionate-use application of the MNTF 6mer known as GM604 for this patient (IND # 120052) using a protocol that followed the Phase 2 A trial. This program was thus dubbed GALS-C.

GALS-C Study Design I . Patient provides informed consent will be evaluated to establish baseline for clinical progression and with blood draw by IV and undergoin a lumbar poncture with an Atramatic LP needle for biamarker tests. Blood and CSF will be collected for analyzing specific protein biomakers in the CSF before drug administration. After the first LP, the 1st stud dose will be given.

2. Patient will receive one IV bolus injection once a day for three times a week durin these 2 weeks for a total of six injections.

3. the beginn ing of 2nd week before the 4ih dose, a blood draw for Safet Labs and the 2nd blood biomarker testing will be performed. 4, After the sixth and last dose wait 60 minutes for evaluation, a blood draw for Safety Labs and the 3rd blood bioraarker testing, a second lumbar puncture will be performed to test for CSF bionmrker s. S, At the 7th visit (week 6), after clinical efficacy evaluations., a blood dra for Safety Labs and the 4th blood bionmrker testing will be performed.

6. At the 8th visit (Week 12), after the clinical efficacy ^' evaluations,, a blood draw for Safety Labs and the 5th biood bioraarker testing and a 3rd LP will be performed.

7. All Blood and CSF samples will be collected, prepared, stored frozen at ~

80C and sent for analysis together to test for biomarkers.

8. Exploratory clinical outcome measures will inekide the rate of decline of the ALS Functional Rating Scale Revised (ALSFRS-R), Forced Vital Capacity (FYC), stabi&atiori or improvement in any clonus or spasticity symptoms., speech, swallowing, drooling, lingual/oral muscle, improved time off vent (self-breathing) and any movement or increase movement, of any muscle groups. To estimate the participant's rate of progression prior to treatment, an ALSFRS-R of 48 will be assumed at date of symptom onset. There will be 2 weeks of active treatment, followed by 10 weeks of assessment without active treatment,

9. Plus patient will receive erne booster dose every 12 weeks for three times after the initial 12 weeks.

10. At the 9th to 1th visits (Week 24, 36 and 48), after the clinical efficacy evaluations, blood dra for Safety Labs and the 7th to 9th blood bionmrker testing will be performed,

1 1. A 4th LP will be performed after the clinical efficacy evaluations at the Ϊ 1 th visit (Week 48),

12. Patient to keep detailed diary to record clinical observations. This compassionate trial follows the similar protocol and same dosing regimen as in the Phase 2A ALS trial. 320 mg of GM604 was administered intravenously as a slow bolus, once a day for 3 times a week for 2 weeks. Patient will receive one booster dose every 12 weeks for three times after the initial 12 weeks. The patient was treated with GM604 for two weeks. After two weeks of active treatment, evaluation will be performed after the last dosing at the end of week 2 arid return visits on week 6 plus evaluation on week 12, 24, 36, 48 and 60. The compassionate patient was treated for 2 weeks with six doses of 320 mg GM604 by intravenous administration and followed by 10 weeks without additional treatment. CSF and blood plasma tests and clinical evaluations were performed in various biomarkers (SOD1, Cystatin C, Tan and TDP-43).

Since the compassionate patient was very advanced, the usual clinical evaluations such as ALS functional rating scale (ALSFRS), forced vita! capacity (FVC), and muscle strength were not used. The evaluation of swallowing and speech are more relevant outcomes. The sample collection of CSF by LP is difficult for the patient. The initial CSF before dosing and the CSF sample after the * dose were collected and analyzed. The PI and the drag Sponsor agreed to hold off the CSF sample draws until further notice

The CSF and plasm samples collected in. both the ALS ^' Phase . A trial (GALS-001 trial) and this compassionate trial (GASL-C) were tested and examined by Iron Horse Diagnostics. Changes were identified in specific biomarkers during GM604 treatment in ALS patients. In this case report, GALS-C measured specific biomarkers for target and efficacy. Among these biomarkers ar superoxide dismutase. 1 (SOD I), Cystatin C, and Taw./ Shahim P et al. 201 . Blood biomarkers for brain injury in concussed professional ice hockey players, J AMA Neurol . 71(6): 684-92; Grad LI et al. 2014. intercellular propagated misfoSding of wild-type Cu/Zn Superoxide Dismutase occurs via exosome-dependent and -independent mechanisms. Proc Nat Acad Set USA. i l l : 3620-3625; otunno MS et al. 2013. An Emerging Role for Misf lded Wild-type SOD! in Sporadic ALS Pathogenesis. Front Cell Neurosci.7:253; Wilson ME et al. 2030. Cystatin C: a candidate biomarker for amyotrophic lateral sclerosis. PLoS One. 5:e! 5133; Okamoto et al, 2008. Bunina bodies in araytrophic lateral sclerosis. Neuropathology: Official Journal of the Japanese Society of Neuropathology. 28: 109- 1 15. ) Comparison of the CSF expressions changing from baseline to end of week 2 for SODl _s Cystatin C and Tan for Compassionate use patient (GALS-C), GM 604 treated (GALS-T) and placebo treated (GALS-P) in Phase 2A are summarized in Table 5. SODJ

SODl was the first gene mutation linked with ALS. This genetic mutation, of which multiple types have een identified, comprises only about 20% of genetic cases of ALS and about 2% of overall cases. However; there is growing evidence that wild-type iirisfoided SODl protein, like its geneticaUy-mutarit counterparts, is capable of assuming a form toxic to motor neurons. Moreover, misfolded wiSODl is capable of inducing extracellular spread of the misfolded form via a prion-like propagation mechanism SODl appears in increased quantity in the plasma of PALS, A single misfolded copy of this protein is sufficient to cause prion-like propagation in sporadic ALS patients. Reduction of elevated levels can control this mechanism and also indicates reduced levels of oxidative stress in the CNS SODl levels in ALS patient biofluids were tested as a biomarker to monitor efficacy of anti-sense oligonucleotide treatment to tow er SOD l levels i» animal models of ALS and human patients in early clinical trials .

The normal range of SODl in the CSF in healthy individuals is 50-200 ng/ml, in plasma i healthy individuals is 10-50 ng ml. In most of the GM6Q treated patients in ALS Phase 2 A trial where most patients had symptom onset for less than two years, their plasma SODl expressions were higher than the normal range at baseline. GM604 treated patients in the Phase 2 A trial had their plasma SODl levels reduced. The CSF SODl expressions at baseline i Phase 2 A patients were at the higher end of -normal range. At the end of two weeks, the GM604 treated patients had CSF SODl levels reduced from 186.6 to 53.17 (-3.75%), while the placebo group had the CSF SODl levels increased from 137,94 to 175.86 (+30,45%). In this advanced stage ALS patient, his CSF SODl level at baseline was 27,22 ng/mL, below normal. After 6 doses in two weeks, his CSF SODl level was 30.996 ng mL, increased 3.84%, closer to the normal range.

Please note thai GM604 can modulate the CS SODl biomarkers in both directi ns Cystatin C

Cystati C is a cysteine protease inhibitor widely expressed in the body. It is one of two proteins known to localize to inclusions in neuronal cyiopiasm often referred to as B nina bodies. The normal range of CSF Cystatin C in healthy subjects is 3.0-8.0 pg ml. Cystatin C levels in the CSF of the GALS-C patient at baseline was 1.97 pg/mL _; much below the normal range. After 6 dose in 2 weeks, the CSF Cystatin C level in GALS-C patient is 2.35 pg/ml, increased 1 % towards the normal range. In the GALS-001 trial we found that Cystatin C levels in both treated and placebo group were at the low end of the normal range, GM604 raised Cystatin C levels in the treatment group (3.11 μ /ηιΙ to 3,15 pg/ml, -H .57¼) but the placebo grou kept dropping (3,23 fig ml to 3.06 jtg ml, - 4.57%).

Tau

Tau has been a biomarker of neurodegeneratioo for many years and is extensively used for Alzheimer's disease. Tau is a protein thai stabilizes microtubules. Microtubules make up the cytoskeleton necessary for axon extension and provide "roadways" for intracellular transport, Tau levels can be used to measure neuronal injury such as in the case of concussion is mainly expressed in neurons of the CSSIS and is crucial in axonal maintenance and transport, it is a major component of abnormal neuronal aggregates in many CNS disorders, including Alzheimer's disease (AD). High concentrations of Tau may be the results of broken microtubules and are evidence of active neuronal degeneration during early stage of neurodegenerative disease such as ALS, In advanced stage ALS patient, there is a. lot less intact Microtubules to be broken to release Tau, and as a result, the Tau concentration may be low. Reducing the overall levels of this protein in patients during active stage of neural degeneration may hold therapeutic reievaiKe. That Tau accumulation can only be reversed at an early stage in its AD pathogenesis highlights the need for ^• therapeutic strategies to slow or even stop that accumulation. Researchers have previously used Tau as biomarker to monitor effects o memantine treatment in ALS patients in a phase 2 clinical trial, it was shown that reduction of Tau levels in ALS patients due to drag treatment correlated to reductions of clinical parameters of ALS disease progression.

The normal range of Tau in the CSF of healthy subjects are 100-350 pg/rnL. In GALS-001 trial CSF Tan levels at baseline were at high end of normal or higher than normal. After 6 dose treatment in 2 weeks, the G 604 treated group had CSF tau level lowered (305.03 pg/ml to 303.58 pg/ra!, -3.16%), while the placebo group had CSF tau level increased 386.85 pg/rai. to 412,96 pg/ml, +6.43%) showing continued disease progression, in the GALS-C case, the compassionate patient is already very advanced in the disease progression, the CSF Tau level at baseline was 60.55 pg niL, very below normal range at baseline. After 6 doses in 2 weeks, the CSF Tan level was 63,33 pg/mL, an increase of 4.59 %, closer to the normal range.

The normal range of TDP 43 in plasma in healthy individual is 0-50 pg/mi. The end stage patient receiving G 604 for compassionate use had baseline TDP-43 of 144.54 pg/mi which was very high. At the end of 6 doses, it was 92.59 pg/ml and at the end of 12 weeks it was 52.53 pg/mi {almost normal), GM604 brought the TDP-43 levels towards or almost within the normal range in 12 weeks.

It was a surprise that the end stage patient's baseline TDP-43 was as high as those of the definit ALS patients within 2 years of ALS onset in phase 2A trial {mean 138.88 pg/ml at baseline). At 12 weeks, the mean of the individual placebo patients change of TDP-43 is +6%, The 6IV16Q4 treated patients had change of -30%, ALS Phase 2A, slope in plasma TDP-43 through week 12 in treated (-3.513 pg/mL/wk) is lower than placebo {0,493 pg/mL/wk), p=0.0078. The TDP 43 levei in the end stage patient was normalized in 12 weeks.

According to the Kyoto University research results over expressed TDP-43 spread out from the nucleus to the outside, combined and become toxic and caused the death of the other motor neurons. {Egawa N et a!. 2012. Drug screening for ALS using patient-specific induced p!uripotent stem cells. Sci Trans! Med 4, 145ral04. TDP-43 in ALS patients has shorter axons in motor neurons than healthy subjects. When TDP-43 was decreased the death of neurons were prevented and the projection of nerves previously short became longer. Therefore the effect of the slowing down of the ALS disease progression is expected. TDP-43 is also a major disease protein found in the brains of ALS and Alzheimer patients.

Ohiktif Observations

The summar of the clinical observation filed with FDA by principal investiga tor Dr. Dawn Motyka is as follows. The clinical observation .results of the GALS-C patient revealed important, improvement from baseline to week 12. During the trial, no adverse side effects were noted. At week 2, patient's speech video definitely showed clearer articulation than baseline. At the end of the two weeks six-dose treatment of G 6Q4, the patient's swallowing volume increased to 20cc from a baseline of lOcc. 2 weeks after the two weeks treatment, patient's swallow volume was increased 150%-200% to 25cc-30cc. Five weeks after treatment, the patient consumed 240ec of water in 20~25ce bursts without leakage. Mouth suction of water column height was increased from 5-8cm to 10- 15cm with both I /8 and 1/4 inch straw. The increased swallow and suctson ability metrics are logical in that the hypoglossal nerve is one of the shortest motor neurons in the body. The patient has also experienced improved muscle tone and increased pain in certain body parts, which was norma! muscular pain in the past and had decreased after the patient had become completely paralyzed.

Biomarker data of -the GALS-001 trial patients randomized, to the placebo cohort all showed -abnormal levels. As the trial progressed their biomarker levels continued in the abnormal direction, demonstrating that without treatment the disease will continue to uniformly become worse.

Example 5

Homeostasis n recent ALS research. Dysfunction in RNA processing and protein homeostasis is an emerging theme. (Ling SC et ai. 2013. Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron, 79(3): 416-438) These two processes are intimatel linked, with disease-initiated perturbation of either leading to -further deviation of both protein and RNA homeostasis through a feed-forward loop including eell-to-eell prion-Hke spread that, may represent the mechanism for relentless disease progression. Therefore it can. be hypothesized that a compound that can restore RNA and protein homeostasis can be a target to stop the teed- forward loop and attenuate the disease progression. The Phase 2A and compassionate use clinical studies showed that the biomarker data with GM604 can bring homeostasis, and suggested that may be how G 604 attenuate ALS disease progression. An important and unexpected aspect of the GALS-C biomarker results compared with GALS001 biomarker results is that GM604 demonstrated modulation of biomarker levels in both directions,, always toward normal healthy levels. When die patient's biomarker level was below normal such as in the case of the GALS-C patient in CSF SODl and Tan, treatment of G ^' 604 increased the respective biomarker level to closer to normal range. On the other hand, in the case of GALS001 trial where most ALS patient had disease onset, within two years and the degeneration was active, the CSF SODL and Tau. in both treated and placebo groups were above normal range. Treatment with GM604 reduced these biomarkers closer to normal range. Both SOD l and Tau biomarkers in the placebo group continue to increase indicating continue disease progression. In the case of CSF Cystatin C which helps neuroprotection, the patients in GALS-C and GALS001 trial are all below or at the low end of normal range. It is desirable to raise the Cystatin C level to closer to normal range. The GALS-C patient had Cystatin C level increased by 19%, the GALS001 trial GM604 treated patients had Cystatin C level increased slightly or maintain steady; but the placebo group had Cystatms C level decreased indicating continue disease progress.

Most bioinarker data in SODl , Tau, TDP-43 for ALS patients in Phase 2 A were above normal range and the GM604 treatment down regulated their expression levels. In this advance ALS patient, his data in CSF SOD! and CSF Tau. were below norma! at baseline. Treatment of GM604 up regulated CSF ^' SOD i and CSF Tau, This surprising observation indicated that GM604 can modulate the same biomarker in both up and down regulation directions. This phenomenon is very unique, and is the hallmark of homeostasis.

Exclusion from homeostasis comparison table: The plasma level for SODl , Tau in the End Stage patient were NOT below normal but were above normal similar to other Phase 2A patients and therefore wer not used in the homeostasis comparison table to show the contrast. Plasma TOP 43 was also above normal similar to the Phase 2A patients, and was lowered from S44 pg m! at baseline to ~ 50 pg/mL (normal) at week 12 similar to the other Phase 2 A patients who had disease onset within two years, and therefore is also not included in the homeostasis comparison table to show the contrast.

Table 5

ΤΌΡ-43 ^' Three well, known ALS bionmrkers SODL Cystatin C and Tan were modulated by GM604 significantly towards normal range after treatment Based on this compassi nate-use study on a late stage patient, ALS biomarker expression and clinical signs were attenuated after 2 weeks of 6 doses of GM604, suggesting that G 604 might present as a potential therapeutic for ALS. The compare/contrast with the Phase 2A data is to show that GM604 regulates protek/eytokke levels both up and down, always in the direction of normal levels, as GM604 is a regulator of neuronal homeostasis.

ALS is devastating neurodegenerative disease. It is invariably fatal with most patients dying within 3 years of diagnosis. Until recently the disease was believed to involve only the motor neurons with the cause a complete mystery. Multiple attempts to teat the disease with single-target tiierapies have all failed. (Lodolph AC et al. 2009. Evidence-based drug treatment in amyotrophic lateral sclerosis and upcoming clinical trials. Ther Adv Neurol Disord, 2(5); 319- 326), Within the last two decades it has become clear that ALS involves multiple pathways, including factors beyond the motor neurons and outside the CNS. Even within just the motor neurons there are multiple identified pathologies occurring during ALS progression. A therapy addressing only a single one of these multiple targets is unlikely to be sufficient to arrest the disease. Note that GM604 cm modulate multiple biomarkers (Multi-target) and in both directions, the hallmark of homeostatic processes of living organisms. The inventors are pleased to .report the positive result of the post hoc analysis of the homeostasis hypothesis even though there is only one patient who has the ALS disease for 10 years.

in this compassionate use study, GM604 has data that shows homeostatic effect.

Not only has GM604 demonstrated statistical efficacy as shown above, but its safety profile is excellent with no significant drug related adverse events in any treatment grou patients (including the inventors' trial programs for other neurodegenerati e diseases).

The available data on GM604 is extremely safe and positive in metrics including: demonstrated safety, statistically-significant efficacy betwee treated and placebo groups i btomarker data; significant efficacy between treated and historical placebo groups in clinical data; and demonstrated ability to regulate homeostasis. For these reasons we believe that the modulations in multiple biomarkers by GM604 can be a prognostic application for ALS diseas progression and the consequential clinical benefits represent a potent treatment for ALS as well as other neurodegenerati ve diseases.

From the foregoing, it will be appreciated that, although specific embodiments of the invention, have been described herein for the purpose of illustration, various modifications- may be made without deviating from the spirit and scope of the invention. Accordingly, the present invention is not limited except as by the appended claims.

All patents, patent applications, publications, scientific articles, web sites, and other documents and materials referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced document and material is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Additionally, all claims in this application, and all priority applications, including but not limited to original claims, are hereby incorporated in their entirety into, and form a part of, the written description of the invention... Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such patents, applications, publications, scientific articles, web sites, electronically available information, and other referenced materials or documents; Applicants reserve the right to physically incorporate into any part of this document, including any part of the written description, the claims referred to above including hut not limited to any original claims.

The specific methods and compositions described herein are- representative of preferred embodiments and are exemplary and not intended as limitations on the scope o the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideratio of this specification, and are encompassed within the spirit of the invention as defined fay the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and ffiodiiicatioiis may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustrati el -described herein: suitably may he practiced n the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential Thus, for example, in each instance herein, in embodiments or examples of Hie present invention, any of the terms "comprising' ^' ' ^' , "consisting essentially of, and "consisting of ma be replaced with either of the other two terms in the specification. Also, the terms "comprising", "including", containing", etc. are to he read expansively and without limitation. The methods md processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein, o in the claims, it is also that as used herein and in the appended claims, the singular forms "a " "an and "the" include plural reference unless the context clearly dictates otherwise. T hus, tor example, a reference to "a host ceil" includes a plurality (for example, a culture or population) of such host ceils, and. so forth. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under -no circumstances may the patent be interpreted to he limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsi ve writing by Applicants.

The terms and expressions that have been employed are used as terms of description and not of limitation, and there is n intent in the use of such .terms and expressions to exclude an equivalent of the features: reported and described or portions thereof, but it is recognized, that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

The invention has been described broad!y and genericaliy herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure aiso form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of arkush groups, those skilled in the art will recognize that the invention is aiso thereby described fn terms of any individual member or subgroup of members of the Markush group.